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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.

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.

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.

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.

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.