Topologically associating domains and chromatin loops depend on cohesin and are regulated by CTCF, WAPL and PDS5 proteins
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE102884 | GEO | 2017/12/07
SECONDARY ACCESSION(S): PRJNA399157
REPOSITORIES: GEO
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Project description:The spatial organization of chromosomes influences many nuclear processes including gene expression. The cohesin complex shapes the 3D genome by looping together CTCF sites along chromosomes. We show here that chromatin loop size can be increased, and that the duration with which cohesin embraces DNA determines the degree to which loops are enlarged. Cohesin’s DNA release factor WAPL restricts the degree of this loop extension and also prevents looping between incorrectly oriented CTCF sites. We reveal that the SCC2/SCC4 complex promotes the extension of chromatin loops and the formation of topologically associated domains (TADs). Our data support the model that cohesin structures chromosomes through the processive enlargement of loops and that TADs reflect polyclonal collections of loops in the making. Finally, we find that whereas cohesin promotes chromosomal looping, it rather limits nuclear compartmentalization. We conclude that the balanced activity of SCC2/SCC4 and WAPL enables cohesin to correctly structure chromosomes.
2017-05-03 | GSE95014 | GEO
Project description:The spatial organization of chromosomes influences many nuclear processes including gene expression. The cohesin complex shapes the 3D genome by looping together CTCF sites along chromosomes. We show here that chromatin loop size can be increased, and that the duration with which cohesin embraces DNA determines the degree to which loops are enlarged. Cohesin’s DNA release factor WAPL restricts the degree of this loop extension and also prevents looping between incorrectly oriented CTCF sites. We reveal that the SCC2/SCC4 complex promotes the extension of chromatin loops and the formation of topologically associated domains (TADs). Our data support the model that cohesin structures chromosomes through the processive enlargement of loops and that TADs reflect polyclonal collections of loops in the making. Finally, we find that whereas cohesin promotes chromosomal looping, it rather limits nuclear compartmentalization. We conclude that the balanced activity of SCC2/SCC4 and WAPL enables cohesin to correctly structure chromosomes.
2017-05-03 | GSE94993 | GEO
Project description:The spatial organization of chromosomes influences many nuclear processes including gene expression. The cohesin complex shapes the 3D genome by looping together CTCF sites along chromosomes. We show here that chromatin loop size can be increased, and that the duration with which cohesin embraces DNA determines the degree to which loops are enlarged. Cohesin’s DNA release factor WAPL restricts the degree of this loop extension and also prevents looping between incorrectly oriented CTCF sites. We reveal that the SCC2/SCC4 complex promotes the extension of chromatin loops and the formation of topologically associated domains (TADs). Our data support the model that cohesin structures chromosomes through the processive enlargement of loops and that TADs reflect polyclonal collections of loops in the making. Finally, we find that whereas cohesin promotes chromosomal looping, it rather limits nuclear compartmentalization. We conclude that the balanced activity of SCC2/SCC4 and WAPL enables cohesin to correctly structure chromosomes.
2017-05-03 | GSE94992 | GEO
Project description:Paternal genomes are compacted during spermiogenesis and de-compacted following fertilization. These processes are fundamental for inheritance but incompletely understood. We analyzed these processes in the frog Xenopus laevis, whose sperm can be assembled into functional pronuclei in egg extracts in vitro. In such extracts, cohesin extrudes DNA into loops, but in vivo cohesin only assembles topologically-associating domains (TADs) at the mid-blastula transition (MBT). Why cohesin assembles TADs only at this stage is unknown. We first analyzed genome architecture in frog sperm and compared it to human and mouse. Our results indicate that sperm genome organization is conserved between frogs and humans and occurs without formation of TADs. TADs can be detected in mouse sperm samples, as reported, but these structures might originate from somatic chromatin contaminations. We therefore discuss the possibility that the absence of TADs might be a general feature of vertebrate sperm. To analyze sperm genome remodeling upon fertilization, we reconstituted male pronuclei in Xenopus egg extracts. In pronuclei, chromatin compartmentalization increases but cohesin does not accumulate at CTCF sites and assemble TADs. However, if pronuclei are formed in the presence of exogenous CTCF, CTCF binds to its consensus sites, cohesin accumulates at these and forms short-range chromatin loops, which are preferentially anchored at CTCF’s N-terminus. These results indicate that TADs are only assembled at MBT because before this stage CTCF sites are not occupied and cohesin only forms short-range chromatin loops.
2023-10-25 | GSE237050 | GEO
Project description:Paternal genomes are compacted during spermiogenesis and de-compacted following fertilization. These processes are fundamental for inheritance but incompletely understood. We analyzed these processes in the frog Xenopus laevis, whose sperm can be assembled into functional pronuclei in egg extracts in vitro. In such extracts, cohesin extrudes DNA into loops, but in vivo cohesin only assembles topologically-associating domains (TADs) at the mid-blastula transition (MBT). Why cohesin assembles TADs only at this stage is unknown. We first analyzed genome architecture in frog sperm and compared it to human and mouse. Our results indicate that sperm genome organization is conserved between frogs and humans and occurs without formation of TADs. TADs can be detected in mouse sperm samples, as reported, but these structures might originate from somatic chromatin contaminations. We therefore discuss the possibility that the absence of TADs might be a general feature of vertebrate sperm. To analyze sperm genome remodeling upon fertilization, we reconstituted male pronuclei in Xenopus egg extracts. In pronuclei, chromatin compartmentalization increases but cohesin does not accumulate at CTCF sites and assemble TADs. However, if pronuclei are formed in the presence of exogenous CTCF, CTCF binds to its consensus sites, cohesin accumulates at these and forms short-range chromatin loops, which are preferentially anchored at CTCF’s N-terminus. These results indicate that TADs are only assembled at MBT because before this stage CTCF sites are not occupied and cohesin only forms short-range chromatin loops.
2023-10-25 | GSE237049 | GEO
Project description:The cohesin complex organizes the genome forming dynamic chromatin loops that impact on all DNA-mediates 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. The 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 fluorescent 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, 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 results of immunoprecipitation reactions in mammalian cells, as suggested previously from results in vitro. Our 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.
2023-01-26 | GSE212149 | GEO
Project description:The cohesin complex organizes the genome forming dynamic chromatin loops that impact on all DNA-mediates 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. The 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 fluorescent 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, 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 results of immunoprecipitation reactions in mammalian cells, as suggested previously from results in vitro. Our 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.
2023-01-26 | GSE212150 | GEO
Project description:The organisation of mammalian genomes into loops and topologically associated domains (TADs) regulates gene expression. The formation and maintenance of loops and TADs depends on the protein complex cohesin, which also holds replicated sister chromatids together from S phase until mitosis. To understand how cohesin functions, it is essential to know how many cohesin complexes and regulators exist in a typical cell and how they are distributed genome-wide. Here, we have used quantitative mass spectrometry, fluorescence-correlation spectroscopy and fluorescence recovery after photobleaching to measure the absolute number and dynamics of core subunits and regulators of the cohesin complex in human cells. We find that 60000 – 134000 cohesin complexes are bound to chromatin per G1 HeLa cell, suggesting that cohesin most likely does not occupy all its binding sites concurrently. We incorporate our data into mathematical models and discuss implications for how cohesin might contribute to genome organisation and sister chromatid cohesion.
2019-11-13 | PXD012712 | Pride
Project description:Eukaryotic genomes are compacted into loops and topologically associating domains (TADs), which contribute to transcription, recombination and genomic stability. Cohesin extrudes DNA into loops that are thought to lengthen until it encounters CTCF boundaries. Little is known whether loop extrusion is impeded by macromolecular machines. We demonstrate that the replicative helicase MCM is a barrier that restricts loops and TADs in G1 phase. Single-nucleus Hi-C of one-cell embryos revealed that MCM loading reduces CTCF-anchored loops and increases TAD boundary insulation, suggesting loop extrusion is impeded before reaching CTCF. Single-molecule imaging provides evidence that MCM are physical barriers that constrain cohesin translocation in vitro. Simulations are consistent with MCM as abundant, random barriers with low permeability. We conclude that distinct loop extrusion barriers contribute to shaping 3D genomes.
2022-03-07 | GSE155971 | GEO
Project description:Cohesin regulates sister chromatids cohesion but also contributes to chromosome folding by promoting the formation of chromatin loops, a process proposed to be mediated by loop extrusion. PDS5 plays a crucial role in regulating the cohesin dynamic on chromatin, mainly through WAPL-mediated release activity and the opponent ESCO1/2-sororin dependent pathway. However, the exact function of PDS5 on cohesin-mediated chromatin looping remains ambiguous. Two versions of PDS5 exist in vertebrates, PDS5A and PDS5B. Here, we construct cells for rapid PDS5A or PDS5B degradation in PLC/PRF/5 cell line with the FKBP12F36V-dTAG degron system to perform loss of function studies. Combining Hi-C, ChIP-seq for cohesin regulators, and RNA-seq data, we describe the redundant and discrete roles of PDS5A and PDS5B in three-dimensional genome organization and gene expression.
2022-10-08 | GSE209846 | GEO