Directory
2022
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3DGenBench: a web-server to benchmark computational models for 3D Genomics.
- 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.
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3DPolyS-LE: an accessible simulation framework to model the interplay between chromatin and loop extrusion.
- Journal : Bioinformatics
- 2022
- SUMMARY: Recent studies suggest that the loop extrusion activity of StructuralMaintenance of Chromosomes complexes is central to proper organization of genomesin vivo. Polymer physics-based modeling of chromosome structure has beeninstrumental to assess which structures such extrusion can create. Only fewlaboratories however have the technical and computational expertise to create insilico models combining dynamic features of chromatin and loop extruders. Here,we present 3DPolyS-LE, a self-contained, easy to use modeling and simulationframework allowing non-specialists to ask how specific properties of loopextruders and boundary elements impact on 3D chromosome structure. 3DPolyS-LEalso provides algorithms to compare predictions with experimental Hi-C data.AVAILABILITY AND IMPLEMENTATION: Software available athttps://gitlab.com/togop/3DPolyS-LE; implemented in Python and Fortran 2003 andsupported on any Unix-based operating system (Linux and Mac OS). SUPPLEMENTARYINFORMATION: Supplementary information are available at Bioinformatics online.
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A Perspective for Ménière's Disease: In Silico Investigations of Dexamethasone as a Direct Modulator of AQP2.
- 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.
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Altered splicing of ATG16-L1 mediates acquired resistance to tyrosine kinase inhibitors of EGFR by blocking autophagy in non-small cell lung cancer.
- 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.
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CD8 memory precursor cell generation is a continuous process
- Journal : iScience
- 2022
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Detection of Homologous Recombination Intermediates via Proximity Ligation and Quantitative PCR in Saccharomyces cerevisiae
- 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.
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DNA:RNA Immunoprecipitation from S. pombe Cells for qPCR and Genome-Wide Sequencing.
- 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.
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Dynamical modeling of the H3K27 epigenetic landscape in mouse embryonic stem cells.
- 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.
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Evidence for close molecular proximity between reverting and undifferentiated cells
- Journal : BMC Biol
- 2022
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Evolutionary divergence of anaphase spindle mechanics in nematode embryos constrained by antagonistic pulling and viscous forces.
- 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.
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FORK-seq: Single-Molecule Profiling of DNA Replication.
- 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.
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Genome-wide mapping of individual replication fork velocities using nanopore sequencing.
- 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.
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Global genome decompaction leads to stochastic activation of gene expression as a first step toward fate commitment in human hematopoietic cells.
- 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.
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Hematopoietic differentiation is characterized by a transient peak of entropy at a single-cell level
- Journal : BMC Biol
- 2022
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Keeping Cell Death Alive: An Introduction into the French Cell Death Research Network.
- Journal : Biomolecules
- 2022
- Since the Nobel Prize award more than twenty years ago for discovering the coreapoptotic pathway in C. elegans, apoptosis and various other forms of regulatedcell death have been thoroughly characterized by researchers around the world.Although many aspects of regulated cell death still remain to be elucidated inspecific cell subtypes and disease conditions, many predicted that research intocell death was inexorably reaching a plateau. However, this was not the casesince the last decade saw a multitude of cell death modalities being described,while harnessing their therapeutic potential reached clinical use in certaincases. In line with keeping research into cell death alive, francophoneresearchers from several institutions in France and Belgium established theFrench Cell Death Research Network (FCDRN). The research conducted by FCDRN is atthe leading edge of emerging topics such as non-apoptotic functions of apoptoticeffectors, paracrine effects of cell death, novel canonical and non-canonicalmechanisms to induce apoptosis in cell death-resistant cancer cells or regulatedforms of necrosis and the associated immunogenic response. Collectively, thesevarious lines of research all emerged from the study of apoptosis and in the nextfew years will increase the mechanistic knowledge into regulated cell death andhow to harness it for therapy.
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Loss of SET1/COMPASS methyltransferase activity reduces lifespan and fertility in Caenorhabditis elegans.
- 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.
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Myotonic dystrophy RNA toxicity alters morphology, adhesion and migration of mouse and human astrocytes
- 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.
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Painters in chromatin: a unified quantitative framework to systematically characterize epigenome regulation and memory.
- 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.
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Paternal transmission of the Wolbachia CidB toxin underlies cytoplasmic incompatibility.
- 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.
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Polymer Modeling of 3D Epigenome Folding: Application to Drosophila.
- 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.
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Prognostic impact of ABCA3 expression in adult and pediatric acute myeloid leukemia: an ALFA-ELAM02 joint study
- Journal : Blood Adv
- 2022
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Real age prediction from the transcriptome with RAPToR
- 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.
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RNA helicase-dependent gene looping impacts messenger RNA processing.
- 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.
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Spatial organization of chromosomes leads to heterogeneous chromatin motion and drives the liquid- or gel-like dynamical behavior of chromatin.
- 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.
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Structural mechanism underpinning Thermus oshimai Pif1-mediated G-quadruplex unfolding.
- 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.
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The trophectoderm acts as a niche for the inner cell mass through C/EBPα-regulated IL-6 signaling
- Journal : Stem Cell Reports
- 2022
- IL-6 has been shown to be required for somatic cell reprogramming into induced pluripotent stem cells (iPSCs). However, how Il6 expression is regulated and whether it plays a role during embryo development remains unknown. Here, we describe that IL-6 is necessary for C/EBPα-enhanced reprogramming of B cells into iPSCs but not for B cell to macrophage transdifferentiation. C/EBPα overexpression activates both Il6 and Il6ra genes in B cells and in PSCs. In embryo development, Cebpa is enriched in the trophectoderm of blastocysts together with Il6, while Il6ra is mostly expressed in the inner cell mass (ICM). In addition, Il6 expression in blastocysts requires Cebpa. Blastocysts secrete IL-6 and neutralization of the cytokine delays the morula to blastocyst transition. The observed requirement of C/EBPα-regulated IL-6 signaling for pluripotency during somatic cell reprogramming thus recapitulates a physiologic mechanism in which the trophectoderm acts as niche for the ICM through the secretion of IL-6.
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Three classes of epigenomic regulators converge to hyperactivate the essential maternal gene deadhead within a heterochromatin mini-domain.
- 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.
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Titration of Apparent In-Cellula Affinities of Protein-Protein Interactions.
- 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.
Link to PubMed entry