Biology subjects

Shirahige, K.

Publications and source records attributed to Shirahige, K..

At least 19 recordsLinked to original sources

bioRxiv2026

DNA catenation is essential for Sister Chromatid Cohesion

From S phase until anaphase, sister chromatids remain physically linked to ensure accurate chromosome segregation. This linkage, called sister chromatid cohesion, must withstand the pulling forces generated by the mitotic spindle and is thought to require entrapment of sister DNAs inside the ring-shaped cohesin complex. DNA catenations that arise naturally during replication could also tether sister chromatids, but whether they contribute to cohesion has remained unresolved. Here, using budding yeast, we show that cohesin-mediated sister DNA entrapment alone cannot withstand spindle-generated pulling forces and that robust sister chromatid cohesion requires DNA catenation. Selective removal of DNA catenations causes catastrophic cohesion loss, delays chromosome biorientation and increases chromosome mis-segregation, even when cohesin rings remain intact. Importantly, DNA catenation similarly underpins force-resistant cohesion in metazoan chromosomes. Our findings fundamentally redefine the physical basis of sister chromatid cohesion by establishing DNA catenation as an evolutionarily conserved component of the force-resistant linkage between sister chromatids that enables accurate chromosome segregation.

molecular biology↗

bioRxiv2026

Region-specific generation and retention of acetylated cohesin shape the genome-wide pattern of sister chromatid cohesion

Sister chromatid cohesion depends on ESCO2-mediated acetylation of the cohesin subunit SMC3, yet how this modification is established and maintained across the genome remains poorly understood. Here, we examined the chromatin-binding dynamics of ESCO2 and its enzymatic activity using quantitative ChIP-seq of human cells synchronously progressing through S phase. ESCO2 bound broadly along chromosomes without forming discrete peaks and preferentially localized to transcriptionally inactive regions, but was not confined to H3K9me3- or H3K27me3-enriched heterochromatin. Consistent with this binding pattern, ESCO2-dependent SMC3 acetylation showed a similarly broad distribution in these regions. In contrast, ESCO1, a paralog of ESCO2, primarily mediated SMC3 acetylation at cohesin peak sites. Notably, most of the broadly distributed, ESCO2-dependent SMC3 acetylation generated during S phase was progressively lost from chromatin after replication, whereas the fraction that persisted into late G2 accumulated in domains enriched for the repressive H3K27me3 modification. Our work identifies selective post-replicative retention of acetylated cohesin as a previously unappreciated step in shaping the genome-wide architecture of sister chromatid cohesion.

molecular biology↗

bioRxiv2025

Cohesin constrains histone modification-driven chromatin dynamics

Gene expressions are regulated by an interplay between epigenetics and spatial genome organization, the deregulation of which has been implicated in the development of cancers, including myeloid neoplasms. However, it is unclear how they coordinately contribute to normal and malignant hematopoiesis. Here, we show that simultaneous dysregulations of histone modifications and chromatin structures caused by mutations of the epigenetic modulator Asxl1 and cohesin subunit Stag2 cooperatively induce ectopic interactions between polycomb-regulated promoters and active enhancers, leading to the aberrant upregulation of hematopoietic stem cell related genes and development of myelodysplastic syndromes (MDS). De-repression of polycomb-regulated genes induces their translocation to the transcriptionally active loci, where active promoters and enhancers are assembled, in the absence of Stag2-mediated chromatin organization. Our findings revealed that cohesin counteracts histone modification-driven chromatin conformations, manifesting the coordinate roles of histone modifiers and cohesin in regulating genome architectures and gene expressions to prevent malignant transformation.

cancer biology↗

bioRxiv2025

Cohesin acts as a transcriptional gatekeeper by restraining pause-release to promote processive elongation

Cohesin organizes 3D chromatin architecture, including promoter-enhancer loops, yet its loss has surprisingly modest effects on steady-state gene expression. We address this paradox by demonstrating that cohesin acts at different stages of transcription. First, it promotes Pol II promoter recruitment by facilitating enhancer-promoter communication that maintains active promoter chromatin states. Second, it delays pause release by transiently associating with the transcriptional machinery during the pause-release transition. Kinetic modelling suggests that reduced Pol II recruitment and enhanced pause release have compensatory effects, contributing to minimal changes in steady-state gene expression across genes upon cohesin loss. In contrast, cohesin depletion impairs robust transcriptional induction in response to external stimuli. Moreover, cohesin ensures sufficient pausing duration as a quality-control-like step to promote elongation complex assembly and transcription processivity. Here, we show that cohesin regulates multiple steps of transcription, offering mechanistic insight into cohesin-related diseases, including cancers and cohesinopathies.

molecular biology↗

bioRxiv2024

Controlling mechanism of the Scc2-cohesin interaction to restrict peri-centromeric DNA loop expansion and facilitate mitotic chromosome segregation

Cohesin exhibits DNA loop extrusion activity when bound to an ATPase activator Scc2 (NIPBL in humans), thereby organizing higher-order chromosome folding. In budding yeast, the majority of the chromosome-bound cohesins lack association with Scc2. It remains unknown how the interaction between Scc2 and cohesin is regulated on the chromosome and what physiological consequences malfunction in this regulatory mechanism causes. Here, we show that simultaneous deletion of Wpl1 and Eco1, two of the known cohesin regulators, resulted in Scc2 co-localization with cohesin around the centromeres of metaphase chromosomes. In these cells, the pericentromeric DNA loops were enlarged to connect the centromere to the cohesin/Scc2 co-bound sites at distances of up to a few hundred kb, indicating highly active loop extrusion by the Scc2-associated cohesin. Furthermore, we demonstrated that {Delta}wpl1 {Delta}eco1 cells exhibited a delay in the progression of mitotic chromosome segregation, a phenotype dependent on the presence of Scc2 in metaphase. These findings suggest that Wpl1 and Eco1 cooperatively regulate Scc2-cohesin interaction on chromosomes, restrict the size of the pericentromeric DNA loops, and facilitate mitotic chromosome segregation.

molecular biology↗

bioRxiv2024

Cohesin regulates promoter-proximal pausing of RNA Polymerase II by limiting recruitment of super elongation complex

Cohesin is a ring-shaped complex, responsible for establishing sister chromatid cohesion and forming topologically associating domains (TADs) and chromatin loops. Loss-of-function mutations in cohesin subunits and its regulatory factors can cause Cornelia de Lange syndrome (CdLS). Because dysregulated gene expression was observed in CdLS, it has long been thought that cohesin plays a regulatory role in transcription. Here, we investigated the effect of acute cohesin depletion on transcription and observed that a small number of genes exhibited differential expression. Analysis of RNA polymerase II (Pol II) distribution revealed that the depletion reduced Pol II promoter binding and pausing simultaneously at the majority of genes. This implies that at most genes, the two decreases counterbalance each other, resulting in unchanged gene expression. Additionally, we find that cohesin loss increased promoter binding of super elongation complex (SEC), which mediates the release of Pol II from paused state. Moreover, the reduction in pausing caused by cohesin depletion was no longer observed when SEC was inhibited. These observations suggest that cohesin regulates Pol II pausing by restricting SEC recruitment to promoters. Together, our study demonstrates the involvement of cohesin in transcriptional regulation, particularly in Pol II pause and release.

molecular biology↗

bioRxiv2024

Strand asymmetry of DNA damage tolerance mechanisms

DNA damage tolerance mechanisms are crucial for timely and accurate chromosomal replication in response to DNA polymerase stalling. Ubiquitylation of the replicative sliding clamp PCNA drives major tolerance pathways, error-free homologous recombination template switching and error-prone translesion synthesis, though their dynamics at forks and pathway choice determinants are poorly understood. Using strand-specific genomics we revealed an asymmetric nature of tolerance pathways, characterized by preferential template switching-driven recombinase engagement of stalled nascent lagging strands and translesion synthesis usage in response to leading strand polymerase stalling. This asymmetry, determined by a strand-dynamic interplay between PCNA-ubiquitin writers and erasers, likely stems from necessities dictated by leading and lagging strand replication mechanisms and has implications for asymmetric mutation inheritance. One-Sentence SummaryDNA damage tolerance mechanisms respond asymmetrically to leading or lagging strand polymerase blocks.

molecular biology↗

bioRxiv2023

Loop-extruding Smc5/6 organizes transcription-induced positive DNA supercoils

The Structural Maintenance of Chromosome (SMC) protein complexes cohesin, condensin and the Smc5/6 complex (Smc5/6) are essential for chromosome function. At the molecular level, these complexes fold DNA by loop extrusion. Accordingly, cohesin creates chromosome loops in interphase, and condensin compacts mitotic chromosomes. However, the role of Smc5/6s recently discovered DNA loop extrusion activity is unknown. Here, we uncover that Smc5/6 associates with transcription-induced positively supercoiled chromosomal DNA at cohesin-dependent chromosome loop boundaries. Mechanistically, single-molecule imaging reveals that dimers of Smc5/6 specifically recognize the tip of positively supercoiled DNA plectonemes, and efficiently initiates loop extrusion to gather the supercoiled DNA into a large plectonemic loop. Finally, Hi-C analysis shows that Smc5/6 links chromosomal regions containing transcription-induced positive supercoiling in cis. Altogether, our findings indicate that Smc5/6 controls the three-dimensional organization of chromosomes by recognizing and initiating loop extrusion on positively supercoiled DNA.

molecular biology↗

bioRxiv2022

Context-dependent 3D genome regulation by cohesin and related factors

Cohesin plays vital roles in chromatin folding and gene expression regulation, cooperating with such factors as cohesin loaders, unloaders, acetyltransferase, and the insulation factor CTCF. Although various models of regulation have been proposed (e.g., loop extrusion), how cohesin and related factors collectively or individually regulate the hierarchical chromatin structure and gene expression remains unclear. In this study, we have depleted cohesin and related factors and then conducted a comprehensive evaluation of the resulting 3D genome, transcriptome and epigenome data. We observed substantial variation in depletion effects among factors at topologically associating domain (TAD) boundaries and on interTAD interactions, which were partly related to epigenomic status. Gene expression changes were highly correlated with direct cohesin binding and gain of TAD boundaries than with the loss of boundaries. Our results suggested that cohesin positively regulates gene expression, whereas other mechanisms (e.g., cohesin turnover and acetylation) add to the diversity of this pattern of dysregulation. Moreover, cohesin was broadly enriched in active compartment A, but not in compartment B, which were retained even after CTCF depletion. Our rich dataset and the subsequent data-driven analysis support the context-specific regulation of chromatin folding by cohesin and related factors.

genomics↗

bioRxiv2021

Large-scale multi-omics analysis suggests specific roles for intragenic cohesin in transcriptional regulation

Cohesin, an essential protein complex for chromosome segregation, regulates transcription through a variety of mechanisms. It is not a trivial task to genome-widely assign the diverse cohesin functions. Moreover, the context-specific roles of cohesin-mediated interactions, especially on intragenic regions, have not been thoroughly investigated. Here we performed a comprehensive characterization of cohesin binding sites in several human cell types. We integrated epigenomic, transcriptomic and chromatin interaction data with and without transcriptional stimulation, to explore context-specific functions of intragenic cohesin related to gene activation. We identified a new subset of cohesin binding sites, decreased intragenic cohesin sites (DICs), which have a different function from previously known ones. The intron-enriched DICs were negatively correlated with transcriptional regulation: a subgroup of DICs were related to enhancer markers and paused RNA polymerase II, whereas others contributed to chromatin architecture. We implemented machine learning and successfully isolated DICs with distinct genomic features. We observed DICs in various cell types, including cells from cohesinopathy patients. These results suggest a previously unidentified function of cohesin at intragenic regions for transcription regulation.

genomics↗

bioRxiv2021

Codependency and mutual exclusivity for gene community detection from sparse single-cell transcriptome data

Single-cell RNA-seq (scRNA-seq) can be used to characterize cellular heterogeneity in thousands of cells. The reconstruction of a gene network based on coexpression patterns is a fundamental task in scRNA-seq analyses, and the mutual exclusivity of gene expression can be critical for understanding such heterogeneity. Here, we propose an approach for detecting communities from a genetic network constructed on the basis of coexpression properties. The community-based comparison of multiple coexpression networks enables the identification of functionally related gene clusters that cannot be fully captured through differential gene expression-based analysis. We also developed a novel metric referred to as the exclusively expressed index (EEI) that identifies mutually exclusive gene pairs from sparse scRNA-seq data. EEI quantifies and ranks the exclusive expression levels of all gene pairs from binary expression patterns while maintaining robustness against a low sequencing depth. We applied our methods to glioblastoma scRNA-seq data and found that gene communities were partially conserved after serum stimulation despite a considerable number of differentially expressed genes. We also demonstrate that the identification of mutually exclusive gene sets with EEI can improve the sensitivity of capturing cellular heterogeneity. Our methods complement existing approaches and provide new biological insights, even for a large, sparse dataset, in the single-cell analysis field.

bioinformatics↗

bioRxiv2020

Transcription and replication organize cohesin-dependent chromosome loops

Genome function and stability is strictly dependent on regulated folding of chromosomes in space and time. Chromosome loop formation by the protein complex cohesin is a central feature of this multilayer organization1. Accumulating evidence indicates that cohesin creates loops by extruding chromosomal DNA through its ring-like structure in a process that is controlled by the cohesin loading factor Scc2 and the unloader Wpl11-4. Cohesins chromosomal positioning is affected by transcription in both yeast and human cells5,6, and the complex localizes in the vicinity of replication forks7. However, if transcription directly influences chromosome looping remains an unresolved question, and the threedimensional organization of replicating chromosomes is unknown. Here we show that transcription and replication machineries create chromosome loop boundaries. We find that drug-induced depletion of chromosome-bound RNA polymerases triggers a rapid expansion of cohesin-dependent chromosome loops in the budding yeast Saccharomyces cerevisiae. New loop boundaries also form at a few highly expressed genes induced by the cellular stress response caused by the drug. The results also reveal that S-phase chromosomes are shaped by cohesin-dependent loops organized by transcription, with additional loop anchors at replication forks. Together, our results show that replication and transcription control the three-dimensional organization of the genome by blocking the progression of loop-forming cohesin. They also open for the possibility that the resulting positioning of loop-forming cohesin in the vicinity of transcription and replication machineries is part of cohesins functions in transcription control, sister chromatid cohesion and maintenance of fork stability.

molecular biology↗

bioRxiv2019

Synovial fibroblasts contribute to the genetic risk of rheumatoid arthritis through the synergistic action of cytokines

ObjectivesSynovial fibroblasts (SFs) produce a variety of pathogenic molecules in the inflamed synovium of rheumatoid arthritis (RA). We aimed to gain insight into the pathogenic mechanisms of SFs through elucidating the genetic contribution to molecular regulatory networks under inflammatory condition. MethodsSFs from RA and osteoarthritis (OA) patients (n=30 each) were stimulated with 8 different cytokines (IFN-, IFN-{gamma}, TNF-, IL-1{beta}, IL-6/sIL-6R, IL-17, TGF-{beta}1, IL-18) or a combination of all 8 (8-mix). Peripheral blood mononuclear cells (PBMCs) from the same patients were fractioned into five major immune cell subsets (CD4+ T cells, CD8+ T cells, B cells, NK cells, monocytes). Integrative analyses including mRNA expression, histone modifications (H3K27ac, H3K4me1, H3K4me3), 3D genome architecture and genetic variations of SNPs were performed. ResultsSFs exposed to synergistically acting cytokines produced markedly higher levels of pathogenic molecules, including CD40 whose expression was significantly affected by a RA risk SNP (rs6074022). Upon chromatin remodeling in activated SFs, RA risk loci were enriched in clusters of enhancers (super-enhancers; SEs) induced by synergistic proinflammatory cytokines. A RA risk SNP (rs28411362), located in a SE under synergistically acting cytokines, formed three-dimensional contact with the promoter of MTF1 gene, whose binding motif showed significant enrichment in stimulation specific-SEs. Consistently, inhibition of MTF1 suppressed cytokine and chemokine production from SFs and ameliorated mice model of arthritis. ConclusionsOur findings established the dynamic landscape of activated SFs, and yielded potential therapeutic targets associated with genetic risk of RA. Key messagesO_ST_ABSWhat is already known about this subject?C_ST_ABSO_LIIn rheumatoid arthritis (RA), a variety of dysregulated molecules from immune cells and mesenchymal cells drive disease progression. Synovial fibroblasts (SFs), the most abundant resident mesenchymal cells in the inflamed synovium, produce a variety of pathogenic molecules including IL-6. C_LIO_LIGenome-wide association studies (GWAS) have identified more than 100 RA susceptibility loci. To gain insight into the pathogenic mechanisms of SFs, understanding the genetic contribution to molecular regulatory networks under inflammatory condition is crucial. C_LI What does this study add?O_LIIntegrated analyses of activated SFs demonstrated that SFs exposed to synergistically acting cytokines produced markedly higher levels of pathogenic molecules. Some of which were significantly affected by RA risk loci in a stimulation-specific manner. C_LIO_LIChromatin remodeling induced by synergistic proinflammatory cytokines were associated with RA heritability. Some transcription factors (MTF1, RUNX1) could be crucial for this structural rearrangement and the formation of inflammatory arthritis. C_LI How might this impact on clinical practice or future developments?O_LIOur findings established the dynamic landscape of activated SFs, and yielded potential therapeutic targets associated with genetic risk of RA. C_LI

immunology↗

bioRxiv2019

Transcription-driven cohesin repositioning rewires chromatin loops in cellular senescence

Senescence is a phenotypic state of stable proliferative arrest, typically occurring in lineage-committed cells and triggered by various stimuli. It is generally accompanied by activation of a secretory program (senescence-associated secretory phenotype, SASP), which modulates both local (tissue microenvironment) and systemic (ageing) homeostasis1,2. Enhancer-promoter interactions play a key role in gene regulation3-5, facilitated by chromatin loops, mostly formed via CCCTC binding factor (CTCF) and cohesin tethering6-8. The three-dimensional chromatin structure of senescent cells has been characterised9-11 mostly in terms of macro-domain structures, but its relevance in gene expression remains elusive. Here, we use Hi-C and capture Hi-C12,13 to show that oncogenic HRAS-induced senescence (RIS) in human diploid fibroblasts (HDFs) is accompanied by extensive enhancer-promoter rewiring, which is closely connected with dynamic cohesin binding to the genome. We find de novo cohesin peaks at the 3 end of a subset of active genes, reminiscent of the transcription-driven cohesin islands recently discovered in mouse embryonic fibroblasts deficient in both CTCF and the cohesin release factor Wings apart-like (Wapl)14. RIS de novo cohesin peaks are also transcription-dependent and enriched for SASP genes, as exemplified by IL1B, where de novo cohesin binding is involved in new loop formation. Cytokine induction is associated with similar cohesin islands appearance and enhancer-promoter rewiring during the terminal differentiation of monocytes to macrophages15, but not upon acute TNF treatment of HDFs16. These results suggest that RIS represents a fate-determined process in which gene expression is regulated beyond the cell type specific 3D chromatin framework, in part through cohesin redistribution.

molecular biology↗

bioRxiv2019

Comprehensive epigenome characterization reveals diverse transcriptional regulation across human vascular endothelial cells

BackgroundEndothelial cells (ECs) make up the innermost layer throughout the entire vasculature. Their phenotypes and physiological functions are initially regulated by developmental signals and extracellular stimuli. The underlying molecular mechanisms responsible for the diverse phenotypes of ECs from different organs are not well understood.\n\nResultsTo characterize the transcriptomic and epigenomic landscape in the vascular system, we cataloged gene expression and active histone marks in nine types of human ECs (generating 148 genome-wide datasets) and carried out a comprehensive analysis with chromatin interaction data. We identified 3,765 EC-specific enhancers, some of which were associated with disease-associated genetic variations. We also identified various candidate marker genes for each EC type. Notably, reflecting the developmental origins of ECs and their roles in angiogenesis, vasculogenesis and wound healing.\n\nConclusionsWhile the importance of several HOX genes for early vascular development and adult angiogenesis in pathological conditions has been reported, a systematic analysis of the regulation and roles of HOX genes in mature tissue cells has been lacking. These datasets provide a valuable resource for understanding the vascular system and associated diseases.

genomics↗

bioRxiv2019

Dissecting the functions of NIPBL using genome editing: The importance of the N-terminus of NIPBL in transcriptional regulation

Cornelia de Lange syndrome (CdLS) is characterized by craniofacial dysmorphisms, intellectual disabilities, growth retardation, and several other systemic abnormalities. CdLS is caused by heterozygous germline mutations in structural and regulatory components of cohesin. Mutations in NIPBL, which encodes regulatory subunit of cohesin, are frequently found in individuals with CdLS. CdLS is associated with a currently unknown mechanism of global transcriptional dysregulation. In this study, NIPBL mutants were generated using the CRISPR/Cas9 system to study this mechanism. Clones with a biallelic frameshift mutation in exon 3 of NIPBL, resulting in a truncated N-terminus, displayed transcriptional dysregulation without sister chromatid separation defects. Detailed transcriptome analysis revealed the overexpression of genes in NIPBL mutants that are typically expressed at low levels in wild type and the reduced expression of genes that are typically expressed at high levels in wild type. This result suggested that NIPBL plays a role in fine-tuning gene expression levels. MAU2 protein, that closely interacts with NIPBL, was nearly absent in these clones. The reduction of MAU2 observed in NIPBL mutants points to the importance of the NIPBL N-terminus/MAU2 interaction in transcriptional regulatory role of NIPBL.

genetics↗

bioRxiv2017

Sensitive and robust assessment of ChIP-seq read distribution using a strand-shift profile

AbstractChromatin immunoprecipitation followed by sequencing (ChIP-seq) can detect read-enriched DNA loci for point-source (e.g., transcription factor binding) and broad-source factors (e.g., various histone modifications). Although numerous quality metrics for ChIP-seq data have been developed, the peaks thus obtained are still difficult to assess with respect to signal-to-noise ratio (S/N) and the percentage of false positives.\n\nWe developed a quality-assessment tool for ChIP-seq data, SSP (strand-shift profile), that quantifies S/N and peak reliability without peak calling. We validated SSP in-depth using [≥] 1,000 publicly available ChIP-seq datasets along with virtual data to demonstrate that SSP is quantifiable and sensitive to different S/Ns for both pointand broad-source factors. Moreover, SSP is consistent among cell types and with respect to variance of sequencing depth, and identifies low-quality samples that cannot be identified by quality metrics currently available. Finally, we show that \"hidden-duplicate reads\" cause aberrantly high S/Ns, and SSP provides an additional metric to avoid them, which can also contribute to estimation of peak mode (pointor broad-source) of samples.\n\nAvailabilityhttps://github.com/rnakato/SSP\"

bioinformatics↗

bioRxiv2017

Functional Annotation of Chemical Libraries across Diverse Biological Processes

Chemical-genetic approaches offer the potential for unbiased functional annotation of chemical libraries. Mutations can alter the response of cells to a compound, revealing chemical-genetic interactions that can elucidate a compounds mode of action. We developed a highly parallel and unbiased yeast chemical-genetic screening system involving three key components. First, in a drug-sensitive genetic background, we constructed an optimized, diagnostic mutant collection that is predictive all major yeast biological processes. Second, we implemented a multiplexed (768-plex) barcode sequencing protocol, enabling assembly of thousands of chemical-genetic profiles. Finally, based on comparison of the chemical-genetic profiles with a compendium of genome-wide genetic interaction profiles, we predicted compound functionality. Applying this high-throughput approach, we screened 7 different compound libraries and annotated their functional diversity. We further validated biological process predictions, prioritized a diverse set of compounds, and identified compounds that appear to have dual modes of action.

systems biology↗