Genome-wide binding profile of Halo-tagged cohesin
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PROVIDER: PRJNA407925 | ENA |
REPOSITORIES: ENA
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Project description:We determined the genome-wide binding profile of CTCF and cohesin subunits Smc1a and Smc3 in wild type mouse ES cells and compared them to a double FLAG-Halo-mCTCF/mRad21-SNAPf-V5 knock-in mES line, generated by CRISPR/Cas9-mediated genome editing to perform imaging experiments.
2024-02-01 | GSE104000 | GEO
Project description:The study investigates the nuclear organization and the binding dynamics of CTCF and cohesin by super-resolution microscopy and ChIP-seq. We determined the genome-wide binding profile of CTCF and Rad21 cohesin subunit in wild type mouse ES cells and compared them to a double FLAG-Halo-mCTCF/mRad21-SNAPf-V5 knock-in mES line, generated by CRISPR/Cas9-mediated genome editing to perform imaging experiments.
2017-05-03 | GSE90994 | GEO
Project description:Cohesin holds sister chromatids together to enable their accurate segregation in mitosis. How, and where, cohesin binds to chromosomes are still poorly understood, and recent genome-wide surveys have revealed an apparent disparity between its chromosomal association patterns in different organisms. Here, we present the high-resolution analysis of cohesin localisation along fission yeast chromosomes. This reveals that several determinants, thought specific for distinct organisms, come together to shape the overall distribution. Cohesin is detected at chromosomal loading sites, characterised by the cohesin loader Mis4/Ssl3, in regions of strong transcriptional activity. Cohesin also responds to transcription by downstream translocation and accumulation at convergent transcriptional terminators surrounding the loading sites. As cells enter mitosis, a fraction of cohesin leaves chromosomes in a cleavage-independent reaction, while a substantial pool of cohesin dissociates when it is cleaved at anaphase onset. As a unique feature, centromeric cohesin spreads out onto chromosome arms during mitosis as the heterochromatin protein Swi6 dissociates from centromeres. Together, this allows us to suggest conserved mechanisms for chromosomal cohesin binding in eukaryotes. After DNA replication in S phase, sister chromatids are held together by the cohesin complex. This allows DNA break repair by homologous recombination in G2 and bipolar attachment of the spindle to sister kinetochores in mitosis. At anaphase onset, sister chromatid cohesion is resolved to trigger chromosome segregation. Cohesin is an essential, conserved protein complex consisting of at least five subunits, Psm1, Psm3, Rad21, Psc3 and Pds5 in fission yeast (orthologs of budding yeast Smc1, Smc3, Scc1, Scc3 and Pds5, respectively). Chromatin immunoprecipitations against three different subunits of the cohesin complex, fused to two different epitope tags (Rad21-Pk9 -> GSM333001, Rad21-HA3 -> GSM333004, Psc3-Pk9 -> GSM333005 and Pds5-Pk9 -> GSM333006), yielded a reproducible binding pattern between approximately half of all available convergently transcribed gene pairs (in the following called convergent sites). The pattern was almost indistinguishable between exponentially growing cell populations (GSM333004, GSM333005, GSM333006) and cells arrested in G2 using the thermosensitive cdc25-22 mutation (GSM333001). To understand why some, but not all, convergent sites are bound by cohesin, we analysed whether fission yeast cohesin responds to Pol II transcription by downstream translocation, like in budding yeast. As an example, we analysed cohesin at the convergent site between the rad21 and pof3 genes. rad21 expression is high during G1 and S phase, but low in G2. In cells arrested in G2 using the cdc25-22 allele, only a small cohesin peak was detectable that largely overlapped with the rad21 ORF (GSM333001). In contrast, in cells arrested in G1 using the cdc10-129 allele, the rad21 ORF was clear of cohesin and instead a large cohesin peak accumulated downstream of rad21 (GSM333007). It has been suggested that transcriptional readthrough at convergent sites promotes double stranded RNA-dependent heterochromatin formation, which in turn underlies cohesin recruitment. We therefore tested whether the heterochromatin protein Swi6, thought to recruit cohesin, played a role in generating the observed cohesin pattern. We grew wild type and swi6Î cells in synthetic medium lacking thiamine, conditions under which the nmt2 gene is transcribed at the nmt2/avn2 convergent site that has been studied as an example. In contrast to the prediction, the cohesin pattern along chromosome arms, including the nmt2/avn2 convergent site, remained unchanged in swi6Î cells (GSM333008). We detected only small amounts of cohesin at the nmt2/avn2 convergent site, a class 1 convergent site following the above classification. At the centromeric repeats, cohesin levels were reduced in the absence of Swi6. These findings are consistent with previous reports implicating Swi6 in cohesin recruitment to centromeric heterochromatin, but not chromosome arms. To compare cohesinâs pattern to its likely sites of chromosomal loading, we analysed the localisation of the cohesin loader subunits Mis4 and Ssl3. The two subunits showed a largely overlapping pattern of binding. Chromatin immunoprecipitation was performed against epitope-tagged Mis4-Pk9 (GSM333163) and Ssl3-Pk9 (GSM334196) from exponentially proliferating cells. As a control, cells without epitope-tagged protein were grown under identical conditions and processed in parallel for chromatin immunoprecipitation with an α-Pk antibody (GSM333230). The untagged control sample yielded trace amounts of immunoprecipitated DNA, which after amplification led to several strong peaks often in intergenic low complexity regions. Several of these peaks overlapped with Mis4/Ssl3 peaks, which were excluded from the analysis. Peaks in low complexity regions were not observed in chromatin immunoprecipitates of cohesin subunits (compare GSM333001). In the search for an underlying determinant of Mis4/Ssl3 binding sites, we noticed a striking correlation with tRNA and ribosomal protein genes. Mis4/Ssl3 binding sites at tRNA genes overlapped with the binding profile of the Pol III transcription factor TFIIIC subunit Sfc6 (GSM334198), while at ribosomal protein genes it colocalised with the forkhead domain containing protein Fhl1 (GSM334307). Fhl1 is a possible fission yeast ortholog of the transcription factor Fhl1 that controls ribosomal protein gene expression in budding yeast. We found Sfc6 also associated with ribosomal protein genes, and Fhl1 with tRNA genes, though with weaker signal intensities. We do not currently know whether Sfc6 contributes to transcriptional control of ribosomal protein genes, and Fhl1 to that of tRNA genes, or whether the weaker levels of association may reflect indirect association, mediated by interactions between ribosomal protein and tRNA gene loci. Similar to budding yeast, Mis4/Ssl3 binding sites are also binding sites for the chromosomal condensin complex (GSM334197), which may mediate such interactions. Having identified the binding sites of the Mis4/Ssl3 cohesin loader, we wanted to analyse its relationship with the cohesin distribution along chromosome arms. If Mis4/Ssl3 cohesin loading sites promote cohesin binding in their surrounding, deletion of a loading site should alter this pattern. To test this, we deleted a 489 bp sequence, containing two adjacent tRNA genes that form a Mis4/Ssl3 and cohesin binding site, on the left arm of chromosome 2. In response to the deletion, Mis4/Ssl3 binding to this locus disappeared (GSM334308). Cohesin was also no longer detected at this site (GSM334309), consistent with the notion that cohesin loading had been disrupted. However, the cohesin distribution at convergent sites surrounding the former loading site remained unchanged. This suggests that establishment of the cohesin pattern did not depend on a specific Mis4/Ssl3 binding site. Despite the tRNA deletion, residual Mis4/Ssl3 remained detectable in the vicinity of this site (GSM334308). Cohesin loading by Mis4/Ssl3 might therefore be less restricted to its peaks of binding than the pattern suggests. Alternatively, other determinants, e.g. gene orientation and their transcriptional activity, might define cohesin distribution at this locus. We next addressed how cohesin on fission yeast chromosomes responds to loss of sister chromatid cohesion during mitosis and whether cohesin removal in mitosis affected certain regions of the chromosome more than others. In order to follow cohesin through mitosis, we arrested a cell population in G2 using the thermosensitive cdc25-22 mutation and released the culture into synchronous mitotic progression at permissive temperature.The cohesin pattern remained largely unchanged throughout G2, metaphase and anaphase, although the height of peaks along chromosome arms decreased. This suggests that cohesin is not removed from a subset of its binding sites during mitosis, but that its association among most binding sites was uniformly reduced. A noticeable change to the cohesin pattern during mitosis became apparent around centromeres. Over a region of approximately 50 kb surrounding the centromeres, cohesin appeared to spread along chromosome arms, showing little preference for convergent sites. The spreading started as cells entered metaphase (GSM334316) and became most pronounced during anaphase (GSM334317). We also observed a similar, although less pronounced, spreading around centromeres in cells arrested in G1 using the cdc10-129 thermosensitive mutation (GSM333007). This suggests that spreading of cohesin around centromeres is cell cycle stage specific. Cohesin is enriched at the centromeric repeat regions (GSM333001), tethered by the heterochromatin protein Swi6, possibly after being loaded at core centromere sequences, where we observed strong Mis4/Ssl3 accumulation (GSM333163 & GSM334196). In mitosis, Swi6 levels are reduced after aurora B kinase-dependent phosphorylation of histone H3, and it reaccumulates as cells enter the subsequent S phase. To test whether loss of Swi6-dependent
2010-05-14 | E-GEOD-13517 | biostudies-arrayexpress
Project description:The study investigates CTCF/cohesin binding and chromatin looping by ChIP-seq and Micro-C. By ChIP-seq, we determined the genome-wide binding profiles of CTCF and Smc1a cohesin subunit in a knock-in mouse ES cell line (wt-CTCF; clone C59) with endogenously tagged wild type CTCF (FLAG-Halo-mCTCF) and Rad21 (mRad21-SNAPf-V5), and compared them to the same ES line expressing a mutant CTCF (ΔRBR-CTCF; clone C59D2), where we replaced a previously described RNA binding region with a short linker (GDGAGLINS) followed by a 3xHA tag (N576_D611del::3xHA). By Micro-C, we compared nucleosome-resolution chromosome folding maps of the same ES cell lines C59 and C59D2 described above, to determine the effect of deleting CTCF RNA binding region on chromatin looping.
2019-05-30 | GSE123636 | GEO
Project description:The functions of cohesin are central to genome integrity, chromosome organization, and transcription regulation through its prevention of premature sister-chromatid separation and the formation of DNA loops. The loading of cohesin onto chromatin depends on the Scc2-Scc4 complex, however, little is known about how it stimulates the cohesin loading activity. Here we determine the large “hook” structure of Scc2 responsible for catalyzing cohesin loading. We identify key Scc2 surfaces that are crucial for DNA binding and for cohesin loading in vivo. Using previously determined structures and modeling, we derive a pseudo-atomic structure of the full-length Scc2-Scc4 complex. Finally, our crosslinking and electron microscopy analyses reveal that Scc2-Scc4 utilizes its modular structure to make multiple contacts with a folded cohesin at an interface created by the cohesin head-hinge interaction.
2017-01-11 | PXD004692 | Pride
Project description:Cohesin is present in almost all active enhancer regions, where it is associated with transcription factors. Cohesin frequently colocalizes with CTCF (CCCTC-binding factor), affecting genomic stability, expression and epigenetic homeostasis. Cohesin subunits are mutated in cancer, but CTCF/cohesin-binding sites (CBSs) in DNA have not been examined for mutations. Here we report frequent mutations at CBSs in cancers displaying a mutational signature where mutations in A•T base pairs predominate. Integration of whole-genome sequencing data from 213 colorectal cancer (CRC) samples and chromatin immunoprecipitation sequencing (ChIP-exo) data identified frequent point mutations at CBSs. In contrast, CRCs showing an ultramutator phenotype caused by defects in the exonuclease domain of DNA polymerase ɛ (POLE) displayed significantly fewer mutations at and adjacent to CBSs. Analysis of public data showed that multiple cancer types accumulate CBS mutations. CBSs are a major mutational hotspot in the noncoding cancer genome.
| EGAS00001003010 | EGA
Project description:Identification of cohesin (Rad21) and CTCF binding sites in ~2% of the mouse genome in B3 pre-B cells stably expressing FLAG-tagged Rad21 using anti-FLAG (Sigma F3165) and anti-CTCF (Upstate 07729) antibodies
2008-01-31 | E-TABM-401 | biostudies-arrayexpress
Project description:In this assay, we aimed to investigate the occupancy of ZFP661 in the mouse genome and its possible role in regulating the occupancies of CTCF and cohesin, which are two key factors in establishing 3D chromatin structures. To achieve this, we performed ChIP-seq in ZFP661-3HA endogenously tagged mESCs (for ZFP661), Zfp661 knockout (KO) mESCs (for CTCF and RAD21), and ZFP661-3HA overexpressing (OE) mESCs (for ZFP661, CTCF, RAD21, KAP1, and H3K9me3). Our results reveal that 1) ZFP661 binding peaks were co-occupied with those of CTCF; 2) ZFP661 binds exclusively inside the CTCF loop anchors; and 3) ZFP661 can suppress cohesin binding at CTCF barriers without altering CTCF binding.
2023-05-09 | GSE231866 | GEO
Project description:ChIP-seq for CTCF and Rad21 in 3 week old mouse brain from reciprocal BxC and CxB crosses. One biological replicate of each. Examination of CTCF and Cohesin binding sites in neonetal mouse brain
2013-06-20 | E-GEOD-35140 | biostudies-arrayexpress
Project description:In mammalian spermatocytes and oocytes, cohesin complexes serve several distinct functions including establishing the meiotic chromosome architecture into axes and loops, providing sister chromatid cohesion, protecting telomeres, and supporting meiotic recombination. High-resolution analysis of cohesin binding to meiotic chromosomes, however, remained to be performed, mainly because of the complexity of germ cell development and meiotic chromatin, and of the difficulty obtaining proper meiocyte populations. Here, we established a ChIP-seq approach to identify and characterize the genome-wide binding profile of cohesin specifically in murine spermatocytes. Chromatin binding of cohesin proteins SMC3, present in all cohesin complexes, and SMC1β representing the major meiotic complexes, were analyzed. Both cohesins were much reduced in coding exons as well as in 5’ and 3’ UTRs. SMC1β cohesin binding sequences coincide in a large fraction with CACA repeats and thus with transcription factor ZSCAN4C binding sites, but also with other TF sites. We observed neither overlap with recombinase DMC1 sites nor with recombination hotspot marker PRDM9 sites. Most interestingly, meiotic cohesin is strongly enriched at 5S ribosomal RNA genes. We hypothesize a role of cohesin in the regulation of meiotic rDNA stability and/or transcription.
2016-10-21 | GSE88972 | GEO