Cohesin Binding Sites Research Articles

Comprehensive multiomics analyses reveal pervasive involvement of aberrant cohesin binding in transcriptional and chromosomal disorder of cancer cells

Chromatin organization, whose malfunction causes various diseases including cancer, is fundamentally controlled by cohesin. While cancer cells have been found with mutated or misexpressed cohesin genes, there is no comprehensive survey about the presence and role of abnormal cohesin binding in cancer cells. Here, we systematically identified ∼1% of cohesin-binding sites (701-2,633) as cancer-aberrant binding sites of cohesin (CASs). We integrated CASs with large-scale transcriptomics, epigenomics, 3D genomics, and clinical information. CASs represent tissue-specific epigenomic signatures enriched for cancer-dysregulated genes with functional and clinical significance. CASs exhibited alterations in chromatin compartments, loops within topologically associated domains, and cis-regulatory elements, indicating that CASs induce dysregulated genes through misguided chromatin structure. Cohesin depletion data suggested that cohesin binding at CASs actively regulates cancer-dysregulated genes. Overall, our comprehensive investigation suggests that aberrant cohesin binding is an essential epigenomic signature responsible for dysregulated chromatin structure and transcription in cancer cells.

Contribution of variant subunits and associated factors to genome-wide distribution and dynamics of cohesin

BackgroundThe cohesin complex organizes the genome-forming dynamic chromatin loops that impact on all DNA-mediated processes. There are two different cohesin complexes in vertebrate somatic cells, carrying the STAG1 or STAG2 subunit, and two versions of the regulatory subunit PDS5, PDS5A and PDS5B. Mice deficient for any of the variant subunits are embryonic lethal, which indicates that they are not functionally redundant. However, their specific behavior at the molecular level is not fully understood.ResultsThe genome-wide distribution of cohesin provides important information with functional consequences. Here, we have characterized the distribution of cohesin subunits and regulators in mouse embryo fibroblasts (MEFs) either wild type or deficient for cohesin subunits and regulators by chromatin immunoprecipitation and deep sequencing. We identify non-CTCF cohesin-binding sites in addition to the commonly detected CTCF cohesin sites and show that cohesin-STAG2 is the preferred variant at these positions. Moreover, this complex has a more dynamic association with chromatin as judged by fluorescence recovery after photobleaching (FRAP), associates preferentially with WAPL and is more easily extracted from chromatin with salt than cohesin-STAG1. We observe that both PDS5A and PDS5B are exclusively located at cohesin-CTCF positions and that ablation of a single paralog has no noticeable consequences for cohesin distribution while double knocked out cells show decreased accumulation of cohesin at all its binding sites. With the exception of a fraction of cohesin positions in which we find binding of all regulators, including CTCF and WAPL, the presence of NIPBL and PDS5 is mutually exclusive, consistent with our immunoprecipitation analyses in mammalian cell extracts and previous results in yeast.ConclusionOur findings support the idea that non-CTCF cohesin-binding sites represent sites of cohesin loading or pausing and are preferentially occupied by the more dynamic cohesin-STAG2. PDS5 proteins redundantly contribute to arrest cohesin at CTCF sites, possibly by preventing binding of NIPBL, but are not essential for this arrest. These results add important insights towards understanding how cohesin regulates genome folding and the specific contributions of the different variants that coexist in the cell.

CohesinDB: a comprehensive database for decoding cohesin-related epigenomes, 3D genomes and transcriptomes in human cells.

Cohesin is a multifunctional protein responsible for transcriptional regulation and chromatin organization. Cohesin binds to chromatin at tens of thousands of distinct sites in a conserved or tissue-specific manner, whereas the function of cohesin varies greatly depending on the epigenetic properties of specific chromatin loci. Cohesin also extensively mediates cis-regulatory modules (CRMs) and chromatin loops. Even though next-generation sequencing technologies have provided a wealth of information on different aspects of cohesin, the integration and exploration of the resultant massive cohesin datasets are not straightforward. Here, we present CohesinDB (https://cohesindb.iqb.u-tokyo.ac.jp), a comprehensive multiomics cohesin database in human cells. CohesinDB includes 2043 epigenomics, transcriptomics and 3D genomics datasets from 530 studies involving 176 cell types. By integrating these large-scale data, CohesinDB summarizes three types of 'cohesin objects': 751 590 cohesin binding sites, 957 868 cohesin-related chromatin loops and 2229 500 cohesin-related CRMs. Each cohesin object is annotated with locus, cell type, classification, function, 3D genomics and cis-regulatory information. CohesinDB features a user-friendly interface for browsing, searching, analyzing, visualizing and downloading the desired information. CohesinDB contributes a valuable resource for all researchers studying cohesin, epigenomics, transcriptional regulation and chromatin organization.

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 assign diverse cohesin functions. Moreover, the context-specific roles of cohesin-mediated interactions, especially on intragenic regions, have not been thoroughly investigated. Here we perform a comprehensive characterization of cohesin binding sites in several human cell types. We integrate epigenomic, transcriptomic and chromatin interaction data to explore the context-specific functions of intragenic cohesin related to gene activation. We identify a specific subset of cohesin binding sites, decreased intragenic cohesin sites (DICs), which are negatively correlated with transcriptional regulation. A subgroup of DICs is enriched with enhancer markers and RNA polymerase II, while the others are more correlated to chromatin architecture. DICs are observed in various cell types, including cells from patients with cohesinopathy. We also implement machine learning to our data and identified genomic features for isolating DICs from all cohesin sites. These results suggest a previously unidentified function of cohesin on intragenic regions for transcriptional regulation.

Chromosomal localization of cohesin is differentially regulated by WIZ, WAPL, and G9a

BackgroundThe cohesin complex is essential for proper chromosome structure and gene expression. Defects in cohesin subunits and regulators cause changes in cohesin complex dynamics and thereby alter three-dimensional genome organization. However, the molecular mechanisms that drive cohesin localization and function remain poorly understood.ResultsIn this study, we observe that loss of WIZ causes changes to cohesin localization that are distinct from loss of the known WIZ binding partner G9a. Whereas loss of WIZ uniformly increases cohesin levels on chromatin at known binding sites and leads to new, ectopic cohesin binding sites, loss of G9a does not. Ectopic cohesin binding on chromatin after the loss of WIZ occurs at regions that are enriched for activating histone modifications and transcription factors motifs. Furthermore, loss of WIZ causes changes in cohesin localization that are distinct from those observed by loss of WAPL, the canonical cohesin unloading factor.ConclusionsThe evidence presented here suggests that WIZ can function independently from its previously identified role with G9a and GLP in heterochromatin formation. Furthermore, while WIZ limits the levels and localization pattern of cohesin across the genome, it appears to function independently of WAPL-mediated cohesin unloading.

The three-dimensional structure of Epstein-Barr virus genome varies by latency type and is regulated by PARP1 enzymatic activity

Epstein-Barr virus (EBV) persists in human B-cells by maintaining its chromatinized episomes within the nucleus. We have previously shown that cellular factor Poly [ADP-ribose] polymerase 1 (PARP1) binds the EBV genome, stabilizes CTCF binding at specific loci, and that PARP1 enzymatic activity correlates with maintaining a transcriptionally active latency program. To better understand PARP1’s role in regulating EBV latency, here we functionally characterize the effect of PARP enzymatic inhibition on episomal structure through in situ HiC mapping, generating a complete 3D structure of the EBV genome. We also map intragenomic contact changes after PARP inhibition to global binding of chromatin looping factors CTCF and cohesin across the EBV genome. We find that PARP inhibition leads to fewer total unique intragenomic interactions within the EBV episome, yet new chromatin loops distinct from the untreated episome are also formed. This study also illustrates that PARP inhibition alters gene expression at the regions where chromatin looping is most effected. We observe that PARP1 inhibition does not alter cohesin binding sites but does increase its frequency of binding at those sites. Taken together, these findings demonstrate that PARP has an essential role in regulating global EBV chromatin structure and latent gene expression.

A Robust Protocol for Investigating the Cohesin Complex by ChIP-Sequencing.

The investigation of cohesin binding sites throughout different mammalian genomes by ChIP-sequencing has been fundamental to discover how cohesin and CTCF collaborate to form chromatin loops and to gain insight in the intricate regulation of cohesin. Here we describe a detailed ChIP protocol that has been successfully used for different cohesin subunits and cohesin regulators in various cell lines.

Assessing acute myeloid leukemia susceptibility in rearrangement-driven patients by DNA breakage at topoisomerase II and CCCTC-binding factor/cohesin binding sites.

An initiating DNA double strand break (DSB) event precedes the formation of cancer-driven chromosomal abnormalities, such as gene rearrangements. Therefore, measuring DNA breaks at rearrangement-participating regions can provide a unique tool to identify and characterize susceptible individuals. Here, we developed a highly sensitive and low-input DNA break mapping method, the first of its kind for patient samples. We then measured genome-wide DNA breakage in normal cells of acute myeloid leukemia (AML) patients with KMT2A (previously MLL) rearrangements, compared to that of nonfusion AML individuals, as a means to evaluate individual susceptibility to gene rearrangements. DNA breakage at the KMT2A gene region was significantly greater in fusion-driven remission individuals, as compared to nonfusion individuals. Moreover, we identified select topoisomerase II (TOP2)-sensitive and CCCTC-binding factor (CTCF)/cohesin-binding sites with preferential DNA breakage in fusion-driven patients. Importantly, measuring DSBs at these sites, in addition to the KMT2A gene region, provided greater predictive power when assessing individual break susceptibility. We also demonstrated that low-dose etoposide exposure further elevated DNA breakage at these regions in fusion-driven AML patients, but not in nonfusion patients, indicating that these sites are preferentially sensitive to TOP2 activity in fusion-driven AML patients. These results support that mapping of DSBs in patients enables discovery of novel break-prone regions and monitoring of individuals susceptible to chromosomal abnormalities, and thus cancer. This will build the foundation for early detection of cancer-susceptible individuals, as well as those preferentially susceptible to therapy-related malignancies caused by treatment with TOP2 poisons.

High-resolution, genome-wide mapping of positive supercoiling in chromosomes.

Supercoiling impacts DNA replication, transcription, protein binding to DNA, and the three-dimensional organization of chromosomes. However, there are currently no methods to directly interrogate or map positive supercoils, so their distribution in genomes remains unknown. Here, we describe a method, GapR-seq, based on the chromatin immunoprecipitation of GapR, a bacterial protein that preferentially recognizes overtwisted DNA, for generating high-resolution maps of positive supercoiling. Applying this method to Escherichia coli and Saccharomyces cerevisiae, we find that positive supercoiling is widespread, associated with transcription, and particularly enriched between convergently oriented genes, consistent with the 'twin-domain' model of supercoiling. In yeast, we also find positive supercoils associated with centromeres, cohesin-binding sites, autonomously replicating sites, and the borders of R-loops (DNA-RNA hybrids). Our results suggest that GapR-seq is a powerful approach, likely applicable in any organism, to investigate aspects of chromosome structure and organization not accessible by Hi-C or other existing methods.

Detecting chromatin interactions between and along sister chromatids with SisterC.

Chromosome segregation requires both compaction and disentanglement of sister chromatids. We describe SisterC, a chromosome conformation capture assay that distinguishes interactions between and along identical sister chromatids. SisterC employs BrdU incorporation during S-phase to label newly replicated strands, followed by Hi-C and then the destruction of BrdU-containing strands by UV/Hoechst treatment. After sequencing of the remaining intact strands, this allows for assignment of Hi-C products as inter- and intra-sister interactions based on the strands that reads are mapped to. We performed SisterC on mitotic S. cerevisiae cells. We find precise alignment of sister chromatids at centromeres. Along arms, sister chromatids are less precisely aligned with inter-sister connections every ~35kb. Inter-sister interactions occur between cohesin binding sites that often are offset by 5 to 25kb. Along sister chromatids, cohesin forms loops of up to 50kb. SisterC allows study of the complex interplay between sister chromatid compaction and their segregation during mitosis.

Role of chromosomal architecture in germinal center B cells and lymphomagenesis.

Chromatin organization during interphase is nonrandom, and dictated by a delicate equilibrium between biophysics, transcription factor expression, and topological regulators of the chromatin. Emerging evidence demonstrate a role for chromosomal conformation at different stages of B-cell development. In the present review, we provide an updated picture of the current knowledge regarding how chromosomal conformation regulates the B-cell phenotype and how disruption of this architecture could lead to B-cell lymphoma. B-cell development requires proper assembly of a rearranged VDJ locus, which will determine antigen receptor specificity. Recently, evidence pointed to a role for topological regulators during VDJ recombination. Research studies also demonstrated a link between shifts in nuclear chromosomal architecture during B-cell activation and in formation of germinal centers, which is required for immunoglobulin affinity maturation. Class-switch recombination was shown to be dependent on the presence of topology regulators. Loss of topological insulation of enhancers may lead to oncogene activation, suggesting that misfolding of chromatin may constitute a new epigenetic mechanism of malignant transformation. Finally, CCCTC-binding factor and cohesin binding sites have shown a higher probability of mutations and translocations in lymphomas, lending further support to the potential role of chromatin architecture in cancer development. Chromosomal conformation is now recognized as a key feature in the development of a robust humoral immune response. Several examples from the literature show that dysregulation of chromosomal architecture may be a foundational event during malignancy. Therefore, understanding the mechanisms that regulate chromosomal folding and drive gene activation are instrumental for a better understanding of immune regulation and lymphomagenesis.

From Cellular Diversity to the Roots of Variability

From Cellular Diversity to the Roots of Variability

Hemimethylation drives chromatin assembly

Molecular Biology Cytosine DNA methylation is a heritable and essential epigenetic mark. During DNA replication, cytosines on mother strands remain methylated, but those on daughter strands are initially unmethylated. These hemimethylated sites are rapidly methylated to maintain faithful methylation patterns. Xu and Corces mapped genome-wide strand-specific DNA methylation sites on nascent chromatin, confirming such maintenance in the vast majority of the DNA methylome (see the Perspective by Sharif and Koseki). However, they also identified a small fraction of sites that were stably hemimethylated and showed their inheritance at CTCF (CCCTC-binding factor)/cohesin binding sites. These inherited hemimethylation sites were required for CTCF and cohesin to establish proper chromatin interactions. Science , this issue p. [1166][1]; see also p. [1102][2] [1]: /lookup/doi/10.1126/science.aan5480 [2]: /lookup/doi/10.1126/science.aat0789

Nascent DNA methylome mapping reveals inheritance of hemimethylation at CTCF/cohesin sites.

The faithful inheritance of the epigenome is critical for cells to maintain gene expression programs and cellular identity across cell divisions. We mapped strand-specific DNA methylation after replication forks and show maintenance of the vast majority of the DNA methylome within 20 minutes of replication and inheritance of some hemimethylated CpG dinucleotides (hemiCpGs). Mapping the nascent DNA methylome targeted by each of the three DNA methyltransferases (DNMTs) reveals interactions between DNMTs and substrate daughter cytosines en route to maintenance methylation or hemimethylation. Finally, we show the inheritance of hemiCpGs at short regions flanking CCCTC-binding factor (CTCF)/cohesin binding sites in pluripotent cells. Elimination of hemimethylation causes reduced frequency of chromatin interactions emanating from these sites, suggesting a role for hemimethylation as a stable epigenetic mark regulating CTCF-mediated chromatin interactions.

Dissecting super-enhancer hierarchy based on chromatin interactions

Recent studies have highlighted super-enhancers (SEs) as important regulatory elements for gene expression, but their intrinsic properties remain incompletely characterized. Through an integrative analysis of Hi-C and ChIP-seq data, here we find that a significant fraction of SEs are hierarchically organized, containing both hub and non-hub enhancers. Hub enhancers share similar histone marks with non-hub enhancers, but are distinctly associated with cohesin and CTCF binding sites and disease-associated genetic variants. Genetic ablation of hub enhancers results in profound defects in gene activation and local chromatin landscape. As such, hub enhancers are the major constituents responsible for SE functional and structural organization.

Structure–function analyses generate novel specificities to assemble the components of multienzyme bacterial cellulosome complexes

The cellulosome is a remarkably intricate multienzyme nanomachine produced by anaerobic bacteria to degrade plant cell wall polysaccharides. Cellulosome assembly is mediated through binding of enzyme-borne dockerin modules to cohesin modules of the primary scaffoldin subunit. The anaerobic bacterium Acetivibrio cellulolyticus produces a highly intricate cellulosome comprising an adaptor scaffoldin, ScaB, whose cohesins interact with the dockerin of the primary scaffoldin (ScaA) that integrates the cellulosomal enzymes. The ScaB dockerin selectively binds to cohesin modules in ScaC that anchors the cellulosome onto the cell surface. Correct cellulosome assembly requires distinct specificities displayed by structurally related type-I cohesin-dockerin pairs that mediate ScaC-ScaB and ScaA-enzyme assemblies. To explore the mechanism by which these two critical protein interactions display their required specificities, we determined the crystal structure of the dockerin of a cellulosomal enzyme in complex with a ScaA cohesin. The data revealed that the enzyme-borne dockerin binds to the ScaA cohesin in two orientations, indicating two identical cohesin-binding sites. Combined mutagenesis experiments served to identify amino acid residues that modulate type-I cohesin-dockerin specificity in A. cellulolyticus Rational design was used to test the hypothesis that the ligand-binding surfaces of ScaA- and ScaB-associated dockerins mediate cohesin recognition, independent of the structural scaffold. Novel specificities could thus be engineered into one, but not both, of the ligand-binding sites of ScaB, whereas attempts at manipulating the specificity of the enzyme-associated dockerin were unsuccessful. These data indicate that dockerin specificity requires critical interplay between the ligand-binding surface and the structural scaffold of these modules.

Translocation Breakpoints Preferentially Occur in Euchromatin and Acrocentric Chromosomes.

Chromosomal translocations drive the development of many hematological and some solid cancers. Several factors have been identified to explain the non-random occurrence of translocation breakpoints in the genome. These include chromatin density, gene density and CCCTC-binding factor (CTCF)/cohesin binding site density. However, such factors are at least partially interdependent. Using 13,844 and 1563 karyotypes from human blood and solid cancers, respectively, our multiple regression analysis only identified chromatin density as the primary statistically significant predictor. Specifically, translocation breakpoints preferentially occur in open chromatin. Also, blood and solid tumors show markedly distinct translocation signatures. Strikingly, translocation breakpoints occur significantly more frequently in acrocentric chromosomes than in non-acrocentric chromosomes. Thus, translocations are probably often generated around nucleoli in the inner nucleoplasm, away from the nuclear envelope. Importantly, our findings remain true both in multivariate analyses and after removal of highly recurrent translocations. Finally, we applied pairwise probabilistic co-occurrence modeling. In addition to well-known highly prevalent translocations, such as those resulting in BCR-ABL1 (BCR-ABL) and RUNX1-RUNX1T1 (AML1-ETO) fusion genes, we identified significantly underrepresented translocations with putative fusion genes, which are probably subject to strong negative selection during tumor evolution. Taken together, our findings provide novel insights into the generation and selection of translocations during cancer development.

Observation of DNA intertwining along authentic budding yeast chromosomes.

DNA replication of circular genomes generates physically interlinked or catenated sister DNAs. These are resolved through transient DNA fracture by type II topoisomerases to permit chromosome segregation during cell division. Topoisomerase II is similarly required for linear chromosome segregation, suggesting that linear chromosomes also remain intertwined following DNA replication. Indeed, chromosome resolution defects are a frequent cause of chromosome segregation failure and consequent aneuploidies. When and where intertwines arise and persist along linear chromosomes are not known, owing to the difficulty of demonstrating intertwining of linear DNAs. Here, we used excision of chromosomal regions as circular "loop outs" to convert sister chromatid intertwines into catenated circles. This revealed intertwining at replication termination and cohesin-binding sites, where intertwines are thought to arise and persist but not to a greater extent than elsewhere in the genome. Intertwining appears to spread evenly along chromosomes but is excluded from heterochromatin. We found that intertwines arise before replication termination, suggesting that replication forks rotate during replication elongation to dissipate torsion ahead of the forks. Our approach provides previously inaccessible insight into the topology of eukaryotic chromosomes and illuminates a process critical for successful chromosome segregation.

Long-Range Chromosome Interactions Mediated by Cohesin Shape Circadian Gene Expression.

Mammalian circadian rhythm is established by the negative feedback loops consisting of a set of clock genes, which lead to the circadian expression of thousands of downstream genes in vivo. As genome-wide transcription is organized under the high-order chromosome structure, it is largely uncharted how circadian gene expression is influenced by chromosome architecture. We focus on the function of chromatin structure proteins cohesin as well as CTCF (CCCTC-binding factor) in circadian rhythm. Using circular chromosome conformation capture sequencing, we systematically examined the interacting loci of a Bmal1-bound super-enhancer upstream of a clock gene Nr1d1 in mouse liver. These interactions are largely stable in the circadian cycle and cohesin binding sites are enriched in the interactome. Global analysis showed that cohesin-CTCF co-binding sites tend to insulate the phases of circadian oscillating genes while cohesin-non-CTCF sites are associated with high circadian rhythmicity of transcription. A model integrating the effects of cohesin and CTCF markedly improved the mechanistic understanding of circadian gene expression. Further experiments in cohesin knockout cells demonstrated that cohesin is required at least in part for driving the circadian gene expression by facilitating the enhancer-promoter looping. This study provided a novel insight into the relationship between circadian transcriptome and the high-order chromosome structure.

Reduced cohesin destabilizes high-level gene amplification by disrupting pre-replication complex bindings in human cancers with chromosomal instability.

Gene amplification is a hallmark of cancer with chromosomal instability although the underlying mechanism by which altered copy numbers are maintained is largely unclear. Cohesin, involved in sister chromatid cohesion, DNA repair, cell cycle progression and transcriptional regulation of key developmental genes, is frequently overexpressed in human cancer. Here we show that cohesin-dependent change in DNA replication controls the copy numbers of amplified genes in cancer cells with chromosomal instability. We found that the down-regulation of elevated cohesin leads to copy number-associated gene expression changes without disturbing chromosomal segregation. Highly amplified genes form typical long-range chromatin interactions, which are stabilized by enriched cohesin. The spatial proximities among cohesin binding sites within amplified genes are decreased by RAD21-knockdown, resulting in the rapid decline of amplified gene expression. After several passages, cohesin depletion inhibits DNA replication initiation by reducing the recruitment of pre-replication complexes such as minichromosome maintenance subunits 7 (MCM7), DNA polymerase α, and CDC45 at replication origins near the amplified regions, and as a result, decreases the DNA copy numbers of highly amplified genes. Collectively, our data demonstrate that cohesin-mediated chromatin organization and DNA replication are important for stabilizing gene amplification in cancer cells with chromosomal instability.