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Les 20 dernières publications

Global genome decompaction leads to stochastic activation of gene expression as a first step toward fate commitment in human hematopoietic cells.

Author(s) : Parmentier R, Racine L, Moussy A, Chantalat S, Sudharshan R, Papili Gao N, Stockholm D, Corre G, Fourel G, Deleuze J, Gunawan R, Paldi A,
Journal : PLoS Biol
2022
When human cord blood-derived CD34+ cells are induced to differentiate, theyundergo rapid and dynamic morphological and molecular transformations that arecritical for fate commitment. In particular, the cells pass through a transitoryphase known as "multilineage-primed" state. These cells are characterized by amixed gene expression profile, different in each cell, with the coexpression ofmany genes characteristic for concurrent cell lineages. The aim of our study isto understand the mechanisms of the establishment and the exit from thistransitory state. We investigated this issue using single-cell RNA sequencing andATAC-seq. Two phases were detected. The first phase is a rapid and globalchromatin decompaction that makes most of the gene promoters in the genomeaccessible for transcription. It results 24 h later in enhanced and pervasivetranscription of the genome leading to the concomitant increase in thecell-to-cell variability of transcriptional profiles. The second phase is theexit from the multilineage-primed phase marked by a slow chromatin closure and asubsequent overall down-regulation of gene transcription. This process isselective and results in the emergence of coherent expression profilescorresponding to distinct cell subpopulations. The typical time scale of theseevents spans 48 to 72 h. These observations suggest that the nonspecificity ofgenome decompaction is the condition for the generation of a highly variablemultilineage expression profile. The nonspecific phase is followed by specificregulatory actions that stabilize and maintain the activity of key genes, whilethe rest of the genome becomes repressed again by the chromatin recompaction.Thus, the initiation of differentiation is reminiscent of a constrainedoptimization process that associates the spontaneous generation of geneexpression diversity to subsequent regulatory actions that maintain the activityof some genes, while the rest of the genome sinks back to the repressive closedchromatin state.

Painters in chromatin: a unified quantitative framework to systematically characterize epigenome regulation and memory.

Author(s) : Abdulla A, Vaillant C, Jost D,
Journal : Nucleic Acids Res
2022
In eukaryotes, many stable and heritable phenotypes arise from the same DNAsequence, owing to epigenetic regulatory mechanisms relying on the molecularcooperativity of 'reader-writer' enzymes. In this work, we focus on thefundamental, generic mechanisms behind the epigenome memory encoded bypost-translational modifications of histone tails. Based on experimentalknowledge, we introduce a unified modeling framework, the painter model,describing the mechanistic interplay between sequence-specific recruitment ofchromatin regulators, chromatin-state-specific reader-writer processes andlong-range spreading mechanisms. A systematic analysis of the model buildingblocks highlights the crucial impact of tridimensional chromatin organization andstate-specific recruitment of enzymes on the stability of epigenomic domains andon gene expression. In particular, we show that enhanced 3D compaction of thegenome and enzyme limitation facilitate the formation of ultra-stable, confinedchromatin domains. The model also captures how chromatin state dynamics impactthe intrinsic transcriptional properties of the region, slower kinetics leadingto noisier expression. We finally apply our framework to analyze experimentaldata, from the propagation of γH2AX around DNA breaks in human cells to themaintenance of heterochromatin in fission yeast, illustrating how the paintermodel can be used to extract quantitative information on epigenomic molecularprocesses.

Dynamical modeling of the H3K27 epigenetic landscape in mouse embryonic stem cells.

Author(s) : Newar K, Abdulla A, Salari H, Fanchon E, Jost D,
Journal : PLoS Comput Biol
2022
The Polycomb system via the methylation of the lysine 27 of histone H3 (H3K27)plays central roles in the silencing of many lineage-specific genes duringdevelopment. Recent experimental evidence suggested that the recruitment ofhistone modifying enzymes like the Polycomb repressive complex 2 (PRC2) atspecific sites and their spreading capacities from these sites are key to theestablishment and maintenance of a proper epigenomic landscape aroundPolycomb-target genes. Here, to test whether such mechanisms, as a minimal set ofqualitative rules, are quantitatively compatible with data, we developed amathematical model that can predict the locus-specific distributions of H3K27modifications based on previous biochemical knowledge. Within the biologicalcontext of mouse embryonic stem cells, our model showed quantitative agreementwith experimental profiles of H3K27 acetylation and methylation aroundPolycomb-target genes in wild-type and mutants. In particular, we demonstratedthe key role of the reader-writer module of PRC2 and of the competition betweenthe binding of activating and repressing enzymes in shaping the H3K27 landscapearound transcriptional start sites. The predicted dynamics of establishment andmaintenance of the repressive trimethylated H3K27 state suggest a slowaccumulation, in perfect agreement with experiments. Our approach represents afirst step towards a quantitative description of PcG regulation in variouscellular contexts and provides a generic framework to better characterizeepigenetic regulation in normal or disease situations.

Detection of Homologous Recombination Intermediates via Proximity Ligation and Quantitative PCR in Saccharomyces cerevisiae

Author(s) : Reitz D, Savocco J, Piazza A, Heyer W,
Journal : J Vis Exp
2022
DNA damage, including DNA double-stranded breaks and inter-strand cross-links, incurred during the S and G2 phases of the cell cycle can be repaired by homologous recombination (HR). In addition, HR represents an important mechanism of replication fork rescue following stalling or collapse. The regulation of the many reversible and irreversible steps of this complex pathway promotes its fidelity. The physical analysis of the recombination intermediates formed during HR enables the characterization of these controls by various nucleoprotein factors and their interactors. Though there are well-established methods to assay specific events and intermediates in the recombination pathway, the detection of D-loop formation and extension, two critical steps in this pathway, has proved challenging until recently. Here, efficient methods for detecting key events in the HR pathway, namely DNA double-stranded break formation, D-loop formation, D-loop extension, and the formation of products via break-induced replication (BIR) in Saccharomyces cerevisiae are described. These assays detect their relevant recombination intermediates and products with high sensitivity and are independent of cellular viability. The detection of D-loops, D-loop extension, and the BIR product is based on proximity ligation. Together, these assays allow for the study of the kinetics of HR at the population level to finely address the functions of HR proteins and regulators at significant steps in the pathway.

Altered splicing of ATG16-L1 mediates acquired resistance to tyrosine kinase inhibitors of EGFR by blocking autophagy in non-small cell lung cancer.

Author(s) : Hatat A, Benoit-Pilven C, Pucciarelli A, de Fraipont F, Lamothe L, Perron P, Rey A, Levra M, Toffart A, Auboeuf D, Eymin B, Gazzeri S,
Journal : Mol Oncol
2022
Despite the initial efficacy of using tyrosine kinase inhibitors of epidermalgrowth factor receptors (EGFR-TKIs) for treating patients with non-small celllung cancer (NSCLC), resistance inevitably develops. Recent studies highlight alink between alternative splicing and cancer drug response. Therefore, we aimedto identify deregulated splicing events that play a role in resistance toEGFR-TKI. By using RNA sequencing, reverse-transcription PCR (RT-PCR), and RNAinterference, we showed that overexpression of a splice variant of the autophagicgene ATG16-L1 that retains exon 8 and encodes the β-isoform of autophagy-relatedprotein 16-1 (ATG16-L1 β) concurs acquired resistance to EGFR-TKI in NSCLC cells.Using matched biopsies, we found increased levels of ATG16-L1 β at the time ofprogression in 3 of 11 NSCLC patients treated with EGFR-TKI. Mechanistically,gefitinib-induced autophagy was impaired in resistant cells that accumulatedATG16-L1 β. Neutralization of ATG16-L1 β restored autophagy in response togefitinib, induced apoptosis, and inhibited the growth of in ovo tumorxenografts. Conversely, overexpression of ATG16-L1 β in parental sensitive cellsprevented gefitinib-induced autophagy and increased cell survival. These resultssupport a role of defective autophagy in acquired resistance to EGFR-TKIs andidentify splicing regulation of ATG16-L1 as a therapeutic vulnerability thatcould be explored for improving EGFR-targeted cancer therapy.

RNA helicase-dependent gene looping impacts messenger RNA processing.

Author(s) : Terrone S, Valat J, Fontrodona N, Giraud G, Claude J, Combe E, Lapendry A, Polvèche H, Ameur L, Duvermy A, Modolo L, Bernard P, Mortreux F, Auboeuf D, Bourgeois C,
Journal : Nucleic Acids Res
2022
DDX5 and DDX17 are DEAD-box RNA helicase paralogs which regulate several aspectsof gene expression, especially transcription and splicing, through incompletelyunderstood mechanisms. A transcriptome analysis of DDX5/DDX17-depleted humancells confirmed the large impact of these RNA helicases on splicing and revealeda widespread deregulation of 3' end processing. In silico analyses andexperiments in cultured cells showed the binding and functional contribution ofthe genome organizing factor CTCF to chromatin sites at or near a subset ofDDX5/DDX17-dependent exons that are characterized by a high GC content and a highdensity of RNA Polymerase II. We propose the existence of an RNAhelicase-dependent relationship between CTCF and the dynamics of transcriptionacross DNA and/or RNA structured regions, that contributes to the processing ofinternal and terminal exons. Moreover, local DDX5/DDX17-dependent chromatin loopsspatially connect RNA helicase-regulated exons with their cognate promoter, andwe provide the first direct evidence that de novo gene looping modifiesalternative splicing and polyadenylation. Overall our findings uncover the impactof DDX5/DDX17-dependent chromatin folding on pre-messenger RNA processing.

Real age prediction from the transcriptome with RAPToR

Author(s) : Bulteau R, Francesconi M,
Journal : Nature Methods
2022
Transcriptomic data is often affected by uncontrolled variation among samples that can obscure and confound the effects of interest. This variation is frequently due to unintended differences in developmental stages between samples. The transcriptome itself can be used to estimate developmental progression, but existing methods require many samples and do not estimate a specimen’s real age. Here we present real-age prediction from transcriptome staging on reference (RAPToR), a computational method that precisely estimates the real age of a sample from its transcriptome, exploiting existing time-series data as reference. RAPToR works with whole animal, dissected tissue and single-cell data for the most common animal models, humans and even for non-model organisms lacking reference data. We show that RAPToR can be used to remove age as a confounding factor and allow recovery of a signal of interest in differential expression analysis. RAPToR will be especially useful in large-scale single-organism profiling because it eliminates the need for accurate staging or synchronisation before profiling.

Myotonic dystrophy RNA toxicity alters morphology, adhesion and migration of mouse and human astrocytes

Author(s) : Dincã D, Lallemant L, González-Barriga A, Cresto N, Braz S, Sicot G, Pillet L, Polvèche H, Magneron P, Huguet-Lachon A, Benyamine H, Azotla-Vilchis C, Agonizantes-Juárez L, Tahraoui-Bories J, Martinat C, Hernández-Hernández O, Auboeuf D, Rouach N, Bourgeois C, Gourdon G, Gomes-Pereira M,
Journal : Nat Commun
2022
Brain dysfunction in myotonic dystrophy type 1 (DM1), the prototype of toxic RNAdisorders, has been mainly attributed to neuronal RNA misprocessing, while littleattention has been given to non-neuronal brain cells. Here, using a transgenicmouse model of DM1 that expresses mutant RNA in various brain cell types(neurons, astroglia, and oligodendroglia), we demonstrate that astrocytes exhibitimpaired ramification and polarization in vivo and defects in adhesion,spreading, and migration. RNA-dependent toxicity and phenotypes are also found inhuman transfected glial cells. In line with the cell phenotypes, molecularanalyses reveal extensive expression and accumulation of toxic RNA in astrocytes,which result in RNA spliceopathy that is more severe than in neurons. Astrocytemissplicing affects primarily transcripts that regulate cell adhesion,cytoskeleton, and morphogenesis, and it is confirmed in human brain tissue. Ourfindings demonstrate that DM1 impacts astrocyte cell biology, possiblycompromising their support and regulation of synaptic function.

DNA:RNA Immunoprecipitation from S. pombe Cells for qPCR and Genome-Wide Sequencing.

Author(s) : Vachez L, Teste C, Vanoosthuyse V,
Journal : Methods Mol Biol
2022
By temporarily distorting the DNA double helix, the moving RNA polymerases can lead to the formation of non-B DNA structures. One of the most abundant and largest non-B DNA structures in the genome is the R-loop, a three-stranded structure forming when the nascent RNA hybridizes with its DNA template, thereby extruding the non-template DNA strand. Growing evidence suggests that at least a subset of R-loops could induce transcription stress and genome instability, although the direct, primary consequences of R-loop formation on the surrounding chromatin are still unclear. To understand the direct impact of R-loops on transcription and genome stability, accurate and quantitative mapping of R-loops is essential. R-loop mapping is commonly achieved using the antibody-based DNA:RNA Immunoprecipitation (DRIP) strategy. While it is reasonably straightforward to obtain robust DRIP enrichments from human cells, this has proved harder in yeast, where DRIP signals are often relatively weak, with a poor signal-to-noise ratio. Although it is unclear whether such weak signals stem from a technical or a biological reality, they make the accurate quantification of DRIP signals all the more important, especially when deep sequencing is used to monitor and quantify the distribution of R-loops genome-wide. Here we propose a DRIP protocol that has been optimized for the mapping and the quantification of R-loops in Schizosaccharomyces pombe but that can also be used in Saccharomyces cerevisiae. As a result, this protocol can be used to generate calibrated DRIP-seq data, where genomic DNA extracted from S. cerevisiae serves as spike-in reference.

Structural mechanism underpinning Thermus oshimai Pif1-mediated G-quadruplex unfolding.

Author(s) : Dai Y, Guo H, Liu N, Chen W, Ai X, Li H, Sun B, Hou X, Rety S, Xi X,
Journal : EMBO Rep
2022
G-quadruplexes (G4s) are unusual stable DNA structures that cause genomicinstability. To overcome the potential barriers formed by G4s, cells have evolveddifferent families of proteins that unfold G4s. Pif1 is a DNA helicase fromsuperfamily 1 (SF1) conserved from bacteria to humans with high G4-unwindingactivity. Here, we present the first X-ray crystal structure of the Thermusoshimai Pif1 (ToPif1) complexed with a G4. Our structure reveals that ToPif1recognizes the entire native G4 via a cluster of amino acids at domains 1B/2Bwhich constitute a G4-Recognizing Surface (GRS). The overall structure of the G4maintains its three-layered propeller-type G4 topology, without significantreorganization of G-tetrads upon protein binding. The three G-tetrads in G4 arerecognized by GRS residues mainly through electrostatic, ionic interactions, andhydrogen bonds formed between the GRS residues and the ribose-phosphate backbone.Compared with previously solved structures of SF2 helicases in complex with G4,our structure reveals how helicases from distinct superfamilies adopt differentstrategies for recognizing and unfolding G4s.

A Perspective for Ménière's Disease: In Silico Investigations of Dexamethasone as a Direct Modulator of AQP2.

Author(s) : Mom R, Robert-Paganin J, Mom T, Chabbert C, Réty S, Auguin D,
Journal : Biomolecules
2022
Ménière's disease is a chronic illness characterized by intermittent episodes ofvertigo associated with fluctuating sensorineural hearing loss, tinnitus andaural pressure. This pathology strongly correlates with a dilatation of the fluidcompartment of the endolymph, so-called hydrops. Dexamethasone is one of thetherapeutic approaches recommended when conventional antivertigo treatments havefailed. Several mechanisms of actions have been hypothesized for the mode ofaction of dexamethasone, such as the anti-inflammatory effect or as a regulatorof inner ear water homeostasis. However, none of them have been experimentallyconfirmed so far. Aquaporins (AQPs) are transmembrane water channels and arehence central in the regulation of transcellular water fluxes. In the presentstudy, we investigated the hypothesis that dexamethasone could impact waterfluxes in the inner ear by targeting AQP2. We addressed this question throughmolecular dynamics simulations approaches and managed to demonstrate a directinteraction between AQP2 and dexamethasone and its significant impact on thechannel water permeability. Through compartmentalization of sodium and potassiumions, a significant effect of Na+ upon AQP2 water permeability was highlighted aswell. The molecular mechanisms involved in dexamethasone binding and in itsregulatory action upon AQP2 function are described.

3DGenBench: a web-server to benchmark computational models for 3D Genomics.

Author(s) : Belokopytova P, Viesná E, Chiliński M, Qi Y, Salari H, Di Stefano M, Esposito A, Conte A, Chiariello A, Teif V, Plewczynski D, Zhang B, Jost D, Fishman V,
Journal : Nucleic Acids Res
2022
Modeling 3D genome organisation has been booming in the last years thanks to theavailability of experimental datasets of genomic contacts. However, the field iscurrently missing the standardisation of methods and metrics to comparepredictions and experiments. We present 3DGenBench, a web server available athttps://inc-cost.eu/benchmarking/, that allows benchmarking computational modelsof 3D Genomics. The benchmark is performed using a manually curated dataset of 39capture Hi-C profiles in wild type and genome-edited mouse cells, and fivegenome-wide Hi-C profiles in human, mouse, and Drosophila cells. 3DGenBenchperforms two kinds of analysis, each supplied with a specific scoring module thatcompares predictions of a computational method to experimental data using severalmetrics. With 3DGenBench, the user obtains model performance scores, allowing anunbiased comparison with other models. 3DGenBench aims to become a reference webserver to test new 3D genomics models and is conceived as an evolving platformwhere new types of analysis will be implemented in the future.

FORK-seq: Single-Molecule Profiling of DNA Replication.

Author(s) : Hennion M, Theulot B, Arbona J, Audit B, Hyrien O,
Journal : Methods Mol Biol
2022
Most genome replication mapping methods profile cell populations, maskingcell-to-cell heterogeneity. Here, we describe FORK-seq, a nanopore sequencingmethod to map replication of single DNA molecules at 200 nucleotide resolutionusing a nanopore current interpretation tool allowing the quantification of BrdUincorporation. Along pulse-chased replication intermediates from Saccharomycescerevisiae, we can orient replication tracks and reproduce population-basedreplication directionality profiles. Additionally, we can map individualinitiation and termination events. Thus, FORK-seq reveals the full extent ofcell-to-cell heterogeneity in DNA replication.

Genome-wide mapping of individual replication fork velocities using nanopore sequencing.

Author(s) : Theulot B, Lacroix L, Arbona J, Millot G, Jean E, Cruaud C, Pellet J, Proux F, Hennion M, Engelen S, Lemainque A, Audit B, Hyrien O, Le Tallec B,
Journal : Nat Commun
2022
Little is known about replication fork velocity variations along eukaryoticgenomes, since reference techniques to determine fork speed either provide nosequence information or suffer from low throughput. Here we presentNanoForkSpeed, a nanopore sequencing-based method to map and extract the velocityof individual forks detected as tracks of the thymidine analoguebromodeoxyuridine incorporated during a brief pulse-labelling of asynchronouslygrowing cells. NanoForkSpeed retrieves previous Saccharomyces cerevisiae meanfork speed estimates (≈2 kb/min) in the BT1 strain exhibiting highly efficientbromodeoxyuridine incorporation and wild-type growth, and precisely quantifiesspeed changes in cells with altered replisome progression or exposed tohydroxyurea. The positioning of >125,000 fork velocities provides a genome-widemap of fork progression based on individual fork rates, showing a uniform forkspeed across yeast chromosomes except for a marked slowdown at known pausingsites.

Titration of Apparent In-Cellula Affinities of Protein-Protein Interactions.

Author(s) : Cluet D, Vergier B, Levy N, Dehau L, Thurman A, Amri I, Spichty M,
Journal : Chembiochem
2022
A genetic assay permits simultaneous quantification of two interacting proteinsand their bound fraction at the single-cell level using flow cytometry. Apparentin-cellula affinities of protein-protein interactions can be extracted from theacquired data through a titration-like analysis. The applicability of thisapproach is demonstrated on a diverse set of interactions with proteins fromdifferent families and organisms and with in-vitro dissociation constants rangingfrom picomolar to micromolar.

Prognostic impact of ABCA3 expression in adult and pediatric acute myeloid leukemia: an ALFA-ELAM02 joint study

Author(s) : Ceraulo A, Lapillonne H, Cheok M, Preudhomme C, Dombret H, Terré C, Lambert J, Leverger G, Bertrand Y, Mortreux F, Wattel E,
Journal : Blood Adv
2022

Evolutionary divergence of anaphase spindle mechanics in nematode embryos constrained by antagonistic pulling and viscous forces.

Author(s) : Khatri D, Brugière T, Athale C, Delattre M,
Journal : Mol Biol Cell
2022
Cellular functions like cell division are remarkably conserved across phyla.However the evolutionary principles of cellular organization that drive it areless well explored. Thus, an essential question remains: to what extent cellularparameters evolve without altering the basic function they sustain? Here we haveobserved 6 different nematode species for which the mitotic spindle is positionedasymmetrically during the first embryonic division. Whereas the C. elegansspindle undergoes oscillations during its displacement, the spindle elongateswithout oscillations in other species. We asked which evolutionary changes inbiophysical parameters could explain differences in spindle motion whilemaintaining a constant output. Using laser microsurgery of the spindle werevealed that all species are subjected to cortical pulling forces, of varyingmagnitudes. Using a viscoelastic model to fit the recoil trajectories and with anindependent measurement of cytoplasmic viscosity, we extracted the values ofcytoplasmic drag, cortical pulling forces and spindle elasticity for all species.We found large variations in cytoplasmic viscosity whereas cortical pullingforces and elasticity were often more constrained. In agreement with previoussimulations, we found that increased viscosity correlates with decreasedoscillation speeds across species. However, the absence of oscillations despitelow viscosity in some species, can only be explained by smaller pulling forces.Consequently, we find that spindle mobility across the species analyzed here ischaracterized by a tradeoff between cytoplasmic viscosity and pulling forcesnormalized by the size of the embryo. Our work provides a framework forunderstanding mechanical constraints on evolutionary diversification of spindlemobility.

Paternal transmission of the Wolbachia CidB toxin underlies cytoplasmic incompatibility.

Author(s) : Horard B, Terretaz K, Gosselin-Grenet A, Sobry H, Sicard M, Landmann F, Loppin B,
Journal : Curr Biol
2022
Wolbachia are widespread endosymbiotic bacteria that manipulate the reproduction ofarthropods through a diversity of cellular mechanisms. In cytoplasmicincompatibility (CI), a sterility syndrome originally discovered in the mosquitoCulex pipiens, uninfected eggs fertilized by sperm from infected males areselectively killed during embryo development following the abortive segregation ofpaternal chromosomes in the zygote. Despite the recent discovery of Wolbachia CIfactor (cif) genes, the mechanism by which they control the fate of paternalchromosomes at fertilization remains unknown. Here, we have analyzed the cytologicaldistribution and cellular impact of CidA and CidB, a pair of Cif proteins from theCulex-infecting Wolbachia strain wPip. We show that expression of CidB in DrosophilaS2R+ cells induces apoptosis unless CidA is co-expressed and associated with itspartner. In transgenic Drosophila testes, both effectors colocalize in germ cellsuntil the histone-to-protamine transition in which only CidB is retained in maturingspermatid nuclei. We further show that CidB is similarly targeted to maturing spermof naturally infected Culex mosquitoes. At fertilization, CidB associates withpaternal DNA regions exhibiting DNA replication stress, as a likely cause ofincomplete replication of paternal chromosomes at the onset of the first mitosis.Importantly, we demonstrate that inactivation of the deubiquitylase activity of CidBdoes not abolish its cell toxicity or its ability to induce CI in Drosophila. Ourstudy thus demonstrates that CI functions as a transgenerational toxin-antidotesystem and suggests that CidB acts by poisoning paternal DNA replication inincompatible crosses.

Three classes of epigenomic regulators converge to hyperactivate the essential maternal gene deadhead within a heterochromatin mini-domain.

Author(s) : Torres-Campana D, Horard B, Denaud S, Benoit G, Loppin B, Orsi G,
Journal : PLoS Genet
2022
The formation of a diploid zygote is a highly complex cellular process that isentirely controlled by maternal gene products stored in the egg cytoplasm. Thishighly specialized transcriptional program is tightly controlled at the chromatinlevel in the female germline. As an extreme case in point, the massive and specificovarian expression of the essential thioredoxin Deadhead (DHD) is criticallyregulated in Drosophila by the histone demethylase Lid and its partner, the histonedeacetylase complex Sin3A/Rpd3, via yet unknown mechanisms. Here, we identified Snr1and Mod(mdg4) as essential for dhd expression and investigated how these epigenomiceffectors act with Lid and Sin3A to hyperactivate dhd. Using Cut&Run chromatinprofiling with a dedicated data analysis procedure, we found that dhd isintriguingly embedded in an H3K27me3/H3K9me3-enriched mini-domain flanked by DNAregulatory elements, including a dhd promoter-proximal element essential for itsexpression. Surprisingly, Lid, Sin3a, Snr1 and Mod(mdg4) impact H3K27me3 and thisregulatory element in distinct manners. However, we show that these effectorsactivate dhd independently of H3K27me3/H3K9me3, and that dhd remains silent in theabsence of these marks. Together, our study demonstrates an atypical and criticalrole for chromatin regulators Lid, Sin3A, Snr1 and Mod(mdg4) to triggertissue-specific hyperactivation within a unique heterochromatin mini-domain.

Spatial organization of chromosomes leads to heterogeneous chromatin motion and drives the liquid- or gel-like dynamical behavior of chromatin.

Author(s) : Salari H, Di Stefano M, Jost D,
Journal : Genome Res
2022
Chromosome organization and dynamics are involved in regulating many fundamentalprocesses such as gene transcription and DNA repair. Experiments unveiled thatchromatin motion is highly heterogeneous inside cell nuclei, ranging from aliquid-like, mobile state to a gel-like, rigid regime. Using polymer modeling, weinvestigate how these different physical states and dynamical heterogeneities mayemerge from the same structural mechanisms. We found that the formation oftopologically associating domains (TADs) is a key driver of chromatin motionheterogeneity. In particular, we showed that the local degree of compaction of theTAD regulates the transition from a weakly compact, fluid state of chromatin to amore compact, gel state exhibiting anomalous diffusion and coherent motion. Our workprovides a comprehensive study of chromosome dynamics and a unified view ofchromatin motion enabling interpretation of the wide variety of dynamical behaviorsobserved experimentally across different biological conditions, suggesting that the"liquid" or "solid" state of chromatin are in fact two sides of the same coin.

2022

3DGenBench: a web-server to benchmark computational models for 3D Genomics.

Author(s) : Belokopytova P, Viesná E, Chiliński M, Qi Y, Salari H, Di Stefano M, Esposito A, Conte A, Chiariello A, Teif V, Plewczynski D, Zhang B, Jost D, Fishman V,
Journal : Nucleic Acids Res
2022
Modeling 3D genome organisation has been booming in the last years thanks to theavailability of experimental datasets of genomic contacts. However, the field iscurrently missing the standardisation of methods and metrics to comparepredictions and experiments. We present 3DGenBench, a web server available athttps://inc-cost.eu/benchmarking/, that allows benchmarking computational modelsof 3D Genomics. The benchmark is performed using a manually curated dataset of 39capture Hi-C profiles in wild type and genome-edited mouse cells, and fivegenome-wide Hi-C profiles in human, mouse, and Drosophila cells. 3DGenBenchperforms two kinds of analysis, each supplied with a specific scoring module thatcompares predictions of a computational method to experimental data using severalmetrics. With 3DGenBench, the user obtains model performance scores, allowing anunbiased comparison with other models. 3DGenBench aims to become a reference webserver to test new 3D genomics models and is conceived as an evolving platformwhere new types of analysis will be implemented in the future.

A Perspective for Ménière's Disease: In Silico Investigations of Dexamethasone as a Direct Modulator of AQP2.

Author(s) : Mom R, Robert-Paganin J, Mom T, Chabbert C, Réty S, Auguin D,
Journal : Biomolecules
2022
Ménière's disease is a chronic illness characterized by intermittent episodes ofvertigo associated with fluctuating sensorineural hearing loss, tinnitus andaural pressure. This pathology strongly correlates with a dilatation of the fluidcompartment of the endolymph, so-called hydrops. Dexamethasone is one of thetherapeutic approaches recommended when conventional antivertigo treatments havefailed. Several mechanisms of actions have been hypothesized for the mode ofaction of dexamethasone, such as the anti-inflammatory effect or as a regulatorof inner ear water homeostasis. However, none of them have been experimentallyconfirmed so far. Aquaporins (AQPs) are transmembrane water channels and arehence central in the regulation of transcellular water fluxes. In the presentstudy, we investigated the hypothesis that dexamethasone could impact waterfluxes in the inner ear by targeting AQP2. We addressed this question throughmolecular dynamics simulations approaches and managed to demonstrate a directinteraction between AQP2 and dexamethasone and its significant impact on thechannel water permeability. Through compartmentalization of sodium and potassiumions, a significant effect of Na+ upon AQP2 water permeability was highlighted aswell. The molecular mechanisms involved in dexamethasone binding and in itsregulatory action upon AQP2 function are described.

Altered splicing of ATG16-L1 mediates acquired resistance to tyrosine kinase inhibitors of EGFR by blocking autophagy in non-small cell lung cancer.

Author(s) : Hatat A, Benoit-Pilven C, Pucciarelli A, de Fraipont F, Lamothe L, Perron P, Rey A, Levra M, Toffart A, Auboeuf D, Eymin B, Gazzeri S,
Journal : Mol Oncol
2022
Despite the initial efficacy of using tyrosine kinase inhibitors of epidermalgrowth factor receptors (EGFR-TKIs) for treating patients with non-small celllung cancer (NSCLC), resistance inevitably develops. Recent studies highlight alink between alternative splicing and cancer drug response. Therefore, we aimedto identify deregulated splicing events that play a role in resistance toEGFR-TKI. By using RNA sequencing, reverse-transcription PCR (RT-PCR), and RNAinterference, we showed that overexpression of a splice variant of the autophagicgene ATG16-L1 that retains exon 8 and encodes the β-isoform of autophagy-relatedprotein 16-1 (ATG16-L1 β) concurs acquired resistance to EGFR-TKI in NSCLC cells.Using matched biopsies, we found increased levels of ATG16-L1 β at the time ofprogression in 3 of 11 NSCLC patients treated with EGFR-TKI. Mechanistically,gefitinib-induced autophagy was impaired in resistant cells that accumulatedATG16-L1 β. Neutralization of ATG16-L1 β restored autophagy in response togefitinib, induced apoptosis, and inhibited the growth of in ovo tumorxenografts. Conversely, overexpression of ATG16-L1 β in parental sensitive cellsprevented gefitinib-induced autophagy and increased cell survival. These resultssupport a role of defective autophagy in acquired resistance to EGFR-TKIs andidentify splicing regulation of ATG16-L1 as a therapeutic vulnerability thatcould be explored for improving EGFR-targeted cancer therapy.

Detection of Homologous Recombination Intermediates via Proximity Ligation and Quantitative PCR in Saccharomyces cerevisiae

Author(s) : Reitz D, Savocco J, Piazza A, Heyer W,
Journal : J Vis Exp
2022
DNA damage, including DNA double-stranded breaks and inter-strand cross-links, incurred during the S and G2 phases of the cell cycle can be repaired by homologous recombination (HR). In addition, HR represents an important mechanism of replication fork rescue following stalling or collapse. The regulation of the many reversible and irreversible steps of this complex pathway promotes its fidelity. The physical analysis of the recombination intermediates formed during HR enables the characterization of these controls by various nucleoprotein factors and their interactors. Though there are well-established methods to assay specific events and intermediates in the recombination pathway, the detection of D-loop formation and extension, two critical steps in this pathway, has proved challenging until recently. Here, efficient methods for detecting key events in the HR pathway, namely DNA double-stranded break formation, D-loop formation, D-loop extension, and the formation of products via break-induced replication (BIR) in Saccharomyces cerevisiae are described. These assays detect their relevant recombination intermediates and products with high sensitivity and are independent of cellular viability. The detection of D-loops, D-loop extension, and the BIR product is based on proximity ligation. Together, these assays allow for the study of the kinetics of HR at the population level to finely address the functions of HR proteins and regulators at significant steps in the pathway.

DNA:RNA Immunoprecipitation from S. pombe Cells for qPCR and Genome-Wide Sequencing.

Author(s) : Vachez L, Teste C, Vanoosthuyse V,
Journal : Methods Mol Biol
2022
By temporarily distorting the DNA double helix, the moving RNA polymerases can lead to the formation of non-B DNA structures. One of the most abundant and largest non-B DNA structures in the genome is the R-loop, a three-stranded structure forming when the nascent RNA hybridizes with its DNA template, thereby extruding the non-template DNA strand. Growing evidence suggests that at least a subset of R-loops could induce transcription stress and genome instability, although the direct, primary consequences of R-loop formation on the surrounding chromatin are still unclear. To understand the direct impact of R-loops on transcription and genome stability, accurate and quantitative mapping of R-loops is essential. R-loop mapping is commonly achieved using the antibody-based DNA:RNA Immunoprecipitation (DRIP) strategy. While it is reasonably straightforward to obtain robust DRIP enrichments from human cells, this has proved harder in yeast, where DRIP signals are often relatively weak, with a poor signal-to-noise ratio. Although it is unclear whether such weak signals stem from a technical or a biological reality, they make the accurate quantification of DRIP signals all the more important, especially when deep sequencing is used to monitor and quantify the distribution of R-loops genome-wide. Here we propose a DRIP protocol that has been optimized for the mapping and the quantification of R-loops in Schizosaccharomyces pombe but that can also be used in Saccharomyces cerevisiae. As a result, this protocol can be used to generate calibrated DRIP-seq data, where genomic DNA extracted from S. cerevisiae serves as spike-in reference.

Dynamical modeling of the H3K27 epigenetic landscape in mouse embryonic stem cells.

Author(s) : Newar K, Abdulla A, Salari H, Fanchon E, Jost D,
Journal : PLoS Comput Biol
2022
The Polycomb system via the methylation of the lysine 27 of histone H3 (H3K27)plays central roles in the silencing of many lineage-specific genes duringdevelopment. Recent experimental evidence suggested that the recruitment ofhistone modifying enzymes like the Polycomb repressive complex 2 (PRC2) atspecific sites and their spreading capacities from these sites are key to theestablishment and maintenance of a proper epigenomic landscape aroundPolycomb-target genes. Here, to test whether such mechanisms, as a minimal set ofqualitative rules, are quantitatively compatible with data, we developed amathematical model that can predict the locus-specific distributions of H3K27modifications based on previous biochemical knowledge. Within the biologicalcontext of mouse embryonic stem cells, our model showed quantitative agreementwith experimental profiles of H3K27 acetylation and methylation aroundPolycomb-target genes in wild-type and mutants. In particular, we demonstratedthe key role of the reader-writer module of PRC2 and of the competition betweenthe binding of activating and repressing enzymes in shaping the H3K27 landscapearound transcriptional start sites. The predicted dynamics of establishment andmaintenance of the repressive trimethylated H3K27 state suggest a slowaccumulation, in perfect agreement with experiments. Our approach represents afirst step towards a quantitative description of PcG regulation in variouscellular contexts and provides a generic framework to better characterizeepigenetic regulation in normal or disease situations.

Evolutionary divergence of anaphase spindle mechanics in nematode embryos constrained by antagonistic pulling and viscous forces.

Author(s) : Khatri D, Brugière T, Athale C, Delattre M,
Journal : Mol Biol Cell
2022
Cellular functions like cell division are remarkably conserved across phyla.However the evolutionary principles of cellular organization that drive it areless well explored. Thus, an essential question remains: to what extent cellularparameters evolve without altering the basic function they sustain? Here we haveobserved 6 different nematode species for which the mitotic spindle is positionedasymmetrically during the first embryonic division. Whereas the C. elegansspindle undergoes oscillations during its displacement, the spindle elongateswithout oscillations in other species. We asked which evolutionary changes inbiophysical parameters could explain differences in spindle motion whilemaintaining a constant output. Using laser microsurgery of the spindle werevealed that all species are subjected to cortical pulling forces, of varyingmagnitudes. Using a viscoelastic model to fit the recoil trajectories and with anindependent measurement of cytoplasmic viscosity, we extracted the values ofcytoplasmic drag, cortical pulling forces and spindle elasticity for all species.We found large variations in cytoplasmic viscosity whereas cortical pullingforces and elasticity were often more constrained. In agreement with previoussimulations, we found that increased viscosity correlates with decreasedoscillation speeds across species. However, the absence of oscillations despitelow viscosity in some species, can only be explained by smaller pulling forces.Consequently, we find that spindle mobility across the species analyzed here ischaracterized by a tradeoff between cytoplasmic viscosity and pulling forcesnormalized by the size of the embryo. Our work provides a framework forunderstanding mechanical constraints on evolutionary diversification of spindlemobility.

FORK-seq: Single-Molecule Profiling of DNA Replication.

Author(s) : Hennion M, Theulot B, Arbona J, Audit B, Hyrien O,
Journal : Methods Mol Biol
2022
Most genome replication mapping methods profile cell populations, maskingcell-to-cell heterogeneity. Here, we describe FORK-seq, a nanopore sequencingmethod to map replication of single DNA molecules at 200 nucleotide resolutionusing a nanopore current interpretation tool allowing the quantification of BrdUincorporation. Along pulse-chased replication intermediates from Saccharomycescerevisiae, we can orient replication tracks and reproduce population-basedreplication directionality profiles. Additionally, we can map individualinitiation and termination events. Thus, FORK-seq reveals the full extent ofcell-to-cell heterogeneity in DNA replication.

Genome-wide mapping of individual replication fork velocities using nanopore sequencing.

Author(s) : Theulot B, Lacroix L, Arbona J, Millot G, Jean E, Cruaud C, Pellet J, Proux F, Hennion M, Engelen S, Lemainque A, Audit B, Hyrien O, Le Tallec B,
Journal : Nat Commun
2022
Little is known about replication fork velocity variations along eukaryoticgenomes, since reference techniques to determine fork speed either provide nosequence information or suffer from low throughput. Here we presentNanoForkSpeed, a nanopore sequencing-based method to map and extract the velocityof individual forks detected as tracks of the thymidine analoguebromodeoxyuridine incorporated during a brief pulse-labelling of asynchronouslygrowing cells. NanoForkSpeed retrieves previous Saccharomyces cerevisiae meanfork speed estimates (≈2 kb/min) in the BT1 strain exhibiting highly efficientbromodeoxyuridine incorporation and wild-type growth, and precisely quantifiesspeed changes in cells with altered replisome progression or exposed tohydroxyurea. The positioning of >125,000 fork velocities provides a genome-widemap of fork progression based on individual fork rates, showing a uniform forkspeed across yeast chromosomes except for a marked slowdown at known pausingsites.

Global genome decompaction leads to stochastic activation of gene expression as a first step toward fate commitment in human hematopoietic cells.

Author(s) : Parmentier R, Racine L, Moussy A, Chantalat S, Sudharshan R, Papili Gao N, Stockholm D, Corre G, Fourel G, Deleuze J, Gunawan R, Paldi A,
Journal : PLoS Biol
2022
When human cord blood-derived CD34+ cells are induced to differentiate, theyundergo rapid and dynamic morphological and molecular transformations that arecritical for fate commitment. In particular, the cells pass through a transitoryphase known as "multilineage-primed" state. These cells are characterized by amixed gene expression profile, different in each cell, with the coexpression ofmany genes characteristic for concurrent cell lineages. The aim of our study isto understand the mechanisms of the establishment and the exit from thistransitory state. We investigated this issue using single-cell RNA sequencing andATAC-seq. Two phases were detected. The first phase is a rapid and globalchromatin decompaction that makes most of the gene promoters in the genomeaccessible for transcription. It results 24 h later in enhanced and pervasivetranscription of the genome leading to the concomitant increase in thecell-to-cell variability of transcriptional profiles. The second phase is theexit from the multilineage-primed phase marked by a slow chromatin closure and asubsequent overall down-regulation of gene transcription. This process isselective and results in the emergence of coherent expression profilescorresponding to distinct cell subpopulations. The typical time scale of theseevents spans 48 to 72 h. These observations suggest that the nonspecificity ofgenome decompaction is the condition for the generation of a highly variablemultilineage expression profile. The nonspecific phase is followed by specificregulatory actions that stabilize and maintain the activity of key genes, whilethe rest of the genome becomes repressed again by the chromatin recompaction.Thus, the initiation of differentiation is reminiscent of a constrainedoptimization process that associates the spontaneous generation of geneexpression diversity to subsequent regulatory actions that maintain the activityof some genes, while the rest of the genome sinks back to the repressive closedchromatin state.

Loss of SET1/COMPASS methyltransferase activity reduces lifespan and fertility in Caenorhabditis elegans.

Author(s) : Caron M, Gely L, Garvis S, Adrait A, Couté Y, Palladino F, Fabrizio P,
Journal : Life Sci Alliance
2022
Changes in histone post-translational modifications are associated with agingthrough poorly defined mechanisms. Histone 3 lysine 4 (H3K4) methylation atpromoters is deposited by SET1 family methyltransferases acting within conservedmultiprotein complexes known as COMPASS. Previous work yielded conflicting resultsabout the requirement for H3K4 methylation during aging. Here, we reassessed therole of SET1/COMPASS-dependent H3K4 methylation in Caenorhabditis elegans lifespanand fertility by generating set-2(syb2085) mutant animals that express acatalytically inactive form of SET-2, the C. elegans SET1 homolog. We show thatset-2(syb2085) animals retain the ability to form COMPASS, but have a marked globalloss of H3K4 di- and trimethylation (H3K4me2/3). Reduced H3K4 methylation wasaccompanied by loss of fertility, as expected; however, in contrast to earlierstudies, set-2(syb2085) mutants displayed a significantly shortened, not extended,lifespan and had normal intestinal fat stores. Other commonly used set-2 mutantswere also short-lived, as was a cfp-1 mutant that lacks the SET1/COMPASSchromatin-targeting component. These results challenge previously held views andestablish that WT H3K4me2/3 levels are essential for normal lifespan in C. elegans.

Myotonic dystrophy RNA toxicity alters morphology, adhesion and migration of mouse and human astrocytes

Author(s) : Dincã D, Lallemant L, González-Barriga A, Cresto N, Braz S, Sicot G, Pillet L, Polvèche H, Magneron P, Huguet-Lachon A, Benyamine H, Azotla-Vilchis C, Agonizantes-Juárez L, Tahraoui-Bories J, Martinat C, Hernández-Hernández O, Auboeuf D, Rouach N, Bourgeois C, Gourdon G, Gomes-Pereira M,
Journal : Nat Commun
2022
Brain dysfunction in myotonic dystrophy type 1 (DM1), the prototype of toxic RNAdisorders, has been mainly attributed to neuronal RNA misprocessing, while littleattention has been given to non-neuronal brain cells. Here, using a transgenicmouse model of DM1 that expresses mutant RNA in various brain cell types(neurons, astroglia, and oligodendroglia), we demonstrate that astrocytes exhibitimpaired ramification and polarization in vivo and defects in adhesion,spreading, and migration. RNA-dependent toxicity and phenotypes are also found inhuman transfected glial cells. In line with the cell phenotypes, molecularanalyses reveal extensive expression and accumulation of toxic RNA in astrocytes,which result in RNA spliceopathy that is more severe than in neurons. Astrocytemissplicing affects primarily transcripts that regulate cell adhesion,cytoskeleton, and morphogenesis, and it is confirmed in human brain tissue. Ourfindings demonstrate that DM1 impacts astrocyte cell biology, possiblycompromising their support and regulation of synaptic function.

Painters in chromatin: a unified quantitative framework to systematically characterize epigenome regulation and memory.

Author(s) : Abdulla A, Vaillant C, Jost D,
Journal : Nucleic Acids Res
2022
In eukaryotes, many stable and heritable phenotypes arise from the same DNAsequence, owing to epigenetic regulatory mechanisms relying on the molecularcooperativity of 'reader-writer' enzymes. In this work, we focus on thefundamental, generic mechanisms behind the epigenome memory encoded bypost-translational modifications of histone tails. Based on experimentalknowledge, we introduce a unified modeling framework, the painter model,describing the mechanistic interplay between sequence-specific recruitment ofchromatin regulators, chromatin-state-specific reader-writer processes andlong-range spreading mechanisms. A systematic analysis of the model buildingblocks highlights the crucial impact of tridimensional chromatin organization andstate-specific recruitment of enzymes on the stability of epigenomic domains andon gene expression. In particular, we show that enhanced 3D compaction of thegenome and enzyme limitation facilitate the formation of ultra-stable, confinedchromatin domains. The model also captures how chromatin state dynamics impactthe intrinsic transcriptional properties of the region, slower kinetics leadingto noisier expression. We finally apply our framework to analyze experimentaldata, from the propagation of γH2AX around DNA breaks in human cells to themaintenance of heterochromatin in fission yeast, illustrating how the paintermodel can be used to extract quantitative information on epigenomic molecularprocesses.

Paternal transmission of the Wolbachia CidB toxin underlies cytoplasmic incompatibility.

Author(s) : Horard B, Terretaz K, Gosselin-Grenet A, Sobry H, Sicard M, Landmann F, Loppin B,
Journal : Curr Biol
2022
Wolbachia are widespread endosymbiotic bacteria that manipulate the reproduction ofarthropods through a diversity of cellular mechanisms. In cytoplasmicincompatibility (CI), a sterility syndrome originally discovered in the mosquitoCulex pipiens, uninfected eggs fertilized by sperm from infected males areselectively killed during embryo development following the abortive segregation ofpaternal chromosomes in the zygote. Despite the recent discovery of Wolbachia CIfactor (cif) genes, the mechanism by which they control the fate of paternalchromosomes at fertilization remains unknown. Here, we have analyzed the cytologicaldistribution and cellular impact of CidA and CidB, a pair of Cif proteins from theCulex-infecting Wolbachia strain wPip. We show that expression of CidB in DrosophilaS2R+ cells induces apoptosis unless CidA is co-expressed and associated with itspartner. In transgenic Drosophila testes, both effectors colocalize in germ cellsuntil the histone-to-protamine transition in which only CidB is retained in maturingspermatid nuclei. We further show that CidB is similarly targeted to maturing spermof naturally infected Culex mosquitoes. At fertilization, CidB associates withpaternal DNA regions exhibiting DNA replication stress, as a likely cause ofincomplete replication of paternal chromosomes at the onset of the first mitosis.Importantly, we demonstrate that inactivation of the deubiquitylase activity of CidBdoes not abolish its cell toxicity or its ability to induce CI in Drosophila. Ourstudy thus demonstrates that CI functions as a transgenerational toxin-antidotesystem and suggests that CidB acts by poisoning paternal DNA replication inincompatible crosses.

Polymer Modeling of 3D Epigenome Folding: Application to Drosophila.

Author(s) : Jost D,
Journal : Methods Mol Biol
2022
Mechanistic modeling in biology allows to investigate, based on first principles, ifputative hypotheses are compatible with observations and to drive furtherexperimental works. Along this line, polymer modeling has been instrumental in 3Dgenomics to better understand the impact of key mechanisms on the spatial genomeorganization. Here, I describe how polymer-based models can be practically used tostudy the role of epigenome in chromosome folding. I illustrate this methodology inthe context of Drosophila epigenome folding.

Prognostic impact of ABCA3 expression in adult and pediatric acute myeloid leukemia: an ALFA-ELAM02 joint study

Author(s) : Ceraulo A, Lapillonne H, Cheok M, Preudhomme C, Dombret H, Terré C, Lambert J, Leverger G, Bertrand Y, Mortreux F, Wattel E,
Journal : Blood Adv
2022

Real age prediction from the transcriptome with RAPToR

Author(s) : Bulteau R, Francesconi M,
Journal : Nature Methods
2022
Transcriptomic data is often affected by uncontrolled variation among samples that can obscure and confound the effects of interest. This variation is frequently due to unintended differences in developmental stages between samples. The transcriptome itself can be used to estimate developmental progression, but existing methods require many samples and do not estimate a specimen’s real age. Here we present real-age prediction from transcriptome staging on reference (RAPToR), a computational method that precisely estimates the real age of a sample from its transcriptome, exploiting existing time-series data as reference. RAPToR works with whole animal, dissected tissue and single-cell data for the most common animal models, humans and even for non-model organisms lacking reference data. We show that RAPToR can be used to remove age as a confounding factor and allow recovery of a signal of interest in differential expression analysis. RAPToR will be especially useful in large-scale single-organism profiling because it eliminates the need for accurate staging or synchronisation before profiling.

RNA helicase-dependent gene looping impacts messenger RNA processing.

Author(s) : Terrone S, Valat J, Fontrodona N, Giraud G, Claude J, Combe E, Lapendry A, Polvèche H, Ameur L, Duvermy A, Modolo L, Bernard P, Mortreux F, Auboeuf D, Bourgeois C,
Journal : Nucleic Acids Res
2022
DDX5 and DDX17 are DEAD-box RNA helicase paralogs which regulate several aspectsof gene expression, especially transcription and splicing, through incompletelyunderstood mechanisms. A transcriptome analysis of DDX5/DDX17-depleted humancells confirmed the large impact of these RNA helicases on splicing and revealeda widespread deregulation of 3' end processing. In silico analyses andexperiments in cultured cells showed the binding and functional contribution ofthe genome organizing factor CTCF to chromatin sites at or near a subset ofDDX5/DDX17-dependent exons that are characterized by a high GC content and a highdensity of RNA Polymerase II. We propose the existence of an RNAhelicase-dependent relationship between CTCF and the dynamics of transcriptionacross DNA and/or RNA structured regions, that contributes to the processing ofinternal and terminal exons. Moreover, local DDX5/DDX17-dependent chromatin loopsspatially connect RNA helicase-regulated exons with their cognate promoter, andwe provide the first direct evidence that de novo gene looping modifiesalternative splicing and polyadenylation. Overall our findings uncover the impactof DDX5/DDX17-dependent chromatin folding on pre-messenger RNA processing.

Spatial organization of chromosomes leads to heterogeneous chromatin motion and drives the liquid- or gel-like dynamical behavior of chromatin.

Author(s) : Salari H, Di Stefano M, Jost D,
Journal : Genome Res
2022
Chromosome organization and dynamics are involved in regulating many fundamentalprocesses such as gene transcription and DNA repair. Experiments unveiled thatchromatin motion is highly heterogeneous inside cell nuclei, ranging from aliquid-like, mobile state to a gel-like, rigid regime. Using polymer modeling, weinvestigate how these different physical states and dynamical heterogeneities mayemerge from the same structural mechanisms. We found that the formation oftopologically associating domains (TADs) is a key driver of chromatin motionheterogeneity. In particular, we showed that the local degree of compaction of theTAD regulates the transition from a weakly compact, fluid state of chromatin to amore compact, gel state exhibiting anomalous diffusion and coherent motion. Our workprovides a comprehensive study of chromosome dynamics and a unified view ofchromatin motion enabling interpretation of the wide variety of dynamical behaviorsobserved experimentally across different biological conditions, suggesting that the"liquid" or "solid" state of chromatin are in fact two sides of the same coin.

Structural mechanism underpinning Thermus oshimai Pif1-mediated G-quadruplex unfolding.

Author(s) : Dai Y, Guo H, Liu N, Chen W, Ai X, Li H, Sun B, Hou X, Rety S, Xi X,
Journal : EMBO Rep
2022
G-quadruplexes (G4s) are unusual stable DNA structures that cause genomicinstability. To overcome the potential barriers formed by G4s, cells have evolveddifferent families of proteins that unfold G4s. Pif1 is a DNA helicase fromsuperfamily 1 (SF1) conserved from bacteria to humans with high G4-unwindingactivity. Here, we present the first X-ray crystal structure of the Thermusoshimai Pif1 (ToPif1) complexed with a G4. Our structure reveals that ToPif1recognizes the entire native G4 via a cluster of amino acids at domains 1B/2Bwhich constitute a G4-Recognizing Surface (GRS). The overall structure of the G4maintains its three-layered propeller-type G4 topology, without significantreorganization of G-tetrads upon protein binding. The three G-tetrads in G4 arerecognized by GRS residues mainly through electrostatic, ionic interactions, andhydrogen bonds formed between the GRS residues and the ribose-phosphate backbone.Compared with previously solved structures of SF2 helicases in complex with G4,our structure reveals how helicases from distinct superfamilies adopt differentstrategies for recognizing and unfolding G4s.

Three classes of epigenomic regulators converge to hyperactivate the essential maternal gene deadhead within a heterochromatin mini-domain.

Author(s) : Torres-Campana D, Horard B, Denaud S, Benoit G, Loppin B, Orsi G,
Journal : PLoS Genet
2022
The formation of a diploid zygote is a highly complex cellular process that isentirely controlled by maternal gene products stored in the egg cytoplasm. Thishighly specialized transcriptional program is tightly controlled at the chromatinlevel in the female germline. As an extreme case in point, the massive and specificovarian expression of the essential thioredoxin Deadhead (DHD) is criticallyregulated in Drosophila by the histone demethylase Lid and its partner, the histonedeacetylase complex Sin3A/Rpd3, via yet unknown mechanisms. Here, we identified Snr1and Mod(mdg4) as essential for dhd expression and investigated how these epigenomiceffectors act with Lid and Sin3A to hyperactivate dhd. Using Cut&Run chromatinprofiling with a dedicated data analysis procedure, we found that dhd isintriguingly embedded in an H3K27me3/H3K9me3-enriched mini-domain flanked by DNAregulatory elements, including a dhd promoter-proximal element essential for itsexpression. Surprisingly, Lid, Sin3a, Snr1 and Mod(mdg4) impact H3K27me3 and thisregulatory element in distinct manners. However, we show that these effectorsactivate dhd independently of H3K27me3/H3K9me3, and that dhd remains silent in theabsence of these marks. Together, our study demonstrates an atypical and criticalrole for chromatin regulators Lid, Sin3A, Snr1 and Mod(mdg4) to triggertissue-specific hyperactivation within a unique heterochromatin mini-domain.

Titration of Apparent In-Cellula Affinities of Protein-Protein Interactions.

Author(s) : Cluet D, Vergier B, Levy N, Dehau L, Thurman A, Amri I, Spichty M,
Journal : Chembiochem
2022
A genetic assay permits simultaneous quantification of two interacting proteinsand their bound fraction at the single-cell level using flow cytometry. Apparentin-cellula affinities of protein-protein interactions can be extracted from theacquired data through a titration-like analysis. The applicability of thisapproach is demonstrated on a diverse set of interactions with proteins fromdifferent families and organisms and with in-vitro dissociation constants rangingfrom picomolar to micromolar.

2021

4D nucleome modeling.

Author(s) : Di Stefano M, Paulsen J, Jost D, Marti-Renom M,
Journal : Curr Opin Genet Dev
2021
The intrinsic dynamic nature of chromosomes is emerging as a fundamental componentin regulating DNA transcription, replication, and damage-repair among other nuclearfunctions. With this increased awareness, reinforced over the last ten years, manynew experimental techniques, mainly based on microscopy and chromosome conformationcapture, have been introduced to study the genome in space and time. Owing to theincreasing complexity of these cutting-edge techniques, computational approacheshave become of paramount importance to interpret, contextualize, and complement suchexperiments with new insights. Hence, it is becoming crucial for experimentalbiologists to have a clear understanding of the diverse theoretical modelingapproaches available and the biological information each of them can provide.

A Novel Screen for Expression Regulators of the Telomeric Protein TRF2 Identified Small Molecules That Impair TRF2 Dependent Immunosuppression and Tumor Growth.

Author(s) : El Maï M, Janho Dit Hreich S, Gaggioli C, Roisin A, Wagner N, Ye J, Jalinot P, Cherfils-Vicini J, Gilson E,
Journal : Cancers (Basel)
2021
Telomeric repeat-binding factor 2 (TRF2) is a subunit of the shelterin proteincomplex, which binds to and protects telomeres from unwanted DNA damage response(DDR) activation. TRF2 expression plays a pivotal role in aging and cancer, beingdownregulated during cellular senescence and overexpressed during oncogenesis.Cancers overexpressing TRF2 often exhibit a poor prognosis. In cancer cells, TRF2plays multiple functions, including telomere protection and non-cell autonomousroles, promoting neo-angiogenesis and immunosuppression. We present here anoriginal screening strategy, which enables identification of small molecules thatdecrease or increase TRF2 expression. By screening a small library of Food andDrug Agency (FDA)-approved drugs, we identified two molecules (AR-A014418 andalexidine·2HCl) that impaired tumor growth, neo-angiogenesis andimmunosuppression by downregulating TRF2 expression in a mouse xenograft model.These results support the chemotherapeutic strategy of downregulating TRF2expression to treat aggressive human tumors and validate this cell-based assaycapable of screening for potential anti-cancer and anti-aging molecules bymodulating TRF2 expression levels.

A single-chain and fast-responding light-inducible Cre recombinase as a novel optogenetic switch.

Author(s) : Duplus-Bottin H, Spichty M, Triqueneaux G, Place C, Mangeot P, Ohlmann T, Vittoz F, Yvert G,
Journal : Elife
2021
Optogenetics enables genome manipulations with high spatiotemporal resolution,opening exciting possibilities for fundamental and applied biological research.Here, we report the development of LiCre, a novel light-inducible Cre recombinase.LiCre is made of a single flavin-containing protein comprising the AsLOV2photoreceptor domain of Avena sativa fused to a Cre variant carrying destabilizingmutations in its N-terminal and C-terminal domains. LiCre can be activated withinminutes of illumination with blue light, without the need of additional chemicals.When compared to existing photoactivatable Cre recombinases based on two splitunits, LiCre displayed faster and stronger activation by light as well as a lowerresidual activity in the dark. LiCre was efficient both in yeast, where it allowedus to control the production of β-carotene with light, and in human cells. Given itssimplicity and performances, LiCre is particularly suited for fundamental andbiomedical research, as well as for controlling industrial bioprocesses.

Abnormal accumulation of lipid droplets in neurons induces the conversion of alpha-Synuclein to proteolytic resistant forms in a Drosophila model of Parkinson's disease.

Author(s) : Girard V, Jollivet F, Knittelfelder O, Celle M, Arsac J, Chatelain G, Van den Brink D, Baron T, Shevchenko A, Kühnlein R, Davoust N, Mollereau B,
Journal : PLoS Genet
2021
Parkinson's disease (PD) is a neurodegenerative disorder characterized byalpha-synuclein (αSyn) aggregation and associated with abnormalities in lipidmetabolism. The accumulation of lipids in cytoplasmic organelles called lipiddroplets (LDs) was observed in cellular models of PD. To investigate thepathophysiological consequences of interactions between αSyn and proteins thatregulate the homeostasis of LDs, we used a transgenic Drosophila model of PD, inwhich human αSyn is specifically expressed in photoreceptor neurons. We first foundthat overexpression of the LD-coating proteins Perilipin 1 or 2 (dPlin1/2), whichlimit the access of lipases to LDs, markedly increased triacylglyclerol (TG) loadedLDs in neurons. However, dPlin-induced-LDs in neurons are independent of lipidanabolic (diacylglycerol acyltransferase 1/midway, fatty acid transportprotein/dFatp) and catabolic (brummer TG lipase) enzymes, indicating thatalternative mechanisms regulate neuronal LD homeostasis. Interestingly, theaccumulation of LDs induced by various LD proteins (dPlin1, dPlin2, CG7900 orKlarsichtLD-BD) was synergistically amplified by the co-expression of αSyn, whichlocalized to LDs in both Drosophila photoreceptor neurons and in human neuroblastomacells. Finally, the accumulation of LDs increased the resistance of αSyn toproteolytic digestion, a characteristic of αSyn aggregation in human neurons. Wepropose that αSyn cooperates with LD proteins to inhibit lipolysis and that bindingof αSyn to LDs contributes to the pathogenic misfolding and aggregation of αSyn inneurons.

Asymmetry is defined during meiosis in the oocyte of the parthenogenetic nematode Diploscapter pachys.

Author(s) : Eweis D, Delattre M, Plastino J,
Journal : Dev Biol
2021
Asymmetric cell division is an essential feature of normal development and certainpathologies. The process and its regulation have been studied extensively in theCaenorhabditis elegans embryo, particularly how symmetry of the actomyosin corticalcytoskeleton is broken by a sperm-derived signal at fertilization, upstream ofpolarity establishment. Diploscapter pachys is the closest parthenogenetic relativeto C. elegans, and D. pachys one-cell embryos also divide asymmetrically. Howeverhow polarity is triggered in the absence of sperm remains unknown. In post-meioticembryos, we find that the nucleus inhabits principally one embryo hemisphere, thefuture posterior pole. When forced to one pole by centrifugation, the nucleusreturns to its preferred pole, although poles appear identical as concerns corticalruffling and actin cytoskeleton. The location of the meiotic spindle also correlateswith the future posterior pole and slight actin enrichment is observed at that polein some early embryos along with microtubule structures emanating from the meioticspindle. Polarized location of the nucleus is not observed in pre-meiotic D. pachysoocytes. All together our results are consistent with the idea that polarity of theD. pachys embryo is attained during meiosis, seemingly based on the location of themeiotic spindle, by a mechanism that may be present but suppressed in C. elegans.

Auxin confers protection against ER stress in Caenorhabditis elegans.

Author(s) : Bhoi A, Palladino F, Fabrizio P,
Journal : Biol Open
2021
Auxins are plant growth regulators that influence most aspects of plant developmentthrough complex mechanisms. The development of an auxin-inducible degradation (AID)system has enabled rapid, conditional protein depletion in yeast and cultured cells.More recently, the system was successfully adapted to C aenorhabditis elegans toachieve auxin-dependent degradation of targets in all tissues and developmentalstages. Whether auxin treatment alone has an impact on nematode physiology is anopen question. Here we show that indole-3-acetic acid (IAA), the auxin most commonlyused to trigger AID in worms, functions through the conserved IRE-1/XBP-1 branch ofthe Unfolded Protein Response (UPR) to promote resistance to endoplasmic reticulum(ER) stress. Because the UPR not only plays a central role in restoring ERhomeostasis, but also promotes lipid biosynthesis and regulates lifespan, we suggestthat extreme caution should be exercised when using the AID system to study theseand related processes.

Baboon Envelope Pseudotyped "Nanoblades" Carrying Cas9/gRNA Complexes Allow Efficient Genome Editing in Human T, B, and CD34(+) Cells and Knock-in of AAV6-Encoded Donor DNA in CD34(+) Cells.

Author(s) : Gutierrez-Guerrero A, Abrey Recalde M, Mangeot P, Costa C, Bernadin O, Périan S, Fusil F, Froment G, Martinez-Turtos A, Krug A, Martin F, Benabdellah K, Ricci E, Giovannozzi S, Gijsbers R, Ayuso E, Cosset F, Verhoeyen E,
Journal : Front Genome Ed
2021
Programmable nucleases have enabled rapid and accessible genome engineering ineukaryotic cells and living organisms. However, their delivery into human bloodcells can be challenging. Here, we have utilized "nanoblades," a new technology thatdelivers a genomic cleaving agent into cells. These are modified murine leukemiavirus (MLV) or HIV-derived virus-like particle (VLP), in which the viral structuralprotein Gag has been fused to Cas9. These VLPs are thus loaded with Cas9 proteincomplexed with the guide RNAs. Highly efficient gene editing was obtained in celllines, IPS and primary mouse and human cells. Here, we showed that nanoblades wereremarkably efficient for entry into human T, B, and hematopoietic stem andprogenitor cells (HSPCs) thanks to their surface co-pseudotyping with baboonretroviral and VSV-G envelope glycoproteins. A brief incubation of human T and Bcells with nanoblades incorporating two gRNAs resulted in 40 and 15% edited deletionin the Wiskott-Aldrich syndrome (WAS) gene locus, respectively. CD34(+) cells(HSPCs) treated with the same nanoblades allowed 30-40% exon 1 drop-out in the WASgene locus. Importantly, no toxicity was detected upon nanoblade-mediated geneediting of these blood cells. Finally, we also treated HSPCs with nanoblades incombination with a donor-encoding rAAV6 vector resulting in up to 40% of stableexpression cassette knock-in into the WAS gene locus. Summarizing, this newtechnology is simple to implement, shows high flexibility for different targetsincluding primary immune cells of human and murine origin, is relatively inexpensiveand therefore gives important prospects for basic and clinical translation in thearea of gene therapy.

Best practices for the visualization, mapping, and manipulation of R-loops.

Author(s) : Chédin F, Hartono S, Sanz L, Vanoosthuyse V,
Journal : EMBO J
2021
R-loops represent an abundant class of large non-B DNA structures in genomes. Eventhough they form transiently and at modest frequencies, interfering with R-loopformation or dissolution has significant impacts on genome stability. Addressing themechanism(s) of R-loop-mediated genome destabilization requires a precisecharacterization of their distribution in genomes. A number of independent methodshave been developed to visualize and map R-loops, but their results are at timesdiscordant, leading to confusion. Here, we review the main existing methodologiesfor R-loop mapping and assess their limitations as well as the robustness ofexisting datasets. We offer a set of best practices to improve the reproducibilityof maps, hoping that such guidelines could be useful for authors and referees alike.Finally, we propose a possible resolution for the apparent contradictions in R-loopmapping outcomes between antibody-based and RNase H1-based mapping approaches.

Chronic Exposure to Paraquat Induces Alpha-Synuclein Pathogenic Modifications in Drosophila.

Author(s) : Arsac J, Sedru M, Dartiguelongue M, Vulin J, Davoust N, Baron T, Mollereau B,
Journal : Int J Mol Sci
2021
Parkinson's disease (PD) is characterized by the progressive accumulation ofneuronal intracellular aggregates largely composed of alpha-Synuclein (αSyn)protein. The process of αSyn aggregation is induced during aging and enhanced byenvironmental stresses, such as the exposure to pesticides. Paraquat (PQ) is anherbicide which has been widely used in agriculture and associated with PD. PQ isknown to cause an increased oxidative stress in exposed individuals but theconsequences of such stress on αSyn conformation remains poorly understood. To studyαSyn pathogenic modifications in response to PQ, we exposed Drosophila expressinghuman αSyn to a chronic PQ protocol. We first showed that PQ exposure and αSynexpression synergistically induced fly mortality. The exposure to PQ was alsoassociated with increased levels of total and phosphorylated forms of αSyn in theDrosophila brain. Interestingly, PQ increased the detection of soluble αSyn inhighly denaturating buffer but did not increase αSyn resistance to proteinase Kdigestion. These results suggest that PQ induces the accumulation of toxic solubleand misfolded forms of αSyn but that these toxic forms do not form fibrils oraggregates that are detected by the proteinase K assay. Collectively, our resultsdemonstrate that Drosophila can be used to study the effect of PQ or otherenvironmental neurotoxins on αSyn driven pathology.

Cohesin regulates homology search during recombinational DNA repair

Author(s) : Piazza A, Bordelet H, Dumont A, Thierry A, Savocco J, Girard F, Koszul R,
Journal : Nature Cell Biology
2021
Homologous recombination repairs DNA double-strand breaks (DSB) using an intact dsDNA molecule as a template. It entails a homology search step, carried out along a conserved RecA/Rad51-ssDNA filament assembled on each DSB end. Whether, how and to what extent a DSB impacts chromatin folding, and how this (re)organization in turns influences the homology search process, remain ill-defined. Here we characterize two layers of spatial chromatin reorganization following DSB formation in Saccharomyces cerevisiae. Although cohesin folds chromosomes into cohesive arrays of ~20-kb-long chromatin loops as cells arrest in G2/M, the DSB-flanking regions interact locally in a resection- and 9-1-1 clamp-dependent manner, independently of cohesin, Mec1ATR, Rad52 and Rad51. This local structure blocks cohesin progression, constraining the DSB region at the base of a loop. Functionally, cohesin promotes DSB–dsDNA interactions and donor identification in cis, while inhibiting them in trans. This study identifies multiple direct and indirect ways by which cohesin regulates homology search during recombinational DNA repair.

Comparison of lipidome profiles of Caenorhabditis elegans-results from an inter-laboratory ring trial.

Author(s) : Spanier B, Laurençon A, Weiser A, Pujol N, Omi S, Barsch A, Korf A, Meyer S, Ewbank J, Paladino F, Garvis S, Aguilaniu H, Witting M,
Journal : Metabolomics
2021
INTRODUCTION: Lipidomic profiling allows 100s if not 1000s of lipids in a sample tobe detected and quantified. Modern lipidomics techniques are ultra-sensitive assaysthat enable the discovery of novel biomarkers in a variety of fields and provide newinsight in mechanistic investigations. Despite much progress in lipidomics, thereremains, as for all high throughput "omics" strategies, the need to developstrategies to standardize and integrate quality control into studies in order toenhance robustness, reproducibility, and usability of studies within specific fieldsand beyond. OBJECTIVES: We aimed to understand how much results from lipid profilingin the model organism Caenorhabditis elegans are influenced by different cultureconditions in different laboratories. METHODS: In this work we have undertaken aninter-laboratory study, comparing the lipid profiles of N2 wild type C. elegans anddaf-2(e1370) mutants lacking a functional insulin receptor. Sample were collectedfrom worms grown in four separate laboratories under standardized growth conditions.We used an UPLC-UHR-ToF-MS system allowing chromatographic separation before MSanalysis. RESULTS: We found common qualitative changes in several marker lipids insamples from the individual laboratories. On the other hand, even in this controlledexperimental system, the exact fold-changes for each marker varied betweenlaboratories. CONCLUSION: Our results thus reveal a serious limitation to thereproducibility of current lipid profiling experiments and reveal challenges to theintegration of such data from different laboratories.

Coupled protein synthesis and ribosome-guided piRNA processing on mRNAs.

Author(s) : Sun Y, Wang R, Du K, Zhu J, Zheng J, Xie L, Pereira A, Zhang C, Ricci E, Li X,
Journal : Nat Commun
2021
PIWI-interacting small RNAs (piRNAs) protect the germline genome and are essentialfor fertility. piRNAs originate from transposable element (TE) RNAs, long non-codingRNAs, or 3´ untranslated regions (3´UTRs) of protein-coding messenger genes, withthe last being the least characterized of the three piRNA classes. Here, wedemonstrate that the precursors of 3´UTR piRNAs are full-length mRNAs and thatpost-termination 80S ribosomes guide piRNA production on 3´UTRs in mice andchickens. At the pachytene stage, when other co-translational RNA surveillancepathways are sequestered, piRNA biogenesis degrades mRNAs right after pioneer roundsof translation and fine-tunes protein production from mRNAs. Although 3´UTR piRNAprecursor mRNAs code for distinct proteins in mice and chickens, they all harborembedded TEs and produce piRNAs that cleave TEs. Altogether, we discover a functionof the piRNA pathway in fine-tuning protein production and reveal a conserved piRNAbiogenesis mechanism that recognizes translating RNAs in amniotes.

Crystal structures of N-terminally truncated telomerase reverse transcriptase from fungi‡.

Author(s) : Zhai L, Rety S, Chen W, Song Z, Auguin D, Sun B, Dou S, Xi X,
Journal : Nucleic Acids Res
2021
Telomerase plays critical roles in cellular aging, in the emergence and/ordevelopment of cancer, and in the capacity for stem-cell renewal, consists of acatalytic telomerase reverse transcriptase (TERT) and a template-encoding RNA(TER). TERs from diverse organisms contain two conserved structural elements: thetemplate-pseudoknot (T-PK) and a helical three-way junction (TWJ).Species-specific features of the structure and function of telomerase makeobtaining a more in-depth understanding of the molecular mechanism of telomeraseparticularly important. Here, we report the first structural studies ofN-terminally truncated TERTs from Candida albicans and Candida tropicalis in apoform and complexed with their respective TWJs in several conformations. We foundthat Candida TERT proteins perform only one round of telomere addition in thepresence or absence of PK/TWJ and display standard reverse transcriptaseactivity. The C-terminal domain adopts at least two extreme conformations andundergoes conformational interconversion, which regulates the catalytic activity.Most importantly, we identified a conserved tertiary structural motif, called theU-motif, which interacts with the reverse transcriptase domain and is crucial forcatalytic activity. Together these results shed new light on the structure andmechanics of fungal TERTs, which show common TERT characteristics, but alsodisplay species-specific features.

Decreased expression of the translation factor eIF3e induces senescence in breast cancer cells via suppression of PARP1 and activation of mTORC1.

Author(s) : Morris C, Durand S, Jalinot P,
Journal : Oncotarget
2021
Altered expression of the translation factor eIF3e is associated with breastcancer occurrence. We have previously shown that eIF3e deficiency leads to animpaired DNA damage response with a marked decrease in DNA repair by homologousrecombination. Here, we explored the possibility to exploit this DNA repairdefect in targeted cancer therapy using PARP inhibitors. Surprisingly,eIF3e-deficient breast cancer cells are resistant to these drugs, in contrast toBRCA1-deficient cells. Studying this, we found that eIF3e-depleted cellssynthesize lowered amounts of PARP1 protein, due to a weakened translation of thecorresponding mRNA, associated with a strong decrease in cellularpoly(ADP-ribosyl)ation. Additionally, we discovered that the mTORC1 signalingpathway is aberrantly activated in response to eIF3e suppression. Together, thesePARP1 and mTORC1 dysfunctions upon eIF3e depletion are causally linked toinduction of cellular senescence associated with a pro-inflammatory secretoryphenotype. This study provides mechanistic insights into how eIF3e protectsagainst breast cancer, with potential novel cancer therapeutic opportunities.While PARP inhibitors appear as inappropriate drugs for eIF3e-deficient breasttumors, our findings suggest that such cancers may benefit from senolytic drugsor mTORC1 inhibitors.

Delivery of the Cas9/sgRNA Ribonucleoprotein Complex in Immortalized and Primary Cells via Virus-like Particles ("Nanoblades").

Author(s) : Mangeot P, Guiguettaz L, Sohier T, Ricci E,
Journal : J Vis Exp
2021
The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas systemhas democratized genome-editing in eukaryotic cells and led to the development ofnumerous innovative applications. However, delivery of the Cas9 protein andsingle-guide RNA (sgRNA) into target cells can be technically challenge. Classicalviral vectors, such as those derived from lentiviruses (LVs) or adeno-associatedviruses (AAVs), allow for efficient delivery of transgenes coding for the Cas9protein and its associated sgRNA in many primary cells and in vivo. Nevertheless,these vectors can suffer from drawbacks such as integration of the transgene in thetarget cell genome, a limited cargo capacity, and long-term expression of the Cas9protein and guide RNA in target cells. To overcome some of these problems, adelivery vector based on the murine Leukemia virus (MLV) was developed to packagethe Cas9 protein and its associated guide RNA in the absence of any codingtransgene. By fusing the Cas9 protein to the C-terminus of the structural proteinGag from MLV, virus-like particles (VLPs) loaded with the Cas9 protein and sgRNA(named "Nanoblades") were formed. Nanoblades can be collected from the culturemedium of producer cells, purified, quantified, and used to transduce target cellsand deliver the active Cas9/sgRNA complex. Nanoblades deliver theirribonucleoprotein (RNP) cargo transiently and rapidly in a wide range of primary andimmortalized cells and can be programmed for other applications, such as transienttranscriptional activation of targeted genes, using modified Cas9 proteins.Nanoblades are capable of in vivo genome-editing in the liver of injected adult miceand in oocytes to generate transgenic animals. Finally, they can be complexed withdonor DNA for "transfection-free" homology-directed repair. Nanoblade preparation issimple, relatively low-cost, and can be easily carried out in any cell biologylaboratory.

Distinct spermiogenic phenotypes underlie sperm elimination in the Segregation Distorter meiotic drive system.

Author(s) : Herbette M, Wei X, Chang C, Larracuente A, Loppin B, Dubruille R,
Journal : PLoS Genet
2021
Segregation Distorter (SD) is a male meiotic drive system in Drosophilamelanogaster. Males heterozygous for a selfish SD chromosome rarely transmit thehomologous SD+ chromosome. It is well established that distortion results from aninteraction between Sd, the primary distorting locus on the SD chromosome and itstarget, a satellite DNA called Rsp, on the SD+ chromosome. However, the molecularand cellular mechanisms leading to post-meiotic SD+ sperm elimination remainunclear. Here we show that SD/SD+ males of different genotypes but with similarlystrong degrees of distortion have distinct spermiogenic phenotypes. In somegenotypes, SD+ spermatids fail to fully incorporate protamines after the removal ofhistones, and degenerate during the individualization stage of spermiogenesis. Incontrast, in other SD/SD+ genotypes, protamine incorporation appears less disturbed,yet spermatid nuclei are abnormally compacted, and mature sperm nuclei areeventually released in the seminal vesicle. Our analyses of different SD+chromosomes suggest that the severity of the spermiogenic defects associates withthe copy number of the Rsp satellite. We propose that when Rsp copy number is veryhigh (> 2000), spermatid nuclear compaction defects reach a threshold that triggersa checkpoint controlling sperm chromatin quality to eliminate abnormal spermatidsduring individualization.

Endogenous Bos taurus RECQL is predominantly monomeric and more active than oligomers.

Author(s) : Liu N, Song Z, Guo H, Yin H, Chen W, Dai Y, Xin B, Ai X, Ji L, Wang Q, Hou X, Dou S, Rety S, Xi X,
Journal : Cell Rep
2021
There is broad consensus that RecQ family helicase is a high-order oligomer thatdissociates into a dimer upon ATP binding. This conclusion is based mainly onstudies of highly purified recombinant proteins, and the oligomeric states ofRecQ helicases in living cells remain unknown. We show here that, in contrast tocurrent models, monomeric RECQL helicase is more abundant than oligomer/dimerforms in living cells. Further characterization of endogenous BtRECQL andisolated monomeric BtRECQL using various approaches demonstrates that bothendogenous and recombinant monomeric BtRECQL effectively function as monomers,displaying higher helicase and ATPase activities than dimers and oligomers.Furthermore, monomeric BtRECQL unfolds intramolecular G-quadruplex DNA asefficiently as human RECQL and BLM helicases. These discoveries have implicationsfor understanding endogenous RECQL oligomeric structures and their regulation. Itis worth revisiting oligomeric states of the other members of the RecQ familyhelicases in living cells.

Integrative Cell Type-Specific Multi-Omics Approaches Reveal Impaired Programs of Glial Cell Differentiation in Mouse Culture Models of DM1

Author(s) : González-Barriga A, Lallemant L, Dincã D, Braz S, Polvèche H, Magneron P, Pionneau C, Huguet-Lachon A, Claude J, Chhuon C, Guerrera I, Bourgeois C, Auboeuf D, Gourdon G, Gomes-Pereira M,
Journal : Front Cell Neurosci
2021
gene. This mutation generates a toxic CUG RNA that interferes with the RNA processing of target genes in multiple tissues. Despite debilitating neurological impairment, the pathophysiological cascade of molecular and cellular events in the central nervous system (CNS) has been less extensively characterized than the molecular pathogenesis of muscle/cardiac dysfunction. Particularly, the contribution of different cell types to DM1 brain disease is not clearly understood. We first used transcriptomics to compare the impact of expanded CUG RNA on the transcriptome of primary neurons, astrocytes and oligodendrocytes derived from DMSXL mice, a transgenic model of DM1. RNA sequencing revealed more frequent expression and splicing changes in glia than neuronal cells. In particular, primary DMSXL oligodendrocytes showed the highest number of transcripts differentially expressed, while DMSXL astrocytes displayed the most severe splicing dysregulation. Interestingly, the expression and splicing defects of DMSXL glia recreated molecular signatures suggestive of impaired cell differentiation: while DMSXL oligodendrocytes failed to upregulate a subset of genes that are naturally activated during the oligodendroglia differentiation, a significant proportion of missplicing events in DMSXL oligodendrocytes and astrocytes increased the expression of RNA isoforms typical of precursor cell stages. Together these data suggest that expanded CUG RNA in glial cells affects preferentially differentiation-regulated molecular events. This hypothesis was corroborated by gene ontology (GO) analyses, which revealed an enrichment for biological processes and cellular components with critical roles during cell differentiation. Finally, we combined exon ontology with phosphoproteomics and cell imaging to explore the functional impact of CUG-associated spliceopathy on downstream protein metabolism. Changes in phosphorylation, protein isoform expression and intracellular localization in DMSXL astrocytes demonstrate the far-reaching impact of the DM1 repeat expansion on cell metabolism. Our multi-omics approaches provide insight into the mechanisms of CUG RNA toxicity in the CNS with cell type resolution, and support the priority for future research on non-neuronal mechanisms and proteomic changes in DM1 brain disease.

Live imaging and biophysical modeling support a button-based mechanism of somatic homolog pairing in Drosophila.

Author(s) : Child M, Bateman J, Jahangiri A, Reimer A, Lammers N, Sabouni N, Villamarin D, McKenzie-Smith G, Johnson J, Jost D, Garcia H,
Journal : Elife
2021
Three-dimensional eukaryotic genome organization provides the structural basis forgene regulation. In Drosophila melanogaster, genome folding is characterized bysomatic homolog pairing, where homologous chromosomes are intimately paired from endto end; however, how homologs identify one another and pair has remained mysterious.Recently, this process has been proposed to be driven by specifically interacting'buttons' encoded along chromosomes. Here, we turned this hypothesis into aquantitative biophysical model to demonstrate that a button-based mechanism can leadto chromosome-wide pairing. We tested our model using live-imaging measurements ofchromosomal loci tagged with the MS2 and PP7 nascent RNA labeling systems. We showsolid agreement between model predictions and experiments in the pairing dynamics ofindividual homologous loci. Our results strongly support a button-based mechanism ofsomatic homolog pairing in Drosophila and provide a theoretical framework forrevealing the molecular identity and regulation of buttons.

Loop extrusion as a mechanism for formation of DNA damage repair foci.

Author(s) : Arnould C, Rocher V, Finoux A, Clouaire T, Li K, Zhou F, Caron P, Mangeot P, Ricci E, Mourad R, Haber J, Noordermeer D, Legube G,
Journal : Nature
2021
The repair of DNA double-strand breaks (DSBs) is essential for safeguarding genomeintegrity. When a DSB forms, the PI3K-related ATM kinase rapidly triggers theestablishment of megabase-sized, chromatin domains decorated with phosphorylatedhistone H2AX (γH2AX), which act as seeds for the formation of DNA-damage responsefoci(1). It is unclear how these foci are rapidly assembled to establish a'repair-prone' environment within the nucleus. Topologically associating domains area key feature of 3D genome organization that compartmentalize transcription andreplication, but little is known about their contribution to DNA repairprocesses(2,3). Here we show that topologically associating domains are functionalunits of the DNA damage response, and are instrumental for the correct establishmentof γH2AX-53BP1 chromatin domains in a manner that involves one-sidedcohesin-mediated loop extrusion on both sides of the DSB. We propose a model inwhich H2AX-containing nucleosomes are rapidly phosphorylated as they actively passby DSB-anchored cohesin. Our work highlights the importance of chromosomeconformation in the maintenance of genome integrity and demonstrates theestablishment of a chromatin modification by loop extrusion.

Modulation of alternative splicing during early infection of human primary B lymphocytes with Epstein-Barr virus (EBV): a novel function for the viral EBNA-LP protein

Author(s) : Manet E, Polvèche H, Mure F, Mrozek-Gorska P, Roisné-Hamelin F, Hammerschmidt W, Auboeuf D, Gruffat H,
Journal : Nucleic Acids Res
2021
Epstein-Barr virus (EBV) is a human herpesvirus associated with human cancers worldwide. Ex vivo, the virus efficiently infects resting human B lymphocytes and induces their continuous proliferation. This process is accompanied by a global reprogramming of cellular gene transcription. However, very little is known on the impact of EBV infection on the regulation of alternative splicing, a pivotal mechanism that plays an essential role in cell fate determination and is often deregulated in cancer. In this study, we have developed a systematic time-resolved analysis of cellular mRNA splice variant expression during EBV infection of resting B lymphocytes. Our results reveal that major modifications of alternative splice variant expression appear as early as day 1 post-infection and suggest that splicing regulation provides-besides transcription-an additional mechanism of gene expression regulation at the onset of B cell activation and proliferation. We also report a role for the viral proteins, EBNA2 and EBNA-LP, in the modulation of specific alternative splicing events and reveal a previously unknown function for EBNA-LP-together with the RBM4 splicing factor-in the alternative splicing regulation of two important modulators of cell proliferation and apoptosis respectively, NUMB and BCL-X.

Mutant Huntingtin stalls ribosomes and represses protein synthesis in a cellular model of Huntington disease.

Author(s) : Eshraghi M, Karunadharma P, Blin J, Shahani N, Ricci E, Michel A, Urban N, Galli N, Sharma M, Ramírez-Jarquín U, Florescu K, Hernandez J, Subramaniam S,
Journal : Nat Commun
2021
The polyglutamine expansion of huntingtin (mHTT) causes Huntington disease (HD) andneurodegeneration, but the mechanisms remain unclear. Here, we found that mHttpromotes ribosome stalling and suppresses protein synthesis in mouse HD striatalneuronal cells. Depletion of mHtt enhances protein synthesis and increases the speedof ribosomal translocation, while mHtt directly inhibits protein synthesis in vitro.Fmrp, a known regulator of ribosome stalling, is upregulated in HD, but itsdepletion has no discernible effect on protein synthesis or ribosome stalling in HDcells. We found interactions of ribosomal proteins and translating ribosomes withmHtt. High-resolution global ribosome footprint profiling (Ribo-Seq) and mRNA-Seqindicates a widespread shift in ribosome occupancy toward the 5' and 3' end andunique single-codon pauses on selected mRNA targets in HD cells, compared tocontrols. Thus, mHtt impedes ribosomal translocation during translation elongation,a mechanistic defect that can be exploited for HD therapeutics.

Mutational sources of trans-regulatory variation affecting gene expression in Saccharomyces cerevisiae.

Author(s) : Duveau F, Vande Zande P, Metzger B, Diaz C, Walker E, Tryban S, Siddiq M, Yang B, Wittkopp P,
Journal : Elife
2021
Heritable variation in a gene's expression arises from mutations impacting cis- andtrans-acting components of its regulatory network. Here, we investigate howtrans-regulatory mutations are distributed within the genome and within a generegulatory network by identifying and characterizing 69 mutations withtrans-regulatory effects on expression of the same focal gene in Saccharomycescerevisiae. Relative to 1766 mutations without effects on expression of this focalgene, we found that these trans-regulatory mutations were enriched in codingsequences of transcription factors previously predicted to regulate expression ofthe focal gene. However, over 90% of the trans-regulatory mutations identifiedmapped to other types of genes involved in diverse biological processes includingchromatin state, metabolism, and signal transduction. These data show how geneticchanges in diverse types of genes can impact a gene's expression in trans, revealingproperties of trans-regulatory mutations that provide the raw material fortrans-regulatory variation segregating within natural populations.

Neuronal perception of the social environment generates an inherited memory that controls the development and generation time of C. elegans

Author(s) : Perez M, Shamalnasab M, Mata-Cabana A, Della Valle S, Olmedo M, Francesconi M, Lehner B,
Journal : Current Biology
2021
An old and controversial question in biology is whether information perceived by the nervous system of an animal can “cross the Weismann barrier” to alter the phenotypes and fitness of their progeny. Here, we show that such intergenerational transmission of sensory information occurs in the model organism, C. elegans, with a major effect on fitness. Specifically, that perception of social pheromones by chemosensory neurons controls the post-embryonic timing of the development of one tissue, the germline, relative to others in the progeny of an animal. Neuronal perception of the social environment thus intergenerationally controls the generation time of this animal.

Organization of DNA Replication Origin Firing in Xenopus Egg Extracts: The Role of Intra-S Checkpoint.

Author(s) : Ciardo D, Haccard O, Narassimprakash H, Arbona J, Hyrien O, Audit B, Marheineke K, Goldar A,
Journal : Genes (Basel)
2021
During cell division, the duplication of the genome starts at multiple positionscalled replication origins. Origin firing requires the interaction of rate-limitingfactors with potential origins during the S(ynthesis)-phase of the cell cycle.Origins fire as synchronous clusters which is proposed to be regulated by theintra-S checkpoint. By modelling the unchallenged, the checkpoint-inhibited and thecheckpoint protein Chk1 over-expressed replication pattern of single DNA moleculesfrom Xenopus sperm chromatin replicated in egg extracts, we demonstrate that thequantitative modelling of data requires: (1) a segmentation of the genome intoregions of low and high probability of origin firing; (2) that regions with highprobability of origin firing escape intra-S checkpoint regulation and (3) thevariability of the rate of DNA synthesis close to replication forks is a necessaryingredient that should be taken in to account in order to describe the dynamic ofreplication origin firing. This model implies that the observed origin clusteringemerges from the apparent synchrony of origin firing in regions with highprobability of origin firing and challenge the assumption that the intra-Scheckpoint is the main regulator of origin clustering.

Physical and Genetic Assays for the Study of DNA Joint Molecules Metabolism and Multi-invasion-Induced Rearrangements in S. cerevisiae

Author(s) : Piazza A, Rajput P, Heyer W,
Journal : Methods in Molecular Biology
2021
DNA double-strand breaks (DSBs) are genotoxic lesions that can be repaired in a templated fashion by homologous recombination (HR). HR is a complex pathway that involves the formation of DNA joint molecules (JMs) containing heteroduplex DNA. Various types of JMs are formed throughout the pathway, including displacement loops (D-loops), multi-invasions (MI), and double Holliday junction intermediates. Dysregulation of JM metabolism in various mutant contexts revealed the propensity of HR to generate repeat-mediated chromosomal rearrangements. Specifically, we recently identified MI-induced rearrangements (MIR), a tripartite recombination mechanism initiated by one end of a DSB that exploits repeated regions to generate rearrangements between intact chromosomal regions. MIR occurs upon MI-JM processing by endonucleases and is suppressed by JM disruption activities. Here, we detail two assays: a physical assay for JM detection in Saccharomyces cerevisiae cells and genetic assays to determine the frequency of MIR in various chromosomal contexts. These assays enable studying the regulation of the HR pathway and the consequences of their defects for genomic instability by MIR.

Polymer modelling unveils the roles of heterochromatin and nucleolar organizing regions in shaping 3D genome organization in Arabidopsis thaliana.

Author(s) : Di Stefano M, Nützmann H, Marti-Renom M, Jost D,
Journal : Nucleic Acids Res
2021
The 3D genome is characterized by a complex organization made of genomic andepigenomic layers with profound implications on gene regulation and cell function.However, the understanding of the fundamental mechanisms driving the crosstalkbetween nuclear architecture and (epi)genomic information is still lacking. Theplant Arabidopsis thaliana is a powerful model organism to address these questionsowing to its compact genome for which we have a rich collection of microscopy,chromosome conformation capture (Hi-C) and ChIP-seq experiments. Using polymermodelling, we investigate the roles of nucleolus formation and epigenomics-driveninteractions in shaping the 3D genome of A. thaliana. By validation of severalpredictions with published data, we demonstrate that self-attracting nucleolarorganizing regions and repulsive constitutive heterochromatin are major mechanismsto regulate the organization of chromosomes. Simulations also suggest thatinterphase chromosomes maintain a partial structural memory of the V-shapes, typicalof (sub)metacentric chromosomes in anaphase. Additionally, self-attraction betweenfacultative heterochromatin regions facilitates the formation of Polycomb bodieshosting H3K27me3-enriched gene-clusters. Since nucleolus and heterochromatin arehighly-conserved in eukaryotic cells, our findings pave the way for a comprehensivecharacterization of the generic principles that are likely to shape and regulate the3D genome in many species.

Recombination-mediated genome rearrangements

Author(s) : Savocco J, Piazza A,
Journal : Current Opinion in Genetics and Development
2021
Homologous recombination (HR) is a universal DNA double-strand break (DSB) repair pathway that uses an intact DNA molecule as a template. Signature HR reactions are homology search and DNA strand invasion catalyzed by the prototypical RecA-ssDNA filament (Rad51 and Dmc1 in eukaryotes), which produces heteroduplex DNA-containing joint molecules (JMs). These reactions uniquely infringe on the DNA strands association established at replication, on the basis of substantial sequence similarity. For that reason, and despite the high fidelity of its templated nature, DSB repair by HR authorizes the alteration of genome structure, guided by repetitive DNA elements. The resulting structural variations (SVs) can involve vast genomic regions, potentially affecting multiple coding sequences and regulatory elements at once, with possible pathological consequences. Here, we discuss recent advances in our understanding of genetic and molecular vulnerabilities of HR leading to SVs, and of the various fidelity-enforcing factors acting across scales on the balancing act of this complex pathway. An emphasis is put on extra-chomosomal DNAs, both product of, and substrate for HR-mediated chromosomal rearrangements.

RNA polymerase backtracking results in the accumulation of fission yeast condensin at active genes.

Author(s) : Rivosecchi J, Jost D, Vachez L, Gautier F, Bernard P, Vanoosthuyse V,
Journal : Life Sci Alliance
2021
The mechanisms leading to the accumulation of the SMC complexes condensins aroundspecific transcription units remain unclear. Observations made in bacteria suggestedthat RNA polymerases (RNAPs) constitute an obstacle to SMC translocation,particularly when RNAP and SMC travel in opposite directions. Here we show infission yeast that gene termini harbour intrinsic condensin-accumulating featureswhatever the orientation of transcription, which we attribute to the frequentbacktracking of RNAP at gene ends. Consistent with this, to relocate backtrackedRNAP2 from gene termini to gene bodies was sufficient to cancel the accumulation ofcondensin at gene ends and to redistribute it evenly within transcription units,indicating that RNAP backtracking may play a key role in positioning condensin.Formalization of this hypothesis in a mathematical model suggests that the inclusionof a sub-population of RNAP with longer dwell-times is essential to fullyrecapitulate the distribution profiles of condensin around active genes. Takentogether, our data strengthen the idea that dense arrays of proteins tightly boundto DNA alter the distribution of condensin on chromosomes.

2020

4D Genome Rewiring during Oncogene-Induced and Replicative Senescence.

Author(s) : Sati S, Bonev B, Szabo Q, Jost D, Bensadoun P, Serra F, Loubiere V, Papadopoulos G, Rivera-Mulia J, Fritsch L, Bouret P, Castillo D, Gelpi J, Orozco M, Vaillant C, Pellestor F, Bantignies F, Marti-Renom M, Gilbert D, Lemaitre J, Cavalli G,
Journal : Mol Cell
2020
To understand the role of the extensive senescence-associated 3D genome reorganization, we generated genome-wide chromatin interaction maps, epigenome, replication-timing, whole-genome bisulfite sequencing, and gene expression profiles from cells entering replicative senescence (RS) or upon oncogene-induced senescence (OIS). We identify senescence-associated heterochromatin domains (SAHDs). Differential intra- versus inter-SAHD interactions lead to the formation of senescence-associated heterochromatin foci (SAHFs) in OIS but not in RS. ThisOIS-specific configuration brings active genes located in genomic regions adjacent to SAHDs in close spatial proximity and favors their expression. We also identify DNMT1 as a factor that induces SAHFs by promoting HMGA2 expression. Upon DNMT1 depletion, OIS cells transition to a 3D genome conformation akin to that of cells in replicative senescence. These data show how multi-omics and imaging canidentify critical features of RS and OIS and discover determinants of acute senescence and SAHF formation.

[Circular RNA, actors and biomarkers of cancers]

Author(s) : Ladet J, Mortreux F,
Journal : Med Sci (Paris)
2020

A quantitative tri-fluorescent yeast two-hybrid system: from flow cytometry to in-cellula affinities

Author(s) : Cluet D, Amri I, Vergier B, Léault J, Audibert A, Grosjean C, Calabrési D, Spichty M,
Journal : Molecular & Cellular Proteomics
2020
We present a technological advancement for the estimation of the affinities of Protein-Protein Interactions (PPIs) in living cells. A novel set of vectors is introduced that enables a quantitative yeast two-hybrid system based on fluorescent fusion proteins. The vectors allow simultaneous quantification of the reaction partners (Bait and Prey) and the reporter at the single-cell level by flow cytometry. We validate the applicability of this system on a small but diverse set of PPIs (eleven protein families from six organisms) with different affinities; the dissociation constants range from 117 pM to 17 µM. After only two hours of reaction, expression of the reporter can be detected even for the weakest PPI. Through a simple gating analysis, it is possible to select only cells with identical expression levels of the reaction partners. As a result of this standardization of expression levels, the mean reporter levels directly reflect the affinities of the studied PPIs. With a set of PPIs with known affinities, it is straightforward to construct an affinity ladder that permits rapid classification of PPIs with thus far unknown affinities. Conventional software can be used for this analysis. To permit automated analysis, we provide a graphical user interface for the Python-based FlowCytometryTools package.

A quantitative tri-fluorescent yeast two-hybrid system: from flow cytometry to in-cellula affinities

Author(s) : Cluet D, Amri I, Vergier B, Léault J, Audibert A, Grosjean C, Calabrési D, Spichty M,
Journal : Molecular & Cellular Proteomics
2020
We present a technological advancement for the estimation of the affinities of Protein-Protein Interactions (PPIs) in living cells. A novel set of vectors is introduced that enables a quantitative yeast two-hybrid system based on fluorescent fusion proteins. The vectors allow simultaneous quantification of the reaction partners (Bait and Prey) and the reporter at the single-cell level by flow cytometry. We validate the applicability of this system on a small but diverse set of PPIs (eleven protein families from six organisms) with different affinities; the dissociation constants range from 117 pM to 17 µM. After only two hours of reaction, expression of the reporter can be detected even for the weakest PPI. Through a simple gating analysis, it is possible to select only cells with identical expression levels of the reaction partners. As a result of this standardization of expression levels, the mean reporter levels directly reflect the affinities of the studied PPIs. With a set of PPIs with known affinities, it is straightforward to construct an affinity ladder that permits rapid classification of PPIs with thus far unknown affinities. Conventional software can be used for this analysis. To permit automated analysis, we provide a graphical user interface for the Python-based FlowCytometryTools package.

A Role for Caenorhabditis elegans COMPASS in Germline Chromatin Organization.

Author(s) : Herbette M, Robert V, Bailly A, Gely L, Feil R, Llères D, Palladino F,
Journal : Cells
2020
Deposition of histone H3 lysine 4 (H3K4) methylation at promoters is catalyzed bythe SET1/COMPASS complex and is associated with context-dependent effects on geneexpression and local changes in chromatin organization. The role of SET1/COMPASS inshaping chromosome architecture has not been investigated. Here we usedCaenorhabditis elegans to address this question through a live imaging approach andgenetic analysis. Using quantitative FRET (Förster resonance energy transfer)-basedfluorescence lifetime imaging microscopy (FLIM) on germ cells expressing histoneseGFP-H2B and mCherry-H2B, we find that SET1/COMPASS influences meiotic chromosomeorganization, with marked effects on the close proximity between nucleosomes. Wefurther show that inactivation of set-2, encoding the C. elegans SET1 homologue, orCFP-1, encoding the chromatin targeting subunit of COMPASS, enhances germlinechromosome organization defects and sterility of condensin-II depleted animals.set-2 loss also aggravates germline defects resulting from conditional inactivationof topoisomerase II, another structural component of chromosomes. Expressionprofiling of set-2 mutant germlines revealed only minor transcriptional changes,suggesting that the observed effects are at least partly independent oftranscription. Altogether, our results are consistent with a role for SET1/COMPASSin shaping meiotic chromosomes in C. elegans, together with the non-histone proteinscondensin-II and topoisomerase. Given the high degree of conservation, our findingsexpand the range of functions attributed to COMPASS and suggest a broader role ingenome organization in different species.

Aurora B and condensin are dispensable for chromosome arm and telomere separation during meiosis II.

Author(s) : Berthezene J, Reyes C, Li T, Coulon S, Bernard P, Gachet Y, Tournier S,
Journal : Mol Biol Cell
2020
In mitosis, while the importance of kinetochore (KT)-microtubule (MT) attachment hasbeen known for many years, increasing evidence suggests that telomere dysfunctionsalso perturb chromosome segregation by contributing to the formation of chromatinbridges at anaphase. Recent evidence suggests that Aurora B kinase ensures properchromosome segregation during mitosis not only by controlling KT-MT attachment butalso by regulating telomere and chromosome arm separation. However, whether and howAurora B governs telomere separation during meiosis has remained unknown. Here, weshow that fission yeast Aurora B localizes at telomeres during meiosis I andpromotes telomere separation independently of the meiotic cohesin Rec8. In meiosisII, Aurora B controls KT-MT attachment but appears dispensable for telomere andchromosome arm separation. Likewise, condensin activity is nonessential in meiosisII for telomere and chromosome arm separation. Thus, in meiosis, the requirementsfor Aurora B are distinct at centromeres and telomeres, illustrating the criticaldifferences in the control of chromosome segregation between mitosis and meiosis II.

Caenorhabditis elegans SET1/COMPASS Maintains Germline Identity by Preventing Transcriptional Deregulation Across Generations.

Author(s) : Robert V, Knutson A, Rechtsteiner A, Garvis S, Yvert G, Strome S, Palladino F,
Journal : Front Cell Dev Biol
2020
Chromatin regulators contribute to the maintenance of the germline transcriptionalprogram. In the absence of SET-2, the Caenorhabditis elegans homolog of theSET1/COMPASS H3 Lys4 (H3K4) methyltransferase, animals show transgenerational lossof germline identity, leading to sterility. To identify transcriptional signaturesassociated with progressive loss of fertility, we performed expression profiling ofset-2 mutant germlines across generations. We identify a subset of genes whosemisexpression is first observed in early generations, a step we refer to as priming;their misexpression then further progresses in late generations, as animals reachsterility. Analysis of misregulated genes shows that down-regulation of germlinegenes, expression of somatic transcriptional programs, and desilencing of theX-chromosome are concurrent events leading to loss of germline identity in bothearly and late generations. Upregulation of transcription factor LIN-15B, the C/EBPhomolog CEBP-1, and TGF-β pathway components strongly contribute to loss offertility, and RNAi inactivation of cebp-1 and TGF-β/Smad signaling delays the onsetof sterility, showing they individually contribute to maintenance of germ cellidentity. Our approach therefore identifies genes and pathways whose misexpressionactively contributes to the loss of germ cell fate. More generally, our data showshow loss of a chromatin regulator in one generation leads to transcriptional changesthat are amplified over subsequent generations, ultimately leading to loss ofappropriate cell fate.

Cell-to-cell expression dispersion of B-cell surface proteins is linked to genetic variants in humans.

Author(s) : Triqueneaux G, Burny C, Symmons O, Janczarski S, Gruffat H, Yvert G,
Journal : Commun Biol
2020
Variability in gene expression across a population of homogeneous cells is known toinfluence various biological processes. In model organisms, natural genetic variantswere found that modify expression dispersion (variability at a fixed mean) but veryfew studies have detected such effects in humans. Here, we analyzed single-cellexpression of four proteins (CD23, CD55, CD63 and CD86) across cell lines derivedfrom individuals of the Yoruba population. Using data from over 30 million cells, wefound substantial inter-individual variation of dispersion. We demonstrate, via denovo cell line generation and subcloning experiments, that this variation exceedsthe variation associated with cellular immortalization. We detected a geneticassociation between the expression dispersion of CD63 and the rs971 SNP. Our resultsshow that human DNA variants can have inherently-probabilistic effects on geneexpression. Such subtle genetic effects may participate to phenotypic variation anddisease outcome.

Chromosome dynamics during interphase: a biophysical perspective.

Author(s) : Tortora M, Salari H, Jost D,
Journal : Curr Opin Genet Dev
2020
The dynamic nature of chromosome organization plays a central role in the regulation of many crucial processes, such as DNA transcription and replication.However, the molecular bases of the link between genomic function, structure anddynamics remain elusive. In this review, we focus on how biophysical modelling can be instrumentally used to rationalize experimental studies of chromosome dynamics, and to probe the impact of putative mechanisms on genome folding kinetics during interphase. We introduce the general connection between chromatin internal organization and dynamics, and outline the potential effects of passiveinteractions mediated by architectural proteins and of active, energy-dependent processes on chromatin motion. Finally, we discuss current ambiguities emerging from in vivo observations, in particular related to ATP depletion and transcriptional activation, and highlight future perspectives.

Developmental variability channels mouse molar evolution.

Author(s) : Hayden L, Lochovska K, Semon M, Renaud S, Delignette-Muller M, Vilcot M, Peterkova R, Hovorakova M, Pantalacci S,
Journal : Elife
2020
Do developmental systems preferentially produce certain types of variation that orient phenotypic evolution along preferred directions? At different scales, from the intra-population to the interspecific, the murine first upper molar shows repeated anterior elongation. Using a novel quantitative approach to compare thedevelopment of two mouse strains with short or long molars, we identified temporal, spatial and functional differences in tooth signaling center activity,that arise from differential tuning of the activation-inhibition mechanisms underlying tooth patterning. By tracing their fate, we could explain why only the upper first molar reacts via elongation of its anterior part. Despite a lack of genetic variation, individuals of the elongated strain varied in tooth length and the temporal dynamics of their signaling centers, highlighting the intrinsic instability of the upper molar developmental system. Collectively, these resultsreveal the variational properties of murine molar development that drive morphological evolution along a line of least resistance.

Diversification and hybrid incompatibility in auto-pseudogamous species of Mesorhabditis nematodes.

Author(s) : Launay C, Félix M, Dieng J, Delattre M,
Journal : BMC Evol Biol
2020
BACKGROUND: Pseudogamy is a reproductive system in which females rely on the spermof males to activate their oocytes, generally parasitizing males of other species,but do not use the sperm DNA. The nematode Mesorhabditis belari uses a specific formof pseudogamy, where females produce their own males as a source of sperm. Malesdevelop from rare eggs with true fertilization, while females arise by gynogenesis.Males thus do not contribute their genome to the female offspring. Here, we exploredthe diversity of reproductive mode within the Mesorhabditis genus and addressedspecies barriers in pseudogamous species. RESULTS: To this end, we established acollection of over 60 Mesorhabditis strains from soil and rotting vegetal matter. Wefound that males from pseudogamous species displayed a reduced size of their body,male tail and sperm cells compared to males of sexual Mesorhabditis species, asexpected for males that face little competition. Using rDNA sequences and crosses,we could define 11 auto-pseudogamous biological species, with closely relatedspecies pairs and a possible single origin of pseudogamy in the Mesorhabditis genus.Most crosses between males and females of different species did not even producefemale progeny. This surprising species barrier in pseudogamous egg activation waspre or postcopulatory depending on the species pair. In the latter case, when hybridembryos were produced, most arrested before the first embryonic cell division.Hybrid incompatibility between auto-pseudogamous species was due to defectiveinteraction between sperm and oocyte as well as defective reconstitution of zygoticcentrosomes. CONCLUSIONS: We established a collection of sexual and pseudo-sexualspecies which offer an ideal framework to explore the origin and consequences oftransition to asexuality. Our results demonstrate that speciation occurs in thepseudogamous state. Whereas genomic conflicts are responsible for hybridincompatibility in sexual species, we here reveal that centrosomes constitute keyorganelles in the establishment of species barrier.

FORK-seq: replication landscape of the Saccharomyces cerevisiae genome by nanopore sequencing.

Author(s) : Hennion M, Arbona J, Lacroix L, Cruaud C, Theulot B, Tallec B, Proux F, Wu X, Novikova E, Engelen S, Lemainque A, Audit B, Hyrien O,
Journal : Genome Biol
2020
Genome replication mapping methods profile cell populations, masking cell-to-cellheterogeneity. Here, we describe FORK-seq, a nanopore sequencing method to mapreplication of single DNA molecules at 200-nucleotide resolution. By quantifyingBrdU incorporation along pulse-chased replication intermediates from Saccharomycescerevisiae, we orient 58,651 replication tracks reproducing population-basedreplication directionality profiles and map 4964 and 4485 individual initiation andtermination events, respectively. Although most events cluster at known origins andfork merging zones, 9% and 18% of initiation and termination events, respectively,occur at many locations previously missed. Thus, FORK-seq reveals the full extent ofcell-to-cell heterogeneity in DNA replication.

Functional diversification in the Nudix hydrolase gene family drives sesquiterpene biosynthesis in Rosa × wichurana.

Author(s) : Sun P, Dégut C, Réty S, Caissard J, Hibrand-Saint Oyant L, Bony A, Paramita S, Conart C, Magnard J, Jeauffre J, Abd-El-Haliem A, Marie-Magdelaine J, Thouroude T, Baltenweck R, Tisné C, Foucher F, Haring M, Hugueney P, Schuurink R, Baudino S,
Journal : Plant J
2020
Roses use a non-canonical pathway involving a Nudix hydrolase, RhNUDX1, tosynthesize their monoterpenes, especially geraniol. Here we report thecharacterization of another expressed NUDX1 gene from the rose cultivar Rosa xwichurana, RwNUDX1-2. In order to study the function of the RwNUDX1-2 protein, weanalyzed the volatile profiles of an F(1) progeny generated by crossingR. chinensis cv. 'Old Blush' with R. x wichurana. A correlation test of thevolatilomes with gene expression data revealed that RwNUDX1-2 is involved in thebiosynthesis of a group of sesquiterpenoids, especially E,E-farnesol, in additionto other sesquiterpenes. In vitro enzyme assays and heterologous in plantafunctional characterization of the RwNUDX1-2 gene corroborated this result. Aquantitative trait locus (QTL) analysis was performed using the data ofE,E-farnesol contents in the progeny and a genetic map was constructed based ongene markers. The RwNUDX1-2 gene co-localized with the QTL for E,E-farnesolcontent, thereby confirming its function in sesquiterpenoid biosynthesis in R. xwichurana. Finally, in order to understand the structural bases for the substratespecificity of rose NUDX proteins, the RhNUDX1 protein was crystallized, and itsstructure was refined to 1.7 Å. By molecular modeling of different rose NUDX1protein complexes with their respective substrates, a structural basis forsubstrate discrimination by rose NUDX1 proteins is proposed.

Genome organization via loop extrusion, insights from polymer physics models.

Author(s) : Ghosh S, Jost D,
Journal : Brief Funct Genomics
2020
Understanding how genomes fold and organize is one of the main challenges in modern biology. Recent high-throughput techniques like Hi-C, in combination withcutting-edge polymer physics models, have provided access to precise informationon 3D chromosome folding to decipher the mechanisms driving such multi-scale organization. In particular, structural maintenance of chromosome (SMC) proteinsplay an important role in the local structuration of chromatin, putatively via aloop extrusion process. Here, we review the different polymer physics models that investigate the role of SMCs in the formation of topologically associated domains (TADs) during interphase via the formation of dynamic loops. We describe the main physical ingredients, compare them and discuss their relevance against experimental observations.

Gradient in cytoplasmic pressure in germline cells controls overlying epithelial cell morphogenesis

Author(s) : Lamiré L, Milani P, Runel G, Kiss A, Arias L, Vergier B, de Bossoreille S, Das P, Cluet D, Boudaoud A, Grammont M,
Journal : PLoS Biol
2020

Guidelines for cell-type heterogeneity quantification based on a comparative analysis of reference-free DNA methylation deconvolution software.

Author(s) : Decamps C, Prive F, Bacher R, Jost D, Waguet A, Houseman E, Lurie E, Lutsik P, Milosavljevic A, Scherer M, Blum M, Richard M,
Journal : BMC Bioinformatics
2020
BACKGROUND: Cell-type heterogeneity of tumors is a key factor in tumor progression and response to chemotherapy. Tumor cell-type heterogeneity, definedas the proportion of the various cell-types in a tumor, can be inferred from DNAmethylation of surgical specimens. However, confounding factors known to associate with methylation values, such as age and sex, complicate accurate inference of cell-type proportions. While reference-free algorithms have been developed to infer cell-type proportions from DNA methylation, a comparative evaluation of the performance of these methods is still lacking. RESULTS: Here we use simulations to evaluate several computational pipelines based on the software packages MeDeCom, EDec, and RefFreeEWAS. We identify that accounting for confounders, feature selection, and the choice of the number of estimated cell types are critical steps for inferring cell-type proportions. We find that removal of methylation probes which are correlated with confounder variables reduces the error of inference by 30-35%, and that selection of cell-type informative probes has similar effect. We show that Cattell's rule based on the scree plot is a powerful tool to determine the number of cell-types. Once the pre-processing steps are achieved, the three deconvolution methods provide comparable results. We observe that all the algorithms' performance improves when inter-sample variation of cell-type proportions is large or when the number of available samples is large. We find that under specific circumstances the methods are sensitive to the initialization method, suggesting that averaging different solutions or optimizing initialization is an avenue for future research. CONCLUSION: Based on the lessons learned, to facilitate pipeline validation and catalyze further pipeline improvement by the community, we develop a benchmark pipeline for inference of cell-type proportions and implement it in the R package medepir.

Histone Variants: The Nexus of Developmental Decisions and Epigenetic Memory.

Author(s) : Loppin B, Berger F,
Journal : Annual Review of Genetics
2020
Nucleosome dynamics and properties are central to all forms of genomic activities.Among the core histones, H3 variants play a pivotal role in modulating nucleosomestructure and function. Here, we focus on the impact of H3 variants on variousfacets of development. The deposition of the replicative H3 variant following DNAreplication is essential for the transmission of the epigenomic information encodedin posttranscriptional modifications. Through this process, replicative H3 maintainscell fate while, in contrast, the replacement H3.3 variant opposes celldifferentiation during early embryogenesis. In later steps of development, H3.3 andspecialized H3 variants are emerging as new, important regulators of terminal celldifferentiation, including neurons and gametes. The specific pathways that regulatethe dynamics of the deposition of H3.3 are paramount during reprogramming eventsthat drive zygotic activation and the initiation of a new cycle of development.

Intragenic recruitment of NF-κB drives splicing modifications upon activation by the oncogene Tax of HTLV-1

Author(s) : Ameur L, Marie P, Thenoz M, Giraud G, Combe E, Claude J, Lemaire S, Fontrodona N, Polveche H, Bastien M, Gessain A, Wattel E, Bourgeois C, Auboeuf D, Mortreux F,
Journal : Nat Commun
2020

PenDA, a rank-based method for personalized differential analysis: Application to lung cancer.

Author(s) : Richard M, Decamps C, Chuffart F, Brambilla E, Rousseaux S, Khochbin S, Jost D,
Journal : PLoS Comput Biol
2020
The hopes of precision medicine rely on our capacity to measure various high-throughput genomic information of a patient and to integrate them for personalized diagnosis and adapted treatment. Reaching these ambitious objectives will require the development of efficient tools for the detection of molecular defects at the individual level. Here, we propose a novel method, PenDA, to perform Personalized Differential Analysis at the scale of a single sample. PenDA is based on the local ordering of gene expressions within individual cases and infers the deregulation status of genes in a sample of interest compared to a reference dataset. Based on realistic simulations of RNA-seq data of tumors, we showed that PenDA outcompetes existing approaches with very high specificity andsensitivity and is robust to normalization effects. Applying the method to lung cancer cohorts, we observed that deregulated genes in tumors exhibit a cancer-type-specific commitment towards up- or down-regulation. Based on the individual information of deregulation given by PenDA, we were able to define two new molecular histologies for lung adenocarcinoma cancers strongly correlated tosurvival. In particular, we identified 37 biomarkers whose up-regulation lead tobad prognosis and that we validated on two independent cohorts. PenDA provides arobust, generic tool to extract personalized deregulation patterns that can thenbe used for the discovery of therapeutic targets and for personalized diagnosis.An open-access, user-friendly R package is available at https://github.com/bcm-uga/penda.

Physicochemical Foundations of Life that Direct Evolution: Chance and Natural Selection are not Evolutionary Driving Forces

Author(s) : Auboeuf D,
Journal : Life (Basel)
2020

Pseudomonas aeruginosa cleaves the decoding center of Caenorhabditis elegans ribosomes.

Author(s) : Vasquez-Rifo A, Ricci E, Ambros V,
Journal : PLoS Biol
2020
Pathogens such as Pseudomonas aeruginosa advantageously modify animal hostphysiology, for example, by inhibiting host protein synthesis. Translationalinhibition of insects and mammalian hosts by P. aeruginosa utilizes the well-knownexotoxin A effector. However, for the infection of Caenorhabditis elegans by P.aeruginosa, the precise pathways and mechanism(s) of translational inhibition arenot well understood. We found that upon exposure to P. aeruginosa PA14, C. elegansundergoes a rapid loss of intact ribosomes accompanied by the accumulation ofribosomes cleaved at helix 69 (H69) of the 26S ribosomal RNA (rRNA), a key part ofribosome decoding center. H69 cleavage is elicited by certain virulent P. aeruginosaisolates in a quorum sensing (QS)-dependent manner and independently of exotoxinA-mediated translational repression. H69 cleavage is antagonized by the 3 major hostdefense pathways defined by the pmk-1, fshr-1, and zip-2 genes. The level of H69cleavage increases with the bacterial exposure time, and it is predominantlylocalized in the worm's intestinal tissue. Genetic and genomic analysis suggeststhat H69 cleavage leads to the activation of the worm's zip-2-mediated defenseresponse pathway, consistent with translational inhibition. Taken together, ourobservations suggest that P. aeruginosa deploys a virulence mechanism to induceribosome degradation and H69 cleavage of host ribosomes. In this manner, P.aeruginosa would impair host translation and block antibacterial responses.

Rdh54/Tid1 Inhibits Rad51-Rad54-Mediated D-loop Formation and Limits D-loop Length

Author(s) : Shah S, Hartono S, Piazza A, Som V, Wright W, Chedin F, Heyer W,
Journal : Elife
2020
Displacement loops (D-loops) are critical intermediates formed during homologous recombination. Rdh54 (a.k.a. Tid1), a Rad54 paralog in Saccharomyces cerevisiae, is well-known for its role with Dmc1 recombinase during meiotic recombination. Yet contrary to Dmc1, Rdh54/Tid1 is also present in somatic cells where its function is less understood. While Rdh54/Tid1 enhances the Rad51 DNA strand invasion activity in vitro, it is unclear how it interplays with Rad54. Here, we show that Rdh54/Tid1 inhibits D-loop formation by Rad51 and Rad54 in an ATPase-independent manner. Using a novel D-loop Mapping Assay, we further demonstrate that Rdh54/Tid1 uniquely restricts the length of Rad51-Rad54-mediated D-loops. The alterations in D-loop properties appear to be important for cell survival and mating-type switch in haploid yeast. We propose that Rdh54/Tid1 and Rad54 compete for potential binding sites within the Rad51 filament, where Rdh54/Tid1 acts as a physical roadblock to Rad54 translocation, limiting D-loop formation and D-loop length.

RNA polymerase II CTD S2P is dispensable for embryogenesis but mediates exit from developmental diapause in C. elegans.

Author(s) : Cassart C, Yague-Sanz C, Bauer F, Ponsard P, Stubbe F, Migeot V, Wery M, Morillon A, Palladino F, Robert V, Hermand D,
Journal : Sci Adv
2020
Serine 2 phosphorylation (S2P) within the CTD of RNA polymerase II is considered aCdk9/Cdk12-dependent mark required for 3'-end processing. However, the relevance ofCTD S2P in metazoan development is unknown. We show that cdk-12 lesions or afull-length CTD S2A substitution results in an identical phenotype in Caenorhabditiselegans Embryogenesis occurs in the complete absence of S2P, but the hatched larvaearrest development, mimicking the diapause induced when hatching occurs in theabsence of food. Genome-wide analyses indicate that when CTD S2P is inhibited, onlya subset of growth-related genes is not properly expressed. These genes correspondto SL2 trans-spliced mRNAs located in position 2 and over within operons. We showthat CDK-12 is required for maximal occupancy of cleavage stimulatory factornecessary for SL2 trans-splicing. We propose that CTD S2P functions as agene-specific signaling mark ensuring the nutritional control of the C. elegansdevelopmental program.

Saccharomyces Cerevisiae Mus81-Mms4 Prevents Accelerated Senescence in Telomerase-Deficient Cells

Author(s) : Scwartz E, Hung S, Meyer D, Piazza A, Yan K, Fu B, Heyer W,
Journal : PLoS Genetics
2020
Alternative lengthening of telomeres (ALT) in human cells is a conserved process that is often activated in telomerase-deficient human cancers. This process exploits components of the recombination machinery to extend telomere ends, thus allowing for increased proliferative potential. Human MUS81 (Mus81 in Saccharomyces cerevisiae) is the catalytic subunit of structure-selective endonucleases involved in recombination and has been implicated in the ALT mechanism. However, it is unclear whether MUS81 activity at the telomere is specific to ALT cells or if it is required for more general aspects of telomere stability. In this study, we use S. cerevisiae to evaluate the contribution of the conserved Mus81-Mms4 endonuclease in telomerase-deficient yeast cells that maintain their telomeres by mechanisms akin to human ALT. Similar to human cells, we find that yeast Mus81 readily localizes to telomeres and its activity is important for viability after initial loss of telomerase. Interestingly, our analysis reveals that yeast Mus81 is not required for the survival of cells undergoing recombination-mediated telomere lengthening, i.e. for ALT itself. Rather we infer from genetic analysis that Mus81-Mms4 facilitates telomere replication during times of telomere instability. Furthermore, combining mus81 mutants with mutants of a yeast telomere replication factor, Rrm3, reveals that the two proteins function in parallel to promote normal growth during times of telomere stress. Combined with previous reports, our data can be interpreted in a consistent model in which both yeast and human MUS81-dependent nucleases participate in the recovery of stalled replication forks within telomeric DNA. Furthermore, this process becomes crucial under conditions of additional replication stress, such as telomere replication in telomerase-deficient cells.

Spen modulates lipid droplet content in adult Drosophila glial cells and protects against paraquat toxicity.

Author(s) : Girard V, Goubard V, Querenet M, Seugnet L, Pays L, Nataf S, Dufourd E, Cluet D, Mollereau B, Davoust N,
Journal : Sci Rep
2020
Glial cells are early sensors of neuronal injury and can store lipids in lipiddroplets under oxidative stress conditions. Here, we investigated the functions ofthe RNA-binding protein, SPEN/SHARP, in the context of Parkinson's disease (PD).Using a data-mining approach, we found that SPEN/SHARP is one of manyastrocyte-expressed genes that are significantly differentially expressed in thesubstantia nigra of PD patients compared with control subjects. Interestingly, thedifferentially expressed genes are enriched in lipid metabolism-associated genes. Ina Drosophila model of PD, we observed that flies carrying a loss-of-function alleleof the ortholog split-ends (spen) or with glial cell-specific, but notneuronal-specific, spen knockdown were more sensitive to paraquat intoxication,indicating a protective role for Spen in glial cells. We also found that Spen is apositive regulator of Notch signaling in adult Drosophila glial cells. Moreover,Spen was required to limit abnormal accumulation of lipid droplets in glial cells ina manner independent of its regulation of Notch signaling. Taken together, ourresults demonstrate that Spen regulates lipid metabolism and storage in glial cellsand contributes to glial cell-mediated neuroprotection.

The Complex Relationship between HTLV-1 and Nonsense-Mediated mRNA Decay (NMD).

Author(s) : Prochasson L, Jalinot P, Mocquet V,
Journal : Pathogens
2020
Before the establishment of an adaptive immune response, retroviruses can betargeted by several cellular host factors at different stages of the viralreplication cycle. This intrinsic immunity relies on a large diversity ofantiviral processes. In the case of HTLV-1 infection, these active innate hostdefense mechanisms are debated. Among these mechanisms, we focused on an RNAdecay pathway called nonsense-mediated mRNA decay (NMD), which can targetmultiple viral RNAs, including HTLV-1 unspliced RNA, as has been recentlydemonstrated. NMD is a co-translational process that depends on the RNA helicaseUPF1 and regulates the expression of multiple types of host mRNAs. RNAsensitivity to NMD depends on mRNA organization and the ribonucleoprotein (mRNP)composition. HTLV-1 has evolved several means to evade the NMD threat, leading toNMD inhibition. In the early steps of infection, NMD inhibition favours theproduction of HTLV-1 infectious particles, which may contribute to the survivalof the fittest clones despite genome instability; however, its direct long-termimpact remains to be investigated.

The Enigma of Centriole Loss in the 1182-4 Cell Line.

Author(s) : Debec A, Loppin B, Zheng C, Liu X, Megraw T,
Journal : Cells
2020
The Drosophila melanogaster cell line 1182-4, which constitutively lacks centrioles,was established many years ago from haploid embryos laid by females homozygous forthe maternal haploid (mh) mutation. This was the first clear example of animal cellsregularly dividing in the absence of this organelle. However, the cause of theacentriolar nature of the 1182-4 cell line remained unclear and could not be clearlyassigned to a particular genetic event. Here, we detail historically thelongstanding mystery of the lack of centrioles in this Drosophila cell line. Recentadvances, such as the characterization of the mh gene and the genomic analysis of1182-4 cells, allow now a better understanding of the physiology of these cells. Bycombining these new data, we propose three reasonable hypotheses of the genesis ofthis remarkable phenotype.

The Lid/KDM5 histone demethylase complex activates a critical effector of the oocyte-to-zygote transition.

Author(s) : Torres-Campana D, Kimura S, Orsi G, Horard B, Benoit G, Loppin B,
Journal : PLoS Genet
2020
Following fertilization of a mature oocyte, the formation of a diploid zygote involves a series of coordinated cellular events that ends with the first embryonic mitosis. In animals, this complex developmental transition is almost entirely controlled by maternal gene products. How such a crucial transcriptional program is established during oogenesis remains poorly understood. Here, we haveperformed an shRNA-based genetic screen in Drosophila to identify genes requiredto form a diploid zygote. We found that the Lid/KDM5 histone demethylase and itspartner, the Sin3A-HDAC1 deacetylase complex, are necessary for sperm nuclear decompaction and karyogamy. Surprisingly, transcriptomic analyses revealed that these histone modifiers are required for the massive transcriptional activation of deadhead (dhd), which encodes a maternal thioredoxin involved in sperm chromatin remodeling. Unexpectedly, while lid knock-down tends to slightly favorthe accumulation of its target, H3K4me3, on the genome, this mark was lost at the dhd locus. We propose that Lid/KDM5 and Sin3A cooperate to establish a local chromatin environment facilitating the unusually high expression of dhd, a key effector of the oocyte-to-zygote transition.

2019

A cohesin/HUSH- and LINC-dependent pathway controls ribosomal DNA double-strand break repair.

Author(s) : Marnef A, Finoux A, Arnould C, Guillou E, Daburon V, Rocher V, Mangeat T, Mangeot P, Ricci E, Legube G,
Journal : Genes Dev
2019
The ribosomal DNA (rDNA) represents a particularly unstable locus undergoing frequent breakage. DNA double-strand breaks (DSBs) within rDNA induce both rDNA transcriptional repression and nucleolar segregation, but the link between the two events remains unclear. Here we found that DSBs induced on rDNA trigger transcriptional repression in a cohesin- and HUSH (human silencing hub) complex-dependent manner throughout the cell cycle. In S/G2 cells, transcriptional repression is further followed by extended resection within the interior of the nucleolus, DSB mobilization at the nucleolar periphery within nucleolar caps, and repair by homologous recombination. We showed that nuclear envelope invaginations frequently connect the nucleolus and that rDNA DSB mobilization, but not transcriptional repression, involves the nuclear envelope-associated LINC complex and the actin pathway. Altogether, our data indicate that rDNA break localization at the nucleolar periphery is not a directconsequence of transcriptional repression but rather is an active process that shares features with the mobilization of persistent DSB in active genes and heterochromatin.

A genome-wide screen identifies IRF2 as a key regulator of caspase-4 in human cells

Author(s) : Benaoudia S, Martin A, Puig Gamez M, Gay G, Lagrange B, Cornut M, Krasnykov K, Claude J, Bourgeois C, Hughes S, Gillet B, Allatif O, Corbin A, Ricci R, Henry T,
Journal : EMBO Rep
2019

Characterizing the interplay between gene nucleotide composition bias and splicing

Author(s) : Lemaire S, Fontrodona N, Aub? F, Claude J, Polv?che H, Modolo L, Bourgeois C, Mortreux F, Auboeuf D,
Journal : Genome Biol
2019

Detecting adaptive convergent amino acid evolution.

Author(s) : Rey C, Lanore V, Veber P, Gueguen L, Lartillot N, Semon M, Boussau B,
Journal : Philos Trans R Soc Lond B Biol Sci
2019
In evolutionary genomics, researchers have taken an interest in identifying substitutions that subtend convergent phenotypic adaptations. This is a difficult question that requires distinguishing foreground convergent substitutions that are involved in the convergent phenotype from background convergent substitutions. Those may be linked to other adaptations, may be neutral or may be the consequence of mutational biases. Furthermore, there is no generally accepted definition of convergent substitutions. Various methods that use different definitions have been proposed in the literature, resulting in different sets ofcandidate foreground convergent substitutions. In this article, we first describe the processes that can generate foreground convergent substitutions in coding sequences, separating adaptive from non-adaptive processes. Second, we review methods that have been proposed to detect foreground convergent substitutions incoding sequences and expose the assumptions that underlie them. Finally, we examine their power on simulations of convergent changes-including in the presence of a change in the efficacy of selection-and on empirical alignments. This article is part of the theme issue 'Convergent evolution in the genomics era: new insights and directions'.

Developmental and comparative transcriptomic identification of iridophore contribution to white barring in clownfish.

Author(s) : Salis P, Lorin T, Lewis V, Rey C, Marcionetti A, Escande M, Roux N, Besseau L, Salamin N, Semon M, Parichy D, Volff J, Laudet V,
Journal : Pigment Cell Melanoma Res
2019
Actinopterygian fishes harbor at least eight distinct pigment cell types, leading to a fascinating diversity of colors. Among this diversity, the cellular origin of the white color appears to be linked to several pigment cell types such as iridophores or leucophores. We used the clownfish Amphiprion ocellaris, which has a color pattern consisting of white bars over a darker body, to characterize thepigment cells that underlie the white hue. We observe by electron microscopy that cells in white bars are similar to iridophores. In addition, the transcriptomic signature of clownfish white bars exhibits similarities with that of zebrafish iridophores. We further show by pharmacological treatments that these cells are necessary for the white color. Among the top differentially expressed genes in white skin, we identified several genes (fhl2a, fhl2b, saiyan, gpnmb, and apoD1a) and show that three of them are expressed in iridophores. Finally, we show by CRISPR/Cas9 mutagenesis that these genes are critical for iridophore developmentin zebrafish. Our analyses provide clues to the genomic underpinning of color diversity and allow identification of new iridophore genes in fish.

Dynamic processing of displacement loops during recombinational DNA repair

Author(s) : Piazza A, Shah S, Wright W, Gore S, Koszul R, Heyer W,
Journal : Molecular cell
2019
Displacement-loops (D-loops) are pivotal intermediates of homologous recombination (HR), a universal DNA double strand break (DSB) repair pathway. We developed a versatile assay for the physical detection of D-loops in vivo, which enabled studying the kinetics of their formation and defining the activities controlling their metabolism. Nascent D-loops are detected within 2 hrs of DSB formation and extended in a delayed fashion in a genetic system designed to preclude downstream repair steps. The majority of nascent D-loops are disrupted by two pathways: one supported by the Srs2 helicase and the other by the Mph1 helicase and the Sgs1-Top3-Rmi1 helicase-topoisomerase complex. Both pathways operate without significant overlap and are delineated by the Rad54 paralog Rdh54 in an ATPase-independent fashion. This study uncovers a layer of quality control of HR relying on nascent D-loop dynamics.

Genome editing in primary cells and in vivo using viral-derived Nanoblades loaded with Cas9-sgRNA ribonucleoproteins.

Author(s) : Mangeot P, Risson V, Fusil F, Marnef A, Laurent E, Blin J, Mournetas V, Massourides E, Sohier T, Corbin A, Aube F, Teixeira M, Pinset C, Schaeffer L, Legube G, Cosset F, Verhoeyen E, Ohlmann T, Ricci E,
Journal : Nat Commun
2019
Programmable nucleases have enabled rapid and accessible genome engineering in eukaryotic cells and living organisms. However, their delivery into target cellscan be technically challenging when working with primary cells or in vivo. Here,we use engineered murine leukemia virus-like particles loaded with Cas9-sgRNA ribonucleoproteins (Nanoblades) to induce efficient genome-editing in cell linesand primary cells including human induced pluripotent stem cells, human hematopoietic stem cells and mouse bone-marrow cells. Transgene-free Nanoblades are also capable of in vivo genome-editing in mouse embryos and in the liver of injected mice. Nanoblades can be complexed with donor DNA for "all-in-one" homology-directed repair or programmed with modified Cas9 variants to mediate transcriptional up-regulation of target genes. Nanoblades preparation process issimple, relatively inexpensive and can be easily implemented in any laboratory equipped for cellular biology.

Global chromatin conformation differences in the Drosophila dosage compensated chromosome X.

Author(s) : Pal K, Forcato M, Jost D, Sexton T, Vaillant C, Salviato E, Mazza E, Lugli E, Cavalli G, Ferrari F,
Journal : Nat Commun
2019
In Drosophila melanogaster the single male chromosome X undergoes an average twofold transcriptional upregulation for balancing the transcriptional output between sexes. Previous literature hypothesised that a global change in chromosome structure may accompany this process. However, recent studies based on Hi-C failed to detect these differences. Here we show that global conformationaldifferences are specifically present in the male chromosome X and detectable using Hi-C data on sex-sorted embryos, as well as male and female cell lines, byleveraging custom data analysis solutions. We find the male chromosome X has more mid-/long-range interactions. We also identify differences at structural domain boundaries containing BEAF-32 in conjunction with CP190 or Chromator. Weakening of these domain boundaries in male chromosome X co-localizes with the binding ofthe dosage compensation complex and its co-factor CLAMP, reported to enhance chromatin accessibility. Together, our data strongly indicate that chromosome X dosage compensation affects global chromosome structure.

Histone Methylation and Memory of Environmental Stress.

Author(s) : Fabrizio P, Garvis S, Palladino F,
Journal : Cells
2019
Cellular adaptation to environmental stress relies on a wide range of tightly controlled regulatory mechanisms, including transcription. Changes in chromatin structure and organization accompany the transcriptional response to stress, andin some cases, can impart memory of stress exposure to subsequent generations through mechanisms of epigenetic inheritance. In the budding yeast Saccharomycescerevisiae, histone post-translational modifications, and in particular histone methylation, have been shown to confer transcriptional memory of exposure to environmental stress conditions through mitotic divisions. Recent evidence from Caenorhabditis elegans also implicates histone methylation in transgenerational inheritance of stress responses, suggesting a more widely conserved role in epigenetic memory.

Homologous recombination and the formation of complex genomic rearrangements

Author(s) : Piazza A, Heyer W,
Journal : Trends in cell biology
2019
The maintenance of genome integrity involves multiple independent DNA damage avoidance and repair mechanisms. Yet, the origin and pathways of the focal chromosomal reshuffling phenomena collectively referred to as chromothripsis remain mechanistically obscure. Here ,we discuss the role, mechanisms, and regulation of HR in the formation of simple and complex chromosomal rearrangements. We emphasize features of the recently characterized Multi-invasions Induced Rearrangement (MIR) pathway, which uniquely amplifies the initial DNA damage. HR intermediates and cellular contexts at risk for genomic stability are discussed along with the emerging roles of various classes of nucleases in the formation of genome rearrangements. Long-read sequencing and improved mapping of repeats should enable better appreciation of the significance of recombination in generating genomic rearrangements.

Interplay between coding and exonic splicing regulatory sequences

Author(s) : Fontrodona N, Aub? F, Claude J, Polv?che H, Lemaire S, Tranchevent L, Modolo L, Mortreux F, Bourgeois C, Auboeuf D,
Journal : Genome Res
2019

Is WDR45 the missing link for ER stress-induced autophagy in beta-propeller associated neurodegeneration?

Author(s) : Mollereau B, Walter L,
Journal : Autophagy
2019
Beta-propeller protein-associated neurodegeneration (BPAN) is caused by mutations inthe autophagy gene WDR45/WIPI4. In human, BPAN is associated with staticencephalopathy in childhood and neurodegeneration in adulthood (SENDA). It has beenproposed that WDR45 mutations cause neurodegeneration due to defective autophagy.Whether these mutations cause a global attenuation or a defect in a subset ofautophagy functions is unknown. Based on a recent study showing that wdr45 knockoutmice exhibit defective autophagy associated with an increased ER stress, we proposethat ER-mediated autophagy, a selective activation of autophagy, is defective inmouse and cellular models of BPAN. We discuss the implication of these findings onthe pathophysiological relevance of the relationship between ER stress and autophagyin BPAN as well as other neurodegenerative diseases exhibiting ER stress anddefective autophagy.

Males as somatic investment in a parthenogenetic nematode.

Author(s) : Grosmaire M, Launay C, Siegwald M, Brugiere T, Estrada-Virrueta L, Berger D, Burny C, Modolo L, Blaxter M, Meister P, Felix M, Gouyon P, Delattre M,
Journal : Science
2019
We report the reproductive strategy of the nematode Mesorhabditis belari This species produces only 9% males, whose sperm is necessary to fertilize and activate the eggs. However, most of the fertilized eggs develop without using the sperm DNA and produce female individuals. Only in 9% of eggs is the male DNA utilized, producing sons. We found that mixing of parental genomes only gives rise to males because the Y-bearing sperm of males are much more competent than the X-bearing sperm for penetrating the eggs. In this previously unrecognized strategy, asexual females produce few sexual males whose genes never reenter thefemale pool. Here, production of males is of interest only if sons are more likely to mate with their sisters. Using game theory, we show that in this context, the production of 9% males by M. belari females is an evolutionary stable strategy.

Modeling Edar expression reveals the hidden dynamics of tooth signaling center patterning

Author(s) : Sadier A, Twarogowska M, Steklíková k, Hayden L, Lambert A, Schneider P, Laudet V, Hovorakova M, Calvez V, Pantalacci S,
Journal : PLOS Biology
2019

Moving forward one step back at a time: reversibility during homologous recombination

Author(s) : Piazza A, Heyer W,
Journal : Current genetics
2019
DNA double-strand breaks (DSBs) are genotoxic lesions whose repair can be templated off an intact DNA duplex through the conserved Homologous Recombination (HR) pathway. Because it mainly consists of a succession of non-covalent associations of molecules, HR is intrinsically reversible. Reversibility serves as an integral property of HR, exploited and tuned at various stages throughout the pathway with anti- and pro-recombinogenic consequences. Here, we focus on the reversibility of displacement loops (D-loops), a central DNA joint molecule intermediate whose dynamics and regulations has recently been physically probed in somatic S. cerevisiae cells. From homology search to repair completion, we discuss putative roles of D-loop reversibility in repair fidelity and outcome.

Physical and functional interaction between SET1/COMPASS complex component CFP-1 and a Sin3S HDAC complex in C. elegans.

Author(s) : Beurton F, Stempor P, Caron M, Appert A, Dong Y, Chen R, Cluet D, Coute Y, Herbette M, Huang N, Polveche H, Spichty M, Bedet C, Ahringer J, Palladino F,
Journal : Nucleic Acids Res
2019
The CFP1 CXXC zinc finger protein targets the SET1/COMPASS complex to non-methylated CpG rich promoters to implement tri-methylation of histone H3 Lys4 (H3K4me3). Although H3K4me3 is widely associated with gene expression, the effects of CFP1 loss vary, suggesting additional chromatin factors contribute tocontext dependent effects. Using a proteomics approach, we identified CFP1 associated proteins and an unexpected direct link between Caenorhabditis elegansCFP-1 and an Rpd3/Sin3 small (SIN3S) histone deacetylase complex. Supporting a functional connection, we find that mutants of COMPASS and SIN3 complex components genetically interact and have similar phenotypic defects including misregulation of common genes. CFP-1 directly binds SIN-3 through a region including the conserved PAH1 domain and recruits SIN-3 and the HDA-1/HDAC subunit to H3K4me3 enriched promoters. Our results reveal a novel role for CFP-1 in mediating interaction between SET1/COMPASS and a Sin3S HDAC complex at promoters.

Pluripotent Stem Cell-Based Drug Screening Reveals Cardiac Glycosides as Modulators of Myotonic Dystrophy Type 1

Author(s) : Maury Y, Poydenot P, Brinon B, Lesueur L, Gide J, Roquevi?re S, C?me J, Polv?che H, Auboeuf D, Alexandre Denis J, Pietu G, Furling D, Lechuga M, Baghdoyan S, Peschanski M, Martinat C,
Journal : iScience
2019

Regulation of Numb during planar cell polarity establishment in the Drosophila eye

Author(s) : Domingos P, Jenny A, Combie K, Alamo D, Mlodzik M, Steller H, Mollereau B,
Journal : Mechanisms of Development
2019
The establishment of planar cell polarity (PCP) in the Drosophila eye requires correct specification of the R3/R4 pair of photoreceptor cells, determined by a Frizzled mediated signaling event that specifies R3 and induces Delta to activate Notch signaling in the neighboring cell, specifying it as R4. Here, we investigated the role of the Notch signaling negative regulator Numb in the specification of R3/R4 fates and PCP establishment in the Drosophila eye. We observed that Numb is transiently upregulated in R3 at the time of R3/R4 specification. This regulation of Numb levels in developing photoreceptors occurs at the post-transcriptional level and is dependent on Dishevelled, an effector of Frizzled signaling, and Lethal Giant Larva. We detected PCP defects in cells homozygous for numb15, but these defects were due to a loss of function mutation in fat (fatQ805⁎) being present in the numb15 chromosome. However, mosaic overexpression of Numb in R4 precursors (only) caused PCP defects and numb loss-of-function alleles had a modifying effect on the defects found in a hypomorphic dishevelled mutation. Our results suggest that Numb levels are upregulated to reinforce the bias of Notch signaling activation in the R3/R4 pair, two post-mitotic cells that are not specified by asymmetric cell division.

Rouse model with transient intramolecular contacts on a timescale of seconds recapitulates folding and fluctuation of yeast chromosomes.

Author(s) : Socol M, Wang R, Jost D, Carrivain P, Vaillant C, Le Cam E, Dahirel V, Normand C, Bystricky K, Victor J, Gadal O, Bancaud A,
Journal : Nucleic Acids Res
2019
DNA folding and dynamics along with major nuclear functions are determined by chromosome structural properties, which remain, thus far, elusive in vivo. Here,we combine polymer modeling and single particle tracking experiments to determine the physico-chemical parameters of chromatin in vitro and in living yeast. We find that the motion of reconstituted chromatin fibers can be recapitulated by the Rouse model using mechanical parameters of nucleosome arrays deduced from structural simulations. Conversely, we report that the Rouse model shows some inconsistencies to analyze the motion and structural properties inferred from yeast chromosomes determined with chromosome conformation capture techniques (specifically, Hi-C). We hence introduce the Rouse model with Transient InternalContacts (RouseTIC), in which random association and dissociation occurs along the chromosome contour. The parametrization of this model by fitting motion and Hi-C data allows us to measure the kinetic parameters of the contact formation reaction. Chromosome contacts appear to be transient; associated to a lifetime of seconds and characterized by an attractive energy of -0.3 to -0.5 kBT. We suggest attributing this energy to the occurrence of histone tail-DNA contacts and notice that its amplitude sets chromosomes in 'theta' conditions, in which they are poised for compartmentalization and phase separation.

Senataxin homologue Sen1 is required for efficient termination of RNA polymerase III transcription.

Author(s) : Rivosecchi J, Larochelle M, Teste C, Grenier F, Malapert A, Ricci E, Bernard P, Bachand F, Vanoosthuyse V,
Journal : EMBO J
2019
R-loop disassembly by the human helicase Senataxin contributes to genome integrity and to proper transcription termination at a subset of RNA polymerase II genes. Whether Senataxin also contributes to transcription termination at other classes of genes has remained unclear. Here, we show that Sen1, one of twofission yeast homologues of Senataxin, promotes efficient termination of RNA polymerase III (RNAP3) transcription in vivo. In the absence of Sen1, RNAP3 accumulates downstream of RNAP3-transcribed genes and produces long exosome-sensitive 3'-extended transcripts. Importantly, neither of these defectswas affected by the removal of R-loops. The finding that Sen1 acts as an ancillary factor for RNAP3 transcription termination in vivo challenges the pre-existing view that RNAP3 terminates transcription autonomously. We propose that Sen1 is a cofactor for transcription termination that has been co-opted by different RNA polymerases in the course of evolution.

Senataxin homologue Sen1 is required for efficient termination of RNA polymerase III transcription.

Author(s) : Rivosecchi J, Larochelle M, Teste C, Grenier F, Malapert A, Ricci E, Bernard P, Bachand F, Vanoosthuyse V,
Journal : EMBO J
2019
R-loop disassembly by the human helicase Senataxin contributes to genome integrity and to proper transcription termination at a subset of RNA polymerase II genes. Whether Senataxin also contributes to transcription termination at other classes of genes has remained unclear. Here, we show that Sen1, one of twofission yeast homologues of Senataxin, promotes efficient termination of RNA polymerase III (RNAP3) transcription in vivo. In the absence of Sen1, RNAP3 accumulates downstream of RNAP3-transcribed genes and produces long exosome-sensitive 3'-extended transcripts. Importantly, neither of these defectswas affected by the removal of R-loops. The finding that Sen1 acts as an ancillary factor for RNAP3 transcription termination in vivo challenges the pre-existing view that RNAP3 terminates transcription autonomously. We propose that Sen1 is a cofactor for transcription termination that has been co-opted by different RNA polymerases in the course of evolution.

Single cell RNA-seq identifies the origins of heterogeneity in efficient cell transdifferentiation and reprogramming

Author(s) : Francesconi M, Di Stefano B, Berenguer C, de Andrés-Aguayo L, Plana-Carmona M, Mendez-Lago M, Guillaumet-Adkins A, Rodriguez-Esteban G, Gut M, Gut I, Heyn H, Lehner B, Graf T,
Journal : eLife
2019

Staphylococcus aureus Small Colony Variants (SCVs): News From a Chronic Prosthetic Joint Infection.

Author(s) : Loss G, Simoes P, Valour F, Cortes M, Gonzaga L, Bergot M, Trouillet-Assant S, Josse J, Diot A, Ricci E, Vasconcelos A, Laurent F,
Journal : Front Cell Infect Microbiol
2019
Small colony variants (SCV) of Staphylococcus aureus have been reported as implicated in chronic infections. Here, we investigated the genomic and transcriptomic changes involved in the evolution from a wild-type to a SCV from in a patient with prosthetic joint infection relapse. The SCV presented a stablephenotype with no classical auxotrophy and the emergence of rifampicin resistance. Whole Genome Sequencing (WGS) analysis showed only the loss of a 42.5 kb phage and 3 deletions, among which one targeting the rpoB gene, known to be the target of rifampicin and to be associated to SCV formation in the context ofa constitutively active stringent response. Transcriptomic analysis highlighted a specific signature in the SCV strain including a complex, multi-level strategy of survival and adaptation to chronicity within the host including a protection from the inflammatory response, an evasion of the immune response, a constitutively activated stringent response and a scavenging of iron sources.

Structural analysis reveals a "molecular calipers" mechanism for a LATERAL ORGAN BOUNDARIES DOMAIN transcription factor protein from wheat.

Author(s) : Chen W, Wei X, Rety S, Huang L, Liu N, Dou S, Xi X,
Journal : J Biol Chem
2019
LATERAL ORGAN BOUNDARIES DOMAIN (LBD) proteins, a family of plant-specific transcription factors harboring a conserved Lateral Organ Boundaries (LOB) domain, are regulators of plant organ development. Recent studies have unraveledadditional pivotal roles of the LBD protein family beyond defining lateral organboundaries, such as pollen development and nitrogen metabolism. The structural basis for the molecular network of LBD-dependent processes remains to be deciphered. Here, we solved the first structure of the homodimeric LOB domain ofRamosa2 from wheat (TtRa2LD) to 1.9 A resolution. Our crystal structure reveals structural features shared with other zinc-finger transcriptional factors, as well as some features unique to LBD proteins. Formation of the TtRa2LD homodimerrelied on hydrophobic interactions of its coiled-coil motifs. Several specific motifs/domains of the LBD protein were also involved in maintaining its overall conformation. The intricate assembly within and between the monomers determined the precise spatial configuration of the two zinc fingers that recognize palindromic DNA sequences. Biochemical, molecular modeling, and small-angle X-ray scattering experiments indicated that dimerization is important for cooperative DNA binding and discrimination of palindromic DNA through a molecular calipers mechanism. Along with previously published data, this study enables us to establish an atomic-scale mechanistic model for LBD proteins as transcriptional regulators in plants.

System-wide Profiling of RNA-Binding Proteins Uncovers Key Regulators of Virus Infection.

Author(s) : Garcia-Moreno M, Noerenberg M, Ni S, Jarvelin A, Gonzalez-Almela E, Lenz C, Bach-Pages M, Cox V, Avolio R, Davis T, Hester S, Sohier T, Li B, Heikel G, Michlewski G, Sanz M, Carrasco L, Ricci E, Pelechano V, Davis I, Fischer B, Mohammed S, Castello A,
Journal : Mol Cell
2019
The compendium of RNA-binding proteins (RBPs) has been greatly expanded by the development of RNA-interactome capture (RIC). However, it remained unknown if the complement of RBPs changes in response to environmental perturbations and whether these rearrangements are important. To answer these questions, we developed "comparative RIC" and applied it to cells challenged with an RNA virus called sindbis (SINV). Over 200 RBPs display differential interaction with RNA upon SINV infection. These alterations are mainly driven by the loss of cellular mRNAs andthe emergence of viral RNA. RBPs stimulated by the infection redistribute to viral replication factories and regulate the capacity of the virus to infect. For example, ablation of XRN1 causes cells to be refractory to SINV, while GEMIN5 moonlights as a regulator of SINV gene expression. In summary, RNA availability controls RBP localization and function in SINV-infected cells.

TCTP and CSN4 control cell cycle progression and development by regulating CULLIN1 neddylation in plants and animals.

Author(s) : Betsch L, Boltz V, Brioudes F, Pontier G, Girard V, Savarin J, Wipperman B, Chambrier P, Tissot N, Benhamed M, Mollereau B, Raynaud C, Bendahmane M, Szécsi J,
Journal : PLoS Genet
2019
Translationally Controlled Tumor Protein (TCTP) controls growth by regulating theG1/S transition during cell cycle progression. Our genetic interaction studies showthat TCTP fulfills this role by interacting with CSN4, a subunit of the COP9Signalosome complex, known to influence CULLIN-RING ubiquitin ligases activity bycontrolling CULLIN (CUL) neddylation status. In agreement with these data,downregulation of CSN4 in Arabidopsis and in tobacco cells leads to delayed G1/Stransition comparable to that observed when TCTP is downregulated. Loss-of-functionof AtTCTP leads to increased fraction of deneddylated CUL1, suggesting that AtTCTPinterferes negatively with COP9 function. Similar defects in cell proliferation andCUL1 neddylation status were observed in Drosophila knockdown for dCSN4 or dTCTP,respectively, demonstrating a conserved mechanism between plants and animals.Together, our data show that CSN4 is the missing factor linking TCTP to the controlof cell cycle progression and cell proliferation during organ development and openperspectives towards understanding TCTP's role in organ development and disordersassociated with TCTP miss-expression.

The extruded non-template strand determines the architecture of R-loops.

Author(s) : Carrasco-Salas Y, Malapert A, Sulthana S, Molcrette B, Chazot-Franguiadakis L, Bernard P, Chedin F, Faivre-Moskalenko C, Vanoosthuyse V,
Journal : Nucleic Acids Res
2019
Three-stranded R-loop structures have been associated with genomic instability phenotypes. What underlies their wide-ranging effects on genome stability remains poorly understood. Here we combined biochemical and atomic force microscopy approaches with single molecule R-loop footprinting to demonstrate that R-loops formed at the model Airn locus in vitro adopt a defined set of three-dimensionalconformations characterized by distinct shapes and volumes, which we call R-loopobjects. Interestingly, we show that these R-loop objects impose specific physical constraints on the DNA, as revealed by the presence of stereotypical angles in the surrounding DNA. Biochemical probing and mutagenesis experiments revealed that the formation of R-loop objects at Airn is dictated by the extruded non-template strand, suggesting that R-loops possess intrinsic sequence-driven properties. Consistent with this, we show that R-loops formed at the fission yeast gene sum3 do not form detectable R-loop objects. Our results reveal that R-loops differ by their architectures and that the organization of the non-template strand is a fundamental characteristic of R-loops, which could explain that only a subset of R-loops is associated with replication-dependent DNA breaks.

The long noncoding RNA CHROME regulates cholesterol homeostasis in primate.

Author(s) : Hennessy E, van Solingen C, Scacalossi K, Ouimet M, Afonso M, Prins J, Koelwyn G, Sharma M, Ramkhelawon B, Carpenter S, Busch A, Chernogubova E, Matic L, Hedin U, Maegdefessel L, Caffrey B, Hussein M, Ricci E, Temel R, Garabedian M, Berger J, Vickers K, Kanke M, Sethupathy P, Teupser D, Holdt L, Moore K,
Journal : Nat Metab
2019
The human genome encodes thousands of long non-coding RNAs (lncRNAs), the majority of which are poorly conserved and uncharacterized. Here we identify a primate-specific lncRNA (CHROME), elevated in the plasma and atherosclerotic plaques of individuals with coronary artery disease, that regulates cellular andsystemic cholesterol homeostasis. LncRNA CHROME expression is influenced by dietary and cellular cholesterol via the sterol-activated liver X receptor transcription factors, which control genes mediating responses to cholesterol overload. Using gain- and loss-of-function approaches, we show that CHROME promotes cholesterol efflux and HDL biogenesis by curbing the actions of a set of functionally related microRNAs that repress genes in those pathways. CHROME knockdown in human hepatocytes and macrophages increases levels of miR-27b, miR-33a, miR-33b and miR-128, thereby reducing expression of their overlapping target gene networks and associated biologic functions. In particular, cells lacking CHROME show reduced expression of ABCA1, which regulates cholesterol efflux and nascent HDL particle formation. Collectively, our findings identify CHROME as a central component of the non-coding RNA circuitry controlling cholesterol homeostasis in humans.

2018

Accurate detection of convergent amino-acid evolution with PCOC.

Author(s) : Rey C, Gueguen L, Semon M, Boussau B,
Journal : Mol Biol Evol
2018
In the history of life, some phenotypes have been acquired several times independently, through convergent evolution. Recently, lots of genome-scale studies have been devoted to identify nucleotides or amino acids that changed ina convergent manner when the convergent phenotypes evolved. These efforts have had mixed results, probably because of differences in the detection methods, andbecause of conceptual differences about the definition of a convergent substitution. Some methods contend that substitutions are convergent only if they occur on all branches where the phenotype changed towards the exact same state at a given nucleotide or amino acid position. Others are much looser in their requirements and define a convergent substitution as one that leads the site at which they occur to prefer a phylogeny in which species with the convergent phenotype group together. Here we suggest to look for convergent shifts in aminoacid preferences instead of convergent substitutions to the exact same amino acid. We define as convergent shifts substitutions that occur on all branches where the phenotype changed and such that they correspond to a change in the type of amino acid preferred at this position. We implement the corresponding model into a method named PCOC. We show on simulations that PCOC better recovers convergent shifts than existing methods in terms of sensitivity and specificity.We test it on a plant protein alignment where convergent evolution has been studied in detail and find that our method recovers several previously identified convergent substitutions and proposes credible new candidates.

Alternative mRNA processing sites decrease genetic variability while increasing functional diversity

Author(s) : Auboeuf D,
Journal : Transcription
2018

ASF1 is required to load histones on the HIRA complex in preparation of paternal chromatin assembly at fertilization.

Author(s) : Horard B, Sapey-Triomphe L, Bonnefoy E, Loppin B,
Journal : Epigenetics Chromatin
2018
BACKGROUND: Anti-Silencing Factor 1 (ASF1) is a conserved H3-H4 histone chaperone involved in both Replication-Coupled and Replication-Independent (RI) nucleosomeassembly pathways. At DNA replication forks, ASF1 plays an important role in regulating the supply of H3.1/2 and H4 to the CAF-1 chromatin assembly complex. ASF1 also provides H3.3-H4 dimers to HIRA and DAXX chaperones for RI nucleosome assembly. The early Drosophila embryo is an attractive system to study chromatinassembly in a developmental context. The formation of a diploid zygote begins with the unique, genome-wide RI assembly of paternal chromatin following sperm protamine eviction. Then, within the same cytoplasm, syncytial embryonic nuclei undergo a series of rapid, synchronous S and M phases to form the blastoderm embryo. Here, we have investigated the implication of ASF1 in these two distinctassembly processes. RESULTS: We show that depletion of the maternal pool of ASF1with a specific shRNA induces a fully penetrant, maternal effect embryo lethal phenotype. Unexpectedly, despite the depletion of ASF1 protein to undetectable levels, we show that asf1 knocked-down (KD) embryos can develop to various stages, thus demonstrating that ASF1 is not absolutely required for the amplification of cleavage nuclei. Remarkably, we found that ASF1 is required forthe formation of the male pronucleus, although ASF1 protein does not reside in the decondensing sperm nucleus. In asf1 KD embryos, HIRA localizes to the male nucleus but is only capable of limited and insufficient chromatin assembly. Finally, we show that the conserved HIRA B domain, which is involved in ASF1-HIRA interaction, is dispensable for female fertility. CONCLUSIONS: We conclude that ASF1 is critically required to load H3.3-H4 dimers on the HIRA complex prior to histone deposition on paternal DNA. This separation of tasks could optimize the rapid assembly of paternal chromatin within the gigantic volume of the egg cell.In contrast, ASF1 is surprisingly dispensable for the amplification of cleavage nuclei, although chromatin integrity is likely compromised in KD embryos.

Assigning function to natural allelic variation via dynamic modeling of gene network induction.

Author(s) : Richard M, Chuffart F, Duplus-Bottin H, Pouyet F, Spichty M, Fulcrand E, Entrevan M, Barthelaix A, Springer M, Jost D, Yvert G,
Journal : Mol Syst Biol
2018
More and more natural DNA variants are being linked to physiological traits. Yet, understanding what differences they make on molecular regulations remains challenging. Important properties of gene regulatory networks can be captured bycomputational models. If model parameters can be "personalized" according to thegenotype, their variation may then reveal how DNA variants operate in the network. Here, we combined experiments and computations to visualize natural alleles of the yeast GAL3 gene in a space of model parameters describing the galactose response network. Alleles altering the activation of Gal3p by galactose were discriminated from those affecting its activity (production/degradation or efficiency of the activated protein). The approach allowed us to correctly predict that a non-synonymous SNP would change the binding affinity of Gal3p with the Gal80p transcriptional repressor. Our results illustrate how personalizing gene regulatory models can be used for the mechanistic interpretation of geneticvariants.

CAARS: comparative assembly and annotation of RNA-Seq data.

Author(s) : Rey C, Veber P, Boussau B, Semon M,
Journal : Bioinformatics
2018
Motivation: RNA sequencing is a widely used approach to obtain transcript sequences in non-model organisms, notably for performing comparative analyses. However, current bioinformatic pipelines do not take full advantage of pre-existing reference data in related species for improving RNA-seq assembly, annotation, and gene family reconstruction. Results: We built an automated pipeline named CAARS to combine novel data from RNA-Seq experiments with existing multi-species gene family alignments. RNA-Seq reads are assembled into transcripts by both de novo and assisted assemblies. Then, CAARS incorporates transcripts into gene families, builds gene alignments and trees, and uses phylogenetic information to classify the genes as orthologs and paralogs of existing genes. We used CAARS to assemble and annotate RNA-Seq data in rodents and fishes using distantly related genomes as reference, a difficult case for this kind of analysis. We showed CAARS assemblies are more complete and accuratethan those assembled by a standard pipeline consisting of de novo assembly coupled with annotation by sequence similarity on a guide species. In addition to annotated transcripts, CAARS provides gene family alignments and trees, annotated with orthology relationships, directly usable for downstream comparative analyses. Availability and implementation: CAARS is implemented in Python and Ocaml and is freely available at https://github.com/carinerey/caars. Supplementary information: Supplementary data are available at Bioinformatics online.

Characterizing meiotic chromosomes' structure and pairing using a designer sequence optimized for Hi-C

Author(s) : Muller H, Scolari V, Agier N, Piazza A, Thierry A, Mercy G, Descorps-Declere S, Lazar-Stefanita L, Espéli O, Llorente B, others,
Journal : Molecular systems biology
2018
In all chromosome conformation capture based experiments the accuracy with which contacts are detected varies considerably because of the uneven distribution of restriction sites along genomes. In addition, repeated sequences as well as homologous regions in isogenic diploid backgrounds remain indistinguishable by the assay because of the ambiguities they introduce during the alignment of the sequencing reads along the genome. As a result, the investigation of homologs during meiosis prophase through 3C studies has been limited. Here, we redesigned and reassembled in yeast a 145kb region with regularly spaced restriction sites for various enzymes. Thanks to this Syn-HiC design, we enhanced the signal to noise ratio and improved our understanding of Hi-C data and definition of resolution. The redesigned sequence is now distinguishable from its native homologous counterpart in an isogenic diploid strain. As a proof of principle, we track the establishment of homolog pairing during meiotic prophase in a synchronized population. This provides new insights on the individualization and pairing of homologs, as well as on their internal restructuration into arrays of loops during meiosis prophase. Overall, we show the interest of redesigned genomic regions to explore complex biological questions otherwise difficult to address.

Complementarity of assembly-first and mapping-first approaches for alternative splicing annotation and differential analysis from RNAseq data

Author(s) : Benoit-Pilven C, Marchet C, Chautard E, Lima L, Lambert M, Sacomoto G, Rey A, Cologne A, Terrone S, Dulaurier L, Claude J, Bourgeois C, Auboeuf D, Lacroix V,
Journal : Sci Rep
2018

Condensin controls cellular RNA levels through the accurate segregation of chromosomes instead of directly regulating transcription.

Author(s) : Hocquet C, Robellet X, Modolo L, Sun X, Burny C, Cuylen-Haering S, Toselli E, Clauder-Munster S, Steinmetz L, Haering C, Marguerat S, Bernard P,
Journal : Elife
2018
Condensins are genome organisers that shape chromosomes and promote their accurate transmission. Several studies have also implicated condensins in gene expression, although any mechanisms have remained enigmatic. Here, we report on the role of condensin in gene expression in fission and budding yeasts. In contrast to previous studies, we provide compelling evidence that condensin plays no direct role in the maintenance of the transcriptome, neither during interphase nor during mitosis. We further show that the changes in gene expression in post-mitotic fission yeast cells that result from condensin inactivation are largely a consequence of chromosome missegregation during anaphase, which notably depletes the RNA-exosome from daughter cells. Crucially, preventing karyotype abnormalities in daughter cells restores a normal transcriptome despite condensin inactivation. Thus, chromosome instability, rather than a direct role of condensin in the transcription process, changes gene expression. This knowledge challenges the concept of gene regulation by canonical condensin complexes.

Crystal structure of Escherichia coli DEAH/RHA helicase HrpB.

Author(s) : Xin B, Chen W, Rety S, Dai Y, Xi X,
Journal : Biochem Biophys Res Commun
2018
RNA helicases are almost ubiquitous important enzymes that take part in multipleaspects of RNA metabolism. Prokaryotes encode fewer RNA helicases than eukaryotes, suggesting that individual prokaryotic RNA helicases may take on multiple roles. The specific functions and molecular mechanisms of bacterial DEAH/RHA helicases are poorly understood, and no structures are available of these bacterial enzymes. Here, we report the first crystal structure of the DEAH/RHA helicase HrpB of Escherichia coli in a complex with ADP*AlF4. It showedan atypical globular structure, consisting of two RecA domains, an HA2 domain and an OB domain, similar to eukaryotic DEAH/RHA helicases. Notably, it showed a unique C-terminal extension that has never been reported before. Activity assaysindicated that EcHrpB binds RNA but not DNA, and does not exhibit unwinding activity in vitro. Thus, within cells, the EcHrpB may function in helicase activity-independent RNA metabolic processes.

Drosophila p53 integrates the antagonism between autophagy and apoptosis in response to stress.

Author(s) : Robin M, Issa A, Santos C, Napoletano F, Petitgas C, Chatelain G, Ruby M, Walter L, Birman S, Domingos P, Calvi B, Mollereau B,
Journal : Autophagy
2018
The tumor suppressor TP53/p53 is a known regulator of apoptosis and macroautophagy/autophagy. However, the molecular mechanism by which TP53 regulates 2 apparently incompatible processes remains unknown. We found that Drosophila lacking p53 displayed impaired autophagic flux, higher caspase activation and mortality in response to oxidative stress compared with wild-typeflies. Moreover, autophagy and apoptosis were differentially regulated by the p53 (p53B) and DeltaNp53 (p53A) isoforms: while the former induced autophagy in differentiated neurons, which protected against cell death, the latter inhibitedautophagy by activating the caspases Dronc, Drice, and Dcp-1. Our results demonstrate that the differential use of p53 isoforms combined with the antagonism between apoptosis and autophagy ensures the generation of an appropriate p53 biological response to stress.

DRP-1-mediated apoptosis induces muscle degeneration in dystrophin mutants.

Author(s) : Scholtes C, Bellemin S, Martin E, Carre-Pierrat M, Mollereau B, Gieseler K, Walter L,
Journal : Sci Rep
2018
Mitochondria are double-membrane subcellular organelles with highly conserved metabolic functions including ATP production. Mitochondria shapes change continually through the combined actions of fission and fusion events rendering mitochondrial network very dynamic. Mitochondria are largely implicated in pathologies and mitochondrial dynamics is often disrupted upon muscle degeneration in various models. Currently, the exact roles of mitochondria in the molecular mechanisms that lead to muscle degeneration remain poorly understood. Here we report a role for DRP-1 in regulating apoptosis induced by dystrophin-dependent muscle degeneration. We found that: (i) dystrophin-dependent muscle degeneration was accompanied by a drastic increase in mitochondrial fragmentation that can be rescued by genetic manipulations of mitochondrial dynamics (ii) the loss of function of the fission gene drp-1 or the overexpression of the fusion genes eat-3 and fzo-1 provoked a reduction of muscle degeneration and an improved mobility of dystrophin mutant worms (iii) the functions of DRP-1 in apoptosis and of others apoptosis executors are important for dystrophin-dependent muscle cell death (iv) DRP-1-mediated apoptosis is alsolikely to induce age-dependent loss of muscle cell. Collectively, our findings point toward a mechanism involving mitochondrial dynamics to respond to trigger(s) of muscle degeneration via apoptosis in Caenorhabditis elegans.

Epigenomics in 3D: importance of long-range spreading and specific interactions in epigenomic maintenance.

Author(s) : Jost D, Vaillant C,
Journal : Nucleic Acids Res
2018
Recent progresses of genome-wide chromatin conformation capture techniques have shown that the genome is segmented into hierarchically organized spatial compartments. However, whether this non-random 3D organization only reflects or indeed contributes-and how-to the regulation of genome function remain to be elucidated. The observation in many species that 3D domains correlate strongly with the 1D epigenomic information along the genome suggests a dynamic coupling between chromatin organization and epigenetic regulation. Here, we posit that chromosome folding may contribute to the maintenance of a robust epigenomic identity via the formation of spatial compartments like topologically-associating domains. Using a novel theoretical framework, the living chromatin model, we show that 3D compartmentalization leads to the spatial colocalization of epigenome regulators, thus increasing their local concentration and enhancing their ability to spread an epigenomic signal at long-range. Interestingly, we find that the presence of 1D insulator elements, like CTCF, may contribute greatly to the stable maintenance of adjacent antagonistic epigenomic domains. We discuss the generic implications of our findings in the light of various biological contextsfrom yeast to human. Our approach provides a modular framework to improve our understanding and to investigate in details the coupling between the structure and function of chromatin.

Evolution of mitotic spindle behavior during the first asymmetric embryonic division of nematodes.

Author(s) : Valfort A, Launay C, Semon M, Delattre M,
Journal : PLoS Biol
2018
Asymmetric cell division is essential to generate cellular diversity. In many animal cells, the cleavage plane lies perpendicular to the mitotic spindle, and it is the spindle positioning that dictates the size of the daughter cells. Although some properties of spindle positioning are conserved between distantly related model species and different cell types, little is known of the evolutionary robustness of the mechanisms underlying this event. We recorded thefirst embryonic division of 42 species of nematodes closely related to Caenorhabditis elegans, which is an excellent model system to study the biophysical properties of asymmetric spindle positioning. Our recordings, corresponding to 128 strains from 27 Caenorhabditis and 15 non-Caenorhabditis species (accessible at http://www.ens-lyon.fr/LBMC/NematodeCell/videos/), constitute a powerful collection of subcellular phenotypes to study the evolution of various cellular processes across species. In the present work, we analyzed our collection to the study of asymmetric spindle positioning. Although all the strains underwent an asymmetric first cell division, they exhibited large intra-and inter-species variations in the degree of cell asymmetry and in several parameters controlling spindle movement, including spindle oscillation, elongation, and displacement. Notably, these parameters changed frequently during evolution with no apparent directionality in the species phylogeny, with the exception of spindle transverse oscillations, which were an evolutionary innovation at the base of the Caenorhabditis genus. These changes were also unrelated to evolutionary variations in embryo size. Importantly, spindle elongation, displacement, and oscillation each evolved independently. This finding contrasts starkly with expectations based on C. elegans studies and reveals previously unrecognized evolutionary changes in spindle mechanics. Collectively, these data demonstrate that, while the essential process of asymmetric cell division has been conserved over the course of nematode evolution, the underlying spindle movement parameters can combine in various ways. Like other developmental processes, asymmetric cell division is subject tosystem drift.

From "the Worm" to "the Worms" and Back Again: The Evolutionary Developmental Biology of Nematodes.

Author(s) : Haag E, Fitch D, Delattre M,
Journal : Genetics
2018
Since the earliest days of research on nematodes, scientists have noted the developmental and morphological variation that exists within and between species. As various cellular and developmental processes were revealed through intense focus on Caenorhabditis elegans, these comparative studies have expanded. Withinthe genus Caenorhabditis, they include characterization of intraspecific polymorphisms and comparisons of distinct species, all generally amenable to thesame laboratory culture methods and supported by robust genomic and experimentaltools. The C. elegans paradigm has also motivated studies with more distantly related nematodes and animals. Combined with improved phylogenies, this work hasled to important insights about the evolution of nematode development. First, while many aspects of C. elegans development are representative of Caenorhabditis, and of terrestrial nematodes more generally, others vary in waysboth obvious and cryptic. Second, the system has revealed several clear examplesof developmental flexibility in achieving a particular trait. This includes developmental system drift, in which the developmental control of homologous traits has diverged in different lineages, and cases of convergent evolution. Overall, the wealth of information and experimental techniques developed in C. elegans is being leveraged to make nematodes a powerful system for evolutionary cellular and developmental biology.

Functions of DEAD box RNA helicases DDX5 and DDX17 in chromatin organization and transcriptional regulation

Author(s) : Giraud G, Terrone S, Bourgeois C,
Journal : BMB Rep
2018

Genomics of cellular proliferation in periodic environmental fluctuations.

Author(s) : Salignon J, Richard M, Fulcrand E, Duplus-Bottin H, Yvert G,
Journal : Mol Syst Biol
2018
Living systems control cell growth dynamically by processing information from their environment. Although responses to a single environmental change have beenintensively studied, little is known about how cells react to fluctuating conditions. Here, we address this question at the genomic scale by measuring therelative proliferation rate (fitness) of 3,568 yeast gene deletion mutants in out-of-equilibrium conditions: periodic oscillations between two environmental conditions. In periodic salt stress, fitness and its genetic variance largely depended on the oscillating period. Surprisingly, dozens of mutants displayed pronounced hyperproliferation under short stress periods, revealing unexpected controllers of growth under fast dynamics. We validated the implication of the high-affinity cAMP phosphodiesterase and of a regulator of protein translocationto mitochondria in this group. Periodic oscillations of extracellular methionine, a factor unrelated to salinity, also altered fitness but to a lesser extent and for different genes. The results illustrate how natural selection acts on mutations in a dynamic environment, highlighting unsuspected genetic vulnerabilities to periodic stress in molecular processes that are conserved across all eukaryotes.

How epigenome drives chromatin folding and dynamics, insights from efficient coarse-grained models of chromosomes.

Author(s) : Ghosh S, Jost D,
Journal : PLoS Comput Biol
2018
The 3D organization of chromosomes is crucial for regulating gene expression andcell function. Many experimental and polymer modeling efforts are dedicated to deciphering the mechanistic principles behind chromosome folding. Chromosomes are long and densely packed-topologically constrained-polymers. The main challenges are therefore to develop adequate models and simulation methods to investigate properly the multi spatio-temporal scales of such macromolecules. Here, we proposed a generic strategy to develop efficient coarse-grained models for self-avoiding polymers on a lattice. Accounting accurately for the polymer entanglement length and the volumic density, we show that our simulation scheme not only captures the steady-state structural and dynamical properties of the system but also tracks the same dynamics at different coarse-graining. This strategy allows a strong power-law gain in numerical efficiency and offers a systematic way to define reliable coarse-grained null models for chromosomes andto go beyond the current limitations by studying long chromosomes during an extended time period with good statistics. We use our formalism to investigate in details the time evolution of the 3D organization of chromosome 3R (20 Mbp) in drosophila during one cell cycle (20 hours). We show that a combination of our coarse-graining strategy with a one-parameter block copolymer model integrating epigenomic-driven interactions quantitatively reproduce experimental data at thechromosome-scale and predict that chromatin motion is very dynamic during the cell cycle.

HTLV-1 Tax plugs and freezes UPF1 helicase leading to nonsense-mediated mRNA decay inhibition.

Author(s) : Fiorini F, Robin J, Kanaan J, Borowiak M, Croquette V, Le Hir H, Jalinot P, Mocquet V,
Journal : Nat Commun
2018
Up-Frameshift Suppressor 1 Homolog (UPF1) is a key factor for nonsense-mediated mRNA decay (NMD), a cellular process that can actively degrade mRNAs. Here, we study NMD inhibition during infection by human T-cell lymphotropic virus type I (HTLV-1) and characterise the influence of the retroviral Tax factor on UPF1 activity. Tax interacts with the central helicase core domain of UPF1 and might plug the RNA channel of UPF1, reducing its affinity for nucleic acids. Furthermore, using a single-molecule approach, we show that the sequential interaction of Tax with a RNA-bound UPF1 freezes UPF1: this latter is less sensitive to the presence of ATP and shows translocation defects, highlighting the importance of this feature for NMD. These mechanistic insights reveal how HTLV-1 hijacks the central component of NMD to ensure expression of its own genome.

Identification of Splicing Factors Involved in DMD Exon Skipping Events Using an In Vitro RNA Binding Assay

Author(s) : Miro J, Bourgeois C, Claustres M, Koenig M, Tuffery-Giraud S,
Journal : Methods Mol Biol
2018

Insights into the structural and mechanistic basis of multifunctional S. cerevisiae Pif1p helicase.

Author(s) : Lu K, Chen W, Rety S, Liu N, Wu W, Dai Y, Li D, Ma H, Dou S, Xi X,
Journal : Nucleic Acids Res
2018
The Saccharomyces cerevisiae Pif1 protein (ScPif1p) is the prototypical member of the Pif1 family of DNA helicases. ScPif1p is involved in the maintenance of mitochondrial, ribosomal and telomeric DNA and suppresses genome instability at G-quadruplex motifs. Here, we report the crystal structures of a truncated ScPif1p (ScPif1p237-780) in complex with different ssDNAs. Our results have revealed that a yeast-specific insertion domain protruding from the 2B domain folds as a bundle bearing an alpha-helix, alpha16. The alpha16 helix regulates the helicase activities of ScPif1p through interactions with the previously identified loop3. Furthermore, a biologically relevant dimeric structure has been identified, which can be further specifically stabilized by G-quadruplex DNA. Basing on structural analyses and mutational studies with DNA binding and unwinding assays, a potential G-quadruplex DNA binding site in ScPif1p monomers is suggested. Our results also show that ScPif1p uses the Q-motif to preferentially hydrolyze ATP, and a G-rich tract is preferentially recognized bymore residues, consistent with previous biochemical observations. These findingsprovide a structural and mechanistic basis for understanding the multifunctionalScPif1p.

Microtubule Feedback and LET-99-Dependent Control of Pulling Forces Ensure Robust Spindle Position.

Author(s) : Bouvrais H, Chesneau L, Pastezeur S, Fairbrass D, Delattre M, Pecreaux J,
Journal : Biophys J
2018
During asymmetric division of the Caenorhabditis elegans zygote, to properly distribute cell fate determinants, the mitotic spindle is asymmetrically localized by a combination of centering and cortical-pulling microtubule-mediated forces, the dynamics of the latter being regulated by mitotic progression. Here,we show a, to our knowledge, novel and additional regulation of these forces by spindle position itself. For that, we observed the onset of transverse spindle oscillations, which reflects the burst of anaphase pulling forces. After delaying anaphase onset, we found that the position at which the spindle starts to oscillate was unchanged compared to control embryos and uncorrelated to anaphaseonset. In mapping the cortical microtubule dynamics, we measured a steep increase in microtubule contact density after the posterior centrosome reached the critical position of 70% of embryo length, strongly suggesting the presence of apositional switch for spindle oscillations. Expanding a previous model based on a force-generator temporal control, we implemented this positional switch and observed that the large increase in microtubule density accounted for the pulling force burst. Thus, we propose that the spindle position influences the cortical availability of microtubules on which the active force generators, controlled bycell cycle progression, can pull. Importantly, we found that this positional control relies on the polarity-dependent LET-99 cortical band, the boundary of which could be probed by microtubules. This dual positional and temporal controlwell accounted for our observation that the oscillation onset position resists changes in cellular geometry and moderate variations in the active force generator number. Finally, our model suggests that spindle position at mitosis end is more sensitive to the polarity factor LET-99, which restricts the region of active force generators to a posterior-most region, than to microtubule number or force generator number/activity. Overall, we show that robustness in spindle positioning originates in cell mechanics rather than biochemical networks.

Multi-invasion-induced rearrangements as a pathway for physiological and pathological recombination

Author(s) : Piazza A, Heyer W,
Journal : Bioessays
2018
Cells mitigate the detrimental consequences of DNA damage on genome stability by attempting high fidelity repair. Homologous recombination (HR) templates DNA double-strand break (DSB) repair on an identical or near identical donor sequence in a process that can in principle access the entire genome. Other physiological processes, such as homolog recognition and pairing during meiosis, also harness the HR machinery using programmed DSB to physically link homologs and generate crossovers. A consequence of the homology search process by a long nucleoprotein filament is the formation of multi-invasions (MI), a joint molecule in which the damaged ssDNA has invaded more than one donor molecule. Processing of MI joint molecules can compromise the integrity of both donor sites and lead to their rearrangement. Here, we detail two general mechanisms for the generation of rearrangements as a pathological consequence of MI processing and discuss the potential relevance for non-allelic homologous recombination. In addition, we discuss that MI-induced crossover formation may be a feature of physiological meiotic recombination.

PDZ domain-binding motif of Tax sustains T-cell proliferation in HTLV-1-infected humanized mice.

Author(s) : Peres E, Blin J, Ricci E, Artesi M, Hahaut V, Van den Broeke A, Corbin A, Gazzolo L, Ratner L, Jalinot P, Duc Dodon M,
Journal : PLoS Pathog
2018
Human T-cell leukemia virus type 1 (HTLV-1) is the etiological agent of adult T-cell leukemia/lymphoma (ATLL), an aggressive malignant proliferation of activated CD4+ T lymphocytes. The viral Tax oncoprotein is critically involved in both HTLV-1-replication and T-cell proliferation, a prerequisite to the development of ATLL. In this study, we investigated the in vivo contribution of the Tax PDZ domain-binding motif (PBM) to the lymphoproliferative process. To that aim, we examined T-cell proliferation in humanized mice (hu-mice) carrying a human hemato-lymphoid system infected with either a wild type (WT) or a Tax PBM-deleted (DeltaPBM) provirus. We observed that the frequency of CD4+ activated T-cells in the peripheral blood and in the spleen was significantly higher in WTthan in DeltaPBM hu-mice. Likewise, human T-cells collected from WT hu-mice and cultivated in vitro in presence of interleukin-2 were proliferating at a higher level than those from DeltaPBM animals. We next examined the association of Tax with the Scribble PDZ protein, a prominent regulator of T-cell polarity, in human T-cells analyzed either after ex vivo isolation or after in vitro culture. We confirmed the interaction of Tax with Scribble only in T-cells from the WT hu-mice. This association correlated with the presence of both proteins in aggregates at the leading edge of the cells and with the formation of long actinfilopods. Finally, data from a comparative genome-wide transcriptomic analysis suggested that the PBM-PDZ association is implicated in the expression of genes regulating proliferation, apoptosis and cytoskeletal organization. Collectively,our findings suggest that the Tax PBM is an auxiliary motif that contributes to the sustained growth of HTLV-1 infected T-cells in vivo and in vitro and is essential to T-cell immortalization.

PDZ domain-binding motif of Tax sustains T-cell proliferation in HTLV-1-infected humanized mice.

Author(s) : Peres E, Blin J, Ricci E, Artesi M, Hahaut V, Van den Broeke A, Corbin A, Gazzolo L, Ratner L, Jalinot P, Duc Dodon M,
Journal : PLoS Pathog
2018
Human T-cell leukemia virus type 1 (HTLV-1) is the etiological agent of adult T-cell leukemia/lymphoma (ATLL), an aggressive malignant proliferation of activated CD4+ T lymphocytes. The viral Tax oncoprotein is critically involved in both HTLV-1-replication and T-cell proliferation, a prerequisite to the development of ATLL. In this study, we investigated the in vivo contribution of the Tax PDZ domain-binding motif (PBM) to the lymphoproliferative process. To that aim, we examined T-cell proliferation in humanized mice (hu-mice) carrying a human hemato-lymphoid system infected with either a wild type (WT) or a Tax PBM-deleted (DeltaPBM) provirus. We observed that the frequency of CD4+ activated T-cells in the peripheral blood and in the spleen was significantly higher in WTthan in DeltaPBM hu-mice. Likewise, human T-cells collected from WT hu-mice and cultivated in vitro in presence of interleukin-2 were proliferating at a higher level than those from DeltaPBM animals. We next examined the association of Tax with the Scribble PDZ protein, a prominent regulator of T-cell polarity, in human T-cells analyzed either after ex vivo isolation or after in vitro culture. We confirmed the interaction of Tax with Scribble only in T-cells from the WT hu-mice. This association correlated with the presence of both proteins in aggregates at the leading edge of the cells and with the formation of long actinfilopods. Finally, data from a comparative genome-wide transcriptomic analysis suggested that the PBM-PDZ association is implicated in the expression of genes regulating proliferation, apoptosis and cytoskeletal organization. Collectively,our findings suggest that the Tax PBM is an auxiliary motif that contributes to the sustained growth of HTLV-1 infected T-cells in vivo and in vitro and is essential to T-cell immortalization.

Physical and functional interaction between SET1/COMPASS complex component CFP-1 and a Sin3 HDAC complex

Author(s) : Beurton F, Stempor P, Caron M, Appert A, Dong Y, Chen R, Cluet D, Coute Y, Herbette M, Huang N, others,
Journal : bioRxiv
2018

Physiological and pathological roles of FATP-mediated lipid droplets in Drosophila and mice retina.

Author(s) : Van Den Brink D, Cubizolle A, Chatelain G, Davoust N, Girard V, Johansen S, Napoletano F, Dourlen P, Guillou L, Angebault-Prouteau C, Bernoud-Hubac N, Guichardant M, Brabet P, Mollereau B,
Journal : PLoS Genet
2018
Increasing evidence suggests that dysregulation of lipid metabolism is associated with neurodegeneration in retinal diseases such as age-related macular degeneration and in brain disorders such as Alzheimer's and Parkinson's diseases. Lipid storage organelles (lipid droplets, LDs), accumulate in many cell types inresponse to stress, and it is now clear that LDs function not only as lipid stores but also as dynamic regulators of the stress response. However, whether these LDs are always protective or can also be deleterious to the cell is unknown. Here, we investigated the consequences of LD accumulation on retinal cell homeostasis under physiological and stress conditions in Drosophila and in mice. In wild-type Drosophila, we show that dFatp is required and sufficient forexpansion of LD size in retinal pigment cells (RPCs) and that LDs in RPCs are required for photoreceptor survival during aging. Similarly, in mice, LD accumulation induced by RPC-specific expression of human FATP1 was non-toxic andpromoted mitochondrial energy metabolism in RPCs and non-autonomously in photoreceptor cells. In contrast, the inhibition of LD accumulation by dFatp knockdown suppressed neurodegeneration in Aats-metFB Drosophila mutants, which carry elevated levels of reactive oxygen species (ROS). This suggests that abnormal turnover of LD may be toxic for photoreceptors cells of the retina under oxidative stress. Collectively, these findings indicate that FATP-mediated LD formation in RPCs promotes RPC and neuronal homeostasis under physiological conditions but could be deleterious for the photoreceptors under pathological conditions.

Promoting the clearance of neurotoxic proteins in neurodegenerative disorders of ageing.

Author(s) : Boland B, Yu W, Corti O, Mollereau B, Henriques A, Bezard E, Pastores G, Rubinsztein D, Nixon R, Duchen M, Mallucci G, Kroemer G, Levine B, Eskelinen E, Mochel F, Spedding M, Louis C, Martin O, Millan M,
Journal : Nat Rev Drug Discov
2018
Neurodegenerative disorders of ageing (NDAs) such as Alzheimer disease, Parkinson disease, frontotemporal dementia, Huntington disease and amyotrophic lateral sclerosis represent a major socio-economic challenge in view of their high prevalence yet poor treatment. They are often called 'proteinopathies' owing to the presence of misfolded and aggregated proteins that lose their physiological roles and acquire neurotoxic properties. One reason underlying the accumulation and spread of oligomeric forms of neurotoxic proteins is insufficient clearance by the autophagic-lysosomal network. Several other clearance pathways are also compromised in NDAs: chaperone-mediated autophagy, the ubiquitin-proteasome system, extracellular clearance by proteases and extrusion into the circulation via the blood-brain barrier and glymphatic system. This article focuses on emerging mechanisms for promoting the clearance of neurotoxic proteins, a strategy that may curtail the onset and slow the progression of NDAs.

2017

[Fertilization: the spermatic nucleus unlocked by an ultra-specialized thioredoxin].

Author(s) : Horard B, Loppin B,
Journal : Med Sci (Paris)
2017

A helical bundle in the N-terminal domain of the BLM helicase mediates dimer and potentially hexamer formation.

Author(s) : Shi J, Chen W, Zhang B, Fan S, Ai X, Liu N, Rety S, Xi X,
Journal : J Biol Chem
2017
Helicases play a critical role in processes such as replication or recombinationby unwinding double-stranded DNA; mutations of these genes can therefore have devastating biological consequences. In humans, mutations in genes of three members of the RecQ family helicases (blm, wrn, and recq4) give rise to three strikingly distinctive clinical phenotypes: Bloom syndrome, Werner syndrome, andRothmund-Thomson syndrome, respectively. However, the molecular basis for these varying phenotypic outcomes is unclear, in part because a full mechanistic description of helicase activity is lacking. Because the helicase core domains are highly conserved, it has been postulated that functional differences among family members might be explained by significant differences in the N-terminal domains, but these domains are poorly characterized. To help fill this gap, we now describe bioinformatics, biochemical, and structural data for three vertebrate BLM proteins. We pair high resolution crystal structures with SAXS analysis to describe an internal, highly conserved sequence we term the dimerization helical bundle in N-terminal domain (DHBN). We show that, despite the N-terminal domain being loosely structured and potentially lacking a definedthree-dimensional structure in general, the DHBN exists as a dimeric structure required for higher order oligomer assembly. Interestingly, the unwinding amplitude and rate decrease as BLM is assembled from dimer into hexamer, and also, the stable DHBN dimer can be dissociated upon ATP hydrolysis. Thus, the structural and biochemical characterizations of N-terminal domains will provide new insights into how the N-terminal domain affects the structural and functional organization of the full BLM molecule.

An miRNA-DNMT1 Axis Is Involved in Azacitidine Resistance and Predicts Survival in Higher-Risk Myelodysplastic Syndrome and Low Blast Count Acute Myeloid Leukemia.

Author(s) : Solly F, Koering C, Mohamed A, Maucort-Boulch D, Robert G, Auberger P, Flandrin-Gresta P, Ades L, Fenaux P, Kosmider O, Tavernier-Tardy E, Cornillon J, Guyotat D, Campos L, Mortreux F, Wattel E,
Journal : Clin Cancer Res
2017
Purpose: Azacitidine inhibits DNA methyltransferases, including DNMT1, and is currently the standard of care for patients with higher-risk myelodysplastic syndrome (HRMDS) or low blast count acute myeloid leukemia (AML).Experimental Design: The expression of 754 miRNAs was compared in azacitidine-resistant and azacitidine-sensitive myelodysplastic syndrome cells. We investigated the role of differentially expressed miRNAs on DNMT1 expression and azacitidine resistance in vitro We next evaluated anti-DNMT1 miRNA expression in pretreatment bone marrow samples derived from 75 patients treated with azacitidine for HRMDS or AML.Results: Seven miRNAs, including 5 that in silico targeted the DNMT1 3' UTR,were repressed in azacitidine-resistant cells in which DNMT1 protein levels weresignificantly higher. Ectopic anti-DNMT1 miRNA expression decreased DNMT1 expression and increased azacitidine sensitivity, whereas specific inhibition ofendogenous anti-DNMT1 miRNAs increased DNMT1 expression and triggered azacitidine resistance. In patients treated with azacitidine, decreased expression of anti-DNMT1 miRNAs was associated with poor outcome. miR-126* had the strongest prognostic impact. Patients with miR-126*low myelodysplastic syndrome had significantly lower response rates (P = 0.04) and higher relapse rates (P = 0.03), as well as shorter progression-free (PFS; P = 0.004) and overall survival(OS; P = 0.004). Multivariate analysis showed that age, miR-126* expression, andrevised International Prognostic Scoring System risk independently predicted PFSand OS. In 15 patient samples collected over time, decreased miRNA expression levels were associated with secondary resistance.Conclusions: A decreased expression of anti-DNMT1 miRNAs might account for azacitidine resistance in HRMDS and AML, and measuring miRNA expression before and during treatment might help predict primary or secondary azacitidine resistance. Clin Cancer Res; 23(12); 3025-34. (c)2016 AACR.

An miRNA-DNMT1 Axis Is Involved in Azacitidine Resistance and Predicts Survival in Higher-Risk Myelodysplastic Syndrome and Low Blast Count Acute Myeloid Leukemia

Author(s) : Solly F, Koering C, Mohamed A, Maucort-Boulch D, Robert G, Auberger P, Flandrin-Gresta P, Ad?s L, Fenaux P, Kosmider O, Tavernier-Tardy E, Cornillon J, Guyotat D, Campos L, Mortreux F, Wattel E,
Journal : Clin Cancer Res
2017

Coupling 1D modifications and 3D nuclear organization: data, models and function.

Author(s) : Jost D, Vaillant C, Meister P,
Journal : Curr Opin Cell Biol
2017
Over the past decade, advances in molecular methods have strikingly improved theresolution at which nuclear genome folding can be analyzed. This revealed a wealth of conserved features organizing the one dimensional DNA molecule into tridimensional nuclear domains. In this review, we briefly summarize the main findings and highlight how models based on polymer physics shed light on the principles underlying the formation of these domains. Finally, we discuss the mechanistic similarities allowing self-organization of these structures and the functional importance of these in the maintenance of transcriptional programs.

Eicosapentaenoic acid modulates the synergistic action of CREB1 and ID/E2A family members in the rat pup brain and mouse embryonic stem cells.

Author(s) : Rossi M, Spichty M, Attorri L, Distante C, Nervi C, Salvati S, Vitelli L,
Journal : Biochim Biophys Acta
2017
The aim of this study was to investigate the molecular mechanism by which eicosapentaenoic acid (EPA) may exert neuroprotective effects through an "EPA-cyclic AMP response element-binding protein (CREB)" signaling pathway. The current study reveals that EPA modulates the exquisite interplay of interaction of CREB1 with the inhibitor of DNA binding (ID) and E2A family members, thereby delivering mechanistic insights into specific neural differentiation program. Inthis scenario, our work provides evidence for the capability of CREB1 to sequester ID:E2A family members in brain tissues and neural differentiating mouse embryonic stem cells (mESCs) through formation of a [CREB1]2:ID2:E47 tetrameric complex.In essence, the molecular function of CREB1 is to dynamically regulate the location-specific assembly or disassembly of basic-helix-loop-helix (bHLH):HLH protein complexes to mediate the activation of neural/glial target genes. Together, these findings support the one-to-many binding mechanism of CREB1 and indicate that EPA treatment potentiates the integration of CREB dependent signaling with HLH/bHLH transcriptional network, adding specificity tothe CREB1-mediated gene regulation during neural/glial differentiation. Our current research on the EPA-CREB axis could reveal new molecular targets for treating neurogenerative disease.

Escherichia coli DNA polymerase I can disrupt G-quadruplex structures during DNA replication.

Author(s) : Teng F, Hou X, Fan S, Rety S, Dou S, Xi X,
Journal : FEBS J
2017
Non-canonical four-stranded G-quadruplex (G4) DNA structures can form in G-rich sequences that are widely distributed throughout the genome. The presence of G4 structures can impair DNA replication by hindering the progress of replicative polymerases (Pols), and failure to resolve these structures can lead to genetic instability. In the present study, we combined different approaches to address the question of whether and how Escherichia coli Pol I resolves G4 obstacles during DNA replication and/or repair. We found that E. coli Pol I-catalyzed DNA synthesis could be arrested by G4 structures at low protein concentrations and the degree of inhibition was strongly dependent on the stability of the G4 structures. Interestingly, at high protein concentrations, E. coli Pol I was able to overcome some kinds of G4 obstacles without the involvement of other molecules and could achieve complete replication of G4 DNA. Mechanistic studies suggested that multiple Pol I proteins might be implicated in G4 unfolding, and the disruption of G4 structures requires energy derived from dNTP hydrolysis. The present work not only reveals an unrealized function of E. coli Pol I, but also presents a possible mechanism by which G4 structures can be resolved during DNA replication and/or repair in E. coli.

Fatty acid transport protein 1 regulates retinoid metabolism and photoreceptor development in mouse retina.

Author(s) : Cubizolle A, Guillou L, Mollereau B, Hamel C, Brabet P,
Journal : PLoS One
2017
In retinal pigment epithelium (RPE), RPE65 catalyzes the isomerization of all-trans-retinyl fatty acid esters to 11-cis-retinol in the visual cycle and controls the rhodopsin regeneration rate. However, the mechanisms by which theseprocesses are regulated are still unclear. Fatty Acid Transport Protein 1 (FATP1) is involved in fatty acid uptake and lipid metabolism in a variety of cell types. FATP1 co-localizes with RPE65 in RPE and inhibits its isomerase activity in vitro. Here, we further investigated the role of FATP1 in the visual cycle usingtransgenic mice that overexpress human FATP1 specifically in the RPE (hFATP1TG mice). The mice displayed no delay in the kinetics of regeneration of the visualchromophore 11-cis-retinal after photobleaching and had no defects in light sensitivity. However, the total retinoid content was higher in the hFATP1TG micethan in wild type mice, and the transgenic mice also displayed an age-related accumulation (up to 40%) of all-trans-retinal and retinyl esters that was not observed in control mice. Consistent with these results, hFATP1TG mice were moresusceptible to light-induced photoreceptor degeneration. hFATP1 overexpression also induced an ~3.5-fold increase in retinosome autofluorescence, as measured by two-photon microscopy. Interestingly, hFATP1TG retina contained ~25% more photoreceptor cells and ~35% longer outer segments than wild type mice, revealing a non-cell-autonomous effect of hFATP1 expressed in the RPE. These data are the first to show that FATP1-mediated fatty acid uptake in the RPE controls both retinoid metabolism in the outer retina and photoreceptor development.

Functional link between DEAH/RHA helicase Prp43 activation and ATP base binding.

Author(s) : Robert-Paganin J, Halladjian M, Blaud M, Lebaron S, Delbos L, Chardon F, Capeyrou R, Humbert O, Henry Y, Henras A, Rety S, Leulliot N,
Journal : Nucleic Acids Res
2017
The DEAH box helicase Prp43 is a bifunctional enzyme from the DEAH/RHA helicase family required both for the maturation of ribosomes and for lariat intron release during splicing. It interacts with G-patch domain containing proteins which activate the enzymatic activity of Prp43 in vitro by an unknown mechanism.In this work, we show that the activation by G-patch domains is linked to the unique nucleotide binding mode of this helicase family. The base of the ATP molecule is stacked between two residues, R159 of the RecA1 domain (R-motif) andF357 of the RecA2 domain (F-motif). Using Prp43 F357A mutants or pyrimidine nucleotides, we show that the lack of stacking of the nucleotide base to the F-motif decouples the NTPase and helicase activities of Prp43. In contrast the R159A mutant (R-motif) showed reduced ATPase and helicase activities. We show that the Prp43 R-motif mutant induces the same phenotype as the absence of the G-patch protein Gno1, strongly suggesting that the processing defects observed in the absence of Gno1 result from a failure to activate the Prp43 helicase. Overall we propose that the stacking between the R- and F-motifs and the nucleotide baseis important for the activity and regulation of this helicase family.

Genome evolution is driven by gene expression-generated biophysical constraints through RNA-directed genetic variation: A hypothesis

Author(s) : Auboeuf D,
Journal : Bioessays
2017

Genomic regions controlling shape variation in the first upper molar of the house mouse.

Author(s) : Pallares L, Ledevin R, Pantalacci S, Turner L, Steingrimsson E, Renaud S,
Journal : Elife
2017
Numerous loci of large effect have been shown to underlie phenotypic variation between species. However, loci with subtle effects are presumably more frequently involved in microevolutionary processes but have rarely been discovered. We explore the genetic basis of shape variation in the first upper molar of hybrid mice between Mus musculus musculus and M. m. domesticus. We performed the first genome-wide association study for molar shape and used 3D surface morphometrics to quantify subtle variation between individuals. We show that many loci of small effect underlie phenotypic variation, and identify five genomic regions associated with tooth shape; one region contained the gene microphthalmia-associated transcription factor Mitf that has previously been associated with tooth malformations. Using a panel of five mutant laboratory strains, we show the effect of the Mitf gene on tooth shape. This is the first report of a gene causing subtle but consistent variation in tooth shape resembling variation in nature.

HBZ-mediated shift of JunD from growth suppressor to tumor promoter in leukemic cells by inhibition of ribosomal protein S25 expression.

Author(s) : Terol M, Gazon H, Lemasson I, Duc-Dodon M, Barbeau B, Cesaire R, Mesnard J, Peloponese J,
Journal : Leukemia
2017
Human T-cell leukemia virus type 1 (HTLV-1) basic-leucine zipper (bZIP) factor (HBZ) is a key player in proliferation and transformation of HTLV-1-infected cells, thus contributing to adult T-cell leukemia (ATL) development. HBZ deregulates gene expression within the host cell by interacting with several cellular partners. Through its C-terminal ZIP domain, HBZ is able to contact andactivate JunD, a transcription factor of the AP-1 family. JunD mRNA is intronless but can generate two protein isoforms by alternative translation initiation: JunD full-length and Delta JunD, an N-terminal truncated form unresponsive to the tumor suppressor menin. Using various cell lines and primary T-lymphocytes, we show that after serum deprivation HBZ induces the expression of Delta JunD isoform. We demonstrate that, unlike JunD, Delta JunD induces proliferation and transformation of cells. To decipher the mechanisms for Delta JunD production, we looked into the translational machinery and observed that HBZ induces nuclear retention of RPS25 mRNA and loss of RPS25 protein expression, a component of thesmall ribosomal subunit. Therefore, HBZ bypasses translational control of JunD uORF and favors the expression of Delta JunD. In conclusion, we provide strong evidences that HBZ induces Delta JunD expression through alteration of the cellular translational machinery and that the truncated isoform Delta JunD has acentral role in the oncogenic process leading to ATL.Leukemia advance online publication, 24 March 2017; doi:10.1038/leu.2017.74.

HTLV-1-induced leukotriene B4 secretion by T cells promotes T cell recruitment and virus propagation.

Author(s) : Percher F, Curis C, Peres E, Artesi M, Rosewick N, Jeannin P, Gessain A, Gout O, Mahieux R, Ceccaldi P, Van den Broeke A, Duc Dodon M, Afonso P,
Journal : Nat Commun
2017
The human T-lymphotropic virus type 1 (HTLV-1) is efficiently transmitted through cellular contacts. While the molecular mechanisms of viral cell-to-cell propagation have been extensively studied in vitro, those facilitating the encounter between infected and target cells remain unknown. In this study, we demonstrate that HTLV-1-infected CD4 T cells secrete a potent chemoattractant, leukotriene B4 (LTB4). LTB4 secretion is dependent on Tax-induced transactivation of the pla2g4c gene, which encodes the cytosolic phospholipase A2 gamma. Inhibition of LTB4 secretion or LTB4 receptor knockdown on target cells reduces T-cell recruitment, cellular contact formation and virus propagation in vitro. Finally, blocking the synthesis of LTB4 in a humanized mouse model of HTLV-1 infection significantly reduces proviral load. This results from a decrease in the number of infected clones while their expansion is not impaired. This study shows the critical role of LTB4 secretion in HTLV-1 transmission both in vitro and in vivo.

IC-Finder: inferring robustly the hierarchical organization of chromatin folding.

Author(s) : Haddad N, Vaillant C, Jost D,
Journal : Nucleic Acids Res
2017
The spatial organization of the genome plays a crucial role in the regulation ofgene expression. Recent experimental techniques like Hi-C have emphasized the segmentation of genomes into interaction compartments that constitute conserved functional domains participating in the maintenance of a proper cell identity. Here, we propose a novel method, IC-Finder, to identify interaction compartments(IC) from experimental Hi-C maps. IC-Finder is based on a hierarchical clustering approach that we adapted to account for the polymeric nature of chromatin. Basedon a benchmark of realistic in silico Hi-C maps, we show that IC-Finder is one of the best methods in terms of reliability and is the most efficient numerically. IC-Finder proposes two original options: a probabilistic description of the inferred compartments and the possibility to explore the various hierarchies of chromatin organization. Applying the method to experimental data in fly and human, we show how the predicted segmentation may depend on the normalization scheme and how 3D compartmentalization is tightly associated with epigenomic information. IC-Finder provides a robust and generic 'all-in-one' tool to uncover the general principles of 3D chromatin folding and their influence on gene regulation. The software is available at http://membres-timc.imag.fr/Daniel.Jost/DJ-TIMC/Software.html.

Identification of Nascent Memory CD8 T Cells and Modeling of Their Ontogeny.

Author(s) : Crauste F, Mafille J, Boucinha L, Djebali S, Gandrillon O, Marvel J, Arpin C,
Journal : Cell Syst
2017
Primary immune responses generate short-term effectors and long-term protective memory cells. The delineation of the genealogy linking naive, effector, and memory cells has been complicated by the lack of phenotypes discriminating effector from memory differentiation stages. Using transcriptomics and phenotypic analyses, we identify Bcl2 and Mki67 as a marker combination that enables the tracking of nascent memory cells within the effector phase. We then use a formalapproach based on mathematical models describing the dynamics of population sizeevolution to test potential progeny links and demonstrate that most cells followa linear naive-->early effector-->late effector-->memory pathway. Moreover, our mathematical model allows long-term prediction of memory cell numbers from a fewearly experimental measurements. Our work thus provides a phenotypic means to identify effector and memory cells, as well as a mathematical framework to investigate their genealogy and to predict the outcome of immunization regimens in terms of memory cell numbers generated.

Identification of protein features encoded by alternative exons using Exon Ontology.

Author(s) : Tranchevent L, Aube F, Dulaurier L, Benoit-Pilven C, Rey A, Poret A, Chautard E, Mortada H, Desmet F, Chakrama F, Moreno-Garcia M, Goillot E, Janczarski S, Mortreux F, Bourgeois C, Auboeuf D,
Journal : Genome Res
2017
Transcriptomic genome-wide analyses demonstrate massive variation of alternativesplicing in many physiological and pathological situations. One major challenge is now to establish the biological contribution of alternative splicing variation in physiological- or pathological-associated cellular phenotypes. Toward this end, we developed a computational approach, named "Exon Ontology," based on terms corresponding to well-characterized protein features organized in an ontology tree. Exon Ontology is conceptually similar to Gene Ontology-based approaches but focuses on exon-encoded protein features instead of gene level functional annotations. Exon Ontology describes the protein features encoded by a selected list of exons and looks for potential Exon Ontology term enrichment. By applyingthis strategy to exons that are differentially spliced between epithelial and mesenchymal cells and after extensive experimental validation, we demonstrate that Exon Ontology provides support to discover specific protein features regulated by alternative splicing. We also show that Exon Ontology helps to unravel biological processes that depend on suites of coregulated alternative exons, as we uncovered a role of epithelial cell-enriched splicing factors in the AKT signaling pathway and of mesenchymal cell-enriched splicing factors in driving splicing events impacting on autophagy. Freely available on the web, Exon Ontology is the first computational resource that allows getting a quick insightinto the protein features encoded by alternative exons and investigating whethercoregulated exons contain the same biological information.

Identification of protein features encoded by alternative exons using Exon Ontology

Author(s) : Tranchevent L, Aub? F, Dulaurier L, Benoit-Pilven C, Rey A, Poret A, Chautard E, Mortada H, Desmet F, Chakrama F, Moreno-Garcia M, Goillot E, Janczarski S, Mortreux F, Bourgeois C, Auboeuf D,
Journal : Genome Res
2017

Maternal age generates phenotypic variation in Caenorhabditis elegans.

Author(s) : Perez M, Francesconi M, Hidalgo-Carcedo C, Lehner B,
Journal : Nature
2017
Genetically identical individuals that grow in the same environment often show substantial phenotypic variation within populations of organisms as diverse as bacteria, nematodes, rodents and humans. With some exceptions, the causes are poorly understood. Here we show that isogenic Caenorhabditis elegans nematodes vary in their size at hatching, speed of development, growth rate, starvation resistance, fecundity, and also in the rate of development of their germline relative to that of somatic tissues. We show that the primary cause of this variation is the age of an individual's mother, with the progeny of young mothers exhibiting several phenotypic impairments. We identify age-dependent changes in the maternal provisioning of the lipoprotein complex vitellogenin to embryos as the molecular mechanism that underlies the variation in multiple traits throughout the life of an animal. The production of sub-optimal progeny by youngmothers may reflect a trade-off between the competing fitness traits of a short generation time and the survival and fecundity of the progeny.

Mechanical constraint from growing jaw facilitates mammalian dental diversity.

Author(s) : Renvoise E, Kavanagh K, Lazzari V, Hakkinen T, Rice R, Pantalacci S, Salazar-Ciudad I, Jernvall J,
Journal : Proc Natl Acad Sci U S A
2017
Much of the basic information about individual organ development comes from studies using model species. Whereas conservation of gene regulatory networks across higher taxa supports generalizations made from a limited number of species, generality of mechanistic inferences remains to be tested in tissue culture systems. Here, using mammalian tooth explants cultured in isolation, we investigate self-regulation of patterning by comparing developing molars of the mouse, the model species of mammalian research, and the bank vole. A distinct patterning difference between the vole and the mouse molars is the alternate cusp offset present in the vole. Analyses of both species using 3D reconstructions ofdeveloping molars and jaws, computational modeling of cusp patterning, and toothexplants cultured with small braces show that correct cusp offset requires constraints on the lateral expansion of the developing tooth. Vole molars cultured without the braces lose their cusp offset, and mouse molars cultured with the braces develop a cusp offset. Our results suggest that cusp offset, which changes frequently in mammalian evolution, is more dependent on the 3D support of the developing jaw than other aspects of tooth shape. This jaw-tooth integration of a specific aspect of the tooth phenotype indicates that organs may outsource specific aspects of their morphology to be regulated by adjacent body parts or organs. Comparative studies of morphologically different species are needed to infer the principles of organogenesis.

microRNAs stimulate translation initiation mediated by HCV-like IRESes.

Author(s) : Mengardi C, Limousin T, Ricci E, Soto-Rifo R, Decimo D, Ohlmann T,
Journal : Nucleic Acids Res
2017
MicroRNAs (miRNAs) are small non-coding RNAs that control gene expression by recognizing and hybridizing to a specific sequence generally located in the 3 untranslated region (UTR) of targeted mRNAs. miRNA-induced inhibition of translation occurs during the initiation step, most probably at the level of ribosome scanning. In this process, the RNA-induced silencing complex interacts both with PABP and the 43S pre-initiation complex to disrupt scanning of the 40Sribosome. However, in some specific cases, miRNAs can stimulate translation. Although the mechanism of miRNA-mediated upregulation is unknown, it appears that the poly(A) tail and the lack of availability of the TNRC6 proteins are amongst major determinants. The genomic RNA of the Hepatitis C Virus is uncapped, non-polyadenylated and harbors a peculiar internal ribosome entry site (IRES) that binds the ribosome directly to the AUG codon. Thus, we have exploited the unique properties of the HCV IRES and other related IRESes (HCV-like) to study how translation initiation can be modulated by miRNAs on these elements. Here, we report that miRNA binding to the 3 UTR can stimulate translation of a reporter gene given that its expression is driven by an HCV-like IRES and that it lacks apoly(A) tail at its 3 extremity.

Multi-invasions are recombination byproducts that induce chromosomal rearrangements

Author(s) : Piazza A, Wright W, Heyer W,
Journal : Cell
2017
Inaccurate repair of broken chromosomes generates structural variants that can fuel evolution and inflict pathology. We describe a novel rearrangement mechanism in which translocation between intact chromosomes is induced by a lesion on a third chromosome. This multi-invasion-induced rearrangement (MIR) stems from a homologous recombination byproduct, where a broken DNA end simultaneously invades two intact donors. No homology is required between the donors, and the intervening sequence from the invading molecule is inserted at the translocation site. MIR is stimulated by increasing homology length and spatial proximity of the donors, and depends on the overlapping activities of the structure-selective endonucleases Mus81-Mms4, Slx1-Slx4, and Yen1. Conversely, the 3’-flap nuclease Rad1-Rad10 and enzymes known to disrupt recombination intermediates (Sgs1-Top3-Rmi1, Srs2 and Mph1) inhibit MIR. Resolution of MIR intermediates propagates secondary chromosome breaks that frequently cause additional rearrangements. MIR features have implications for the formation of simple and complex rearrangements underlying human pathologies.

Nerve Growth Factor Signaling from Membrane Microdomains to the Nucleus: Differential Regulation by Caveolins.

Author(s) : Spencer A, Yu L, Guili V, Reynaud F, Ding Y, Ma J, Jullien J, Koubi D, Gauthier E, Cluet D, Falk J, Castellani V, Yuan C, Rudkin B,
Journal : Int J Mol Sci
2017
Membrane microdomains or "lipid rafts" have emerged as essential functional modules of the cell, critical for the regulation of growth factor receptor-mediated responses. Herein we describe the dichotomy between caveolin-1and caveolin-2, structural and regulatory components of microdomains, in modulating proliferation and differentiation. Caveolin-2 potentiates while caveolin-1 inhibits nerve growth factor (NGF) signaling and subsequent cell differentiation. Caveolin-2 does not appear to impair NGF receptor trafficking but elicits prolonged and stronger activation of MAPK (mitogen-activated proteinkinase), Rsk2 (ribosomal protein S6 kinase 2), and CREB (cAMP response element binding protein). In contrast, caveolin-1 does not alter initiation of the NGF signaling pathway activation; rather, it acts, at least in part, by sequesteringthe cognate receptors, TrkA and p75NTR, at the plasma membrane, together with the phosphorylated form of the downstream effector Rsk2, which ultimately prevents CREB phosphorylation. The non-phosphorylatable caveolin-1 serine 80 mutant (S80V), no longer inhibits TrkA trafficking or subsequent CREB phosphorylation. MC192, a monoclonal antibody towards p75NTR that does not block NGF binding, prevents exit of both NGF receptors (TrkA and p75NTR) from lipid rafts. The results presented herein underline the role of caveolin and receptor signaling complex interplay in the context of neuronal development and tumorigenesis.

Non-Canonical G-quadruplexes cause the hCEB1 minisatellite instability in Saccharomyces cerevisiae

Author(s) : Piazza A, Cui X, Adrian M, Samazan F, Heddi B, Phan A, Nicolas A,
Journal : Elife
2017
G-quadruplexes (G4) are polymorphic four-stranded structures formed by certain G-rich nucleic acids in vitro, but the sequence and structural features dictating their formation and function in vivo remains uncertain. Here we report a structure-function analysis of the complex hCEB1 G4-forming sequence. We isolated four G4 conformations in vitro, all of which bear unusual structural features: Form 1 bears a V-shaped loop and a snapback guanine; Form 2 contains a terminal G-triad; Form 3 bears a zero-nucleotide loop; and Form 4 is a zero-nucleotide loop monomer or an interlocked dimer. In vivo, Form 1 and Form 2 differently account for 2/3rd of the genomic instability of hCEB1 in two G4-stabilizing conditions. Form 3 and an unidentified form contribute to the remaining instability, while Form 4 has no detectable effect. This work underscores the structural polymorphisms originated from a single highly G-rich sequence and demonstrates the existence of non-canonical G4s in cells, thus broadening the definition of G4-forming sequences.

p53-dependent programmed necrosis controls germ cell homeostasis during spermatogenesis.

Author(s) : Napoletano F, Gibert B, Yacobi-Sharon K, Vincent S, Favrot C, Mehlen P, Girard V, Teil M, Chatelain G, Walter L, Arama E, Mollereau B,
Journal : PLoS Genet
2017
The importance of regulated necrosis in pathologies such as cerebral stroke and myocardial infarction is now fully recognized. However, the physiological relevance of regulated necrosis remains unclear. Here, we report a conserved role for p53 in regulating necrosis in Drosophila and mammalian spermatogenesis. We found that Drosophila p53 is required for the programmed necrosis that occurs spontaneously in mitotic germ cells during spermatogenesis. This form of necrosis involved an atypical function of the initiator caspase Dronc/Caspase 9, independent of its catalytic activity. Prevention of p53-dependent necrosis resulted in testicular hyperplasia, which was reversed by restoring necrosis in spermatogonia. In mouse testes, p53 was required for heat-induced germ cell necrosis, indicating that regulation of necrosis is a primordial function of p53conserved from invertebrates to vertebrates. Drosophila and mouse spermatogenesis will thus be useful models to identify inducers of necrosis to treat cancers that are refractory to apoptosis.

Perspectives: Using polymer modeling to understand the formation and function of nuclear compartments

Author(s) : Haddad N, Jost D, Vaillant C,
Journal : Chromosome Research
2017

Recombination, meiotic expression and human codon usage.

Author(s) : Pouyet F, Mouchiroud D, Duret L, Semon M,
Journal : Elife
2017
Synonymous codon usage (SCU) varies widely among human genes. In particular, genes involved in different functional categories display a distinct codon usage, which was interpreted as evidence that SCU is adaptively constrained to optimizetranslation efficiency in distinct cellular states. We demonstrate here that SCUis not driven by constraints on tRNA abundance, but by large-scale variation in GC-content, caused by meiotic recombination, via the non-adaptive process of GC-biased gene conversion (gBGC). Expression in meiotic cells is associated witha strong decrease in recombination within genes. Differences in SCU among functional categories reflect differences in levels of meiotic transcription, which is linked to variation in recombination and therefore in gBGC. Overall, the gBGC model explains 70% of the variance in SCU among genes. We argue that the strong heterogeneity of SCU induced by gBGC in mammalian genomes precludes any optimization of the tRNA pool to the demand in codon usage.

TET2 exon 2 skipping is an independent favorable prognostic factor for cytogenetically normal acute myelogenous leukemia (AML): TET2 exon 2 skipping in AML.

Author(s) : Mohamed A, Balsat M, Koering C, Maucort-Boulch D, Boissel N, Payen-Gay L, Cheok M, Mortada H, Auboeuf D, Pinatel C, El-Hamri M, Tigaud I, Hayette S, Dumontet C, Cros E, Flandrin-Gresta P, Nibourel O, Preudhomme C, Thomas X, Nicolini F, Solly F, Guyotat D, Campos L, Michallet M, Ceraulo A, Mortreux F, Wattel E,
Journal : Leuk Res
2017
In AML, approximately one-third of expressed genes are abnormally spliced, including aberrant TET2 exon 2 expression. In a discovery cohort (n=99), TET2 exon 2 skipping (TET2E2S) was found positively associated with a significant reduction in the cumulative incidence of relapse (CIR). Age, cytogenetics, and TET2E2S were independent prognostic factors for disease-free survival (DFS), andfavorable effects on outcomes predominated in cytogenetic normal (CN)-AML and younger patients. Using the same cutoff in a validation cohort of 86 CN-AML patients, TET2E2Shigh patients were found to be younger than TET2low patients without a difference in the rate of complete remission. However, TET2E2Shigh patients exhibited a significantly lower CIR (p<10-4). TET2E2S and FLT3-ITD, butnot age or NPM1 mutation status were independent prognostic factors for DFS and event-free survival (EFS), while TET2E2S was the sole prognostic factor that we identified for overall survival (OS). In both the intermediate-1 and favorable ELN genetic categories, TET2E2S remained significantly associated with prolongedsurvival. There was no correlation between TET2E2S status and outcomes in 34 additional AML patients who were unfit for IC. Therefore our results suggest that assessments of TET2 exon 2 splicing status might improve risk stratification in CN-AML patients treated with IC.

TET2 exon 2 skipping is an independent favorable prognostic factor for cytogenetically normal acute myelogenous leukemia (AML): TET2 exon 2 skipping in AML

Author(s) : Mohamed A, Balsat M, Koering C, Maucort-Boulch D, Boissel N, Payen-Gay L, Cheok M, Mortada H, Auboeuf D, Pinatel C, El-Hamri M, Tigaud I, Hayette S, Dumontet C, Cros E, Flandrin-Gresta P, Nibourel O, Preudhomme C, Thomas X, Nicolini F, Solly F, Guyotat D, Campos L, Michallet M, Ceraulo A, Mortreux F, Wattel E,
Journal : Leuk Res
2017

2016

[An intimate look at the viral replication cycle through ribosome profiling].

Author(s) : Blin J, Ricci E,
Journal : Med Sci (Paris)
2016
Next Generation Sequencing (NGS) techniques have revolutionized most biomedical research fields over the past decade by allowing a broader vision on biological processes that occur at the molecular level. Among these, ribosome profiling or footprinting is a powerful tool to study mRNA translation in a transcriptome-wide manner. Ribosome profiling has been used to study the impact of translational control of gene expression under many different cellular conditions including viral infections. Indeed, translation is a critical step during the viral replication cycle in which the infected cell is embezzled to produce viral proteins. Ribosome profiling tools can provide new insights on viral translationby monitoring ribosome binding to viral and cellular RNAs with a high definitionduring the time course of an infection. Here, we describe the potential uses of ribosome profiling for the understanding of viral translational control and the impact of viral infection on host gene expression. We also discuss the main limitations and biases related to the technique that need to be taken into account for its use.

A Long Noncoding RNA lincRNA-EPS Acts as a Transcriptional Brake to Restrain Inflammation.

Author(s) : Atianand M, Hu W, Satpathy A, Shen Y, Ricci E, Alvarez-Dominguez J, Bhatta A, Schattgen S, McGowan J, Blin J, Braun J, Gandhi P, Moore M, Chang H, Lodish H, Caffrey D, Fitzgerald K,
Journal : Cell
2016
Long intergenic noncoding RNAs (lincRNAs) are important regulators of gene expression. Although lincRNAs are expressed in immune cells, their functions in immunity are largely unexplored. Here, we identify an immunoregulatory lincRNA, lincRNA-EPS, that is precisely regulated in macrophages to control the expression of immune response genes (IRGs). Transcriptome analysis of macrophages from lincRNA-EPS-deficient mice, combined with gain-of-function and rescue experiments, revealed a specific role for this lincRNA in restraining IRG expression. Consistently, lincRNA-EPS-deficient mice manifest enhanced inflammation and lethality following endotoxin challenge in vivo. lincRNA-EPS localizes at regulatory regions of IRGs to control nucleosome positioning and repress transcription. Further, lincRNA-EPS mediates these effects by interacting with heterogeneous nuclear ribonucleoprotein L via a CANACA motif located in its3' end. Together, these findings identify lincRNA-EPS as a repressor of inflammatory responses, highlighting the importance of lincRNAs in the immune system.

Adaptive preconditioning in neurological diseases - therapeutic insights from proteostatic perturbations.

Author(s) : Mollereau B, Rzechorzek N, Roussel B, Sedru M, Van den Brink D, Bailly-Maitre B, Palladino F, Medinas D, Domingos P, Hunot S, Chandran S, Birman S, Baron T, Vivien D, Duarte C, Ryoo H, Steller H, Urano F, Chevet E, Kroemer G, Ciechanover A, Calabrese E, Kaufman R, Hetz C,
Journal : Brain Res
2016
In neurological disorders, both acute and chronic neural stress can disrupt cellular proteostasis, resulting in the generation of pathological protein. However in most cases, neurons adapt to these proteostatic perturbations by activating a range of cellular protective and repair responses, thus maintainingcell function. These interconnected adaptive mechanisms comprise a 'proteostasisnetwork' and include the unfolded protein response, the ubiquitin proteasome system and autophagy. Interestingly, several recent studies have shown that these adaptive responses can be stimulated by preconditioning treatments, which conferresistance to a subsequent toxic challenge - the phenomenon known as hormesis. In this review we discuss the impact of adaptive stress responses stimulated in diverse human neuropathologies including Parkinsons disease, Wolfram syndrome, brain ischemia, and brain cancer. Further, we examine how these responses and the molecular pathways they recruit might be exploited for therapeutic gain. This article is part of a Special Issue entitled SI:ER stress.

Adaptive preconditioning in neurological diseases - therapeutic insights from proteostatic perturbations.

Author(s) : Mollereau B, Rzechorzek N, Roussel B, Sedru M, Van den Brink D, Bailly-Maitre B, Palladino F, Medinas D, Domingos P, Hunot S, Chandran S, Birman S, Baron T, Vivien D, Duarte C, Ryoo H, Steller H, Urano F, Chevet E, Kroemer G, Ciechanover A, Calabrese E, Kaufman R, Hetz C,
Journal : Brain Res
2016
In neurological disorders, both acute and chronic neural stress can disrupt cellular proteostasis, resulting in the generation of pathological protein. However in most cases, neurons adapt to these proteostatic perturbations by activating a range of cellular protective and repair responses, thus maintainingcell function. These interconnected adaptive mechanisms comprise a 'proteostasisnetwork' and include the unfolded protein response, the ubiquitin proteasome system and autophagy. Interestingly, several recent studies have shown that these adaptive responses can be stimulated by preconditioning treatments, which conferresistance to a subsequent toxic challenge - the phenomenon known as hormesis. In this review we discuss the impact of adaptive stress responses stimulated in diverse human neuropathologies including Parkinsons disease, Wolfram syndrome, brain ischemia, and brain cancer. Further, we examine how these responses and the molecular pathways they recruit might be exploited for therapeutic gain. This article is part of a Special Issue entitled SI:ER stress.

Adenomatous polyposis coli (APC) regulates miR17-92 cluster through beta-catenin pathway in colorectal cancer.

Author(s) : Li Y, Lauriola M, Kim D, Francesconi M, D'Uva G, Shibata D, Malafa M, Yeatman T, Coppola D, Solmi R, Cheng J,
Journal : Oncogene
2016
Adenomatous polyposis coli (APC) mutation is the most common genetic change in sporadic colorectal cancer (CRC). Although deregulations of miRNAs have been frequently reported in this malignancy, APC-regulated miRNAs have not been extensively documented. Here, by using an APC-inducible cell line and array analysis, we identified a total of 26 deregulated miRNAs. Among them, members ofmiR-17-92 cluster were dramatically inhibited by APC and induced by enforced expression of beta-catenin. Furthermore, we demonstrate that activated beta-catenin resulted from APC loss binds to and activates the miR-17-92 promoter. Notably, enforced expression of miR-19a overrides APC tumor suppressoractivity, and knockdown of miR-19a in cancer cells with compromised APC functionreduced their aggressive features in vitro. Finally, we observed that expressionof miR-19a significantly correlates with beta-catenin levels in colorectal cancer specimens, and it is associated to the aggressive stage of tumor progression. Thus, our study reveals that miR-17-92 cluster is directly regulated by APC/beta-catenin pathway and could be a potential therapeutic target in colon cancers with aberrant APC/beta-catenin signaling.

Biogenesis and function of tRNA fragments during sperm maturation and fertilization in mammals.

Author(s) : Sharma U, Conine C, Shea J, Boskovic A, Derr A, Bing X, Belleannee C, Kucukural A, Serra R, Sun F, Song L, Carone B, Ricci E, Li X, Fauquier L, Moore M, Sullivan R, Mello C, Garber M, Rando O,
Journal : Science
2016
Several recent studies link parental environments to phenotypes in subsequent generations. In this work, we investigate the mechanism by which paternal diet affects offspring metabolism. Protein restriction in mice affects small RNA (sRNA) levels in mature sperm, with decreased let-7 levels and increased amountsof 5' fragments of glycine transfer RNAs (tRNAs). In testicular sperm, tRNA fragments are scarce but increase in abundance as sperm mature in the epididymis. Epididymosomes (vesicles that fuse with sperm during epididymal transit) carry RNA payloads matching those of mature sperm and can deliver RNAs to immature sperm in vitro. Functionally, tRNA-glycine-GCC fragments repress genes associated with the endogenous retroelement MERVL, in both embryonic stem cells and embryos. Our results shed light on sRNA biogenesis and its dietary regulation during posttesticular sperm maturation, and they also link tRNA fragments to regulationof endogenous retroelements active in the preimplantation embryo.

Bis(mesitoyl)phosphinic acid: photo-triggered release of metaphosphorous acid in solution.

Author(s) : Fast D, Zalibera M, Lauer A, Eibel A, Schweigert C, Kelterer A, Spichty M, Neshchadin D, Voll D, Ernst H, Liang Y, Dietliker K, Unterreiner A, Barner-Kowollik C, Grutzmacher H, Gescheidt G,
Journal : Chem Commun (Camb)
2016
Bis(mesitoyl)phosphinic acid and its sodium salt display a unique photo-induced reactivity: both derivatives stepwise release two mesitoyl radicals and, remarkably, metaphosphorous acid (previously postulated as transient species in the gas phase), providing a new phosphorus-based reagent.

C/EBPalpha creates elite cells for iPSC reprogramming by upregulating Klf4 and increasing the levels of Lsd1 and Brd4.

Author(s) : Di Stefano B, Collombet S, Jakobsen J, Wierer M, Sardina J, Lackner A, Stadhouders R, Segura-Morales C, Francesconi M, Limone F, Mann M, Porse B, Thieffry D, Graf T,
Journal : Nat Cell Biol
2016
Reprogramming somatic cells into induced pluripotent stem cells (iPSCs) is typically inefficient and has been explained by elite-cell and stochastic models. We recently reported that B cells exposed to a pulse of C/EBPalpha (Balpha' cells) behave as elite cells, in that they can be rapidly and efficiently reprogrammed into iPSCs by the Yamanaka factors OSKM. Here we show that C/EBPalpha post-transcriptionally increases the abundance of several hundred proteins, including Lsd1, Hdac1, Brd4, Med1 and Cdk9, components of chromatin-modifying complexes present at super-enhancers. Lsd1 was found to be required for B cell gene silencing and Brd4 for the activation of the pluripotency program. C/EBPalpha also promotes chromatin accessibility in pluripotent cells and upregulates Klf4 by binding to two haematopoietic enhancers. Balpha' cells share many properties with granulocyte/macrophage progenitors, naturally occurring elite cells that are obligate targets for leukaemic transformation, whose formation strictly requires C/EBPalpha.

Candidate gene prioritization with Endeavour

Author(s) : Tranchevent L, Ardeshirdavani A, ElShal S, Alcaide D, Aerts J, Auboeuf D, Moreau Y,
Journal : Nucleic Acids Res
2016

Crystal structures of the BsPif1 helicase reveal that a major movement of the 2B SH3 domain is required for DNA unwinding.

Author(s) : Chen W, Dai Y, Duan X, Liu N, Shi W, Li N, Li M, Dou S, Dong Y, Rety S, Xi X,
Journal : Nucleic Acids Res
2016
Pif1 helicases are ubiquitous members of the SF1B family and are essential for maintaining genome stability. It was speculated that Pif1-specific motifs may fold in specific structures, conferring distinct activities upon it. Here, we report the crystal structures of the Pif1 helicase from Bacteroides spp with andwithout adenosine triphosphate (ATP) analog/ssDNA. BsPif1 shares structural similarities with RecD2 and Dda helicases but has specific features in the 1B and 2B domains. The highly conserved Pif1 family specific sequence motif interacts with and constraints a putative pin-loop in domain 1B in a precise conformation.More importantly, we found that the 2B domain which contains a specific extendedhairpin undergoes a significant rotation and/or movement upon ATP and DNA binding, which is absolutely required for DNA unwinding. We therefore propose a mechanism for DNA unwinding in which the 2B domain plays a predominant role. Thefact that the conformational change regulates Pif1 activity may provide insight into the puzzling observation that Pif1 becomes highly processive during break-induced replication in association with Poldelta, while the isolated Pif1 has low processivity.

Exploiting Single-Cell Quantitative Data to Map Genetic Variants Having Probabilistic Effects.

Author(s) : Chuffart F, Richard M, Jost D, Burny C, Duplus-Bottin H, Ohya Y, Yvert G,
Journal : PLoS Genet
2016
Despite the recent progress in sequencing technologies, genome-wide association studies (GWAS) remain limited by a statistical-power issue: many polymorphisms contribute little to common trait variation and therefore escape detection. The small contribution sometimes corresponds to incomplete penetrance, which may result from probabilistic effects on molecular regulations. In such cases, genetic mapping may benefit from the wealth of data produced by single-cell technologies. We present here the development of a novel genetic mapping method that allows to scan genomes for single-cell Probabilistic Trait Loci that modifythe statistical properties of cellular-level quantitative traits. Phenotypic values are acquired on thousands of individual cells, and genetic association isobtained from a multivariate analysis of a matrix of Kantorovich distances. No prior assumption is required on the mode of action of the genetic loci involved and, by exploiting all single-cell values, the method can reveal non-deterministic effects. Using both simulations and yeast experimental datasets, we show that it can detect linkages that are missed by classical genetic mapping. A probabilistic effect of a single SNP on cell shape was detected and validated. The method also detected a novel locus associated with elevated gene expression noise of the yeast galactose regulon. Our results illustrate how single-cell technologies can be exploited to improve the genetic dissection of certain common traits. The method is available as an open source Rpackage called ptlmapper.

Ferritin Assembly in Enterocytes of Drosophila melanogaster.

Author(s) : Rosas-Arellano A, Vasquez-Procopio J, Gambis A, Blowes L, Steller H, Mollereau B, Missirlis F,
Journal : Int J Mol Sci
2016
Ferritins are protein nanocages that accumulate inside their cavity thousands ofoxidized iron atoms bound to oxygen and phosphates. Both characteristic types ofeukaryotic ferritin subunits are present in secreted ferritins from insects, buthere dimers between Ferritin 1 Heavy Chain Homolog (Fer1HCH) and Ferritin 2 Light Chain Homolog (Fer2LCH) are further stabilized by disulfide-bridge in the 24-subunit complex. We addressed ferritin assembly and iron loading in vivo using novel transgenic strains of Drosophila melanogaster. We concentrated on the intestine, where the ferritin induction process can be controlled experimentallyby dietary iron manipulation. We showed that the expression pattern of Fer2LCH-Gal4 lines recapitulated iron-dependent endogenous expression of the ferritin subunits and used these lines to drive expression from UAS-mCherry-Fer2LCH transgenes. We found that the Gal4-mediated induction of mCherry-Fer2LCH subunits was too slow to effectively introduce them into newly formed ferritin complexes. Endogenous Fer2LCH and Fer1HCH assembled and stored excess dietary iron, instead. In contrast, when flies were genetically manipulated to co-express Fer2LCH and mCherry-Fer2LCH simultaneously, both subunits were incorporated with Fer1HCH in iron-loaded ferritin complexes. Our study provides fresh evidence that, in insects, ferritin assembly and iron loading in vivo are tightly regulated.

Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition).

Author(s) : Klionsky D, al,
Journal : Autophagy
2016

Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition).

Author(s) : Klionsky D, al,
Journal : Autophagy
2016

HBx triggers either cellular senescence or cell proliferation depending on cellular phenotype

Author(s) : Idrissi M, Hachem H, Koering C, Merle P, Th?noz M, Mortreux F, Wattel E,
Journal : J Viral Hepat
2016

HBx triggers either cellular senescence or cell proliferation depending on cellular phenotype.

Author(s) : Idrissi M, Hachem H, Koering C, Merle P, Thenoz M, Mortreux F, Wattel E,
Journal : J Viral Hepat
2016
Replicative senescence is a hallmark of chronic liver diseases including chronichepatitis B virus (HBV) infection, whereas HBV-encoded oncoproteins HBx and preS2 have been found to overcome senescence. HBx possesses a C-terminal truncation mainly in hepatocellular carcinomas but also in noncancerous liver tissues. Here, by cell counting, BrdU incorporation, MTT proliferation assay, cell cycle analysis, SA-betagal staining and Western blotting in primary and malignant cells, we investigated the effect of HBx C-terminal mutants on cellular senescence. HBx C-terminal mutants were found to trigger cellular senescence in primary MRC5 cells, and malignant liver cells Huh7, and SK-Hep1. In contrast, these mutants promoted the proliferation of HepG2 malignant liver cells. The pro-senescent effect of HBx relied on an increased p16(INK4a) and p21(Waf1/Cip1)expression, and a decreased phosphorylation of Rb. Together, these results suggest that the two main variants of HBx present in HBV-infected liver possess opposite effects on cellular senescence that depend on the phenotype of infectedcells.

IL-2 sensitivity and exogenous IL-2 concentration gradient tune the productive contact duration of CD8(+) T cell-APC: a multiscale modeling study.

Author(s) : Gao X, Arpin C, Marvel J, Prokopiou S, Gandrillon O, Crauste F,
Journal : BMC Syst Biol
2016
BACKGROUND: The CD8(+) T cell immune response fights acute infections by intracellular pathogens and, by generating an immune memory, enables immune responses against secondary infections. Activation of the CD8(+) T cell immune response involves a succession of molecular events leading to modifications of CD8(+) T cell population. To understand the endogenous and exogenous mechanisms controlling the activation of CD8(+) T cells and to investigate the influence ofearly molecular events on the long-term cell population behavior, we developed amultiscale computational model. It integrates three levels of description: a Cellular Potts model describing the individual behavior of CD8(+) T cells, a system of ordinary differential equations describing a decision-making molecularregulatory network at the intracellular level, and a partial differential equation describing the diffusion of IL-2 in the extracellular environment. RESULTS: We first calibrated the model parameters based on in vivo data and showed the model's ability to reproduce early dynamics of CD8(+) T cells in murine lymph nodes after influenza infection, both at the cell population and intracellular levels. We then showed the model's ability to reproduce the proliferative responses of CD5(hi) and CD5(lo) CD8(+) T cells to exogenous IL-2 under a weak TCR stimulation. This stressed the role of short-lasting molecular events and the relevance of explicitly describing both intracellular and cellular scale dynamics. Our results suggest that the productive contact duration of CD8(+) T cell-APC is influenced by the sensitivity of individual CD8(+) T cells to the activation signal and by the IL-2 concentration in the extracellular environment. CONCLUSIONS: The multiscale nature of our model allows the reproduction and explanation of some acquired characteristics and functions of CD8(+) T cells, and of their responses to multiple stimulation conditions, that would not be accessible in a classical description of cell population dynamics that would not consider intracellular dynamics.

INT6/EIF3E Controls the RNF8-Dependent Ubiquitylation Pathway and Facilitates DNA Double-Strand Break Repair in Human Cells.

Author(s) : Morris C, Tomimatsu N, Burma S, Jalinot P,
Journal : Cancer Res
2016
Unrepaired DNA double-strand breaks (DSB) are the most destructive chromosomal lesions driving genomic instability, a core hallmark of cancer. Here, we identify the antioncogenic breast cancer factor INT6/EIF3E as an essential regulator of DSB repair that promotes homologous recombination (HR)-mediated repair and, to alesser extent, nonhomologous end-joining repair. INT6 silencing impaired the accrual of the ubiquitin ligase RNF8 at DSBs and the formation of ubiquitin conjugates at DSB sites, especially Lys63-linked polyubiquitin chains, resultingin impaired recruitment of BRCA1, BRCA2, and RAD51, which are all involved in HRrepair. In contrast, INT6 deficiency did not affect the accumulation of RNF168, 53BP1, or RPA at DSBs. In INT6-silenced cells, there was also an alteration in DNA damage-induced localization of MDC1, a key target for ATM phosphorylation, which is a prerequisite for RNF8 recruitment. The attenuated DNA damage localization of RNF8 resulting from INT6 depletion could be attributed to the defective retention of ATM previously reported by us. Our findings deepen insights into how INT6 protects against breast cancer by showing how it functions in DSB repair, with potential clinical implications for cancer therapy. Cancer Res; 76(20); 6054-65. (c)2016 AACR.

Is It worth Considering Circulating microRNAs in Multiple Sclerosis?

Author(s) : Jagot F, Davoust N,
Journal : Front Immunol
2016
New evidence has highlighted that miRNA production and trafficking can be dysregulated in both autoimmmune and neurological disorders. Multiple sclerosis (MS) in particular is an autoimmune pathology leading to neurodegeneration. Profiling studies performed on cells derived from MS patients have described a dysregulated network of miRNAs in both immune and neural cells. Interestingly, new evidence has emerged showing that circulating miRNAs are also dysregulated in MS body fluids, including plasma/serum and cerebrospinal fluid. This review summarizes the current scientific theories on the function of this altered circulating miRNA network. It builds up new insights about miRNA transfer mechanisms including extracellular vesicle trafficking involved in cell-to-cell communication and the possible physiopathological functions of these transfers in MS. Finally, this review proposes that monitoring altered miRNA expression levels could serve as a potential biomarker read-out of MS subtype and severity.

Looking Beyond the Genes: The Interplay Between Signaling Pathways and Mechanics in the Shaping and Diversification of Epithelial Tissues.

Author(s) : Urdy S, Goudemand N, Pantalacci S,
Journal : Curr Top Dev Biol
2016
The core of Evo-Devo lies in the intuition that the way tissues grow during embryonic development, the way they sustain their structure and function throughout lifetime, and the way they evolve are closely linked. Epithelial tissues are ubiquitous in metazoans, covering the gut and internal branched organs, as well as the skin and its derivatives (ie, teeth). Here, we discuss invitro, in vivo, and in silico studies on epithelial tissues to illustrate the conserved, dynamical, and complex aspects of their development. We then explore the implications of the dynamical and nonlinear nature of development on the evolution of their size and shape at the phenotypic and genetic levels. In rare cases, when the interplay between signaling and mechanics is well understood at the cell level, it is becoming clear that the structure of development leads to covariation of characters, an integration which in turn provides some predictable structure to evolutionary changes. We suggest that such nonlinear systems are prone to genetic drift, cryptic genetic variation, and context-dependent mutational effects. We argue that experimental and theoretical studies at the cell level are critical to our understanding of the phenotypic and genetic evolution of epithelial tissues, including carcinomas.

Modeling and Identification of Amnioserosa Cell Mechanical Behavior by Using Mass-Spring Lattices.

Author(s) : Dureau M, Alessandri A, Bagnerini P, Vincent S,
Journal : IEEE/ACM Trans Comput Biol Bioinform
2016
Various mechanical models of live amnioserosa cells during Drosophila melanogaster 's dorsal closure are proposed. Such models account for specific biomechanical oscillating behaviors and depend on a different set of parameters.The identification of the parameters for each of the proposed models is accomplished according to a least-squares approach in such a way to best fit thecellular dynamics extracted from live images. For the purpose of comparison, theresulting models after identification are validated to allow for the selection of the most appropriate description of such a cell dynamics. The proposed methodology is general and it may be applied to other planar biological processes.

Mutation allele burden remains unchanged in chronic myelomonocytic leukaemia responding to hypomethylating agents

Author(s) : Merlevede J, Droin N, Qin T, Meldi K, Yoshida K, Morabito M, Chautard E, Auboeuf D, Fenaux P, Braun T, Itzykson R, de Botton S, Quesnel B, Commes T, Jourdan E, Vainchenker W, Bernard O, Pata-Merci N, Solier S, Gayevskiy V, Dinger M, Cowley M, Selimoglu-Buet D, Meyer V, Artiguenave F, Deleuze J, Preudhomme C, Stratton M, Alexandrov L, Padron E, Ogawa S, Koscielny S, Figueroa M, Solary E,
Journal : Nat Commun
2016

Nucleosome eviction in mitosis assists condensin loading and chromosome condensation.

Author(s) : Toselli-Mollereau E, Robellet X, Fauque L, Lemaire S, Schiklenk C, Klein C, Hocquet C, Legros P, N'Guyen L, Mouillard L, Chautard E, Auboeuf D, Haering C, Bernard P,
Journal : EMBO J
2016
Condensins associate with DNA and shape mitotic chromosomes. Condensins are enriched nearby highly expressed genes during mitosis, but how this binding is achieved and what features associated with transcription attract condensins remain unclear. Here, we report that condensin accumulates at or in the immediate vicinity of nucleosome-depleted regions during fission yeast mitosis. Two transcriptional coactivators, the Gcn5 histone acetyltransferase and the RSC chromatin-remodelling complex, bind to promoters adjoining condensin-binding sites and locally evict nucleosomes to facilitate condensin binding and allow efficient mitotic chromosome condensation. The function of Gcn5 is closely linked to condensin positioning, since neither the localization of topoisomerase II northat of the cohesin loader Mis4 is altered in gcn5 mutant cells. We propose thatnucleosomes act as a barrier for the initial binding of condensin and that nucleosome-depleted regions formed at highly expressed genes by transcriptional coactivators constitute access points into chromosomes where condensin binds free genomic DNA.

Oncogene- and drug resistance-associated alternative exon usage in acute myeloid leukemia (AML)

Author(s) : Mohamed A, Balsat M, Thenoz M, Koering C, Payen-Gay L, Cheok M, Mortada H, Auboeuf D, Pinatel C, El-Hamri M, Dumontet C, Cros E, Flandrin-Gresta P, Nibourel O, Preudhomme C, Michallet M, Thomas X, Nicolini F, Solly F, Guyotat D, Campos L, Wattel E, Mortreux F,
Journal : Oncotarget
2016

Oncogene- and drug resistance-associated alternative exon usage in acute myeloid leukemia (AML).

Author(s) : Mohamed A, Balsat M, Thenoz M, Koering C, Payen-Gay L, Cheok M, Mortada H, Auboeuf D, Pinatel C, El-Hamri M, Dumontet C, Cros E, Flandrin-Gresta P, Nibourel O, Preudhomme C, Michallet M, Thomas X, Nicolini F, Solly F, Guyotat D, Campos L, Wattel E, Mortreux F,
Journal : Oncotarget
2016
In addition to spliceosome gene mutations, oncogene expression and drug resistance in AML might influence exon expression. We performed exon-array analysis and exon-specific PCR (ESPCR) to identify specific landscapes of exon expression that are associated with DEK and WT1 oncogene expression and the resistance of AML cells to AraC, doxorubicin or azacitidine. Data were obtained for these five conditions through exon-array analysis of 17 cell lines and 24 patient samples and were extended through qESPCR of samples from 152 additional AML cases. More than 70% of AEUs identified by exon-array were technically validated through ESPCR. In vitro, 1,130 to 5,868 exon events distinguished the 5 conditions from their respective controls while in vivo 6,560 and 9,378 events distinguished chemosensitive and chemoresistant AML, respectively, from normal bone marrow. Whatever the cause of this effect, 30 to 80% of mis-spliced mRNAs involved genes unmodified at the whole transcriptional level. These AEUs unmasked new functional pathways that are distinct from those generated by transcriptional deregulation. These results also identified new putative pathways that could help increase the understanding of the effects mediated by DEK or WT1, which may allow the targeting of these pathways to prevent resistance of AML cells to chemotherapeutic agents.

Putative RNA-directed adaptive mutations in cancer evolution

Author(s) : Auboeuf D,
Journal : Transcription
2016

Putative RNA-Directed Adaptive Mutations in Cancer Evolution.

Author(s) : Auboeuf D,
Journal : Transcription
2016
Understanding the molecular mechanisms behind the capacity of cancer cells to adapt to the tumor microenvironment and to anticancer therapies is a major challenge. In this context, cancer is believed to be an evolutionary process where random mutations and the selection process shape the mutational pattern and phenotype of cancer cells. This article challenges the notion of randomness of some cancer-associated mutations by describing molecular mechanisms involving stress-mediated biogenesis of mRNA-derived small RNAs able to target and increase the local mutation rate of the genomic loci they originate from. It is proposed that the probability of some mutations at specific loci could be increased in a stress-specific and RNA-depending manner. This would increase the probability ofgenerating mutations that could alleviate stress situations, such as those triggered by anticancer drugs. Such a mechanism is made possible because tumor- and anticancer drug-associated stress situations trigger both cellular reprogramming and inflammation, which leads cancer cells to express molecular tools allowing them to "attack" and mutate their own genome in an RNA-directed manner.

Rapid evolution of a Y-chromosome heterochromatin protein underlies sex chromosome meiotic drive.

Author(s) : Helleu Q, Gerard P, Dubruille R, Ogereau D, Prud'homme B, Loppin B, Montchamp-Moreau C,
Journal : Proc Natl Acad Sci U S A
2016
Sex chromosome meiotic drive, the non-Mendelian transmission of sex chromosomes,is the expression of an intragenomic conflict that can have extreme evolutionaryconsequences. However, the molecular bases of such conflicts remain poorly understood. Here, we show that a young and rapidly evolving X-linked heterochromatin protein 1 (HP1) gene, HP1D2, plays a key role in the classical Paris sex-ratio (SR) meiotic drive occurring in Drosophila simulans Driver HP1D2alleles prevent the segregation of the Y chromatids during meiosis II, causing female-biased sex ratio in progeny. HP1D2 accumulates on the heterochromatic Y chromosome in male germ cells, strongly suggesting that it controls the segregation of sister chromatids through heterochromatin modification. We show that Paris SR drive is a consequence of dysfunctional HP1D2 alleles that fail toprepare the Y chromosome for meiosis, thus providing evidence that the rapid evolution of genes controlling the heterochromatin structure can be a significant source of intragenomic conflicts.

2015

A DPP-mediated feed-forward loop canalizes morphogenesis during Drosophila dorsal closure.

Author(s) : Ducuing A, Keeley C, Mollereau B, Vincent S,
Journal : J Cell Biol
2015
Development is robust because nature has selected various mechanisms to buffer the deleterious effects of environmental and genetic variations to deliver phenotypic stability. Robustness relies on smart network motifs such as feed-forward loops (FFLs) that ensure the reliable interpretation of developmental signals. In this paper, we show that Decapentaplegic (DPP) and JNKform a coherent FFL that controls the specification and differentiation of leading edge cells during Drosophila melanogaster dorsal closure (DC). We provide molecular evidence that through repression by Brinker (Brk), the DPP branch of the FFL filters unwanted JNK activity. High-throughput live imaging revealed that this DPP/Brk branch is dispensable for DC under normal conditions but is required when embryos are subjected to thermal stress. Our results indicate that the wiring of DPP signaling buffers against environmental challenges and canalizes cell identity. We propose that the main function of DPP pathway during Drosophila DC is to ensure robust morphogenesis, a distinct function from its well-established ability to spread spatial information.

An optimized kit-free method for making strand-specific deep sequencing libraries from RNA fragments.

Author(s) : Heyer E, Ozadam H, Ricci E, Cenik C, Moore M,
Journal : Nucleic Acids Res
2015
Deep sequencing of strand-specific cDNA libraries is now a ubiquitous tool for identifying and quantifying RNAs in diverse sample types. The accuracy of conclusions drawn from these analyses depends on precise and quantitative conversion of the RNA sample into a DNA library suitable for sequencing. Here, we describe an optimized method of preparing strand-specific RNA deep sequencing libraries from small RNAs and variably sized RNA fragments obtained from ribonucleoprotein particle footprinting experiments or fragmentation of long RNAs. Our approach works across a wide range of input amounts (400 pg to 200 ng), is easy to follow and produces a library in 2-3 days at relatively low reagent cost, all while giving the user complete control over every step. Because all enzymatic reactions were optimized and driven to apparent completion, sequence diversity and species abundance in the input sample are well preserved.

C. elegans outside the Petri dish.

Author(s) : Frézal L, Félix M,
Journal : Elife
2015
The roundworm Caenorhabditis elegans has risen to the status of a top model organismfor biological research in the last fifty years. Among laboratory animals, this tinynematode is one of the simplest and easiest organisms to handle. And its lifeoutside the laboratory is beginning to be unveiled. Like other model organisms, C.elegans has a boom-and-bust lifestyle. It feasts on ephemeral bacterial blooms indecomposing fruits and stems. After resource depletion, its young larvae enter amigratory diapause stage, called the dauer. Organisms known to be associated with C.elegans include migration vectors (such as snails, slugs and isopods) and pathogens(such as microsporidia, fungi, bacteria and viruses). By deepening our understandingof the natural history of C. elegans, we establish a broader context and improvedtools for studying its biology.

C. elegans outside the Petri dish.

Author(s) : Frézal L, Félix M,
Journal : Elife
2015
The roundworm Caenorhabditis elegans has risen to the status of a top model organismfor biological research in the last fifty years. Among laboratory animals, this tinynematode is one of the simplest and easiest organisms to handle. And its lifeoutside the laboratory is beginning to be unveiled. Like other model organisms, C.elegans has a boom-and-bust lifestyle. It feasts on ephemeral bacterial blooms indecomposing fruits and stems. After resource depletion, its young larvae enter amigratory diapause stage, called the dauer. Organisms known to be associated with C.elegans include migration vectors (such as snails, slugs and isopods) and pathogens(such as microsporidia, fungi, bacteria and viruses). By deepening our understandingof the natural history of C. elegans, we establish a broader context and improvedtools for studying its biology.

Chaperoning 5S RNA assembly.

Author(s) : Madru C, Lebaron S, Blaud M, Delbos L, Pipoli J, Pasmant E, Rety S, Leulliot N,
Journal : Genes Dev
2015
In eukaryotes, three of the four ribosomal RNAs (rRNAs)-the 5.8S, 18S, and 25S/28S rRNAs-are processed from a single pre-rRNA transcript and assembled intoribosomes. The fourth rRNA, the 5S rRNA, is transcribed by RNA polymerase III and is assembled into the 5S ribonucleoprotein particle (RNP), containing ribosomal proteins Rpl5/uL18 and Rpl11/uL5, prior to its incorporation into preribosomes. In mammals, the 5S RNP is also a central regulator of the homeostasis of the tumor suppressor p53. The nucleolar localization of the 5S RNP and its assembly into preribosomes are performed by a specialized complex composed of Rpf2 and Rrs1 in yeast or Bxdc1 and hRrs1 in humans. Here we report the structural and functional characterization of the Rpf2-Rrs1 complex alone, in complex with the 5S RNA, and within pre-60S ribosomes. We show that the Rpf2-Rrs1 complex contains a specialized 5S RNA E-loop-binding module, contacts the Rpl5 protein, and also contacts the ribosome assembly factor Rsa4 and the 25S RNA. We propose that the Rpf2-Rrs1 complex establishes a network of interactions that guide the incorporation of the 5S RNP in preribosomes in the initial conformation prior toits rotation to form the central protuberance found in the mature large ribosomal subunit.

Chromatids segregate without centrosomes during Caenorhabditis elegans mitosis in a Ran- and CLASP-dependent manner.

Author(s) : Nahaboo W, Zouak M, Askjaer P, Delattre M,
Journal : Mol Biol Cell
2015
During mitosis, chromosomes are connected to a microtubule-based spindle. Current models propose that displacement of the spindle poles and/or the activity of kinetochore microtubules generate mechanical forces that segregate sister chromatids. Using laser destruction of the centrosomes during Caenorhabditis elegans mitosis, we show that neither of these mechanisms is necessary to achieve proper chromatid segregation. Our results strongly suggest that an outward forcegenerated by the spindle midzone, independently of centrosomes, is sufficient tosegregate chromosomes in mitotic cells. Using mutant and RNAi analysis, we show that the microtubule-bundling protein SPD-1/MAP-65 and BMK-1/kinesin-5 act as a brake opposing the force generated by the spindle midzone. Conversely, we identify a novel role for two microtubule-growth and nucleation agents, Ran and CLASP, in the establishment of the centrosome-independent force during anaphase.Their involvement raises the interesting possibility that microtubule polymerization of midzone microtubules is continuously required to sustain chromosome segregation during mitosis.

Cooling-Induced ER Stress is Good for Your Brain.

Author(s) : Mollereau B,
Journal : EBioMedicine
2015

Discovery and characterization of auxiliary proteins encoded by type 3 simian T-cell lymphotropic viruses.

Author(s) : Turpin J, Journo C, Ko N, Sinet F, Carpentier A, Galioot A, Edwards D, Vandamme A, Gazzolo L, Duc Dodon M, Gessain A, Kashanchi F, Balansard I, Lacoste R, Mahieux R,
Journal : J Virol
2015
Human T-cell lymphotropic virus type 1 (HTLV-1) and HTLV-2 encode auxiliary proteins that play important roles in viral replication, viral latency, and immune escape. The presence of auxiliary protein-encoding open reading frames (ORFs) in HTLV-3, the latest HTLV to be discovered, is unknown. Simian T-cell lymphotropic virus type 3 (STLV-3) is almost identical to HTLV-3. Given the lackof HTLV-3-infected cell lines, we took advantage of STLV-3-infected cells and ofan STLV-3 molecular clone to search for the presence of auxiliary transcripts. Using reverse transcriptase PCR (RT-PCR), we first uncovered the presence of three unknown viral mRNAs encoding putative proteins of 5, 8, and 9 kDa and confirmed the presence of the previously reported RorfII transcript. The existence of these viral mRNAs was confirmed by using splice site-specific RT-PCR with ex vivo samples. We showed that p5 is distributed throughout the cell and does not colocalize with a specific organelle. The p9 localization is similar tothat of HTLV-1 p12 and induced a strong decrease in the calreticulin signal, similarly to HTLV-1 p12. Although p8, RorfII, and Rex-3 share an N-terminal sequence that is predicted to contain a nucleolar localization signal (NoLS), only p8 is found in the nucleolus. The p8 location in the nucleolus is linked toa bipartite NoLS. p8 and, to a lesser extent, p9 repressed viral expression but did not alter Rex-3-dependent mRNA export. Using a transformation assay, we finally showed that none of the STLV-3 auxiliary proteins had the ability to induce colony formation, while both Tax-3 and antisense protein of HTLV-3 (APH-3) promoted cellular transformation. Altogether, these results complete the characterization of the newly described primate T-lymphotropic virus type 3 (PTLV-3). IMPORTANCE: Together with their simian counterparts, HTLVs form the primate T-lymphotropic viruses. HTLVs arose from interspecies transmission between nonhuman primates and humans. HTLV-1 and HTLV-2 encode auxiliary proteins that play important roles in viral replication, viral latency, and immune escape. The presence of ORFs encoding auxiliary proteins in HTLV-3 or STLV-3 genomes wasunknown. Using in silico analyses, ex vivo samples, or in vitro experiments, we have uncovered the presence of 3 previously unknown viral mRNAs encoding putative proteins and confirmed the presence of a previously reported viral transcript. We characterized the intracellular localization of the four proteins. We showed that two of these proteins repress viral expression but that none of them have the ability to induce colony formation. However, both Tax and the antisense protein APH-3 promote cell transformation. Our results allowed us to characterize 4 new retroviral proteins for the first time.

Does transcription play a role in creating a condensin binding site?

Author(s) : Bernard P, Vanoosthuyse V,
Journal : Transcription
2015
The highly conserved condensin complex is essential for the condensation and integrity of chromosomes through cell division. Published data argue that high levels of transcription contribute to specify some condensin-binding sites on chromosomes but the exact role of transcription in this process remains elusive.Here we discuss our recent data addressing the role of transcription in establishing a condensin-binding site.

Effect of replication on epigenetic memory and consequences on gene transcription.

Author(s) : Zerihun M, Vaillant C, Jost D,
Journal : Phys Biol
2015
Gene activity in eukaryotes is in part regulated at the level of chromatin through the assembly of local chromatin states that are more or less permissive to transcription. How do these chromatin states achieve their functions and whether or not they contribute to the epigenetic inheritance of the transcriptional program remain to be elucidated. In cycling cells, stability is indeed strongly challenged by the periodic occurrence of replication and cell division. To address this question, we perform simulations of the stochastic dynamics of chromatin states when driven out-of-equilibrium by periodic perturbations. We show how epigenetic memory is significantly affected by the cell cycle length. In addition, we develop a simple model to connect the epigenetic state to the transcriptional state and gene activity. In particular, it suggests that replication may induce transcriptional bursting at repressive loci. Finally, we discuss how our findings-effect of replication and link to gene transcription-have original and deep implications to various biological contextsof epigenetic memory.

Expression of dengue virus NS3 protein in Drosophila alters its susceptibility to infection.

Author(s) : Querenet M, Danjoy M, Mollereau B, Davoust N,
Journal : Fly (Austin)
2015
We developed a Drosophila model in which the dengue virus NS3 protein is expressed in a tissue specific and inducible manner. Dengue virus NS3 is a multifunctional protein playing a major role during viral replication. Both protease and helicase domains of NS3 are interacting with human and insect host proteins including innate immune components of the host machinery. We characterized the NS3 transgenic flies showing that NS3 expression did not affect fly development. To further study the links between NS3 and the innate immune response, we challenge the flies with gram-positive and gram-negative bacteria. Interestingly, the Drosophila transgenic flies expressing NS3 were more susceptible to bacterial infections than control flies. However ubiquitous or immune-specific NS3 expression affected neither the life span nor the response to a non-infectious stress of the flies. In conclusion, we generated a new in vivo system to study the functional impact of DENV NS3 protein on the innate immune response.

Fatty acid transport proteins in disease: New insights from invertebrate models.

Author(s) : Dourlen P, Sujkowski A, Wessells R, Mollereau B,
Journal : Prog Lipid Res
2015
The dysregulation of lipid metabolism has been implicated in various diseases, including diabetes, cardiopathies, dermopathies, retinal and neurodegenerative diseases. Mouse models have provided insights into lipid metabolism. However, progress in the understanding of these pathologies is hampered by the multiplicity of essential cellular processes and genes that modulate lipid metabolism. Drosophila and Caenorhabditis elegans have emerged as simple geneticmodels to improve our understanding of these metabolic diseases. Recent studies have characterized fatty acid transport protein (fatp) mutants in Drosophila andC. elegans, establishing new models of cardiomyopathy, retinal degeneration, fatstorage disease and dermopathies. These models have generated novel insights into the physiological role of the Fatp protein family in vivo in multicellular organisms, and are likely to contribute substantially to progress in understanding the etiology of various metabolic disorders. Here, we describe anddiscuss the mechanisms underlying invertebrate fatp mutant models in the light of the current knowledge relating to FATPs and lipid disorders in vertebrates.

From Immunodeficiency to Humanization: The Contribution of Mouse Models to Explore HTLV-1 Leukemogenesis.

Author(s) : Peres E, Bagdassarian E, This S, Villaudy J, Rigal D, Gazzolo L, Duc Dodon M,
Journal : Viruses
2015
The first discovered human retrovirus, Human T-Lymphotropic Virus type 1 (HTLV-1), is responsible for an aggressive form of T cell leukemia/lymphoma. Mouse models recapitulating the leukemogenesis process have been helpful for understanding the mechanisms underlying the pathogenesis of this retroviral-induced disease. This review will focus on the recent advances in thegeneration of immunodeficient and human hemato-lymphoid system mice with a particular emphasis on the development of mouse models for HTLV-1-mediated pathogenesis, their present limitations and the challenges yet to be addressed.

Genome-wide screen identifies a novel p97/CDC-48-dependent pathway regulating ER-stress-induced gene transcription.

Author(s) : Marza E, Taouji S, Barroso K, Raymond A, Guignard L, Bonneu M, Pallares-Lupon N, Dupuy J, Fernandez-Zapico M, Rosenbaum J, Palladino F, Dupuy D, Chevet E,
Journal : EMBO Rep
2015
The accumulation of misfolded proteins in the endoplasmic reticulum (ER) activates the Unfolded Protein Response (UPR(ER)) to restore ER homeostasis. TheAAA(+) ATPase p97/CDC-48 plays key roles in ER stress by promoting both ER protein degradation and transcription of UPR(ER) genes. Although the mechanisms associated with protein degradation are now well established, the molecular events involved in the regulation of gene transcription by p97/CDC-48 remain unclear. Using a reporter-based genome-wide RNAi screen in combination with quantitative proteomic analysis in Caenorhabditis elegans, we have identified RUVB-2, a AAA(+) ATPase, as a novel repressor of a subset of UPR(ER) genes. We show that degradation of RUVB-2 by CDC-48 enhances expression of ER stress response genes through an XBP1-dependent mechanism. The functional interplay between CDC-48 and RUVB-2 in controlling transcription of select UPR(ER) genes appears conserved in human cells. Together, these results describe a novel role for p97/CDC-48, whereby its role in protein degradation is integrated with its role in regulating expression of ER stress response genes.

Histone storage and deposition in the early Drosophila embryo.

Author(s) : Horard B, Loppin B,
Journal : Chromosoma
2015
Drosophila development initiates with the formation of a diploid zygote followedby the rapid division of embryonic nuclei. This syncytial phase of development occurs almost entirely under maternal control and ends when the blastoderm embryo cellularizes and activates its zygotic genome. The biosynthesis and storage of histones in quantity sufficient for chromatin assembly of several thousands of genome copies represent a unique challenge for the developing embryo. In this article, we have reviewed our current understanding of the mechanisms involved in the production, storage, and deposition of histones in the fertilized egg and during the exponential amplification of cleavage nuclei.

How Retroviruses Escape the Nonsense-Mediated mRNA Decay.

Author(s) : Mocquet V, Durand S, Jalinot P,
Journal : AIDS Res Hum Retroviruses
2015
Many posttranscriptional processes are known to regulate gene expression and some of them can act as an antiviral barrier. The nonsense-mediated mRNA decay (NMD) was first identified as an mRNA quality control pathway that triggers rapid decay of mRNA containing premature stop codons due to mutations. NMD is now consideredas a general posttranscriptional regulation pathway controlling the expression of a large set of cellular genes. In addition to premature stop codons, many other features including alternative splicing, 5' uORF, long 3' UTR, selenocystein codons, and frameshift are able to promote NMD. Interestingly, many viral mRNAs exhibit some of these features suggesting that virus expression and replication might be sensitive to NMD. Several studies, including recent ones, have shown that this is the case for retroviruses; however, it also appears that retroviruses have developed strategies to overcome NMD in order to protect theirgenome and ensure a true expression of their genes. As a consequence of NMD inhibition, these viruses also affect the expression of host genes that are prone to NMD, and therefore can potentially trigger pathological effects on infected cells. Here, we review recent studies supporting this newly uncovered function of the NMD pathway as a defense barrier that viruses must overcome in order to replicate.

In Vitro and In Vivo Modulation of Alternative Splicing by the Biguanide Metformin

Author(s) : Laustriat D, Gide J, Barrault L, Chautard E, Benoit C, Auboeuf D, Boland A, Battail C, Artiguenave F, Deleuze J, B?nit P, Rustin P, Franc S, Charpentier G, Furling D, Bassez G, Nissan X, Martinat C, Peschanski M, Baghdoyan S,
Journal : Mol Ther Nucleic Acids
2015

In Vitro and In Vivo Modulation of Alternative Splicing by the Biguanide Metformin.

Author(s) : Laustriat D, Gide J, Barrault L, Chautard E, Benoit C, Auboeuf D, Boland A, Battail C, Artiguenave F, Deleuze J, Benit P, Rustin P, Franc S, Charpentier G, Furling D, Bassez G, Nissan X, Martinat C, Peschanski M, Baghdoyan S,
Journal : Mol Ther Nucleic Acids
2015
Major physiological changes are governed by alternative splicing of RNA, and itsmisregulation may lead to specific diseases. With the use of a genome-wide approach, we show here that this splicing step can be modified by medication anddemonstrate the effects of the biguanide metformin, on alternative splicing. Themechanism of action involves AMPK activation and downregulation of the RBM3 RNA-binding protein. The effects of metformin treatment were tested on myotonic dystrophy type I (DM1), a multisystemic disease considered to be a spliceopathy.We show that this drug promotes a corrective effect on several splicing defects associated with DM1 in derivatives of human embryonic stem cells carrying the causal mutation of DM1 as well as in primary myoblasts derived from patients. The biological effects of metformin were shown to be compatible with typical therapeutic dosages in a clinical investigation involving diabetic patients. Thedrug appears to act as a modifier of alternative splicing of a subset of genes and may therefore have novel therapeutic potential for many more diseases besides those directly linked to defective alternative splicing.

Integrative analysis of RNA, translation, and protein levels reveals distinct regulatory variation across humans.

Author(s) : Cenik C, Cenik E, Byeon G, Grubert F, Candille S, Spacek D, Alsallakh B, Tilgner H, Araya C, Tang H, Ricci E, Snyder M,
Journal : Genome Res
2015
Elucidating the consequences of genetic differences between humans is essential for understanding phenotypic diversity and personalized medicine. Although variation in RNA levels, transcription factor binding, and chromatin have been explored, little is known about global variation in translation and its genetic determinants. We used ribosome profiling, RNA sequencing, and mass spectrometry to perform an integrated analysis in lymphoblastoid cell lines from a diverse group of individuals. We find significant differences in RNA, translation, and protein levels suggesting diverse mechanisms of personalized gene expression control. Combined analysis of RNA expression and ribosome occupancy improves theidentification of individual protein level differences. Finally, we identify genetic differences that specifically modulate ribosome occupancy--many of thesedifferences lie close to start codons and upstream ORFs. Our results reveal a new level of gene expression variation among humans and indicate that genetic variants can cause changes in protein levels through effects on translation.

Intellectual disability-associated dBRWD3 regulates gene expression through inhibition of HIRA/YEM-mediated chromatin deposition of histone H3.3.

Author(s) : Chen W, Shih H, Liu K, Shih Z, Chen L, Tsai T, Chen M, Liu H, Tan B, Chen C, Lee H, Loppin B, Ait-Ahmed O, Wu J,
Journal : EMBO Rep
2015
Many causal mutations of intellectual disability have been found in genes involved in epigenetic regulations. Replication-independent deposition of the histone H3.3 variant by the HIRA complex is a prominent nucleosome replacement mechanism affecting gene transcription, especially in postmitotic neurons. However, how HIRA-mediated H3.3 deposition is regulated in these cells remains unclear. Here, we report that dBRWD3, the Drosophila ortholog of the intellectual disability gene BRWD3, regulates gene expression through H3.3, HIRA, and its associated chaperone Yemanuclein (YEM), the fly ortholog of mammalian Ubinuclein1. In dBRWD3 mutants, increased H3.3 levels disrupt gene expression, dendritic morphogenesis, and sensory organ differentiation. Inactivation of yem or H3.3 remarkably suppresses the global transcriptome changes and various developmental defects caused by dBRWD3 mutations. Our work thus establishes a previously unknown negative regulation of H3.3 and advances our understanding ofBRWD3-dependent intellectual disability.

Photoswitchable Inhibitors of Microtubule Dynamics Optically Control Mitosis and Cell Death.

Author(s) : Borowiak M, Nahaboo W, Reynders M, Nekolla K, Jalinot P, Hasserodt J, Rehberg M, Delattre M, Zahler S, Vollmar A, Trauner D, Thorn-Seshold O,
Journal : Cell
2015
Small molecules that interfere with microtubule dynamics, such as Taxol and the Vinca alkaloids, are widely used in cell biology research and as clinical anticancer drugs. However, their activity cannot be restricted to specific target cells, which also causes severe side effects in chemotherapy. Here, we introducethe photostatins, inhibitors that can be switched on and off in vivo by visible light, to optically control microtubule dynamics. Photostatins modulate microtubule dynamics with a subsecond response time and control mitosis in living organisms with single-cell spatial precision. In longer-term applications in cell culture, photostatins are up to 250 times more cytotoxic when switched on with blue light than when kept in the dark. Therefore, photostatins are both valuabletools for cell biology, and are promising as a new class of precision chemotherapeutics whose toxicity may be spatiotemporally constrained using light.

Predicting pathogen-specific CD8 T cell immune responses from a modeling approach.

Author(s) : Crauste F, Terry E, Mercier I, Mafille J, Djebali S, Andrieu T, Mercier B, Kaneko G, Arpin C, Marvel J, Gandrillon O,
Journal : J Theor Biol
2015
The primary CD8 T cell immune response constitutes a major mechanism to fight aninfection by intra-cellular pathogens. We aim at assessing whether pathogen-specific dynamical parameters of the CD8 T cell response can be identified, based on measurements of CD8 T cell counts, using a modeling approach. We generated experimental data consisting in CD8 T cell counts kinetics during the response to three different live intra-cellular pathogens: two viruses (influenza, vaccinia) injected intranasally, and one bacteria (Listeria monocytogenes) injected intravenously. All pathogens harbor the same antigen (NP68), but differ in their interaction with the host. In parallel, we developeda mathematical model describing the evolution of CD8 T cell counts and pathogen amount during an immune response. This model is characterized by 9 parameters and includes relevant feedback controls. The model outputs were compared with the three data series and an exhaustive estimation of the parameter values was performed. By focusing on the ability of the model to fit experimental data and to produce a CD8 T cell population mainly composed of memory cells at the end ofthe response, critical parameters were identified. We show that a small number of parameters (2-4) define the main features of the CD8 T cell immune response and are characteristic of a given pathogen. Among these parameters, two are related to the effector CD8 T cell mediated control of cell and pathogen death. The parameter associated with memory cell death is shown to play no relevant role during the main phases of the CD8 T cell response, yet it becomes essential whenlooking at the predictions of the model several months after the infection.

Protection of Drosophila chromosome ends through minimal telomere capping.

Author(s) : Dubruille R, Loppin B,
Journal : J Cell Sci
2015
In Drosophila, telomere-capping proteins have the remarkable capacity to recognize chromosome ends in a sequence-independent manner. This epigenetic protection is essential to prevent catastrophic ligations of chromosome extremities. Interestingly, capping proteins occupy a large telomere chromatin domain of several kilobases; however, the functional relevance of this to end protection is unknown. Here, we investigate the role of the large capping domainby manipulating HOAP (encoded by caravaggio) capping-protein expression in the male germ cells, where telomere protection can be challenged without compromising viability. We show that the exhaustion of HOAP results in a dramatic reduction of other capping proteins at telomeres, including K81 [encoded by ms(3)K81], which is essential for male fertility. Strikingly however, we demonstrate that, although capping complexes are barely detected in HOAP-depleted male germ cells,telomere protection and male fertility are not dramatically affected. Our study thus demonstrates that efficient protection of Drosophila telomeres can be achieved with surprisingly low amounts of capping complexes. We propose that these complexes prevent fusions by acting at the very extremity of chromosomes, reminiscent of the protection conferred by extremely short telomeric arrays in yeast or mammalian systems.

RAR/RXR binding dynamics distinguish pluripotency from differentiation associated cis-regulatory elements.

Author(s) : Chatagnon A, Veber P, Morin V, Bedo J, Triqueneaux G, Semon M, Laudet V, d'Alche-Buc F, Benoit G,
Journal : Nucleic Acids Res
2015
In mouse embryonic cells, ligand-activated retinoic acid receptors (RARs) play akey role in inhibiting pluripotency-maintaining genes and activating some major actors of cell differentiation. To investigate the mechanism underlying this dual regulation, we performed joint RAR/RXR ChIP-seq and mRNA-seq time series during the first 48 h of the RA-induced Primitive Endoderm (PrE) differentiation process in F9 embryonal carcinoma (EC) cells. We show here that this dual regulation is associated with RAR/RXR genomic redistribution during the differentiation process. In-depth analysis of RAR/RXR binding sites occupancy dynamics and composition show that in undifferentiated cells, RAR/RXR interact with genomic regions characterized by binding of pluripotency-associated factors and high prevalence of the non-canonical DR0-containing RA response element. By contrast,in differentiated cells, RAR/RXR bound regions are enriched in functional Sox17 binding sites and are characterized with a higher frequency of the canonical DR5motif. Our data offer an unprecedentedly detailed view on the action of RA in triggering pluripotent cell differentiation and demonstrate that RAR/RXR action is mediated via two different sets of regulatory regions tightly associated withcell differentiation status.

Reconstructing and analysing cellular states, space and time from gene expression profiles of many cells and single cells.

Author(s) : Francesconi M, Lehner B,
Journal : Mol Biosyst
2015
Genome-wide gene expression profiling is a fast, cheap and standardised analysisthat provides a high dimensional measurement of the state of a biological sample. In this review we describe computational methods that can be applied to identifyand interpret sources of variance in gene expression in whole organisms, organs,tissues or single cells. This allows the identification of constituent cell types and states in complex mixtures, the reconstruction of temporal trajectories of development, differentiation and progression, and the reconstruction of spatial patterning. When applied to genetically variable samples, these methods allow the efficient investigation of how genetic variation influences gene expression and biological processes in space and time.

Regulation of DEAH/RHA helicases by G-patch proteins.

Author(s) : Robert-Paganin J, Rety S, Leulliot N,
Journal : Biomed Res Int
2015
RNA helicases from the DEAH/RHA family are present in all the processes of RNA metabolism. The function of two helicases from this family, Prp2 and Prp43, is regulated by protein partners containing a G-patch domain. The G-patch is a glycine-rich domain discovered by sequence alignment, involved in protein-protein and protein-nucleic acid interaction. Although it has been shown to stimulate the helicase's enzymatic activities, the precise role of the G-patch domain remains unclear. The role of G-patch proteins in the regulation of Prp43 activity has been studied in the two biological processes in which it is involved: splicing and ribosome biogenesis. Depending on the pathway, the activity of Prp43 is modulated by different G-patch proteins. A particular feature of the structure of DEAH/RHA helicases revealed by the Prp43 structure is the OB-fold domain in C-terminal part. The OB-fold has been shown to be a platform responsible for theinteraction with G-patch proteins and RNA. Though there is still no structural data on the G-patch domain, in the current model, the interaction between the helicase, the G-patch protein, and RNA leads to a cooperative binding of RNA andconformational changes of the helicase.

Repression of somatic cell fate in the germline.

Author(s) : Robert V, Garvis S, Palladino F,
Journal : Cell Mol Life Sci
2015
Germ cells must transmit genetic information across generations, and produce gametes while also maintaining the potential to form all cell types after fertilization. Preventing the activation of somatic programs is, therefore, crucial to the maintenance of germ cell identity. Studies in Caenorhabditis elegans, Drosophila melanogaster, and mouse have revealed both similarities and differences in how somatic gene expression is repressed in germ cells, thereby preventing their conversion into somatic tissues. This review will focus on recent developments in our understanding of how global or gene-specific transcriptional repression, chromatin regulation, and translational repression operate in the germline to maintain germ cell identity and repress somatic differentiation programs.

2014

'Particle genetics': treating every cell as unique.

Author(s) : Yvert G,
Journal : Trends Genet
2014
Genotype-phenotype relations are usually inferred from a deterministic point of view. For example, quantitative trait loci (QTL), which describe regions of the genome associated with a particular phenotype, are based on a mean trait difference between genotype categories. However, living systems comprise huge numbers of cells (the 'particles' of biology). Each cell can exhibit substantialphenotypic individuality, which can have dramatic consequences at the organismallevel. Now, with technology capable of interrogating individual cells, it is time to consider how genotypes shape the probability laws of single cell traits. The possibility of mapping single cell probabilistic trait loci (PTL), which link genomic regions to probabilities of cellular traits, is a promising step in thisdirection. This approach requires thinking about phenotypes in probabilistic terms, a concept that statistical physicists have been applying to particles fora century. Here, I describe PTL and discuss their potential to enlarge our understanding of genotype-phenotype relations.

A genetic screen for functional partners of condensin in fission yeast.

Author(s) : Robellet X, Fauque L, Legros P, Mollereau E, Janczarski S, Parrinello H, Desvignes J, Thevenin M, Bernard P,
Journal : G3 (Bethesda)
2014
Mitotic chromosome condensation is a prerequisite for the accurate segregation of chromosomes during cell division, and the conserved condensin complex a central player of this process. However, how condensin binds chromatin and shapes mitotic chromosomes remain poorly understood. Recent genome-wide binding studies showingthat in most species condensin is enriched near highly expressed genes suggest aconserved link between condensin occupancy and high transcription rates. To gaininsight into the mechanisms of condensin binding and mitotic chromosome condensation, we searched for factors that collaborate with condensin through a synthetic lethal genetic screen in the fission yeast Schizosaccharomyces pombe. We isolated novel mutations affecting condensin, as well as mutations in four genes not previously implicated in mitotic chromosome condensation in fission yeast. These mutations cause chromosome segregation defects similar to those provoked by defects in condensation. We also identified a suppressor of the cut3-477 condensin mutation, which largely rescued chromosome segregation duringanaphase. Remarkably, of the five genes identified in this study, four encode transcription co-factors. Our results therefore provide strong additional evidence for a functional connection between chromosome condensation and transcription.

A recently evolved class of alternative 3'-terminal exons involved in cell cycle regulation by topoisomerase inhibitors

Author(s) : Dutertre M, Chakrama F, Combe E, Desmet F, Mortada H, Polay Espinoza M, Gratadou L, Auboeuf D,
Journal : Nat Commun
2014

A recently evolved class of alternative 3'-terminal exons involved in cell cycle regulation by topoisomerase inhibitors.

Author(s) : Dutertre M, Chakrama F, Combe E, Desmet F, Mortada H, Polay Espinoza M, Gratadou L, Auboeuf D,
Journal : Nat Commun
2014
Alternative 3'-terminal exons, which use intronic polyadenylation sites, are generally less conserved and expressed at lower levels than the last exon of genes. Here we discover a class of human genes, in which the last exon appeared recently during evolution, and the major gene product uses an alternative 3'-terminal exon corresponding to the ancestral last exon of the gene. This novel class of alternative 3'-terminal exons are downregulated on a large scale by doxorubicin, a cytostatic drug targeting topoisomerase II, and play a role in cell cycle regulation, including centromere-kinetochore assembly. The RNA-binding protein HuR/ELAVL1 is a major regulator of this specific set of alternative 3'-terminal exons. HuR binding to the alternative 3'-terminal exon in the pre-messenger RNA promotes its splicing, and is reduced by topoisomerase inhibitors. These findings provide new insights into the evolution, function andmolecular regulation of alternative 3'-terminal exons.

Automated high-throughput quantification of mitotic spindle positioning from DIC movies of Caenorhabditis embryos.

Author(s) : Cluet D, Stebe P, Riche S, Spichty M, Delattre M,
Journal : PLoS One
2014
The mitotic spindle is a microtubule-based structure that elongates to accurately segregate chromosomes during anaphase. Its position within the cell also dictates the future cell cleavage plan, thereby determining daughter cell orientation within a tissue or cell fate adoption for polarized cells. Therefore, the mitotic spindle ensures at the same time proper cell division and developmental precision. Consequently, spindle dynamics is the matter of intensive research. Among the different cellular models that have been explored, the one-cell stage C. elegans embryo has been an essential and powerful system to dissect the molecular and biophysical basis of spindle elongation and positioning. Indeed, in this large and transparent cell, spindle poles (or centrosomes) can be easily detected from simple DIC microscopy by human eyes. To perform quantitative and high-throughput analysis of spindle motion, we developed a computer program ACT for Automated-Centrosome-Tracking from DIC movies of C. elegans embryos. We therefore offer an alternative to the image acquisition and processing of transgenic lines expressing fluorescent spindle markers. Consequently, experiments on large sets of cells can be performed with a simple setup using inexpensive microscopes. Moreover, analysis of any mutant or wild-type backgrounds is accessible because laborious rounds of crosses with transgenic lines become unnecessary. Last, our program allows spindle detection in other nematode species, offering the same quality of DIC images but for which techniques of transgenesis are not accessible. Thus, our program also opens the way towards a quantitative evolutionary approach of spindle dynamics. Overall, our computer program is a unique macro for the image- and movie-processing platform ImageJ. It is user-friendly and freely available under an open-source licence. ACT allows batch-wise analysis of large sets of mitosis events. Within 2 minutes, a single movie is processed and the accuracy of the automated tracking matches the precision of the human eye.

Automated high-throughput quantification of mitotic spindle positioning from DIC movies of Caenorhabditis embryos.

Author(s) : Cluet D, Stebe P, Riche S, Spichty M, Delattre M,
Journal : PLoS One
2014
The mitotic spindle is a microtubule-based structure that elongates to accurately segregate chromosomes during anaphase. Its position within the cell also dictates the future cell cleavage plan, thereby determining daughter cell orientation within a tissue or cell fate adoption for polarized cells. Therefore, the mitotic spindle ensures at the same time proper cell division and developmental precision. Consequently, spindle dynamics is the matter of intensive research. Among the different cellular models that have been explored, the one-cell stage C. elegans embryo has been an essential and powerful system to dissect the molecular and biophysical basis of spindle elongation and positioning. Indeed, in this large and transparent cell, spindle poles (or centrosomes) can be easily detected from simple DIC microscopy by human eyes. To perform quantitative and high-throughput analysis of spindle motion, we developed a computer program ACT for Automated-Centrosome-Tracking from DIC movies of C. elegans embryos. We therefore offer an alternative to the image acquisition and processing of transgenic lines expressing fluorescent spindle markers. Consequently, experiments on large sets of cells can be performed with a simple setup using inexpensive microscopes. Moreover, analysis of any mutant or wild-type backgrounds is accessible because laborious rounds of crosses with transgenic lines become unnecessary. Last, our program allows spindle detection in other nematode species, offering the same quality of DIC images but for which techniques of transgenesis are not accessible. Thus, our program also opens the way towards a quantitative evolutionary approach of spindle dynamics. Overall, our computer program is a unique macro for the image- and movie-processing platform ImageJ. It is user-friendly and freely available under an open-source licence. ACT allows batch-wise analysis of large sets of mitosis events. Within 2 minutes, a single movie is processed and the accuracy of the automated tracking matches the precision of the human eye.

Bifurcation in epigenetics: implications in development, proliferation, and diseases.

Author(s) : Jost D,
Journal : Phys Rev E Stat Nonlin Soft Matter Phys
2014
Cells often exhibit different and stable phenotypes from the same DNA sequence. Robustness and plasticity of such cellular states are controlled by diverse transcriptional and epigenetic mechanisms, among them the modification of biochemical marks on chromatin. Here, we develop a stochastic model that describes the dynamics of epigenetic marks along a given DNA region. Through mathematical analysis, we show the emergence of bistable and persistent epigenetic states from the cooperative recruitment of modifying enzymes. We alsofind that the dynamical system exhibits a critical point and displays, in the presence of asymmetries in recruitment, a bifurcation diagram with hysteresis. These results have deep implications for our understanding of epigenetic regulation. In particular, our study allows one to reconcile within the same formalism the robust maintenance of epigenetic identity observed in differentiated cells, the epigenetic plasticity of pluripotent cells during differentiation, and the effects of epigenetic misregulation in diseases. Moreover, it suggests a possible mechanism for developmental transitions where the system is shifted close to the critical point to benefit from high susceptibility to developmental cues.

C. elegans epigenetic regulation in development and aging.

Author(s) : Gonzalez-Aguilera C, Palladino F, Askjaer P,
Journal : Brief Funct Genomics
2014
The precise developmental map of the Caenorhabditis elegans cell lineage, as well as a complete genome sequence and feasibility of genetic manipulation make this nematode species highly attractive to study the role of epigenetics during development. Genetic dissection of phenotypical traits, such as formation of egg-laying organs or starvation-resistant dauer larvae, has illustrated how chromatin modifiers may regulate specific cell-fate decisions and behavioral programs. Moreover, the transparent body of C. elegans facilitates non-invasive microscopy to study tissue-specific accumulation of heterochromatin at the nuclear periphery. We also review here recent findings on how small RNA molecules contribute to epigenetic control of gene expression that can be propagated for several generations and eventually determine longevity.

Chromatin redistribution of the DEK oncoprotein represses hTERT transcription in leukemias

Author(s) : Karam M, Thenoz M, Capraro V, Robin J, Pinatel C, Lancon A, Galia P, Sibon D, Thomas X, Ducastelle-Lepretre S, Nicolini F, El-Hamri M, Chelghoun Y, Wattel E, Mortreux F,
Journal : Neoplasia
2014

Chromatin redistribution of the DEK oncoprotein represses hTERT transcription in leukemias.

Author(s) : Karam M, Thenoz M, Capraro V, Robin J, Pinatel C, Lancon A, Galia P, Sibon D, Thomas X, Ducastelle-Lepretre S, Nicolini F, El-Hamri M, Chelghoun Y, Wattel E, Mortreux F,
Journal : Neoplasia
2014
Although numerous factors have been found to modulate hTERT transcription, the mechanism of its repression in certain leukemias remains unknown. We show here that DEK represses hTERT transcription through its enrichment on the hTERT promoter in cells from chronic and acute myeloid leukemias, chronic lymphocytic leukemia, but not acute lymphocytic leukemias where hTERT is overexpressed. We isolated DEK from the hTERT promoter incubated with nuclear extracts derived from fresh acute myelogenous leukemia (AML) cells and from cells expressing Tax, an hTERT repressor encoded by the human T cell leukemia virus type 1. In addition to the recruitment of DEK, the displacement of two potent known hTERT transactivators from the hTERT promoter characterized both AML cells and Tax-expressing cells. Reporter and chromatin immunoprecipitation assays permitted to map the region that supports the repressive effect of DEK on hTERT transcription, which was proportionate to the level of DEK-promoter association but not with the level of DEK expression. Besides hTERT repression, this contextof chromatin redistribution of DEK was found to govern about 40% of overall transcriptional modifications, including those of cancer-prone genes. In conclusion, DEK emerges as an hTERT repressor shared by various leukemia subtypes and seems involved in the deregulation of numerous genes associated with leukemogenesis.

CPF-associated phosphatase activity opposes condensin-mediated chromosome condensation.

Author(s) : Vanoosthuyse V, Legros P, van der Sar S, Yvert G, Toda K, Le Bihan T, Watanabe Y, Hardwick K, Bernard P,
Journal : PLoS Genet
2014
Functional links connecting gene transcription and condensin-mediated chromosomecondensation have been established in species ranging from prokaryotes to vertebrates. However, the exact nature of these links remains misunderstood. Here we show in fission yeast that the 3' end RNA processing factor Swd2.2, a component of the Cleavage and Polyadenylation Factor (CPF), is a negative regulator of condensin-mediated chromosome condensation. Lack of Swd2.2 does notaffect the assembly of the CPF but reduces its association with chromatin. This causes only limited, context-dependent effects on gene expression and transcription termination. However, CPF-associated Swd2.2 is required for the association of Protein Phosphatase 1 PP1(Dis2) with chromatin, through an interaction with Ppn1, a protein that we identify as the fission yeast homologueof vertebrate PNUTS. We demonstrate that Swd2.2, Ppn1 and PP1Dis2 form an independent module within the CPF, which provides an essential function in the absence of the CPF-associated Ssu72 phosphatase. We show that Ppn1 and Ssu72, like Swd2.2, are also negative regulators of condensin-mediated chromosome condensation. We conclude that Swd2.2 opposes condensin-mediated chromosome condensation by facilitating the function of the two CPF-associated phosphatasesPP1 and Ssu72.

Differential spatial and structural organization of the X chromosome underlies dosage compensation in C. elegans.

Author(s) : Sharma R, Jost D, Kind J, Gomez-Saldivar G, van Steensel B, Askjaer P, Vaillant C, Meister P,
Journal : Genes Dev
2014
The adjustment of X-linked gene expression to the X chromosome copy number (dosage compensation [DC]) has been widely studied as a model of chromosome-widegene regulation. In Caenorhabditis elegans, DC is achieved by twofold down-regulation of gene expression from both Xs in hermaphrodites. We show that in males, the single X chromosome interacts with nuclear pore proteins, while inhermaphrodites, the DC complex (DCC) impairs this interaction and alters X localization. Our results put forward a structural model of DC in which X-specific sequences locate the X chromosome in transcriptionally active domainsin males, while the DCC prevents this in hermaphrodites.

Disturbance of endoplasmic reticulum proteostasis in neurodegenerative diseases.

Author(s) : Hetz C, Mollereau B,
Journal : Nat Rev Neurosci
2014
The unfolded protein response (UPR) is a homeostatic mechanism by which cells regulate levels of misfolded proteins in the endoplasmic reticulum (ER). Although it is well characterized in non-neuronal cells, a proliferation of papers over the past few years has revealed a key role for the UPR in normal neuronal function and as an important driver of neurodegenerative diseases. A complex scenario is emerging in which distinct UPR signalling modules have specific and even opposite effects on neurodegeneration depending on the disease context. Here, we provide an overview of the most recent findings addressing the biological relevance of ER stress in the nervous system.

Drosophila protamine-like Mst35Ba and Mst35Bb are required for proper sperm nuclear morphology but are dispensable for male fertility.

Author(s) : Tirmarche S, Kimura S, Sapey-Triomphe L, Sullivan W, Landmann F, Loppin B,
Journal : G3 (Bethesda)
2014
During spermiogenesis, histones are massively replaced with protamines. A previous report showed that Drosophila males homozygous for a genomic deletion covering several genes including the protamine-like genes Mst35Ba/b are surprisingly fertile. Here, we have precisely deleted the Mst35B locus by homologous recombination, and we confirm the dispensability of Mst35Ba/b for fertility.

Endothelial, epithelial, and fibroblast cells exhibit specific splicing programs independently of their tissue of origin

Author(s) : Mallinjoud P, Villemin J, Mortada H, Polay Espinoza M, Desmet F, Samaan S, Chautard E, Tranchevent L, Auboeuf D,
Journal : Genome Res
2014

Endothelial, epithelial, and fibroblast cells exhibit specific splicing programs independently of their tissue of origin.

Author(s) : Mallinjoud P, Villemin J, Mortada H, Polay Espinoza M, Desmet F, Samaan S, Chautard E, Tranchevent L, Auboeuf D,
Journal : Genome Res
2014
Alternative splicing is the main mechanism of increasing the proteome diversity coded by a limited number of genes. It is well established that different tissues or organs express different splicing variants. However, organs are composed of common major cell types, including fibroblasts, epithelial, and endothelial cells. By analyzing large-scale data sets generated by The ENCODE Project Consortium and after extensive RT-PCR validation, we demonstrate that each of the three major cell types expresses a specific splicing program independently of its organ origin. Furthermore, by analyzing splicing factor expression across samples, publicly available splicing factor binding site data sets (CLIP-seq), and exon array data sets after splicing factor depletion, we identified several splicing factors, including ESRP1 and 2, MBNL1, NOVA1, PTBP1, and RBFOX2, that contribute to establishing these cell type-specific splicing programs. All of the analyzed data sets are freely available in a user-friendly web interface named FasterDB, which describes all known splicing variants of human and mouse genes and their splicing patterns across several dozens of normal and cancer cells as well as across tissues. Information regarding splicing factors that potentially contribute to individual exon regulation is also provided via a dedicated CLIP-seq and exon array data visualization interface. To the best of our knowledge, FasterDB is the first database integrating such a variety of large-scale data sets to enable functional genomics analyses at exon-level resolution.

Getting the better of ER stress.

Author(s) : Mollereau B, Manie S, Napoletano F,
Journal : J Cell Commun Signal
2014
Research over the past few years has highlighted the ability of the unfolded protein response (UPR) to minimize the deleterious effects of accumulated misfolded proteins under both physiological and pathological conditions. The endoplasmic reticulum (ER) adapts to endogenous and exogenous stressors by expanding its protein-folding capacity and by stimulating protective processes such as autophagy and antioxidant responses. Although it is clear that severe ERstress can elicit cell death, several recent studies have shown that low levels of ER stress may actually be beneficial to cells by eliciting an adaptive UPR that 'preconditions' the cell to a subsequent lethal insult; this process is called ER hormesis. The findings have important implications for the treatment of a wide variety of diseases associated with defective proteostasis, including neurodegenerative diseases, diabetes, and cancer. Here, we review the physiological and pathological functions of the ER, with a particular focus on the molecular mechanisms that lead to ER hormesis and cellular protection, and discuss the implications for disease treatment.

HIV-2 genomic RNA accumulates in stress granules in the absence of active translation.

Author(s) : Soto-Rifo R, Valiente-Echeverria F, Rubilar P, Garcia-de-Gracia F, Ricci E, Limousin T, Decimo D, Mouland A, Ohlmann T,
Journal : Nucleic Acids Res
2014
During the post-transcriptional events of the HIV-2 replication cycle, the full-length unspliced genomic RNA (gRNA) is first used as an mRNA to synthesize Gag and Gag-Pol proteins and then packaged into progeny virions. However, the mechanisms responsible for the coordinate usage of the gRNA during these two mutually exclusive events are poorly understood. Here, we present evidence showing that HIV-2 expression induces stress granule assembly in cultured cells.This contrasts with HIV-1, which interferes with stress granules assembly even upon induced cellular stress. Moreover, we observed that the RNA-binding proteinand stress granules assembly factor TIAR associates with the gRNA to form a TIAR-HIV-2 ribonucleoprotein (TH2RNP) complex localizing diffuse in the cytoplasm or aggregated in stress granules. Although the assembly of TH2RNP in stress granules did not require the binding of the Gag protein to the gRNA, we observedthat increased levels of Gag promoted both translational arrest and stress granule assembly. Moreover, HIV-2 Gag also localizes to stress granules in the absence of a 'packageable' gRNA. Our results indicate that the HIV-2 gRNA is compartmentalized in stress granules in the absence of active translation prior to being selected for packaging by the Gag polyprotein.

How does evolution tune biological noise?

Author(s) : Richard M, Yvert G,
Journal : Front Genet
2014
Part of molecular and phenotypic differences between individual cells, between body parts, or between individuals can result from biological noise. This sourceof variation is becoming more and more apparent thanks to the recent advances indynamic imaging and single-cell analysis. Some of these studies showed that the link between genotype and phenotype is not strictly deterministic. Mutations canchange various statistical properties of a biochemical reaction, and thereby theprobability of a trait outcome. The fact that they can modulate phenotypic noisebrings up an intriguing question: how may selection act on these mutations? In this review, we approach this question by first covering the evidence that biological noise is under genetic control and therefore a substrate for evolution. We then sequentially inspect the possibilities of negative, neutral, and positive selection for mutations increasing biological noise. Finally, we hypothesize on the specific case of H2A.Z, which was shown to both buffer phenotypic noise and modulate transcriptional efficiency.

How mRNA is misspliced in acute myelogenous leukemia (AML)?

Author(s) : Mohamed A, Th?noz M, Solly F, Balsat M, Mortreux F, Wattel E,
Journal : Oncotarget
2014

How mRNA is misspliced in acute myelogenous leukemia (AML)?

Author(s) : Mohamed A, Thenoz M, Solly F, Balsat M, Mortreux F, Wattel E,
Journal : Oncotarget
2014
Approximately one-third of expressed genes are misspliced in AML, opening the possibility that additional factors than splicing factor mutations might cause RNA missplicing in these diseases. AML cells harbor a constellation of epigenetic modifications and regularly express large amounts of WT1 transcripts. Histone acetylation/methylation and DNA CpG methylation favor either exon skipping or inclusion, mainly through interfering with RNA Pol II-mediated elongation. This can result either from the binding of various factors on Pol II or alternativelyfrom the recruitment of DNA binding factors that create roadblocks to Pol II-induced elongation. WT1 exhibits pleiotropic effects on mRNA splicing, which mainly result from the binding properties of WT1 via its zinc fingers domains toDNA, RNA, and proteins. Through the repression of the kinase SRPK1, WT1 modifiesthe splicing of VEGF, which plays important roles in hematopoiesis and angiogenesis. At the protein level, WT1 interacts with the splicing factors U2AF2, WTAP, and RPM4. Therefore, AML cells appear to have acquired numerous properties known to interfere with mRNA splicing. The challenge is now to elucidate these links in order to trigger mRNA splicing at the therapeutic level.

HTLV-1 bZIP factor HBZ promotes cell proliferation and genetic instability by activating OncomiRs

Author(s) : Vernin C, Thenoz M, Pinatel C, Gessain A, Gout O, Delfau-Larue M, Nazaret N, Legras-Lachuer C, Wattel E, Mortreux F,
Journal : Cancer Res
2014

HTLV-1-infected CD4+ T-cells display alternative exon usages that culminate in adult T-cell leukemia.

Author(s) : Thenoz M, Vernin C, Mortada H, Karam M, Pinatel C, Gessain A, Webb T, Auboeuf D, Wattel E, Mortreux F,
Journal : Retrovirology
2014
BACKGROUND: Reprogramming cellular gene transcription sustains HTLV-1 viral persistence that ultimately leads to the development of adult T-cell leukemia/lymphoma (ATLL). We hypothesized that besides these quantitative transcriptional effects, HTLV-1 qualitatively modifies the pattern of cellular gene expression. RESULTS: Exon expression analysis shows that patients' untransformed and malignant HTLV-1(+) CD4(+) T-cells exhibit multiple alternate exon usage (AEU) events. These affect either transcriptionally modified or unmodified genes, culminate in ATLL, and unveil new functional pathways involvedin cancer and cell cycle. Unsupervised hierarchical clustering of array data permitted to isolate exon expression patterns of 3977 exons that discriminate uninfected, infected, and transformed CD4(+) T-cells. Furthermore, untransformedinfected CD4+ clones and ATLL samples shared 486 exon modifications distributed in 320 genes, thereby indicating a role of AEUs in HTLV-1 leukemogenesis. Exposing cells to splicing modulators revealed that Sudemycin E reduces cell viability of HTLV-1 transformed cells without affecting primary control CD4+ cells and HTLV-1 negative cell lines, suggesting that the huge excess of AEU might provide news targets for treating ATLL. CONCLUSIONS: Taken together, thesedata reveal that HTLV-1 significantly modifies the structure of cellular transcripts and unmask new putative leukemogenic pathways and possible therapeutic targets.

HTLV-1-infected CD4+ T-cells display alternative exon usages that culminate in adult T-cell leukemia

Author(s) : Th?noz M, Vernin C, Mortada H, Karam M, Pinatel C, Gessain A, Webb T, Auboeuf D, Wattel E, Mortreux F,
Journal : Retrovirology
2014

Human T-cell leukemia virus type 3 (HTLV-3) and HTLV-4 antisense-transcript-encoded proteins interact and transactivate Jun family-dependent transcription via their atypical bZIP motif.

Author(s) : Larocque E, Andre-Arpin C, Borowiak M, Lemay G, Switzer W, Duc Dodon M, Mesnard J, Barbeau B,
Journal : J Virol
2014
Human T-cell leukemia virus types 3 and 4 (HTLV-3 and HTLV-4) are recently isolated retroviruses. We have previously characterized HTLV-3- and HTLV-4-encoded antisense genes, termed APH-3 and APH-4, respectively, which, in contrast to HBZ, the HTLV-1 homologue, do not contain a typical bZIP domain (M. Larocque E Halin, S. Landry, S. J. Marriott, W. M. Switzer, and B. Barbeau, J. Virol. 85:12673-12685, 2011, doi:10.1128/JVI.05296-11). As HBZ differentially modulates the transactivation potential of various Jun family members, the effect of APH-3 and APH-4 on JunD-, c-Jun-, and JunB-mediated transcriptional activation was investigated. We first showed that APH-3 and APH-4 upregulated the transactivation potential of all tested Jun family members. Using an human telomerase catalytic subunit (hTERT) promoter construct, our results also highlighted that, unlike HBZ, which solely modulates hTERT expression via JunD, both APH-3 and APH-4 acted positively on the transactivation of the hTERT promoter mediated by tested Jun factors. Coimmunoprecipitation experiments demonstrated that these Jun proteins interacted with APH-3 and APH-4. Although no activation domain was identified for APH proteins, the activation domain of c-Jun was very important in the observed upregulation of its activation potential. We further showed that APH-3 and APH-4 required their putative bZIP-like domains and corresponding leucine residues for interaction and modulation of the transactivation potential of Jun factors. Our results demonstrate that HTLV-encoded antisense proteins behave differently, and that the bZIP-like domains of both APH-3 and APH-4 have retained their interaction potential for Jun members. These studies are important in assessing the differences between HBZ and other antisense proteins, which might further contribute to determining the roleof HBZ in HTLV-1-associated diseases. IMPORTANCE HBZ, the antisense transcript-encoded protein from HTLV-1, is now well recognized as a potential factor for adult T-cell leukemia/lymphoma development. In order to better appreciate the mechanism of action of HBZ, comparison to antisense proteins fromother HTLV viruses is important. Little is known in relation to the seemingly nonpathogenic HTLV-3 and HTLV-4 viruses, and studies of their antisense proteinsare limited to our previously reported study (M. Larocque E Halin, S. Landry, S.J. Marriott, W. M. Switzer, and B. Barbeau, J. Virol. 85:12673-12685, 2011, doi:10.1128/JVI.05296-11). Here, we demonstrate that Jun transcription factors are differently affected by APH-3 and APH-4 compared to HBZ. These intriguing findings suggest that these proteins act differently on viral replication but also on cellular gene expression, and that highlighting their differences of action might lead to important information allowing us to understand the link between HTLV-1 HBZ and ATL in infected individuals.

Immunological alterations and associated diseases in mandrills (Mandrillus sphinx) naturally co-infected with SIV and STLV.

Author(s) : Souquiere S, Makuwa M, Salle B, Lepelletier Y, Mortreux F, Hermine O, Kazanji M,
Journal : Virology
2014
Mandrills are naturally infected with simian T-cell leukaemia virus type 1 (STLV-1) and simian immunodeficiency virus (SIV)mnd. In humans, dual infection with human immunodeficiency virus (HIV) and human T-cell lymphotropic virus type1 (HTLV-1) may worsen their clinical outcome. We evaluated the effect of co-infection in mandrills on viral burden, changes in T-cell subsets and clinical outcome. The SIV viral load was higher in SIV-infected mandrills than in co-infected animals, whereas the STLV-1 proviral load was higher in co-infected than in mono-infected groups. Dually infected mandrills had a statistically significantly lower CD4+ T-cell count, a lower proportion of naive CD8+ T cells and a higher proportion of central memory cells. CD4(+) and CD8(+) T cells from SIV-infected animals had a lower percentage of Ki67 than those from the other groups. Co-infected monkeys had higher percentages of activated CD4(+) and CD8(+) T cells. Two co-infected mandrills with high immune activation and clonal integration of STLV provirus showed pathological manifestations (infective dermatitis and generalised scabies) rarely encountered in nonhuman primates.

2013

A long noncoding RNA mediates both activation and repression of immune response genes.

Author(s) : Carpenter S, Aiello D, Atianand M, Ricci E, Gandhi P, Hall L, Byron M, Monks B, Henry-Bezy M, Lawrence J, O'Neill L, Moore M, Caffrey D, Fitzgerald K,
Journal : Science
2013
An inducible program of inflammatory gene expression is central to antimicrobialdefenses. This response is controlled by a collaboration involving signal-dependent activation of transcription factors, transcriptional co-regulators, and chromatin-modifying factors. We have identified a long noncoding RNA (lncRNA) that acts as a key regulator of this inflammatory response. Pattern recognition receptors such as the Toll-like receptors induce the expression of numerous lncRNAs. One of these, lincRNA-Cox2, mediates both the activation and repression of distinct classes of immune genes. Transcriptional repression of target genes is dependent on interactions of lincRNA-Cox2 with heterogeneous nuclear ribonucleoprotein A/B and A2/B1. Collectively, these studies unveil a central role of lincRNA-Cox2 as a broad-acting regulatory component of the circuit that controls the inflammatory response.

Absolute requirement of cholesterol binding for Hedgehog gradient formation in Drosophila.

Author(s) : Ducuing A, Mollereau B, Axelrod J, Vincent S,
Journal : Biol Open
2013
How morphogen gradients are shaped is a major question in developmental biology,but remains poorly understood. Hedgehog (Hh) is a locally secreted ligand that reaches cells at a distance and acts as a morphogen to pattern the Drosophila wing and the vertebrate neural tube. The proper patterning of both structures relies on the precise control over the slope of Hh activity gradient. A number of hypotheses have been proposed to explain Hh movement and hence graded activity of Hh. A crux to all these models is that the covalent binding of cholesterol to HhN-terminus is essential to achieve the correct slope of the activity gradient. Still, the behavior of cholesterol-free Hh (Hh-N) remains controversial: cholesterol has been shown to either increase or restrict Hh range depending on the experimental setting. Here, in fly embryos and wing imaginal discs, we show that cholesterol-free Hh diffuses at a long-range. This unrestricted diffusion of cholesterol-free Hh leads to an absence of gradient while Hh signaling strength remains uncompromised. These data support a model where cholesterol addition restricts Hh diffusion and can transform a leveled signaling activity into a gradient. In addition, our data indicate that the receptor Patched is not able to sequester cholesterol-free Hh. We propose that a morphogen gradient does not necessarily stem from the active transfer of a poorly diffusing molecule, but can be achieved by the restriction of a highly diffusible ligand.

Antiretroviral therapy promotes an inflammatory-like pattern of human T-cell lymphotropic virus type 1 (HTLV-1) replication in human immunodeficiency virus type 1/HTLV-1 co-infected individuals.

Author(s) : Pomier C, Rabaaoui S, Pouliquen J, Couppie P, El Guedj M, Nacher M, Lacoste V, Wattel E, Kazanji M, Mortreux F,
Journal : J Gen Virol
2013
Upon antiretroviral therapy (ART) human immunodeficiency virus (HIV)/human T-cell lymphotropic virus type 1 (HTLV-1) co-infected individuals frequently develop neurological disorders through hitherto unknown mechanisms. Here, we show that effective anti-HIV ART increases HTLV-1 proviral load through a polyclonal integration pattern of HTLV-1 in both CD4(+) and CD8(+) T-cell subsets that is reminiscent of that typically associated with HTLV-1-related inflammatory conditions. These data indicate that preventing ART-triggered clonal expansion of HTLV-1-infected cells in co-infected individuals deserves investigation.

Antiretroviral therapy promotes an inflammatory-like pattern of human T-cell lymphotropic virus type 1 (HTLV-1) replication in human immunodeficiency virus type 1/HTLV-1 co-infected individuals

Author(s) : Pomier C, Rabaaoui S, Pouliquen J, Couppi? P, El Guedj M, Nacher M, Lacoste V, Wattel E, Kazanji M, Mortreux F,
Journal : J Gen Virol
2013

Biophysical and genetic analysis of iron partitioning and ferritin function in Drosophila melanogaster.

Author(s) : Gutierrez L, Zubow K, Nield J, Gambis A, Mollereau B, Lazaro F, Missirlis F,
Journal : Metallomics
2013
Metals have vital functions as prosthetic groups in enzymes, but in labile form they can propagate oxidative stress. The primary function of ferritin is to store bioavailable iron in the form of ferrihydrite. In animals, ferritin is also usedto traffic and recycle iron, and to modulate intestinal iron absorption. However, the effect of ferritin accumulation on cellular iron bioavailability remains poorly understood. Moreover, putative in vivo interactions of ferritin with other metal ions have been proposed, but their physiological relevance remains unclear. Here, heterozygous mutant and overexpression ferritin strains of Drosophila melanogaster were subjected to dietary iron manipulations to study the dynamics of iron partition between ferritin and other proteins. Quantitative magnetic analysis of whole fly samples indicated that iron loading of the ferritin core varied in the different genotypes. Total paramagnetic iron content, a likely correlate of bioavailable iron, was reduced in flies overexpressing ferritin when compared with control white flies. Further, three-dimensional maps of the ferritin protein shell and iron core were obtained from single particle transmission electron microscopy imaging and confirmed the similarity between Drosophila and Trichoplusia ferritin structures. Purified Drosophila ferritin also contained small amounts of zinc and manganese. Flies that overexpressed ferritin accumulated in their bodies half the amount of manganese compared to their respective controls. Our results indicate that ferritin may be involved inthe homeostasis of other divalent metals, besides iron, and that overexpression of ferritin, sometimes employed to rescue neurodegenerative models of disease, serves to limit divalent metal bio-availability in cells.

Calcineurin A versus NS5A-TP2/HD domain containing 2: a case study of site-directed low-frequency random mutagenesis for dissecting target specificity of peptide aptamers.

Author(s) : Dibenedetto S, Cluet D, Stebe P, Baumle V, Leault J, Terreux R, Bickle M, Chassey B, Mikaelian I, Colas P, Spichty M, Zoli M, Rudkin B,
Journal : Mol Cell Proteomics
2013
We previously identified a peptide aptamer (named R5G42) via functional selection for its capacity to slow cell proliferation. A yeast two-hybrid screen of human cDNA libraries, using R5G42 as "bait," allowed the identification of two bindingproteins with very different functions: calcineurin A (CnA) (PP2B/PPP3CA), a protein phosphatase well characterized for its role in the immune response, and NS5A-TP2/HD domain containing 2, a much less studied protein induced subsequent to hepatitis C virus non-structural protein 5A expression in HepG2 hepatocellular carcinoma cells, with no known activity. Our objective in the present study was to dissect the dual target specificity of R5G42 in order to have tools with which to better characterize the actions of the peptide aptamers toward their individual targets. This was achieved through the selection of random mutants ofthe variable loop, derived from R5G42, evaluating their specificity toward CnA and NS5A-TP2 and analyzing their sequence. An interdisciplinary approach involving biomolecular computer simulations with integration of the sequence data and yeast two-hybrid binding phenotypes of these mutants yielded two structurally distinct conformers affording the potential molecular basis of the binding diversity of R5G42. Evaluation of the biological impact of CnA- versus NS5A-TP2-specific peptide aptamers indicated that although both contributed to the anti-proliferative effect of R5G42, CnA-binding was essential to stimulate the nuclear translocation of nuclear factor of activated T cells, indicative of the activation of endogenous CnA. By dissecting the target specificity of R5G42,we have generated novel tools with which to study each target individually. Apta-C8 is capable of directly activating CnA independent of binding to NS5A-TP2and will be an important tool in studying the role of CnA activation in the regulation of different signaling pathways, whereas Apta-E1 will allow dissection of the function of NS5A-TP2, serving as an example of the usefulness of peptide aptamer technology for investigating signaling pathways.

Calcineurin A versus NS5A-TP2/HD domain containing 2: a case study of site-directed low-frequency random mutagenesis for dissecting target specificity of peptide aptamers.

Author(s) : Dibenedetto S, Cluet D, Stebe P, Baumle V, Leault J, Terreux R, Bickle M, Chassey B, Mikaelian I, Colas P, Spichty M, Zoli M, Rudkin B,
Journal : Mol Cell Proteomics
2013
We previously identified a peptide aptamer (named R5G42) via functional selection for its capacity to slow cell proliferation. A yeast two-hybrid screen of human cDNA libraries, using R5G42 as "bait," allowed the identification of two bindingproteins with very different functions: calcineurin A (CnA) (PP2B/PPP3CA), a protein phosphatase well characterized for its role in the immune response, and NS5A-TP2/HD domain containing 2, a much less studied protein induced subsequent to hepatitis C virus non-structural protein 5A expression in HepG2 hepatocellular carcinoma cells, with no known activity. Our objective in the present study was to dissect the dual target specificity of R5G42 in order to have tools with which to better characterize the actions of the peptide aptamers toward their individual targets. This was achieved through the selection of random mutants ofthe variable loop, derived from R5G42, evaluating their specificity toward CnA and NS5A-TP2 and analyzing their sequence. An interdisciplinary approach involving biomolecular computer simulations with integration of the sequence data and yeast two-hybrid binding phenotypes of these mutants yielded two structurally distinct conformers affording the potential molecular basis of the binding diversity of R5G42. Evaluation of the biological impact of CnA- versus NS5A-TP2-specific peptide aptamers indicated that although both contributed to the anti-proliferative effect of R5G42, CnA-binding was essential to stimulate the nuclear translocation of nuclear factor of activated T cells, indicative of the activation of endogenous CnA. By dissecting the target specificity of R5G42,we have generated novel tools with which to study each target individually. Apta-C8 is capable of directly activating CnA independent of binding to NS5A-TP2and will be an important tool in studying the role of CnA activation in the regulation of different signaling pathways, whereas Apta-E1 will allow dissection of the function of NS5A-TP2, serving as an example of the usefulness of peptide aptamer technology for investigating signaling pathways.

Cause-specific telomere factors deregulation in hepatocellular carcinoma

Author(s) : El Idrissi M, Hervieu V, Merle P, Mortreux F, Wattel E,
Journal : J Exp Clin Cancer Res
2013

Deep sequencing reveals abundant noncanonical retroviral microRNAs in B-cell leukemia/lymphoma

Author(s) : Rosewick N, Momont M, Durkin K, Takeda H, Caiment F, Cleuter Y, Vernin C, Mortreux F, Wattel E, Burny A, Georges M, Van den Broeke A,
Journal : Proc Natl Acad Sci U S A
2013

Differential miRNA expression profiles in proliferating or differentiated keratinocytes in response to gamma irradiation.

Author(s) : Joly-Tonetti N, Vinuelas J, Gandrillon O, Lamartine J,
Journal : BMC Genomics
2013
BACKGROUND: MicroRNAs (miRNAs), a group of short non-coding RNAs that negativelyregulate gene expression, have recently emerged as potential modulators of cellular response to ionizing radiations both in vitro and in vivo in various cell types and tissues. However, in epidermal cells, the involvement of the miRNA machinery in the cellular response to ionizing radiations remains to be clarified. Indeed, understanding the mechanisms of cutaneous radiosensitivity isan important issue since skin is the most exposed organ to ionizing radiations and among the most sensitive. RESULTS: We settled up an expression study of miRNAs in primary human skin keratinocytes using a microfluidic system of qPCR assay, which permits to assess the expression of almost 700 annotated miRNAs. The keratinocytes were cultured to a proliferative or a differentiated state mimicking basal or suprabasal layers of human epidermis. These cells were irradiated at 10 mGy or 6 Gy and RNA was extracted 3 hours after irradiation. Wefound that proliferative cells irradiated at 6 Gy display a global fall of miRNAexpression whereas differentiated cells exposed to the same dose display a global increase of miRNAs expression. We identified twenty miRNAs weakly but significantly modulated after 6 Gy irradiation, whereas only 2 miRNAs were modulated after low-dose irradiation in proliferating cells. To go further into the biological meaning of this miRNA response, we over-expressed some of the responding miRNA in proliferating cells: we observed a significant decrease of cell viability 72 hours after irradiation. Functional annotation of their predicted targets revealed that G-protein related pathways might be regulated bythese responding miRNAs. CONCLUSIONS: Our results reveal that human primary keratinocytes exposed to ionizing irradiation expressed a miRNA pattern stronglyrelated to the differentiation status of irradiated cells. We also demonstrate that some miRNAs play a role in the radiation response to ensure the short-term survival of irradiated keratinocytes.

Drosophila p53 isoforms differentially regulate apoptosis and apoptosis-induced proliferation.

Author(s) : Dichtel-Danjoy M, Ma D, Dourlen P, Chatelain G, Napoletano F, Robin M, Corbet M, Levet C, Hafsi H, Hainaut P, Ryoo H, Bourdon J, Mollereau B,
Journal : Cell Death Differ
2013
Irradiated or injured cells enter apoptosis, and in turn, promote proliferation of surrounding unaffected cells. In Drosophila, apoptotic cells have an active role in proliferation, where the caspase Dronc and p53 induce mitogen expressionand growth in the surrounding tissues. The Drosophila p53 gene structure is conserved and encodes at least two protein isoforms: a full-length isoform (Dp53) and an N-terminally truncated isoform (DDeltaNp53). Historically, DDeltaNp53 wasthe first p53 isoform identified and was thought to be responsible for all p53 biological activities. It was shown that DDeltaNp53 induces apoptosis by inducing the expression of IAP antagonists, such as Reaper. Here we investigated the roles of Dp53 and DDeltaNp53 in apoptosis and apoptosis-induced proliferation. We found that both isoforms were capable of activating apoptosis, but that they each induced distinct IAP antagonists. Expression of DDeltaNp53 induced Wingless (Wg)expression and enhanced proliferation in both 'undead cells' and in 'genuine' apoptotic cells. In contrast to DDeltaNp53, Dp53 did not induce Wg expression inthe absence of the endogenous p53 gene. Thus, we propose that DDeltaNp53 is the main isoform that regulates apoptosis-induced proliferation. Understanding the roles of Drosophila p53 isoforms in apoptosis and in apoptosis-induced proliferation may shed new light on the roles of p53 isoforms in humans, with important implications in cancer biology.

Drosophila Yemanuclein and HIRA cooperate for de novo assembly of H3.3-containing nucleosomes in the male pronucleus.

Author(s) : Orsi G, Algazeery A, Meyer R, Capri M, Sapey-Triomphe L, Horard B, Gruffat H, Couble P, Ait-Ahmed O, Loppin B,
Journal : PLoS Genet
2013
The differentiation of post-meiotic spermatids in animals is characterized by a unique reorganization of their nuclear architecture and chromatin composition. In many species, the formation of sperm nuclei involves the massive replacement of nucleosomes with protamines, followed by a phase of extreme nuclear compaction. At fertilization, the reconstitution of a nucleosome-based paternal chromatin after the removal of protamines requires the deposition of maternally provided histones before the first round of DNA replication. This process exclusively uses the histone H3 variant H3.3 and constitutes a unique case of genome-wide replication-independent (RI) de novo chromatin assembly. We had previously shownthat the histone H3.3 chaperone HIRA plays a central role for paternal chromatinassembly in Drosophila. Although several conserved HIRA-interacting proteins have been identified from yeast to human, their conservation in Drosophila, as well as their actual implication in this highly peculiar RI nucleosome assembly process,is an open question. Here, we show that Yemanuclein (YEM), the Drosophila memberof the Hpc2/Ubinuclein family, is essential for histone deposition in the male pronucleus. yem loss of function alleles affect male pronucleus formation in a way remarkably similar to Hira mutants and abolish RI paternal chromatin assembly. In addition, we demonstrate that HIRA and YEM proteins interact and are mutually dependent for their targeting to the decondensing male pronucleus. Finally, we show that the alternative ATRX/XNP-dependent H3.3 deposition pathwayis not involved in paternal chromatin assembly, thus underlining the specific implication of the HIRA/YEM complex for this essential step of zygote formation.

Endogenous nuclear RNAi mediates behavioral adaptation to odor.

Author(s) : Juang B, Gu C, Starnes L, Palladino F, Goga A, Kennedy S, L'Etoile N,
Journal : Cell
2013
Most eukaryotic cells express small regulatory RNAs. The purpose of one class, the somatic endogenous siRNAs (endo-siRNAs), remains unclear. Here, we show thatthe endo-siRNA pathway promotes odor adaptation in C. elegans AWC olfactory neurons. In adaptation, the nuclear Argonaute NRDE-3, which acts in AWC, is loaded with siRNAs targeting odr-1, a gene whose downregulation is required for adaptation. Concomitant with increased odr-1 siRNA in AWC, we observe increased binding of the HP1 homolog HPL-2 at the odr-1 locus in AWC and reduced odr-1 mRNA in adapted animals. Phosphorylation of HPL-2, an in vitro substrate of the EGL-4kinase that promotes adaption, is necessary and sufficient for behavioral adaptation. Thus, environmental stimulation amplifies an endo-siRNA negative feedback loop to dynamically repress cognate gene expression and shape behavior.This class of siRNA may act broadly as a rheostat allowing prolonged stimulationto dampen gene expression and promote cellular memory formation. PAPERFLICK:

Establishing links between endoplasmic reticulum-mediated hormesis and cancer.

Author(s) : Mollereau B,
Journal : Mol Cell Biol
2013

Evolutionary comparisons reveal a positional switch for spindle pole oscillations in Caenorhabditis embryos.

Author(s) : Riche S, Zouak M, Argoul F, Arneodo A, Pecreaux J, Delattre M,
Journal : J Cell Biol
2013
During the first embryonic division in Caenorhabditis elegans, the mitotic spindle is pulled toward the posterior pole of the cell and undergoes vigorous transverse oscillations. We identified variations in spindle trajectories by analyzing the outwardly similar one-cell stage embryo of its close relative Caenorhabditis briggsae. Compared with C. elegans, C. briggsae embryos exhibit an anterior shifting of nuclei in prophase and reduced anaphase spindle oscillations. By combining physical perturbations and mutant analysis in both species, we show that differences can be explained by interspecies changes in the regulation of the cortical Galpha-GPR-LIN-5 complex. However, we found that in both species (1) a conserved positional switch controls the onset of spindle oscillations, (2) GPR posterior localization may set this positional switch, and(3) the maximum amplitude of spindle oscillations is determined by the time spent in the oscillating phase. By investigating microevolution of a subcellular process, we identify new mechanisms that are instrumental to decipher spindle positioning.

Genome-wide analysis of thyroid hormone receptors shared and specific functions in neural cells.

Author(s) : Chatonnet F, Guyot R, Benoit G, Flamant F,
Journal : Proc Natl Acad Sci U S A
2013
TRalpha1 and TRbeta1, the two main thyroid hormone receptors in mammals, are transcription factors that share similar properties. However, their respective functions are very different. This functional divergence might be explained in two ways: it can reflect different expression patterns or result from different intrinsic properties of the receptors. We tested this second hypothesis by comparing the repertoires of 3,3',5-triiodo-L-thyronine (T3)-responsive genes oftwo neural cell lines, expressing either TRalpha1 or TRbeta1. Using transcriptome analysis, we found that a substantial fraction of the T3 target genes display a marked preference for one of the two receptors. So when placed alone in identical situations, the two receptors have different repertoires of target genes. Chromatin occupancy analysis, performed at a genome-wide scale, revealed that TRalpha1 and TRbeta1 cistromes were also different. However, receptor-selective regulation of T3 target genes did not result from receptor-selective chromatin occupancy of their promoter regions. We conclude that modification of TRalpha1 and TRbeta1 intrinsic properties contributes in a large part to the divergent evolution of the receptors' function, at least during neurodevelopment.

hCAF1/CNOT7 regulates interferon signalling by targeting STAT1

Author(s) : Chapat C, Kolytcheff C, Le Romancer M, Auboeuf D, De La Grange P, Chettab K, Sentis S, Corbo L,
Journal : EMBO J
2013

HTLV-1 bZIP factor impedes the menin tumor suppressor and upregulates JunD-mediated transcription of the hTERT gene.

Author(s) : Borowiak M, Kuhlmann A, Girard S, Gazzolo L, Mesnard J, Jalinot P, Dodon M,
Journal : Carcinogenesis
2013
Telomerase activity in cancer cells is dependent on the transcriptional regulation of the human telomerase reverse transcriptase (hTERT) gene, encoding the catalytic subunit of human telomerase. We have shown previously that HTLV-1 basic leucine zipper (HBZ), a viral regulatory protein encoded by the human retrovirus, human T-cell leukemia virus, type 1 (HTLV-1) cooperates with JunD toenhance hTERT transcription in adult T-cell leukemia (ATL) cells. Menin, the product of the tumor-suppressor MEN-1 gene, also interacts with JunD, represses its transcriptional activity and downregulates telomerase expression. The main objective of this study was to examine how menin and HBZ get involved in the regulation of hTERT transcription. In this study, we report that JunD and menin form a repressor complex of hTERT transcription in HBZ-negative cells. Conversely, in HBZ-positive cells, the formation of a JunD/HBZ/menin ternary complex and the recruitment of p300 histone acetyl transferase activity by HBZ lead to a decreased activity of the JunD-menin suppressor unit that correlates with the activation of hTERT transcription. Silencing HBZ or menin expression inATL cells confirms that these proteins are differentially involved in telomeraseregulation. These results propose that HBZ, by impeding the tumor-suppressor activity of menin, functions as a leukemogenic cofactor to upregulate gene transcription and promote JunD-mediated leukemogenesis.

HTLV-1-associated inflammatory myopathies: low proviral load and moderate inflammation in 13 patients from West Indies and West Africa

Author(s) : Desdouits M, Cassar O, Maisonobe T, Desrames A, Aouba A, Hermine O, Mikol J, Polivka M, Penisson-Besnier I, Marcorelles P, Zagnoli F, Papo T, Lacour A, Amoura Z, Haroche J, Cherin P, Teixeira A, Benveniste O, Herson S, Morin A, Mortreux F, Wattel E, Huerre M, Cumont M, Martin-Latil S, Butler-Browne G, Gout O, Taylor G, Gessain A, Ozden S, Ceccaldi P,
Journal : J Clin Virol
2013

miRNA repression of translation in vitro takes place during 43S ribosomal scanning.

Author(s) : Ricci E, Limousin T, Soto-Rifo R, Rubilar P, Decimo D, Ohlmann T,
Journal : Nucleic Acids Res
2013
microRNAs (miRNAs) regulate gene expression at multiple levels by repressing translation, stimulating deadenylation and inducing the premature decay of target messenger RNAs (mRNAs). Although the mechanism by which miRNAs repress translation has been widely studied, the precise step targeted and the molecularinsights of such repression are still evasive. Here, we have used our newly designed in vitro system, which allows to study miRNA effect on translation independently of deadenylation. By using specific inhibitors of various stages of protein synthesis, we first show that miRNAs target exclusively the early steps of translation with no effect on 60S ribosomal subunit joining, elongation or termination. Then, by using viral proteases and IRES-driven mRNA constructs, we found that translational inhibition takes place during 43S ribosomal scanning and requires both the poly(A) binding protein and eIF4G independently from their physical interaction.

Mosaicism of HTLV-1 5' LTR CpG methylation in the absence of malignancy

Author(s) : Sibon D, Zane L, Idrissi M, Delfau-Larue M, Gessain A, Gout O, Mortreux F, Wattel E,
Journal : Virus Res
2013

Natural sequence variants of yeast environmental sensors confer cell-to-cell expression variability.

Author(s) : Fehrmann S, Bottin-Duplus H, Leonidou A, Mollereau E, Barthelaix A, Wei W, Steinmetz L, Yvert G,
Journal : Mol Syst Biol
2013
Living systems may have evolved probabilistic bet hedging strategies that generate cell-to-cell phenotypic diversity in anticipation of environmental catastrophes, as opposed to adaptation via a deterministic response to environmental changes. Evolution of bet hedging assumes that genotypes segregating in natural populations modulate the level of intraclonal diversity, which so far has largely remained hypothetical. Using a fluorescent P(met17)-GFPreporter, we mapped four genetic loci conferring to a wild yeast strain an elevated cell-to-cell variability in the expression of MET17, a gene regulated by the methionine pathway. A frameshift mutation in the Erc1p transmembrane transporter, probably resulting from a release of laboratory strains from negative selection, reduced P(met17)-GFP expression variability. At a second locus, cis-regulatory polymorphisms increased mean expression of the Mup1p methionine permease, causing increased expression variability in trans. These results demonstrate that an expression quantitative trait locus (eQTL) can simultaneously have a deterministic effect in cis and a probabilistic effect in trans. Our observations indicate that the evolution of transmembrane transportergenes can tune intraclonal variation and may therefore be implicated in both reactive and anticipatory strategies of adaptation.

Quantifying the contribution of chromatin dynamics to stochastic gene expression reveals long, locus-dependent periods between transcriptional bursts.

Author(s) : Vinuelas J, Kaneko G, Coulon A, Vallin E, Morin V, Mejia-Pous C, Kupiec J, Beslon G, Gandrillon O,
Journal : BMC Biol
2013
BACKGROUND: A number of studies have established that stochasticity in gene expression may play an important role in many biological phenomena. This therefore calls for further investigations to identify the molecular mechanisms at stake, in order to understand and manipulate cell-to-cell variability. In this work, we explored the role played by chromatin dynamics in the regulation of stochastic gene expression in higher eukaryotic cells. RESULTS: For this purpose, we generated isogenic chicken-cell populations expressing a fluorescent reporterintegrated in one copy per clone. Although the clones differed only in the genetic locus at which the reporter was inserted, they showed markedly differentfluorescence distributions, revealing different levels of stochastic gene expression. Use of chromatin-modifying agents showed that direct manipulation ofchromatin dynamics had a marked effect on the extent of stochastic gene expression. To better understand the molecular mechanism involved in these phenomena, we fitted these data to a two-state model describing the opening/closing process of the chromatin. We found that the differences between clones seemed to be due mainly to the duration of the closed state, and that theagents we used mainly seem to act on the opening probability. CONCLUSIONS: In this study, we report biological experiments combined with computational modeling, highlighting the importance of chromatin dynamics in stochastic gene expression. This work sheds a new light on the mechanisms of gene expression in higher eukaryotic cells, and argues in favor of relatively slow dynamics with long (hours to days) periods of quiet state.

Single-cell phenomics reveals intra-species variation of phenotypic noise in yeast.

Author(s) : Yvert G, Ohnuki S, Nogami S, Imanaga Y, Fehrmann S, Schacherer J, Ohya Y,
Journal : BMC Syst Biol
2013
BACKGROUND: Most quantitative measures of phenotypic traits represent macroscopic contributions of large numbers of cells. Yet, cells of a tissue do not behave similarly, and molecular studies on several organisms have shown that regulations can be highly stochastic, sometimes generating diversified cellular phenotypes within tissues. Phenotypic noise, defined here as trait variability among isogenic cells of the same type and sharing a common environment, has therefore received a lot of attention. Given the potential fitness advantage provided by phenotypic noise in fluctuating environments, the possibility that it is directly subjected to evolutionary selection is being considered. For selection to act, phenotypic noise must differ between contemporary genotypes. Whether this is thecase or not remains, however, unclear because phenotypic noise has very rarely been quantified in natural populations. RESULTS: Using automated image analysis,we describe here the phenotypic diversity of S. cerevisiae morphology at single-cell resolution. We profiled hundreds of quantitative traits in more than1,000 cells of 37 natural strains, which represent various geographical and ecological origins of the species. We observed abundant trait variation between strains, with no correlation with their ecological origin or population history.Phenotypic noise strongly depended on the strain background. Noise variation waslargely trait-specific (specific strains showing elevated noise for subset of traits) but also global (a few strains displaying elevated noise for many unrelated traits). CONCLUSIONS: Our results demonstrate that phenotypic noise does differ quantitatively between natural populations. This supports the possibility that, if noise is adaptive, microevolution may tune it in the wild. This tuning may happen on specific traits or by varying the degree of global phenotypic buffering.

TAF15 is important for cellular proliferation and regulates the expression of a subset of cell cycle genes through miRNAs.

Author(s) : Ballarino M, Jobert L, Dembele D, de la Grange P, Auboeuf D, Tora L,
Journal : Oncogene
2013
TAF15 (formerly TAFII68) is a member of the FET (FUS, EWS, TAF15) family of RNA-and DNA-binding proteins whose genes are frequently translocated in sarcomas. Byperforming global gene expression profiling, we found that TAF15 knockdown affects the expression of a large subset of genes, of which a significant percentage is involved in cell cycle and cell death. In agreement, TAF15 depletion had a growth-inhibitory effect and resulted in increased apoptosis. Among the TAF15-regulated genes, targets of microRNAs (miRNAs) generated from the onco-miR-17 locus were overrepresented, with CDKN1A/p21 being the top miRNAs-targeted gene. Interestingly, the levels of onco-miR-17 locus coded miRNAs (miR-17-5p and miR-20a) were decreased upon TAF15 depletion and shown to affect the post-transcriptional regulation of TAF15-dependent genes, such as CDKN1A/p21. Thus, our results demonstrate that TAF15 is required to regulate gene expressionof cell cycle regulatory genes post-transcriptionally through a pathway involving miRNAs. The findings that high TAF15 levels are needed for rapid cellular proliferation and that endogenous TAF15 levels decrease during differentiation strongly suggest that TAF15 is a key regulator of maintaining a highly proliferative rate of cellular homeostasis.

TAF15 is important for cellular proliferation and regulates the expression of a subset of cell cycle genes through miRNAs

Author(s) : Ballarino M, Jobert L, Demb?l? D, de la Grange P, Auboeuf D, Tora L,
Journal : Oncogene
2013

The RNA helicase DDX5/p68 is a key factor promoting c-fos expression at different levels from transcription to mRNA export

Author(s) : Zonta E, Bittencourt D, Samaan S, Germann S, Dutertre M, Auboeuf D,
Journal : Nucleic Acids Res
2013

2012

[Mice are not Men and yet... how humanized mice inform us about human infectious diseases].

Author(s) : Cachat A, Villaudy J, Rigal D, Gazzolo L, Duc Dodon M,
Journal : Med Sci (Paris)
2012
The study of human pathologies is often limited by the absence of animal models which are robust, cost-effective and reproduce the hallmarks of human infections. While mice have been frequently employed to study human diseases, many of important pathogens display unique human tropism. These last two decades the graft of human progenitor cells or tissues into -immunodeficient mice has allowed the elaboration of so called humanized mice. Humanized mouse technology has maderapid progress, and it is now possible to achieve high levels of human chimerismin various organs and tissues, particularly the immune system and the liver. Thereview briefly summarizes the different models of humanized mice available for in vivo experiments. With a focus on lymphotropic, monocytotropic and hepatotropic viruses, we here discuss the current status and future prospects of these modelsfor studying the pathogenesis of infectious diseases. Furthermore, they provide a powerful tool for the development of innovative therapies.

A mathematical model to study the dynamics of epithelial cellular networks.

Author(s) : Abate A, Vincent S, Dobbe R, Silletti A, Master N, Axelrod J, Tomlin C,
Journal : IEEE/ACM Trans Comput Biol Bioinform
2012
Epithelia are sheets of connected cells that are essential across the animal kingdom. Experimental observations suggest that the dynamical behavior of many single-layered epithelial tissues has strong analogies with that of specific mechanical systems, namely large networks consisting of point masses connected through spring-damper elements and undergoing the influence of active and dissipating forces. Based on this analogy, this work develops a modeling framework to enable the study of the mechanical properties and of the dynamic behavior of large epithelial cellular networks. The model is built first by creating a network topology that is extracted from the actual cellular geometry as obtained from experiments, then by associating a mechanical structure and dynamics to the network via spring-damper elements. This scalable approach enables running simulations of large network dynamics: the derived modeling framework in particular is predisposed to be tailored to study general dynamics (for example, morphogenesis) of various classes of single-layered epithelial cellular networks. In this contribution, we test the model on a case study of the dorsal epithelium of the Drosophila melanogaster embryo during early dorsal closure (and, less conspicuously, germband retraction).

A unified phylogeny-based nomenclature for histone variants.

Author(s) : Talbert P, Ahmad K, Almouzni G, Ausio J, Berger F, Bhalla P, Bonner W, Cande W, Chadwick B, Chan S, Cross G, Cui L, Dimitrov S, Doenecke D, Eirin-Lopez J, Gorovsky M, Hake S, Hamkalo B, Holec S, Jacobsen S, Kamieniarz K, Khochbin S, Ladurner A, Landsman D, Latham J, Loppin B, Malik H, Marzluff W, Pehrson J, Postberg J, Schneider R, Singh M, Smith M, Thompson E, Torres-Padilla M, Tremethick D, Turner B, Waterborg J, Wollmann H, Yelagandula R, Zhu B, Henikoff S,
Journal : Epigenetics Chromatin
2012
Histone variants are non-allelic protein isoforms that play key roles in diversifying chromatin structure. The known number of such variants has greatly increased in recent years, but the lack of naming conventions for them has led to a variety of naming styles, multiple synonyms and misleading homographs that obscure variant relationships and complicate database searches. We propose here a unified nomenclature for variants of all five classes of histones that uses consistent but flexible naming conventions to produce names that are informativeand readily searchable. The nomenclature builds on historical usage and incorporates phylogenetic relationships, which are strong predictors of structure and function. A key feature is the consistent use of punctuation to represent phylogenetic divergence, making explicit the relationships among variant subtypes that have previously been implicit or unclear. We recommend that by default new histone variants be named with organism-specific paralog-number suffixes that lack phylogenetic implication, while letter suffixes be reserved for structurally distinct clades of variants. For clarity and searchability, we encourage the useof descriptors that are separate from the phylogeny-based variant name to indicate developmental and other properties of variants that may be independent of structure.

Alternative splicing and cancer

Author(s) : Auboeuf D, Carmo-Fonseca M, Valcarcel J, Biamonti G,
Journal : J Nucleic Acids
2012

Analysis of co-transcriptional RNA processing by RNA-ChIP assay

Author(s) : Bittencourt D, Auboeuf D,
Journal : Methods Mol Biol
2012

Chance at the heart of the cell.

Author(s) : Gandrillon O, Kolesnik-Antoine D, Kupiec J, Beslon G,
Journal : Prog Biophys Mol Biol
2012

Different effects of the TAR structure on HIV-1 and HIV-2 genomic RNA translation.

Author(s) : Soto-Rifo R, Limousin T, Rubilar P, Ricci E, Decimo D, Moncorge O, Trabaud M, Andre P, Cimarelli A, Ohlmann T,
Journal : Nucleic Acids Res
2012
The 5'-untranslated region (5'-UTR) of the genomic RNA of human immunodeficiencyviruses type-1 (HIV-1) and type-2 (HIV-2) is composed of highly structured RNA motifs essential for viral replication that are expected to interfere with Gag and Gag-Pol translation. Here, we have analyzed and compared the properties by which the viral 5'-UTR drives translation from the genomic RNA of both human immunodeficiency viruses. Our results showed that translation from the HIV-2 gRNA was very poor compared to that of HIV-1. This was rather due to the intrinsic structural motifs in their respective 5'-UTR without involvement of any viral protein. Further investigation pointed to a different role of TAR RNA, which wasmuch inhibitory for HIV-2 translation. Altogether, these data highlight important structural and functional differences between these two human pathogens.

Drosophila fatty acid transport protein regulates rhodopsin-1 metabolism and is required for photoreceptor neuron survival.

Author(s) : Dourlen P, Bertin B, Chatelain G, Robin M, Napoletano F, Roux M, Mollereau B,
Journal : PLoS Genet
2012
Tight regulation of the visual response is essential for photoreceptor function and survival. Visual response dysregulation often leads to photoreceptor cell degeneration, but the causes of such cell death are not well understood. In thisstudy, we investigated a fatty acid transport protein (fatp) null mutation that caused adult-onset and progressive photoreceptor cell death. Consistent with fatp having a role in the retina, we showed that fatp is expressed in adult photoreceptors and accessory cells and that its re-expression in photoreceptors rescued photoreceptor viability in fatp mutants. The visual response in young fatp-mutant flies was abnormal with elevated electroretinogram amplitudes associated with high levels of Rhodopsin-1 (Rh1). Reducing Rh1 levels in rh1 mutants or depriving flies of vitamin A rescued photoreceptor cell death in fatpmutant flies. Our results indicate that fatp promotes photoreceptor survival by regulating Rh1 abundance.

Dual role of the ddx5/ddx17 RNA helicases in the control of the pro-migratory NFAT5 transcription factor

Author(s) : Germann S, Gratadou L, Zonta E, Dardenne E, Gaudineau B, Foug?re M, Samaan S, Dutertre M, Jauliac S, Auboeuf D,
Journal : Oncogene
2012

Dual role of the ddx5/ddx17 RNA helicases in the control of the pro-migratory NFAT5 transcription factor.

Author(s) : Germann S, Gratadou L, Zonta E, Dardenne E, Gaudineau B, Fougere M, Samaan S, Dutertre M, Jauliac S, Auboeuf D,
Journal : Oncogene
2012
Ddx5 and ddx17 are two highly related RNA helicases involved in both transcription and splicing. These proteins coactivate transcription factors involved in cancer such as the estrogen receptor alpha, p53 and beta-catenin. Ddx5 and ddx17 are part of the splicing machinery and can modulate alternative splicing, the main mechanism increasing the proteome diversity. Alternative splicing also has a role in gene expression level regulation when it is coupled to the nonsense-mediated mRNA decay (NMD) pathway. In this work, we report that ddx5 and ddx17 have a dual role in the control of the pro-migratory NFAT5 transcription factor. First, ddx5 and ddx17 act as transcriptional coactivators of NFAT5 and are required for activating NFAT5 target genes involved in tumor cell migration. Second, at the splicing level, ddx5 and ddx17 increase the inclusion of NFAT5 exon 5. As exon 5 contains a pre-mature translation termination codon, its inclusion leads to the regulation of NFAT5 mRNAs by the NMD pathway and to a decrease in NFAT5 protein level. Therefore, we demonstratedfor the first time that a transcriptional coregulator can simultaneously regulate the transcriptional activity and alternative splicing of a transcription factor.This dual regulation, where ddx5 and ddx17 enhance the transcriptional activity of NFAT5 although reducing its protein expression level, suggests a critical role for ddx5 and ddx17 in tumor cell migration through the fine regulation of NFAT5 pathway.

ER stress inhibits neuronal death by promoting autophagy.

Author(s) : Fouillet A, Levet C, Virgone A, Robin M, Dourlen P, Rieusset J, Belaidi E, Ovize M, Touret M, Nataf S, Mollereau B,
Journal : Autophagy
2012
Endoplasmic reticulum (ER) stress has been implicated in neurodegenerative diseases but its relationship and role in disease progression remain unclear. Using genetic and pharmacological approaches, we showed that mild ER stress ("preconditioning") is neuroprotective in Drosophila and mouse models of Parkinson disease. In addition, we found that the combination of mild ER stress and apoptotic signals triggers an autophagic response both in vivo and in vitro.We showed that when autophagy is impaired, ER-mediated protection is lost. We further demonstrated that autophagy inhibits caspase activation and apoptosis. Based on our findings, we conclude that autophagy is required for the neuroprotection mediated by mild ER stress, and therefore ER preconditioning haspotential therapeutic value for the treatment of neurodegenerative diseases.

Genetic modifiers of chromatin acetylation antagonize the reprogramming of epi-polymorphisms.

Author(s) : Abraham A, Nagarajan M, Veyrieras J, Bottin H, Steinmetz L, Yvert G,
Journal : PLoS Genet
2012
Natural populations are known to differ not only in DNA but also in their chromatin-associated epigenetic marks. When such inter-individual epigenomic differences (or "epi-polymorphisms") are observed, their stability is usually not known: they may or may not be reprogrammed over time or upon environmental changes. In addition, their origin may be purely epigenetic, or they may result from regulatory variation encoded in the DNA. Studying epi-polymorphisms requires, therefore, an assessment of their nature and stability. Here we estimate the stability of yeast epi-polymorphisms of chromatin acetylation, and we provide a genome-by-epigenome map of their genetic control. A transient epi-drug treatment was able to reprogram acetylation variation at more than one thousand nucleosomes, whereas a similar amount of variation persisted, distinguishing "labile" from "persistent" epi-polymorphisms. Hundreds of geneticloci underlied acetylation variation at 2,418 nucleosomes either locally (in cis) or distantly (in trans), and this genetic control overlapped only partially withthe genetic control of gene expression. Trans-acting regulators were not necessarily associated with genes coding for chromatin modifying enzymes. Strikingly, "labile" and "persistent" epi-polymorphisms were associated with poor and strong genetic control, respectively, showing that genetic modifiers contribute to persistence. These results estimate the amount of natural epigenomic variation that can be lost after transient environmental exposures, and they reveal the complex genetic architecture of the DNA-encoded determinism of chromatin epi-polymorphisms. Our observations provide a basis for the development of population epigenetics.

Guidelines for the use and interpretation of assays for monitoring autophagy.

Author(s) : Klionsky D, al,
Journal : Autophagy
2012
In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitorthe numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process vs. those that measure flux through the autophagy pathway (i.e., the complete process); thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from stimuli that result in increased autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. Here, we present a set of guidelines for the selection andinterpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the systembeing used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field.

HTLV-1 positive and negative T cells cloned from infected individuals display telomerase and telomere genes deregulation that predominate in activated but untransformed CD4+ T cells

Author(s) : Zane L, Sibon D, Capraro V, Galia P, Karam M, Delfau-Larue M, Gilson E, Gessain A, Gout O, Hermine O, Mortreux F, Wattel E,
Journal : Int J Cancer
2012

INT6 interacts with MIF4GD/SLIP1 and is necessary for efficient histone mRNA translation.

Author(s) : Neusiedler J, Mocquet V, Limousin T, Ohlmann T, Morris C, Jalinot P,
Journal : RNA
2012
The INT6/EIF3E protein has been implicated in mouse and human breast carcinogenesis. This subunit of the eIF3 translation initiation factor that includes a PCI domain exhibits specific features such as presence in the nucleusand ability to interact with other important cellular protein complexes like the26S proteasome and the COP9 signalosome. It has been previously shown that INT6 was not essential for bulk translation, and this protein is considered to regulate expression of specific mRNAs. Based on the results of a two-hybrid screen performed with INT6 as bait, we characterize in this article the MIF4GD/SLIP1 protein as an interactor of this eIF3 subunit. MIF4GD was previously shown to associate with SLBP, which binds the stem-loop located at the 3' end ofthe histone mRNAs, and to be necessary for efficient translation of these cell cycle-regulated mRNAs that lack a poly(A) tail. In line with the interaction of both proteins, we show using the RNA interference approach that INT6 is also essential to S-phase histone mRNA translation. This was observed by analyzing expression of endogenous histones and by testing heterologous constructs placingthe luciferase reporter gene under the control of the stem-loop element of various histone genes. With such a reporter plasmid, silencing and overexpression of INT6 exerted opposite effects. In agreement with these results, INT6 and MIF4GD were observed to colocalize in cytoplasmic foci. We conclude from these data that INT6, by establishing interactions with MIF4GD and SLBP, plays an important role in translation of poly(A) minus histone mRNAs.

INT6/EIF3E interacts with ATM and is required for proper execution of the DNA damage response in human cells.

Author(s) : Morris C, Tomimatsu N, Richard D, Cluet D, Burma S, Khanna K, Jalinot P,
Journal : Cancer Res
2012
Altered expression of the INT6 gene, encoding the e subunit of the translationalinitiation factor eIF3, occurs in human breast cancers, but how INT6 relates to carcinogenesis remains unestablished. Here, we show that INT6 is involved in theDNA damage response. INT6 was required for cell survival following gamma-irradiation and G(2)-M checkpoint control. RNA interference-mediated silencing of INT6 reduced phosphorylation of the checkpoint kinases CHK1 and CHK2 after DNA damage. In addition, INT6 silencing prevented sustained accumulation of ataxia telangiectasia mutated (ATM) at DNA damage sites in cells treated with gamma-radiation or the radiomimetic drug neocarzinostatin. Mechanistically, thisresult could be explained by interaction of INT6 with ATM, which together with INT6 was recruited to the sites of DNA damage. Finally, INT6 silencing also reduced ubiquitylation events that promote retention of repair proteins at DNA lesions. Accordingly, accumulation of the repair factor BRCA1 was defective in the absence of INT6. Our findings reveal unexpected and striking connections of INT6 with ATM and BRCA1 and suggest that the protective action of INT6 in the onset of breast cancers relies on its involvement in the DNA damage response.

Mathematical model of the primary CD8 T cell immune response: stability analysis of a nonlinear age-structured system.

Author(s) : Terry E, Marvel J, Arpin C, Gandrillon O, Crauste F,
Journal : J Math Biol
2012
The primary CD8 T cell immune response, due to a first encounter with a pathogen, happens in two phases: an expansion phase, with a fast increase of T cell count,followed by a contraction phase. This contraction phase is followed by the generation of memory cells. These latter are specific of the antigen and will allow a faster and stronger response when encountering the antigen for the second time. We propose a nonlinear mathematical model describing the T CD8 immune response to a primary infection, based on three nonlinear ordinary differential equations and one nonlinear age-structured partial differential equation, describing the evolution of CD8 T cell count and pathogen amount. We discuss in particular the roles and relevance of feedback controls that regulate the response. First we reduce our system to a system with a nonlinear differential equation with a distributed delay. We study the existence of two steady states, and we analyze the asymptotic stability of these steady states. Second we study the system with a discrete delay, and analyze global asymptotic stability of steady states. Finally, we show some simulations that we can obtain from the model and confront them to experimental data.

Modeling erythroblastic islands: using a hybrid model to assess the function of central macrophage.

Author(s) : Fischer S, Kurbatova P, Bessonov N, Gandrillon O, Volpert V, Crauste F,
Journal : J Theor Biol
2012
The production and regulation of red blood cells, erythropoiesis, occurs in the bone marrow where erythroid cells proliferate and differentiate within particular structures, called erythroblastic islands. A typical structure of these islands consists of a macrophage (white cell) surrounded by immature erythroid cells (progenitors), with more mature cells on the periphery of the island, ready to leave the bone marrow and enter the bloodstream. A hybrid model, coupling a continuous model (ordinary differential equations) describing intracellular regulation through competition of two key proteins, to a discrete spatial model describing cell-cell interactions, with growth factor diffusion in the medium described by a continuous model (partial differential equations), is proposed toinvestigate the role of the central macrophage in normal erythropoiesis. Intracellular competition of the two proteins leads the erythroid cell to eitherproliferation, differentiation, or death by apoptosis. This approach allows considering spatial aspects of erythropoiesis, involved for instance in the occurrence of cellular interactions or the access to external factors, as well as dynamics of intracellular and extracellular scales of this complex cellular process, accounting for stochasticity in cell cycle durations and orientation ofthe mitotic spindle. The analysis of the model shows a strong effect of the central macrophage on the stability of an erythroblastic island, when assuming the macrophage releases pro-survival cytokines. Even though it is not clear whether or not erythroblastic island stability must be required, investigation of the model concludes that stability improves responsiveness of the model, hence stressing out the potential relevance of the central macrophage in normal erythropoiesis.

Monitoring single-cell bioenergetics via the coarsening of emulsion droplets.

Author(s) : Boitard L, Cottinet D, Kleinschmitt C, Bremond N, Baudry J, Yvert G, Bibette J,
Journal : Proc Natl Acad Sci U S A
2012
Microorganisms are widely used to generate valuable products, and their efficiency is a major industrial focus. Bioreactors are typically composed of billions of cells, and available measurements only reflect the overall performance of the population. However, cells do not equally contribute, and process optimization would therefore benefit from monitoring this intrapopulation diversity. Such monitoring has so far remained difficult because of the inability to probe concentration changes at the single-cell level. Here, we unlock this limitation by taking advantage of the osmotically driven water flux between a droplet containing a living cell toward surrounding empty droplets, within a concentrated inverse emulsion. With proper formulation, excreted products are far more soluble within the continuous hydrophobic phase compared to initial nutrients (carbohydrates and salts). Fast diffusion of products induces an osmotic mismatch, which further relaxes due to slower diffusion of water throughhydrophobic interfaces. By measuring droplet volume variations, we can deduce the metabolic activity down to isolated single cells. As a proof of concept, we present the first direct measurement of the maintenance energy of individual yeast cells. This method does not require any added probes and can in principle apply to any osmotically sensitive bioactivity, opening new routes for screening, and sorting large libraries of microorganisms and biomolecules.

Novel roles of Caenorhabditis elegans heterochromatin protein HP1 and linker histone in the regulation of innate immune gene expression.

Author(s) : Studencka M, Konzer A, Moneron G, Wenzel D, Opitz L, Salinas-Riester G, Bedet C, Kruger M, Hell S, Wisniewski J, Schmidt H, Palladino F, Schulze E, Jedrusik-Bode M,
Journal : Mol Cell Biol
2012
Linker histone (H1) and heterochromatin protein 1 (HP1) are essential componentsof heterochromatin which contribute to the transcriptional repression of genes. It has been shown that the methylation mark of vertebrate histone H1 is specifically recognized by the chromodomain of HP1. However, the exact biological role of linker histone binding to HP1 has not been determined. Here, we investigate the function of the Caenorhabditis elegans H1 variant HIS-24 and theHP1-like proteins HPL-1 and HPL-2 in the cooperative transcriptional regulation of immune-relevant genes. We provide the first evidence that HPL-1 interacts with HIS-24 monomethylated at lysine 14 (HIS-24K14me1) and associates in vivo with promoters of genes involved in antimicrobial response. We also report an increase in overall cellular levels and alterations in the distribution of HIS-24K14me1 after infection with pathogenic bacteria. HIS-24K14me1 localization changes frombeing mostly nuclear to both nuclear and cytoplasmic in the intestinal cells of infected animals. Our results highlight an antimicrobial role of HIS-24K14me1 and suggest a functional link between epigenetic regulation by an HP1/H1 complex andthe innate immune system in C. elegans.

Nucleosome-depleted chromatin gaps recruit assembly factors for the H3.3 histone variant.

Author(s) : Schneiderman J, Orsi G, Hughes K, Loppin B, Ahmad K,
Journal : Proc Natl Acad Sci U S A
2012
Most nucleosomes that package eukaryotic DNA are assembled during DNA replication, but chromatin structure is routinely disrupted in active regions ofthe genome. Replication-independent nucleosome replacement using the H3.3 histone variant efficiently repackages these regions, but how histones are recruited to these sites is unknown. Here, we use an inducible system that produces nucleosome-depleted chromatin at the Hsp70 genes in Drosophila to define steps in the mechanism of nucleosome replacement. We find that the Xnp chromatin remodeler and the Hira histone chaperone independently bind nucleosome-depleted chromatin.Surprisingly, these two factors are only displaced when new nucleosomes are assembled. H3.3 deposition assays reveal that Xnp and Hira are required for efficient nucleosome replacement, and double-mutants are lethal. We propose thatXnp and Hira recognize exposed DNA and serve as a binding platform for the efficient recruitment of H3.3 predeposition complexes to chromatin gaps. These results uncover the mechanisms by which eukaryotic cells actively prevent the exposure of DNA in the nucleus.

Programmed genome rearrangements: in lampreys, all cells are not equal.

Author(s) : Semon M, Schubert M, Laudet V,
Journal : Curr Biol
2012
How can organisms silence deleterious gene loci? A recent study has shed light on a very brute mechanism in a jawless vertebrate: the irreversible deletion of massive chunks of genomic DNA.

Repeated evolution of testis-specific new genes: the case of telomere-capping genes in Drosophila.

Author(s) : Dubruille R, Marais G, Loppin B,
Journal : Int J Evol Biol
2012
Comparative genome analysis has allowed the identification of various mechanismsinvolved in gene birth. However, understanding the evolutionary forces driving new gene origination still represents a major challenge. In particular, an intriguing and not yet fully understood trend has emerged from the study of new genes: many of them show a testis-specific expression pattern, which has remained poorly understood. Here we review the case of such a new gene, which involves a telomere-capping gene family in Drosophila. hiphop and its testis-specific paralog K81 are critical for the protection of chromosome ends in somatic cells and male gametes, respectively. Two independent functional studies recently proposed that these genes evolved under a reproductive-subfunctionalization regime. The 2011 release of new Drosophila genome sequences from the melanogaster group of species allowed us to deepen our phylogenetic analysis of the hiphop/K81 family. This work reveals an unsuspected dynamic of gene birth and death within the group, with recurrent duplication events through retroposition mechanisms. Finally, we discuss the plausibility of different evolutionary scenarios that could explain the diversification of this gene family.

Retinoic acid induces Sertoli cell paracrine signals for spermatogonia differentiation but cell autonomously drives spermatocyte meiosis.

Author(s) : Raverdeau M, Gely-Pernot A, Feret B, Dennefeld C, Benoit G, Davidson I, Chambon P, Mark M, Ghyselinck N,
Journal : Proc Natl Acad Sci U S A
2012
Direct evidence for a role of endogenous retinoic acid (RA), the active metabolite of vitamin A in the initial differentiation and meiotic entry of spermatogonia, and thus in the initiation of spermatogenesis is still lacking. RA is synthesized by dedicated enzymes, the retinaldehyde dehydrogenases (RALDH), and binds to and activates nuclear RA receptors (RARA, RARB, and RARG) either within the RA-synthesizing cells or in the neighboring cells. In the present study, we have used a combination of somatic genetic ablations and pharmacological approaches in vivo to show that during the first, prepubertal, spermatogenic cycle (i) RALDH-dependent synthesis of RA by Sertoli cells (SC), the supporting cells of the germ cell (GC) lineage, is indispensable to initiatedifferentiation of A aligned into A1 spermatogonia; (ii) RARA in SC mediates theeffects of RA, possibly through activating Mafb expression, a gene whose Drosophila homolog is mandatory to GC differentiation; (iii) RA synthesized by premeiotic spermatocytes cell autonomously induces meiotic initiation through controlling the RAR-dependent expression of Stra8. Furthermore, we show that RA of SC origin is no longer necessary for the subsequent spermatogenic cycles but essential to spermiation. Altogether, our data establish that the effects of RA in vivo on spermatogonia differentiation are indirect, via SC, but direct on meiotic initiation in spermatocytes, supporting thereby the notion that, contrary to the situation in the female, RA is necessary to induce meiosis in the male.

Retinoic acid receptors recognize the mouse genome through binding elements with diverse spacing and topology.

Author(s) : Moutier E, Ye T, Choukrallah M, Urban S, Osz J, Chatagnon A, Delacroix L, Langer D, Rochel N, Moras D, Benoit G, Davidson I,
Journal : J Biol Chem
2012
Retinoic acid receptors (RARs) heterodimerize with retinoid X receptors (RXRs) and bind to RA response elements (RAREs) in the regulatory regions of their target genes. Although previous studies on limited sets of RA-regulated genes have defined canonical RAREs as direct repeats of the consensus RGKTCA separatedby 1, 2, or 5 nucleotides (DR1, DR2, DR5), we show that in mouse embryoid bodiesor F9 embryonal carcinoma cells, RARs occupy a large repertoire of sites with DR0, DR8, and IR0 (inverted repeat 0) elements. Recombinant RAR-RXR binds these non-canonical spacings in vitro with comparable affinities to DR2 and DR5. Most DR8 elements comprise three half-sites with DR2 and DR0 spacings. This specific half-site organization constitutes a previously unrecognized but frequent signature of RAR binding elements. In functional assays, DR8 and IR0 elements act as independent RAREs, whereas DR0 does not. Our results reveal an unexpected diversity in the spacing and topology of binding elements for the RAR-RXR heterodimer. The differential ability of RAR-RXR bound to DR0 compared to DR2, DR5, and DR8 to mediate RA-dependent transcriptional activation indicates that half-site spacing allosterically regulates RAR function.

Splicing programs and cancer

Author(s) : Germann S, Gratadou L, Dutertre M, Auboeuf D,
Journal : J Nucleic Acids
2012

Splicing switch of an epigenetic regulator by RNA helicases promotes tumor-cell invasiveness

Author(s) : Dardenne E, Pierredon S, Driouch K, Gratadou L, Lacroix-Triki M, Espinoza M, Zonta E, Germann S, Mortada H, Villemin J, Dutertre M, Lidereau R, Vagner S, Auboeuf D,
Journal : Nat Struct Mol Biol
2012

Stimulation of gross chromosomal rearrangements by the human CEB1 and CEB25 minisatellites in Saccharomyces cerevisiae depends on G-quadruplexes or Cdc13

Author(s) : Piazza A, Serero A, Boule J, Legoix-Ne P, Lopes J, Nicolas A,
Journal : PLoS genetics
2012
Genomes contain tandem repeats that are at risk of internal rearrangements and a threat to genome integrity. Here, we investigated the behavior of the human subtelomeric minisatellites HRAS1, CEB1, and CEB25 in Saccharomyces cerevisiae. In mitotically growing wild-type cells, these GC–rich tandem arrays stimulate the rate of gross chromosomal rearrangements (GCR) by 20, 1,620, and 276,000-fold, respectively. In the absence of the Pif1 helicase, known to inhibit GCR by telomere addition and to unwind G-quadruplexes, the GCR rate is further increased in the presence of CEB1, by 385-fold compared to the pif1Δ control strain. The behavior of CEB1 is strongly dependent on its capacity to form G-quadruplexes, since the treatment of WT cells with the Phen-DC3 G-quadruplex ligand has a 52-fold stimulating effect while the mutation of the G-quadruplex-forming motif reduced the GCR rate 30-fold in WT and 100-fold in pif1Δ cells. The GCR events are telomere additions within CEB1. Differently, the extreme stimulation of CEB25 GCR depends on its affinity for Cdc13, which binds the TG-rich ssDNA telomere overhang. This property confers a biased orientation-dependent behavior to CEB25, while CEB1 and HRAS1 increase GCR similarly in either orientation. Furthermore, we analyzed the minisatellites‚ distribution in the human genome and discuss their potential role to trigger subtelomeric rearrangements.

2011

A conceptually improved TD-DFT approach for predicting the maximum absorption wavelength of cyanine dyes

Author(s) : Meguellati K, Ladame S, Spichty M,
Journal : Dyes and Pigments
2011
Cyanine dyes have found valuable applications in modern bioresearch because of their biocompatibility, high molar absorptivity and moderate fluorescence quantum yield. Of special value for sensing and labeling applications is the fact they can cover a very large spectral range (from blue to infra-red). To design and select the most appropriate dyes for a given application the computational prediction of the absorption wavelength (prior to the costly chemical synthesis) serves as a valuable guidance. However, predicting absorption and emission wavelengths of such compounds remains a challenging task. Herein, we report a fast and highly accurate computational approach which allows the prediction of the maximum absorption wavelength for a wide range of cyanine dyes, including symmetrical and unsymmetrical, trimethine and pentamethine cyanine dyes but also unusual imino-based analogues. In addition to the vertical excitation energy (calculated from time-dependent density functional theory), the approach makes use of a novel correction term that is based on the ground-state zero-point vibrational energy (ZPVE). The correction term is statistically significant (F-test), and it reduces the average error and maximal error of the prediction by a factor of two. We anticipate that the concept of including the ZPVE into the calculation of the maximum absorption wavelength can be used also for other families of dyes to improve their predictability.

A conserved splicing mechanism of the LMNA gene controls premature aging

Author(s) : Lopez-Mejia I, Vautrot V, De Toledo M, Behm-Ansmant I, Bourgeois C, Navarro C, Osorio F, Freije J, St?venin J, De Sandre-Giovannoli A, Lopez-Otin C, L?vy N, Branlant C, Tazi J,
Journal : Hum Mol Genet
2011

A conserved splicing mechanism of the LMNA gene controls premature aging.

Author(s) : Lopez-Mejia I, Vautrot V, De Toledo M, Behm-Ansmant I, Bourgeois C, Navarro C, Osorio F, Freije J, Stevenin J, De Sandre-Giovannoli A, Lopez-Otin C, Levy N, Branlant C, Tazi J,
Journal : Hum Mol Genet
2011
Hutchinson-Gilford progeria syndrome (HGPS) is a rare genetic disorder phenotypically characterized by many features of premature aging. Most cases of HGPS are due to a heterozygous silent mutation (c.1824C>T; p.Gly608Gly) that enhances the use of an internal 5' splice site (5'SS) in exon 11 of the LMNA pre-mRNA and leads to the production of a truncated protein (progerin) with a dominant negative effect. Here we show that HGPS mutation changes the accessibility of the 5'SS of LMNA exon 11 which is sequestered in a conserved RNA structure. Our results also reveal a regulatory role of a subset of serine-arginine (SR)-rich proteins, including serine-arginine rich splicing factor 1 (SRSF1) and SRSF6, on utilization of the 5'SS leading to lamin A or progerin production and a modulation of this regulation in the presence of the c.1824C>T mutation is shown directly on HGPS patient cells. Mutant mice carryingthe equivalent mutation in the LMNA gene (c.1827C>T) also accumulate progerin and phenocopy the main cellular alterations and clinical defects of HGPS patients. RNAi-induced depletion of SRSF1 in the HGPS-like mouse embryonic fibroblasts (MEFs) allowed progerin reduction and dysmorphic nuclei phenotype correction, whereas SRSF6 depletion aggravated the HGPS-like MEF's phenotype. We demonstratethat changes in the splicing ratio between lamin A and progerin are key factors for lifespan since heterozygous mice harboring the mutation lived longer than homozygous littermates but less than the wild-type. Genetic and biochemical datatogether favor the view that physiological progerin production is under tight control of a conserved splicing mechanism to avoid precocious aging.

Activation of a microRNA response in trans reveals a new role for poly(A) in translational repression.

Author(s) : Ricci E, Limousin T, Soto-Rifo R, Allison R, Poyry T, Decimo D, Jackson R, Ohlmann T,
Journal : Nucleic Acids Res
2011
Here, we report that the untreated rabbit reticulocyte lysate contains over 300 different endogenous microRNAs together with the major components of the RNA-induced silencing complex and thus can be used as a model in vitro system tostudy the effects of microRNAs on gene expression. By using this system, we wereable to show that microRNA hybridization to its target resulted in a very rapid and strong inhibition of expression that was exerted exclusively at the level oftranslation initiation with no involvement of transcript degradation or deadenylation. Moreover, we demonstrate that the magnitude of microRNA-induced repression can only be recapitulated in the context of a competitive translatingenvironment. By using a wide spectrum of competitor cellular and viral RNAs, we could further show that competition was not exerted at the level of general components of the translational machinery, but relied exclusively on the presence of the poly(A) tail with virtually no involvement of the cap structure.

Biological functions of p53 isoforms through evolution: lessons from animal and cellular models.

Author(s) : Marcel V, Dichtel-Danjoy M, Sagne C, Hafsi H, Ma D, Ortiz-Cuaran S, Olivier M, Hall J, Mollereau B, Hainaut P, Bourdon J,
Journal : Cell Death Differ
2011
The TP53 tumour-suppressor gene is expressed as several protein isoforms generated by different mechanisms, including use of alternative promoters, splicing sites and translational initiation sites, that are conserved through evolution and within the TP53 homologues, TP63 and TP73. Although first described in the eighties, the importance of p53 isoforms in regulating the suppressive functions of p53 has only become evident in the last 10 years, by analogy with observations that p63 and p73 isoforms appeared indispensable to fully understand the biological functions of TP63 and TP73. This review summarizes recent advances in the field of 'p53 isoforms', including new data on p63 and p73 isoforms. Details of the alternative mechanisms that produce p53 isoforms and cis- and trans-regulators identified are provided. The main focus is on their biological functions (apoptosis, cell cycle, aging and so on) in cellular and animal models, including mouse, zebrafish and Drosophila. Finally, the deregulation of p53 isoform expression in human cancers is reviewed. Based on these latest results, several developments are expected in the future: the identification of drugs modulating p53 isoform expression; the generation of animal models and the evaluation of the use of p53 isoform as biomarkers in human cancers.

Caenorhabditis elegans chromatin-associated proteins SET-2 and ASH-2 are differentially required for histone H3 Lys 4 methylation in embryos and adult germ cells.

Author(s) : Xiao Y, Bedet C, Robert V, Simonet T, Dunkelbarger S, Rakotomalala C, Soete G, Korswagen H, Strome S, Palladino F,
Journal : Proc Natl Acad Sci U S A
2011
Methylation of histone H3 lysine 4 (H3K4me), a mark associated with gene activation, is mediated by SET1 and the related mixed lineage leukemia (MLL) histone methyltransferases (HMTs) across species. Mammals contain seven H3K4 HMTs, Set1A, Set1B, and MLL1-MLL5. The activity of SET1 and MLL proteins relies on protein-protein interactions within large multisubunit complexes that includethree core components: RbBP5, Ash2L, and WDR5. It remains unclear how the composition and specificity of these complexes varies between cell types and during development. Caenorhabditis elegans contains one SET1 protein, SET-2, oneMLL-like protein, SET-16, and single homologs of RbBP5, Ash2L, and WDR5. Here weshow that SET-2 is responsible for the majority of bulk H3K4 methylation at all developmental stages. However, SET-2 and absent, small, or homeotic discs 2 (ASH-2) are differentially required for tri- and dimethylation of H3K4 (H3K4me3 and -me2) in embryos and adult germ cells. In embryos, whereas efficient H3K4me3requires both SET-2 and ASH-2, H3K4me2 relies mostly on ASH-2. In adult germ cells by contrast, SET-2 serves a major role whereas ASH-2 is dispensable for H3K4me3 and most H3K4me2. Loss of SET-2 results in progressive sterility over several generations, suggesting an important function in the maintenance of a functional germ line. This study demonstrates that individual subunits of SET1-related complexes can show tissue specificity and developmental regulation and establishes C. elegans as a model to study SET1-related complexes in a multicellular organism.

Caenorhabditis elegans Heterochromatin protein 1 (HPL-2) links developmental plasticity, longevity and lipid metabolism.

Author(s) : Meister P, Schott S, Bedet C, Xiao Y, Rohner S, Bodennec S, Hudry B, Molin L, Solari F, Gasser S, Palladino F,
Journal : Genome Biol
2011
BACKGROUND: Heterochromatin protein 1 (HP1) family proteins have a well-characterized role in heterochromatin packaging and gene regulation. Their function in organismal development, however, is less well understood. Here we used genome-wide expression profiling to assess novel functions of the Caenorhabditis elegans HP1 homolog HPL-2 at specific developmental stages. RESULTS: We show that HPL-2 regulates the expression of germline genes, extracellular matrix components and genes involved in lipid metabolism. Comparison of our expression data with HPL-2 ChIP-on-chip profiles reveals that a significant number of genes up- and down-regulated in the absence of HPL-2 are bound by HPL-2. Germline genes are specifically up-regulated in hpl-2 mutants, consistent with the function of HPL-2 as a repressor of ectopic germ cell fate. In addition, microarray results and phenotypic analysis suggest that HPL-2 regulates the dauer developmental decision, a striking example of phenotypic plasticity in which environmental conditions determine developmental fate. HPL-2acts in dauer at least partly through modulation of daf-2/IIS and TGF-beta signaling pathways, major determinants of the dauer program. hpl-2 mutants also show increased longevity and altered lipid metabolism, hallmarks of the long-lived, stress resistant dauers. CONCLUSIONS: Our results suggest that the worm HP1 homologue HPL-2 may coordinately regulate dauer diapause, longevity andlipid metabolism, three processes dependent on developmental input and environmental conditions. Our findings are of general interest as a paradigm of how chromatin factors can both stabilize development by buffering environmental variation, and guide the organism through remodeling events that require plasticity of cell fate regulation.

Cholesterol synthesis-related enzyme oxidosqualene cyclase is required to maintain self-renewal in primary erythroid progenitors.

Author(s) : Mejia-Pous C, Damiola F, Gandrillon O,
Journal : Cell Prolif
2011
OBJECTIVES: Molecular mechanisms controlling cell fate decision making in self-renewing cells are poorly understood. A previous transcriptomic study, carried out in primary avian erythroid progenitor cells (T2ECs), revealed that the gene encoding oxidosqualene cyclase (OSC/LSS), an enzyme involved in cholesterol biosynthesis, is significantly up-regulated in self-renewing cells. The aim of the present work is to understand whether this up-regulation is required for self-renewal maintenance and what are the mechanisms involved. MATERIALS AND METHODS: To investigate OSC function, we studied effects of its enzymatic activity inhibition using Ro48-8071, a specific OSC inhibitor. In addition, we completed this pharmacological approach by RNAi-mediated OSC/LSS knockdown. The study of OSC inhibition was carried out on both self-renewing anddifferentiating cells to observe any state-dependent effect. RESULTS: Our data show that OSC acts both by protecting self-renewing T2EC cells from apoptosis and by blocking their differentiation program, as OSC inhibition is sufficient to trigger spontaneous commitment of self-renewing cells towards an early differentiation state. This is self-renewal specific, as OSC inhibition has no effect on erythroid progenitors that have already differentiated. CONCLUSIONS: Taken together, our results suggest that OSC/LSS expression and activity are required to maintain cell self-renewal and may be involved in the self-renewal versus differentiation/apoptosis decision making, by keeping cells in a self-renewal state.

Differential evolvability along lines of least resistance of upper and lower molars in island house mice.

Author(s) : Renaud S, Pantalacci S, Auffray J,
Journal : PLoS One
2011
Variation within a population is a key feature in evolution, because it can increase or impede response to selection, depending on whether or not the intrapopulational variance is correlated to the change under selection. Hence, main directions of genetic variance have been proposed to constitute "lines of least resistance to evolution" along which evolution would be facilitated. Yet, the screening of selection occurs at the phenotypic level, and the phenotypic variance is not only the product of the underlying genetic variance, but also ofdevelopmental processes. It is thus a key issue for interpreting short and long term evolutionary patterns to identify whether main directions of phenotypic variance indeed constitute direction of facilitated evolution, and whether this is favored by developmental processes preferably generating certain phenotypes. We tackled these questions by a morphometric quantification of the directions ofvariance, compared to the direction of evolution of the first upper and lower molars of wild continental and insular house mice. The main phenotypic variance indeed appeared as channeling evolution between populations. The upper molar emerged as highly evolvable, because a strong allometric component contributed to its variance. This allometric relationship drove a repeated but independent evolution of a peculiar upper molar shape whenever size increased. This repeatedevolution, together with knowledge about the molar development, suggest that themain direction of phenotypic variance correspond here to a "line of least developmental resistance" along which evolution between population is channeled.

Epigenetic maintenance of telomere identity in Drosophila: buckle up for the sperm ride.

Author(s) : Dubruille R, Loppin B,
Journal : Cell Cycle
2011
A critical function of telomeres is to prevent the ligation of chromosome ends by DNA repair enzymes. In most eukaryotes, telomeric DNA consists in large arrays of G-rich tandem repeats that are recognized by DNA binding capping proteins. Drosophila telomeres are unusual as they lack short tandem repeats. However, Drosophila capping proteins can bind chromosome extremities in a DNA sequence-independent manner. This epigenetic protection of fly telomeres has been essentially studied in somatic cells where capping proteins such as HOAP or HP1 are essential in preventing chromosome end-to-end fusions. HipHop and K81 are two recently identified paralogous capping proteins with complementary expression patterns. While HipHop is involved in telomere capping in somatic cells, K81 hasspecialized in the protection of telomeres in post-meiotic male germ cells. Remarkably, K81 is required for the stabilization of HOAP and HP1 at telomeres during the massive paternal chromatin remodeling that occurs during spermiogenesis and at fertilization. We thus propose that the maintenance of capping proteins at Drosophila sperm telomeres is crucial for the transmission of telomere identity to the diploid zygote. :

Epigenetics in C. elegans: facts and challenges.

Author(s) : Wenzel D, Palladino F, Jedrusik-Bode M,
Journal : Genesis
2011
Epigenetics is defined as the study of heritable changes in gene expression thatare not accompanied by changes in the DNA sequence. Epigenetic mechanisms include histone post-translational modifications, histone variant incorporation, non-coding RNAs, and nucleosome remodeling and exchange. In addition, the functional compartmentalization of the nucleus also contributes to epigenetic regulation of gene expression. Studies on the molecular mechanisms underlying epigenetic phenomena and their biological function have relied on various model systems, including yeast, plants, flies, and cultured mammalian cells. Here we will expose the reader to the current understanding of epigenetic regulation in the roundworm C. elegans. We will review recent models of nuclear organization and its impact on gene expression, the biological role of enzymes modifying corehistones, and the function of chromatin-associated factors, with special emphasis on Polycomb (PcG) and Trithorax (Trx-G) group proteins. We will discuss how the C. elegans model has provided novel insight into mechanisms of epigenetic regulation as well as suggest directions for future research.

Finding modulators of stochasticity levels by quantitative genetics.

Author(s) : Fehrmann S, Yvert G,
Journal : Methods Mol Biol
2011
Although bakers and wine makers constantly select, compare, and hunt for new wild strains of Saccharomyces cerevisiae, yeast geneticists have long focused on a few "standard" strains to ensure reproducibility and easiness of experimentation. And so far, the wonderful natural resource of wild genetic variation has been poorlyexploited in most academic laboratories. We describe here how one can use this resource to investigate the molecular sources of stochasticity in a gene regulatory network. The approach is general enough to be applied to any network of interest, as long as the experimental read-out offers robust statistics. For a given network, a typical study first identifies two backgrounds A and B displaying different levels of stochasticity and then study the network in A x Bprogeny. Taking advantage of microarrays or resequencing technologies, genotyping of appropriate segregants can then lead to the genomic regions housing modulators of stochasticity. The powerful toolbox available to manipulate the yeast genome offers several ways to narrow these regions further and to unambiguously demonstrate the regulatory consequences of DNA polymorphisms.

G-quadruplex-induced instability during leading-strand replication

Author(s) : Lopes J, Piazza A, Bermejo R, Kriegsman B, Colosio A, Teulade-Fichou M, Foiani M, Nicolas A,
Journal : The EMBO journal
2011
G‐quadruplexes are four‐stranded nucleic acid structures whose biological functions remain poorly understood. In the yeast S. cerevisiae, we report that G‐quadruplexes form and, if not properly processed, pose a specific challenge to replication. We show that the G‐quadruplex‐prone CEB1 tandem array is tolerated when inserted near ARS305 replication origin in wild‐type cells but is very frequently destabilized upon treatment with the potent Phen‐DC3 G‐quadruplex ligand, or in the absence of the G‐quadruplex‐unwinding Pif1 helicase, only when the G‐rich strand is the template of leading‐strand replication. The orientation‐dependent instability is associated with the formation of Rad51–Rad52‐dependent X‐shaped intermediates during replication detected by two‐dimensional (2D) gels, and relies on the presence of intact G‐quadruplex motifs in CEB1 and on the activity of ARS305. The asymmetrical behaviour of G‐quadruplex prone sequences during replication has implications for their evolutionary dynamics within genomes, including the maintenance of G‐rich telomeres.

Genome-wide in silico identification of new conserved and functional retinoic acid receptor response elements (direct repeats separated by 5 bp).

Author(s) : Lalevee S, Anno Y, Chatagnon A, Samarut E, Poch O, Laudet V, Benoit G, Lecompte O, Rochette-Egly C,
Journal : J Biol Chem
2011
The nuclear retinoic acid receptors interact with specific retinoic acid (RA) response elements (RAREs) located in the promoters of target genes to orchestrate transcriptional networks involved in cell growth and differentiation. Here we describe a genome-wide in silico analysis of consensus DR5 RAREs based on the recurrent RGKTSA motifs. More than 15,000 DR5 RAREs were identified and analyzedfor their localization and conservation in vertebrates. We selected 138 elementslocated +/-10 kb from transcription start sites and gene ends and conserved across more than 6 species. We also validated the functionality of these RAREs by analyzing their ability to bind retinoic acid receptors (ChIP sequencing experiments) as well as the RA regulation of the corresponding genes (RNA sequencing and quantitative real time PCR experiments). Such a strategy provideda global set of high confidence RAREs expanding the known experimentally validated RAREs repertoire associated to a series of new genes involved in cell signaling, development, and tumor suppression. Finally, the present work provides a valuable knowledge base for the analysis of a wider range of RA-target genes in different species.

Identification of evolutionarily conserved exons as regulated targets for the splicing activator tra2β in development

Author(s) : Grellscheid S, Dalgliesh C, Storbeck M, Best A, Liu Y, Jakubik M, Mende Y, Ehrmann I, Curk T, Rossbach K, Bourgeois C, St?venin J, Grellscheid D, Jackson M, Wirth B, Elliott D,
Journal : PLoS Genet
2011

Identification of evolutionarily conserved exons as regulated targets for the splicing activator tra2beta in development.

Author(s) : Grellscheid S, Dalgliesh C, Storbeck M, Best A, Liu Y, Jakubik M, Mende Y, Ehrmann I, Curk T, Rossbach K, Bourgeois C, Stevenin J, Grellscheid D, Jackson M, Wirth B, Elliott D,
Journal : PLoS Genet
2011
Alternative splicing amplifies the information content of the genome, creating multiple mRNA isoforms from single genes. The evolutionarily conserved splicing activator Tra2beta (Sfrs10) is essential for mouse embryogenesis and implicated in spermatogenesis. Here we find that Tra2beta is up-regulated as the mitotic stem cell containing population of male germ cells differentiate into meiotic and post-meiotic cells. Using CLIP coupled to deep sequencing, we found that Tra2beta binds a high frequency of exons and identified specific G/A rich motifs as frequent targets. Significantly, for the first time we have analysed the splicing effect of Sfrs10 depletion in vivo by generating a conditional neuronal-specificSfrs10 knock-out mouse (Sfrs10(fl/fl); Nestin-Cre(tg/+)). This mouse has defectsin brain development and allowed correlation of genuine physiologically Tra2betaregulated exons. These belonged to a novel class which were longer than average size and importantly needed multiple cooperative Tra2beta binding sites for efficient splicing activation, thus explaining the observed splicing defects in the knockout mice. Regulated exons included a cassette exon which produces a meiotic isoform of the Nasp histone chaperone that helps monitor DNA double-strand breaks. We also found a previously uncharacterised poison exon identifying a new pathway of feedback control between vertebrate Tra2 proteins. Both Nasp-T and the Tra2a poison exon are evolutionarily conserved, suggesting they might control fundamental developmental processes. Tra2beta protein isoforms lacking the RRM were able to activate specific target exons indicating an additional functional role as a splicing co-activator. Significantly the N-terminal RS1 domain conserved between flies and humans was essential for the splicing activator function of Tra2beta. Versions of Tra2beta lacking this N-terminal RS1 domain potently repressed the same target exons activated by full-length Tra2beta protein.

Identification of human, rat and chicken ribosomal proteins by a combination of two-dimensional polyacrylamide gel electrophoresis and mass spectrometry.

Author(s) : Nguyen-Lefebvre A, Gonin-Giraud S, Scherl A, Arboit P, Granger L, Sanchez J, Diaz J, Gandrillon O, Madjar J,
Journal : J Proteomics
2011
To identify the exact spot position of human, rat and chicken ribosomal proteins(RP) separated by two-dimensional polyacrylamide gel electrophoresis (2-DE), a 2-DE system was designed to separate RP with a pI>8.6 according to their charge in the first dimension and to their molecular mass in the second dimension. Individual proteins were excised from the gels and identified by mass spectrometry after digestion by trypsin. In addition, a mixture of purified RP from these three species was also analyzed by tandem mass tag spectrometry. By combining those two methods 74 RP from human, 76 from rat and 67 from chicken were identified according to the nomenclature initially defined for rat liver RPand by using the Swiss-Prot/trEMBL databases. Whereas human and rat RP were welldescribed, most of RP from chicken were not characterized in databases, since 35out of 67 chicken RP identified in this study were not listed yet. We propose here the first comprehensive description of chicken RP and their comparison to those from human and rat.

Integrated genome-scale prediction of detrimental mutations in transcription networks.

Author(s) : Francesconi M, Jelier R, Lehner B,
Journal : PLoS Genet
2011
A central challenge in genetics is to understand when and why mutations alter the phenotype of an organism. The consequences of gene inhibition have been systematically studied and can be predicted reasonably well across a genome. However, many sequence variants important for disease and evolution may alter gene regulation rather than gene function. The consequences of altering a regulatory interaction (or "edge") rather than a gene (or "node") in a network have not been as extensively studied. Here we use an integrative analysis and evolutionary conservation to identify features that predict when the loss of a regulatory interaction is detrimental in the extensively mapped transcription network of budding yeast. Properties such as the strength of an interaction, location and context in a promoter, regulator and target gene importance, and the potential for compensation (redundancy) associate to some extent with interaction importance. Combined, however, these features predict quite well whether the loss of a regulatory interaction is detrimental across many promoters and for many different transcription factors. Thus, despite the potential for regulatory diversity, common principles can be used to understand and predict when changes in regulation are most harmful to an organism.

microRNA complements in deuterostomes: origin and evolution of microRNAs.

Author(s) : Campo-Paysaa F, Semon M, Cameron R, Peterson K, Schubert M,
Journal : Evol Dev
2011
Although numerous studies have emphasized the role of microRNAs (miRNAs) in the control of many different cellular processes, they might also exert a profound effect on the macroevolution of animal body plans. It has been hypothesized that, because miRNAs increase genic precision and are continuously being added to metazoan genomes through geologic time, miRNAs might be instrumental for canalization of development and morphological evolution. Nonetheless, an outstanding question remains: how are new miRNAs constantly evolving? To addressthis question, we assessed the miRNA complements of four deuterostome species, chosen because of their sequenced genomes and well-resolved phylogeny. Our comparative analysis shows that each of these four species is characterized by aunique repertoire of miRNAs, with few instances of miRNA loss. Moreover, we findthat almost half of the miRNAs identified in this study are located in intronic regions of protein coding genes, suggesting that new miRNAs might arise from intronic regions in a process we term intronic exaptation. We also show that miRNAs often occur within cotranscribed clusters, and describe the biological function of one of these conserved clusters, the miR-1/miR-133 cluster. Taken together, our work shows that miRNAs can easily emerge within already transcribed regions of DNA, whether it be introns or preexisting clusters of miRNAs and/or miRNAs and protein coding genes, and because of their regulatory roles, these novel players change the structure of gene regulatory networks, with potential macroevolutionary results.

Misregulated alternative splicing of BIN1 is associated with T tubule alterations and muscle weakness in myotonic dystrophy

Author(s) : Fugier C, Klein A, Hammer C, Vassilopoulos S, Ivarsson Y, Toussaint A, Tosch V, Vignaud A, Ferry A, Messaddeq N, Kokunai Y, Tsuburaya R, de la Grange P, Dembele D, Francois V, Precigout G, Boulade-Ladame C, Hummel M, Lopez de Munain A, Sergeant N, Laquerri?re A, Thibault C, Deryckere F, Auboeuf D, Garcia L, Zimmermann P, Udd B, Schoser B, Takahashi M, Nishino I, Bassez G, Laporte J, Furling D, Charlet-Berguerand N,
Journal : Nat Med
2011

Misregulation of miR-1 processing is associated with heart defects in myotonic dystrophy

Author(s) : Rau F, Freyermuth F, Fugier C, Villemin J, Fischer M, Jost B, Dembele D, Gourdon G, Nicole A, Duboc D, Wahbi K, Day J, Fujimura H, Takahashi M, Auboeuf D, Dreumont N, Furling D, Charlet-Berguerand N,
Journal : Nat Struct Mol Biol
2011

Molecular design of a splicing switch responsive to the RNA binding protein Tra2beta.

Author(s) : Grellscheid S, Dalgliesh C, Rozanska A, Grellscheid D, Bourgeois C, Stevenin J, Elliott D,
Journal : Nucleic Acids Res
2011
Tra2beta regulates a number of splicing switches including activation of the human testis-specific exon HIPK3-T in the Homeodomain Interacting Protein Kinase3 gene. By testing HIPK3-T exons of different intrinsic strengths, we found Tra2beta most efficiently activated splicing inclusion of intrinsically weak exons, although these were spliced at a lower overall level. Both the RRM and N-terminal RS-rich region of Tra2beta were required for splicing activation. Bioinformatic searches for splicing enhancers and repressors mapped four physically distinct exonic splicing enhancers (ESEs) within HIPK3-T, each containing the known Tra2beta AGAA-rich binding site. Surprisingly disruption ofeach single ESE prevented Tra2beta-mediated activation, although single mutated exons could still bind Tra2beta protein by gel shifts and functional splicing analyses. Titration experiments indicate an additive model of HIPK3-T splicing activation, requiring availability of an array of four distinct ESEs to enable splicing activation. To enable this efficient Tra2beta-mediated splicing switch to operate, a closely adjacent downstream and potentially competitive stronger 5'-splice site is actively repressed. Our data indicate that a novel arrangementof multiple mono-specific AGAA-rich ESEs coupled to a weak 5'-splice site functions as a responsive gauge. This gauge monitors changes in the specific nuclear concentration of the RNA binding protein Tra2beta, and co-ordinately regulates HIPK3-T exon splicing inclusion.

Molecular design of a splicing switch responsive to the RNA binding protein Tra2β

Author(s) : Grellscheid S, Dalgliesh C, Rozanska A, Grellscheid D, Bourgeois C, St?venin J, Elliott D,
Journal : Nucleic Acids Res
2011

Novel genes differentially expressed between posterior and median silk gland identified by SAGE-aided transcriptome analysis.

Author(s) : Royer C, Briolay J, Garel A, Brouilly P, Sasanuma S, Sasanuma M, Shimomura M, Keime C, Gandrillon O, Huang Y, Chavancy G, Mita K, Couble P,
Journal : Insect Biochem Mol Biol
2011
Serial analysis of gene expression (SAGE) profiles, from posterior and median cells of the silk gland of Bombyx mori, were analyzed and compared, so as to identify their respective distinguishing functions. The annotation of the SAGE libraries was performed with a B. mori reference tag collection, which was extracted from a novel set of Bombyx ESTs, sequenced from the 3' side. Most of the tags appeared at similar relative concentration within the two libraries, and corresponded with region-specific and highly abundant silk proteins. Strikingly,in addition to tags from silk protein mRNAs, 19 abundant tags were found (>/= 0.1%), in the median cell library, which were absent in the posterior cell tag collection. With the exception of tags from SP1 mRNA, no PSG specific tags were found in this subset class. The analysis of some of the MSG-specific transcripts, suggested that middle silk gland cells have diversified functions, in addition to their well characterized role in silk sericins synthesis and secretion.

Outcome of children treated with haematopoietic-stem cell transplantations from donors expressing the rare C77G variant of the PTPRC (CD45) gene

Author(s) : Samaan S, Gu?rin-El Khourouj V, Auboeuf D, Peltier L, P?dron B, Ouach?e-Chardin M, Gourgouillon N, Baruchel A, Dalle J, Sterkers G,
Journal : Br J Haematol
2011

Pharmacological inhibition of Frizzled-7 displays anti-tumor properties in hepatocellular carcinoma.

Author(s) : Nambotin S, Lefrancois L, Sainsily X, Berthillon P, Kim M, Wands J, Chevallier M, Jalinot P, Scoazec J, Trepo C, Zoulim F, Merle P,
Journal : J Hepatol
2011
BACKGROUND & AIMS: We previously reported the frequent overexpression of the FZD7 membrane receptor in hepatocellular carcinoma (HCC) and its role for controllingcancer phenotype. Herein, this study aimed at assessing the anticancer properties of compounds inhibiting FZD7 activity by disrupting its binding with the cytosolic Dishevelled (DVL) adaptator. METHODS: We have designed small interfering peptides (RHPDs) that are able to enter within cells and to competitively antagonize the binding of FZD7 to the PDZ domain of DVL. Their anti-neoplastic properties were assessed in vitro on a panel of human HCC cell lines and in vivo on the SV40-TAg transgenic mouse model of HCC. RESULTS: We have shown that RHPDs decrease cell viability via apoptosis depending on their affinity for PDZ, with a therapeutic index between cancerous and non-cancerous cells. RHPD properties were linked to beta-catenin degradation and PKCdelta activation. In transgenic mice, intra-tumor injection of RHPDs inhibited HCC progression. CONCLUSIONS: We have completed a proof-of-concept showing that in vitro and in vivo the pharmacological inhibition of FZD7 displays anti-cancerousproperties against HCC. The mechanisms can involve beta-catenin and PKCdelta modulations. Further studies are warranted to design protocols showing the compatibility with systemic in vivo applications.

Real-time imaging of cotranscriptional splicing reveals a kinetic model that reduces noise: implications for alternative splicing regulation

Author(s) : Schmidt U, Basyuk E, Robert M, Yoshida M, Villemin J, Auboeuf D, Aitken S, Bertrand E,
Journal : J Cell Biol
2011

Telomere deregulations possess cytogenetic, phenotype, and prognostic specificities in acute leukemias

Author(s) : Capraro V, Zane L, Poncet D, Perol D, Galia P, Preudhomme C, Bonnefoy-Berard N, Gilson E, Thomas X, El-Hamri M, Chelghoun Y, Michallet M, Wattel E, Mortreux F, Sibon D,
Journal : Exp Hematol
2011

The emerging role of pre-messenger RNA splicing in stress responses: sending alternative messages and silent messengers

Author(s) : Dutertre M, Sanchez G, Barbier J, Corcos L, Auboeuf D,
Journal : RNA Biol
2011

2010

[Adult T-cell leukemia induced by HTLV-1: before and after HBZ].

Author(s) : Duc Dodon M, Mesnard J, Barbeau B,
Journal : Med Sci (Paris)
2010
Adult T-cell leukemia (ATL) is an often fatal leukemia of CD4+ T lymphocytes associated with a complex retrovirus, human T-cell leukemia virus type 1 (HTLV-1). Although the viral Tax protein is involved in the proliferation of infected cells during the preleukemic stages, Tax expression is not systematically detected in primary leukemic cells. In 2002, we described the characterization of a novel viral protein that we have termed HBZ for HTLV-1 bZIP factor. This viral factor is encoded on the antisense strand of HTLV-1 proviral DNA, demonstrating the existence of antisense transcription from a promoter located in the 3' LTR. HBZ can negatively control the expression of the other viral proteins by blocking the interaction between Tax and ATF/CREB factors and the recruitment of CBP/p300 by Tax on the promoter. Moreover, recent studies found that the viral HBZ gene was always expressed in leukemic cells, suggestingits involvement in the progression of the infected cells towards malignancy.

[Quality proceedings in point-of-care testing: tetanus test example]

Author(s) : Descamps-Pouchelle I, Mortreux F, Sergent-Roumier A, Gressier B,
Journal : Ann Biol Clin (Paris)
2010

A multi-scale model of erythropoiesis.

Author(s) : Demin I, Crauste F, Gandrillon O, Volpert V,
Journal : J Biol Dyn
2010
In this paper, a multi-scale mathematical model of erythropoiesis is proposed inwhich erythroid progenitors are supposed to be able to self-renew. Three cellular processes control erythropoiesis: self-renewal, differentiation and apoptosis. We describe these processes and regulatory networks that govern them. Two proteins (ERK and Fas) are considered as the basic proteins participating in this regulation. All erythroid progenitors are divided into several sub-populations depending on their maturity level. Feedback regulations by erythropoietin, glucocorticoids and Fas ligand (FasL) are introduced in the model. The model consists of a system of ordinary differential equations describing intracellularprotein concentration evolution and cell population dynamics. We study steady states and their stability. We carry out computer simulations of an anaemia situation and analyse the results.

A novel concept in antiangiogenic and antitumoral therapy: multitarget destabilization of short-lived mRNAs by the zinc finger protein ZFP36L1.

Author(s) : Planel S, Salomon A, Jalinot P, Feige J, Cherradi N,
Journal : Oncogene
2010
Angiogenesis inhibitors have shown clinical benefits in patients with advanced cancer, but further therapeutic improvement is needed. We have previously shown that the zinc finger protein 36, C3H type-like 1 (ZFP36L1) enhances vascular endothelial growth factor (VEGF) mRNA decay through its interaction with AU-richelements within VEGF 3'-untranslated region. In this study, we evaluated the possibility to develop an antiangiogenic and antitumoral strategy using the mRNA-destabilizing activity of ZFP36L1. We engineered a cell-penetrating ZFP36L1, by fusing it to the protein transduction domains (PTDs) TAT derived from HIV, orthe polyarginine peptides R7 or R9. PTD-ZFP36L1 fusion proteins were expressed in bacterial cells and affinity-purified to homogeneity. TAT-, R7- and R9-ZFP36L1 were efficiently internalized into living cells and decreased both endogenous VEGF mRNA half-life and VEGF protein levels in vitro. Importantly, a single injection of R9-TIS11b fusion protein into a high-VEGF expressing tissue in vivo(in this study, the mouse adrenal gland) markedly decreased VEGF expression. We further evaluated the effect of R9-ZFP36L1 on tumor growth using Lewis Lung Carcinoma (LL/2) cells implanted subcutaneously into nude mice. Intratumoral injection of R9-ZFP36L1 significantly reduced tumor growth and markedly decreased the expression of multiple angiogenic and inflammatory cytokines, including VEGF, acidic fibroblast growth factor, tumor necrosis factor alpha, interleukin (IL)-1alpha and IL-6, with a concomitant obliteration of tumor vascularization. These findings indicate that R9-ZFP36L1 fusion protein may represent a novel antiangiogenic and antitumoral agent, and supports the emerging idea that modulation of mRNA stability represents a promising therapeutic approach to treat cancer.

A two-step Notch-dependant mechanism controls the selection of the polar cell pair in Drosophila oogenesis.

Author(s) : Vachias C, Couderc J, Grammont M,
Journal : Development
2010
Organisers control the patterning and growth of many tissues and organs. Correctly regulating the size of these organisers is crucial for proper differentiation to occur. Organiser activity in the epithelium of the Drosophilaovarian follicle resides in a pair of cells called polar cells. It is known thatthese two cells are selected from a cluster of equivalent cells. However, the mechanisms responsible for this selection are still unclear. Here, we present evidence that the selection of the two cells is not random but, by contrast, depends on an atypical two-step Notch-dependent mechanism. We show that this sequential process begins when one cell becomes refractory to Notch activation and is selected as the initial polar cell. This cell then produces a Delta signal that induces a high level of Notch activation in one other cell within the cluster. This Notch activity prevents elimination by apoptosis, allowing its selection as the second polar cell. Therefore, the mechanism used to select precisely two cells from among an equivalence group involves an inductive Delta signal that originates from one cell, itself unable to respond to Notch activation, and results in one other cell being selected to adopt the same fate.Given its properties, this two-step Notch-dependent mechanism represents a novelaspect of Notch action.

Alternative splicing and breast cancer

Author(s) : Dutertre M, Vagner S, Auboeuf D,
Journal : RNA Biol
2010

Antagonistic factors control the unproductive splicing of SC35 terminal intron.

Author(s) : Dreumont N, Hardy S, Behm-Ansmant I, Kister L, Branlant C, Stevenin J, Bourgeois C,
Journal : Nucleic Acids Res
2010
Alternative splicing is regulated in part by variations in the relative concentrations of a variety of factors, including serine/arginine-rich (SR) proteins. The SR protein SC35 self-regulates its expression by stimulating unproductive splicing events in the 3' untranslated region of its own pre-mRNA. Using various minigene constructs containing the terminal retained intron and flanking exons, we identified in the highly conserved last exon a number of exonic splicing enhancer elements responding specifically to SC35, and showed aninverse correlation between affinity of SC35 and enhancer strength. The enhancerregion, which is included in a long stem loop, also contains repressor elements,and is recognized by other RNA-binding proteins, notably hnRNP H protein and TARDNA binding protein (TDP-43). Finally, in vitro and in cellulo experiments indicated that hnRNP H and TDP-43 antagonize the binding of SC35 to the terminalexon and specifically repress the use of SC35 terminal 3' splice site. Our studyprovides new information about the molecular mechanisms of SC35-mediated splicing activation. It also highlights the existence of a complex network of self- and cross-regulatory mechanisms between splicing regulators, which controls their homeostasis and offers many ways of modulating their concentration in response to the cellular environment.

Antagonistic factors control the unproductive splicing of SC35 terminal intron

Author(s) : Dreumont N, Hardy S, Behm-Ansmant I, Kister L, Branlant C, St?venin J, Bourgeois C,
Journal : Nucleic Acids Res
2010

Clonal expansion of HTLV-1 positive CD8+ cells relies on cIAP-2 but not on c-FLIP expression

Author(s) : Zane L, Sibon D, Legras C, Lachuer J, Wierinckx A, Mehlen P, Delfau-Larue M, Gessain A, Gout O, Pinatel C, Lan?on A, Mortreux F, Wattel E,
Journal : Virology
2010

Clonal expansion of HTLV-1 positive CD8+ cells relies on cIAP-2 but not on c-FLIP expression.

Author(s) : Zane L, Sibon D, Legras C, Lachuer J, Wierinckx A, Mehlen P, Delfau-Larue M, Gessain A, Gout O, Pinatel C, Lancon A, Mortreux F, Wattel E,
Journal : Virology
2010
Here we investigate the mechanisms by which HTLV-1 infection prevents the cell death of CD8(+) T cells in vivo. We show that upon natural infection, cloned CD8(+) but not CD4(+) cells from patients without malignancy become resistant toFas-mediated cell death and acquire an antiapoptotic transcriptome that includesthe overexpression of cIAP-2 and c-FLIP(L). CD8(+) lymphocyte-restricted cIAP-2 overexpression correlates with resistance to Fas-mediated apoptosis and depends on tax expression via NF-KappaB. In contrast, in the same CD8(+) cells, the HTLV-1-dependent overexpression of c-FLIP(L) does not correlate with resistance to Fas-mediated cell death nor with tax expression. In the present model, infected CD8(+) clones are the only cell subtype in which cIAP-2 expression correlates with resistance to cell death. These results support a role for Tax-dependent cIAP-2 expression in preventing the death of naturally infected CD8(+) cells and thereby in their clonal expansion in vivo.

Complex patterns of gene expression in human T cells during in vivo aging.

Author(s) : Remondini D, Salvioli S, Francesconi M, Pierini M, Mazzatti D, Powell J, Zironi I, Bersani F, Castellani G, Franceschi C,
Journal : Mol Biosyst
2010
Human aging is associated with complex alterations that contribute to remodelling of physiological processes and ultimately manifests in loss of tissue/organ function. Peripheral blood T cells do not escape this phenomenon and undergo profound remodelling with aging. Thus, investigating the effects of aging on T cells transcriptomics and identifying the underlying regulatory mechanisms can be of extreme importance to understand the aging process in the Immune System (IS).To this aim, we performed an analysis of gene expression data of T cells collected from peripheral blood of 25 healthy human donors of different age from25 to more than 95 years, in order to characterize changes that occur throughoutthe entire adult lifespan. By means of microarray analysis, we observed large groups of genes exhibiting non-monotonic expression patterns over time: such behaviour, that could not be observed in typical "two-group" experiments (e.g. young vs. old people) highlights similarities in gene expression profiles of young and "successfully aged" individuals. Genes whose expression profiles change during lifespan were grouped into three main patterns (eigenmodes) to which different biological functions were significantly associated. The analysis of KEGG pathways to which these genes belong indicated that the biological processes altered in T cell aging are not only those typically associated with immune cells (Jak-STAT signalling, T cell receptor signalling, cytokine-cytokine receptor interactions, etc.) but also some not specific of immune cells, such as long-term depression, PPAR and mTOR signalling, glucose and glutathione metabolism, suggesting that T cell aging may be representative of a more generalised aging phenomenon. Thus, the T cell may represent a useful cellular model to study organismal aging. We further searched for over-represented transcription factor binding sites (TFBSs) in the promoter regions of genes clustered by similarity of their age-related patterns to evidence possible co-regulation. A comparison between over-representation of TFBSs and the time course of the corresponding transcription factor (TF) expression levels revealed that a restricted group of TFs may play a central role in driving aging-specific changes in gene expressionof T cells.

Conformational free-energy difference of a miniprotein from nonequilibrium simulations

Author(s) : Spichty M, Cecchini M, Karplus M,
Journal : The Journal of Physical Chemistry Letters
2010
Conformational free-energy differences are essential thermodynamic quantities for understanding the function of many biomolecules. They are accessible from computer simulations, but their accurate calculation is a challenging task. Here nonequilibrium computer simulations and the differential fluctuation theorem are used to evaluate the free-energy difference between two conformational states of a structured miniprotein, the β-hairpin of protein G, with an implicit treatment of the solvent. A molecular dynamics-based protocol is employed for the simulation of rapid switches between the conformational states in both the forward and the reverse direction. From the work performed on the system in the individual switches, the conformational free-energy difference is determined by use of the differential fluctuation theorem. The results are in excellent agreement with reference calculations from a long molecular dynamics simulation and from the confinement method. The nonequilibrium approach is a computationally efficient method for the calculation of conformational free-energy differences for biological systems.

Cotranscriptional exon skipping in the genotoxic stress response

Author(s) : Dutertre M, Sanchez G, De Cian M, Barbier J, Dardenne E, Gratadou L, Dujardin G, Le Jossic-Corcos C, Corcos L, Auboeuf D,
Journal : Nat Struct Mol Biol
2010

Drosophila I-R hybrid dysgenesis is associated with catastrophic meiosis and abnormal zygote formation.

Author(s) : Orsi G, Joyce E, Couble P, McKim K, Loppin B,
Journal : J Cell Sci
2010
The Drosophila I-R type of hybrid dysgenesis is a sterility syndrome (SF sterility) associated with the mobilization of the I retrotransposon in female germ cells. SF sterility results from a maternal-effect embryonic lethality whose origin has remained unclear since its discovery about 40 years ago. Here, we show that meiotic divisions in SF oocytes are catastrophic and systematically fail toproduce a functional female pronucleus at fertilization. As a consequence, most embryos from SF females rapidly arrest their development with aneuploid or damaged nuclei, whereas others develop as non-viable, androgenetic haploid embryos. Finally, we show that, in contrast to mutants affecting the biogenesis of piRNAs, SF egg chambers do not accumulate persistent DNA double-strand breaks, suggesting that I-element activity might perturb the functional organization of meiotic chromosomes without triggering an early DNA damage response.