Directory
2019
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A cohesin/HUSH- and LINC-dependent pathway controls ribosomal DNA double-strand break repair.
- 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.
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A genome-wide screen identifies IRF2 as a key regulator of caspase-4 in human cells
- Journal : EMBO Rep
- 2019
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An automated Bayesian pipeline for rapid analysis of single-molecule binding data
- Journal : Nat Commun
- 2019
- Single-molecule binding assays enable the study of how molecular machines assemble and function. Current algorithms can identify and locate individual molecules, but require tedious manual validation of each spot. Moreover, no solution for high-throughput analysis of single-molecule binding data exists. Here, we describe an automated pipeline to analyze single-molecule data over a wide range of experimental conditions. In addition, our method enables state estimation on multivariate Gaussian signals. We validate our approach using simulated data, and benchmark the pipeline by measuring the binding properties of the well-studied, DNA-guided DNA endonuclease, TtAgo, an Argonaute protein from the Eubacterium Thermus thermophilus. We also use the pipeline to extend our understanding of TtAgo by measuring the protein's binding kinetics at physiological temperatures and for target DNAs containing multiple, adjacent binding sites.
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Calibration, Selection and Identifiability Analysis of a Mathematical Model of the in vitro Erythropoiesis in Normal and Perturbed Contexts
- Journal : In Silico Biol
- 2019
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Characterizing the interplay between gene nucleotide composition bias and splicing
- Journal : Genome Biol
- 2019
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Detecting adaptive convergent amino acid evolution.
- 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'.
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Developmental and comparative transcriptomic identification of iridophore contribution to white barring in clownfish.
- 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.
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Dynamic processing of displacement loops during recombinational DNA repair
- 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.
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Erythroid differentiation displays a peak of energy consumption concomitant with glycolytic metabolism rearrangements
- Journal : PLoS One
- 2019
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Genome editing in primary cells and in vivo using viral-derived Nanoblades loaded with Cas9-sgRNA ribonucleoproteins.
- 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.
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Global chromatin conformation differences in the Drosophila dosage compensated chromosome X.
- 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.
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High-Throughput Analysis Reveals Rules for Target RNA Binding and Cleavage by AGO2
- Journal : Mol Cell
- 2019
- Argonaute proteins loaded with microRNAs (miRNAs) or small interfering RNAs (siRNAs) form the RNA-induced silencing complex (RISC), which represses target RNA expression. Predicting the biological targets, specificity, and efficiency of both miRNAs and siRNAs has been hamstrung by an incomplete understanding of the sequence determinants of RISC binding and cleavage. We applied high-throughput methods to measure the association kinetics, equilibrium binding energies, and single-turnover cleavage rates of mouse AGO2 RISC. We find that RISC readily tolerates insertions of up to 7 nt in its target opposite the central region of the guide. Our data uncover specific guide:target mismatches that enhance the rate of target cleavage, suggesting novel siRNA design strategies. Using these data, we derive quantitative models for RISC binding and target cleavage and show that our in vitro measurements and models predict knockdown in an engineered cellular system.
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Histone Methylation and Memory of Environmental Stress.
- 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.
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Homologous recombination and the formation of complex genomic rearrangements
- 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.
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Interplay between coding and exonic splicing regulatory sequences
- Journal : Genome Res
- 2019
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Is WDR45 the missing link for ER stress-induced autophagy in beta-propeller associated neurodegeneration?
- 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.
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Males as somatic investment in a parthenogenetic nematode.
- 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.
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MicroRNAs tame CRISPR-Cas9
- Journal : Nat Cell Biol
- 2019
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Model-Based Assessment of the Role of Uneven Partitioning of Molecular Content on Heterogeneity and Regulation of Differentiation in CD8 T-Cell Immune Responses
- Journal : Front Immunol
- 2019
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Modeling Edar expression reveals the hidden dynamics of tooth signaling center patterning
- Journal : PLOS Biology
- 2019
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Moving forward one step back at a time: reversibility during homologous recombination
- 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.
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Physical and functional interaction between SET1/COMPASS complex component CFP-1 and a Sin3S HDAC complex in C. elegans.
- 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.
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Pluripotent Stem Cell-Based Drug Screening Reveals Cardiac Glycosides as Modulators of Myotonic Dystrophy Type 1
- Journal : iScience
- 2019
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Regulation of Numb during planar cell polarity establishment in the Drosophila eye
- 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.
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Rouse model with transient intramolecular contacts on a timescale of seconds recapitulates folding and fluctuation of yeast chromosomes.
- 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.
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Senataxin homologue Sen1 is required for efficient termination of RNA polymerase III transcription.
- 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.
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Senataxin homologue Sen1 is required for efficient termination of RNA polymerase III transcription.
- 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.
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Single cell RNA-seq identifies the origins of heterogeneity in efficient cell transdifferentiation and reprogramming
- Journal : eLife
- 2019
Link to PubMed entry