Project description:We apply ChIA-Drop, a single-molecule ligation-free mapping technique, to visualize loop formation over time. Our results demonstrated a cohesin-centric framework that coordinates with CTCF and RNAPII, respectively. At the loading (NIPBL binding) site, cohesin is highly correlated with transcriptional activities and translocates bi-directionally toward CTCF motif sites, where CTCF likely provides the anchoring point for cohesin to actively reel the chromatin template according to the motif forward orientation. Furthermore, cohesin specifically co-localizes with RNAPII and together mediates multiplex chromatin interactions that involve promoters, enhancers and super-enhancers. Intriguingly, single-molecule mapping data revealed that individual constituents of super-enhancers interact with target genes singularly and in cascade through intermediate regulatory elements, suggesting a probability-based mechanism for transcription regulation.
Project description:Most animal genomes are partitioned into Topologically Associating Domains (TADs), created by cohesin-mediated loop extrusion and defined by convergently oriented CTCF sites. The dynamics of loop extrusion and its regulation remains poorly characterized in vivo. Here, we tracked TAD anchors in living human cells to visualize and quantify cohesin-dependent loop extrusion across multiple endogenous genomic regions. We show that TADs are dynamic structures whose anchors are brought in proximity about once per hour and for 6-19 min (~16% of the time). TADs are continuously subjected to extrusion by multiple cohesin complexes, extruding loops at ~0.1 kb/s. Remarkably, despite strong differences of Hi-C patterns between the chromatin regions, their dynamics is consistent with the same density, residence time and speed of cohesin. Our results suggest that TAD dynamics is governed primarily by CTCF site location and affinity, which allows genome-wide predictive models of cohesin-dependent interactions.
Project description:Cohesin plays vital roles in chromatin folding and gene expression regulation, cooperating with such factors as cohesin loaders, unloaders, and the insulation factor CTCF. Although 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. 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 related to epigenomic status.
Project description:Cohesin plays vital roles in chromatin folding and gene expression regulation, cooperating with such factors as cohesin loaders, unloaders, and the insulation factor CTCF. Although 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. 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 related to epigenomic status.
Project description:Cohesin plays vital roles in chromatin folding and gene expression regulation, cooperating with such factors as cohesin loaders, unloaders, and the insulation factor CTCF. Although 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. 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 related to epigenomic status.
Project description:Cohesin is implicated in establishing tissue-specific DNA loops that target enhancers to promoters, and also localizes to sites bound by the insulator protein CTCF, which blocks enhancer-promoter communication. However, cohesin-associated interactions have not been characterized on a genome-wide scale. Here we performed chromatin interaction analysis with paired-end tag sequencing (ChIA-PET) of the cohesin subunit SMC1A in developing mouse limb. We identified 2,264 SMC1A interactions, of which 1,491 (65%) involved sites co-occupied by CTCF. SMC1A participates in tissue- specific enhancer-promoter interactions and interactions that demarcate regions of correlated regulatory output. In contrast to previous studies, we also identified interactions between promoters and distal sites that are maintained in multiple tissues, but are poised in embryonic stem cells and resolve to tissue-specific activated or repressed chromatin states in the mouse embryo. Our results reveal the diversity of cohesin- associated interactions in the genome and highlight their role in establishing the regulatory architecture of development. Smc1a ChIA-PET, RNA-seq, chromatin state maps (H3K27ac, H3K27me3, H3K4m2), and CTCF and Smc1a binding in mouse embryonic limb bud (E11.5)
Project description:Depletion of architectural factors globally alters chromatin structure, but only modestly affects gene expression. We revisit the structure-function relationship using the inactive X chromosome (Xi) as a model. We investigate cohesin imbalances by forcing its depletion or retention using degron-tagged RAD21 (cohesin subunit) or WAPL (cohesin release factor). Interestingly, cohesin loss disrupts Xi superstructure, unveiling superloops between escapee genes, with minimal effect on gene repression. By contrast, forced cohesin retention markedly affects Xi superstructure and compromises spreading of Xist RNA-Polycomb complexes, attenuating Xi silencing. Effects are greatest at distal chromosomal ends, where looping contacts with the Xist locus are weakened. Surprisingly, cohesin loss created an ?Xi superloop? and cohesin retention created ?Xi megadomains? on the active X. Across the genome, a proper cohesin balance protects against aberrant inter-chromosomal interactions and tempers Polycomb-mediated repression. We conclude that a balance of cohesin eviction and retention regulates X-inactivation and inter-chromosomal interactions across the genome.
Project description:Chromosome conformation capture approaches have shown that interphase chromatin is organized into an architectural framework of Mb-sized compartments and sub-Mb-sized topological domains. Cohesin controls chromosome topology to facilitate DNA repair and chromosome segregation in cycling cells, and also associates with active enhancers and promoters and with CTCF to form long-range interactions important for gene regulation. We find that architectural compartments - a major feature of interphase chromatin organization – are maintained in non-cycling mouse thymocytes after genetic depletion of cohesin in vivo. Cohesin was however required for specific long-range interactions within permissive (A-type) compartments, where cohesin-regulated genes reside. Cohesin depletion diminished interactions between cohesin-bound sites, while alternative interactions between chromatin features associated with transcriptional activation and repression became more prominent, with corresponding changes in gene expression. Our findings indicate that cohesin-mediated long-range interactions facilitate discrete gene expression states within pre-existing chromosomal compartments.