The Cohesin Complex Cooperates with Pluripotency Transcription Factors in the Maintenance of Embryonic Stem Cell Identity
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ABSTRACT:
ORGANISM(S): Mus musculus
SUBMITTER: Maciej Paszkowski-Rogacz
PROVIDER: E-GEOD-24030 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
Publications
RAD21 cooperates with pluripotency transcription factors in the maintenance of embryonic stem cell identity.
Nitzsche Anja A Paszkowski-Rogacz Maciej M Matarese Filomena F Janssen-Megens Eva M EM Hubner Nina C NC Schulz Herbert H de Vries Ingrid I Ding Li L Huebner Norbert N Mann Matthias M Stunnenberg Hendrik G HG Buchholz Frank F
PloS one 20110512 5
For self-renewal, embryonic stem cells (ESCs) require the expression of specific transcription factors accompanied by a particular chromosome organization to maintain a balance between pluripotency and the capacity for rapid differentiation. However, how transcriptional regulation is linked to chromosome organization in ESCs is not well understood. Here we show that the cohesin component RAD21 exhibits a functional role in maintaining ESC identity through association with the pluripotency transc ...[more]
Similar Datasets
Project description:Embryonic stem cells (ESCs) cells run a self-renewal gene expression program, requiring the expression of certain transcription factors accompanied by a particular chromosome organization to maintain a balance between pluripotency and the capacity for rapid differentiation. However, how transcriptional regulation is linked to chromosome organization in ESCs remains enigmatic. Here we show that Cohesin exhibits a functional role in maintaining ESC identity through association with the pluripotency transcriptional network. ChIP-seq analyses of the cohesin subunit Rad21 reveal an ESC specific cohesin binding pattern that is characterized by a CTCF independent colocalization of cohesin with pluripotency related transcription factors. Upon ESC differentiation, these binding sites disappear and instead new CTCF independent Rad21 binding sites emerge, which are enriched for binding sites of transcription factors implicated in early differentiation. Furthermore, knock-down of cohesin subunits causes expression changes that are reminiscent of the depletion of key pluripotency transcription factors, demonstrating the functional relevance of the cohesin - pluripotency transcriptional network association. Finally, we show that Nanog physically interacts with the cohesin interacting proteins Stag1 and Wapl, further substantiating this association. Based on these findings we propose that a dynamic placement of cohesin by pluripotency transcription factors contributes to a chromosome organization supporting the ESC expression program. This SuperSeries is composed of the SubSeries listed below.
2011-05-19 | GSE24030 | GEO
Project description:Sister chromatid cohesion is mediated by cohesin but the process of cohesion establishment during S phase is still enigmatic. Recent data indicate that in mammalian cells, cohesin binding to chromatin is dynamic in G1 but becomes stabilized during S phase. Whether the regulation of chromosomal cohesin turn-over is integral to the process of cohesion establishment is unknown. Here, we provide evidence that fission yeast cohesin also displays dynamic behaviour. Cohesin association with G1 chromosomes requires continued activity of the cohesin loader Mis4/Ssl3, implying that repeated loading cycles maintain cohesin binding. Cohesin retention on G1 chromosomes was improved by deletion of wpl, the fission yeast ortholog of mammalian WAPL, suggestive of a conserved mechanism that controls cohesin stability on chromosomes. wpl is non-essential, indicating that a change in wpl-dependent cohesin turnover is not integral to the mechanism of cohesion establishment. Instead we find that cohesin instability is down-regulated during S phase in a reaction independent of DNA replication. Hence, cohesin stabilization might be a pre-requisite for cohesion establishment rather than its consequence. Keywords: ChIP-chip Experiments in budding and fission yeast have shown that the cohesin loading factors are dispensable for viability in G2, when cohesion has been established (Bernard et al., 2006; Ciosk et al., 2000). In fission yeast, inactivation of the loading machinery at that time no longer affects cohesin binding to chromosomes (Bernard et al., 2006). In mammalian cells, about one-third of nuclear cohesin becomes stably bound to chromatin in G2 (Gerlich et al., 2006). Since the binding of cohesin to chromosomes appears labile in G1, but stabilized in G2, we asked how cohesin becomes stable during the intervening S phase. Spreads showed that Rad21 was only slightly decreased in HU arrested cells after inactivation of the cohesin loading factors Mis4 or Ssl3. In this series we analyzed whether cohesin association was equally stabilised at all its association sites along chromosome arms. Rad21 binding was therefore analyzed on a chromosome-wide scale by ChIP followed by hybridization to an oligonucleotide tiling array covering chromosomes 2 and 3. We compared the Rad21 binding pattern in HU arrested wild-type versus ssl3-29 cells after the shift to the restrictive temperature. Four 50 kb regions from chromosome 2 are shown in Figure 5, and the complete chromosome 2 in Supplemental Fig.2 (based on samples GSM209708 & GSM209722 compared to the SUP sample GSM209740, provisional accession numbers). This showed that cohesin peaks remained indistinguishable in their relative height and positions whether or not Ssl3 was inactivated. We conclude that, unlike in G1, the loading machinery is dispensable for the stable binding of cohesin to chromosomes in S phase cells. The experiment was repeated twice with slightly changed parameters (16B12 vs 12CA5 anti-HA antibody, 8.5h vs 9h HU arrest at 20C, 3h at 36C vs 3h at 37C inactivation in HU, see samples for details).
2007-11-30 | E-GEOD-8450 | biostudies-arrayexpress
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:Eukaryotic genomes are folded into three-dimensional structures that govern diverse hromosomal procsses. Studeis in Drosophila and mammals have revealed large self-associating tomological domains whose borders are enriched in cohesin/CTCF factors that are required for long-range intrations. However, mechanisms governing higher-order folding of chromatin fivbers and the exact function of cohesin in this process remain poor understood. Here we perform Hi-C to explore the organization of the Schizosaccharomyces pombe genome at high-resolution, which despite its small size comprises fundamental features found in higher eukaryotes. Our analyses reveal that in addition to determinants of Rabl-like chromosome architecture, smaller locally interacting regions of chromatin, referred to as globules, are a distinctive features of S. pombe chromosome organization. This feature of chromatin architecture requires a function of cohesin distinct from its role in sister chromatid cohesion. Cohesin is enriched at globule boundaries and its loss causes disruption of local globule structure and global chromosome territories. Heterochromatin, which selectively loads cohesin at specific loci including pericentromric and subtelomeric domains, is dispensable for globule formation but uniquely impacts genome organization through chromatin compaction by enforcing Rabl configuration. microarray CGH analysis in mutant fission yeast cell; Copy number change in rad21-K1 were examined by comparative genomic hybridization analysis.
2014-10-22 | E-GEOD-56847 | biostudies-arrayexpress
Project description:ChIP-Seq targeting the major cohesin core subunit RAD21 to represent cohesin occupancy and binding sites in cohesin-mutated (STA2 or RAD21 mutations) and wildtpye adult AMLs.
| EGAD00001011199 | EGA
Project description:MTD project_description Inflammation and decreased stem cell function characterize organism aging, yet the relationship between these factors remains incompletely understood. This study shows that aged hematopoietic stem and progenitor cells exhibit increased ground-stage NF-κB activity, which enhances their responsiveness to undergo differentiation and loss of self-renewal in response to inflammation. The study identifies Rad21/cohesin as a critical mediator of NF-κB signals, by increasing chromatin accessibility of inter-/intra-genic and enhancer regions. Rad21/NF-κB are required for normal differentiation, but limit self-renewal of hematopoietic stem cells (HSCs) during aging and inflammation in an NF-κB dependent manner. HSCs from aged mice fail to downregulate Rad21/cohesin and inflammation/differentiation inducing signals in the resolution phase after acute inflammation. and The inhibition of cohesin/NF-κB is sufficient to revert the hypersensitivity of aged HSPCs to inflammation-induced differentiation. During aging, myeloid-biased HSCs with disrupted and naturally occurring reduced expression of Rad21/cohesin are increasingly selected over lymphoid-biased HSCs. Together, Rad21/cohesin mediated NF-κB signaling limits HSPC function during aging and selects for cohesin deficient HSCs with myeloid skewed differentiation.
2018-11-28 | PXD011537 | Pride
Project description:The Cohesin complex has recently been described to regulate gene expression. We wanted to determine the gene expression profile specific in mouse ES cells after depletion of the Cohesin subunit Rad21. We used microarrays to detail the global programme of gene expression underlying depletion of Rad21 and identified distinct early development related genes up-regulated and many pluripotency related genes downregulated. Rad21 was depleted in R1/E ES cells for 48h using esiRNAs against Rad21. An esiRNA against non-targeting Luciferase was used as a negative control
2011-05-19 | E-GEOD-23923 | biostudies-arrayexpress
Project description:The cohesin complex regulates sister chromatid cohesion, chromosome organization, gene expression, and DNA repair. Here we report that endogenous human cohesin interacts with a panoply of splicing factors and RNA binding proteins, including diverse components of the U4/U6.U5 tri-snRNP complex and several splicing factors that are commonly mutated in cancer. The interactions are enhanced during mitosis, and the interacting splicing factors and RNA binding proteins follow the cohesin cycle and prophase pathway of regulated interactions with chromatin. Depletion of cohesin-interacting splicing factors results in stereotyped cell cycle arrests and alterations in genomic organization. These data support the hypothesis that splicing factors and RNA binding proteins control cell cycle progression and genomic organization via regulated interactions with cohesin and chromatin.
2018-05-17 | GSE114539 | GEO
Project description:One bottleneck in understanding the principles of 3D chromatin structures is caused by the paucity of known regulators. Cohesin is essential for 3D chromatin organization, and its interacting partners are candidate regulators. Here, we performed proteomic profiling of the Cohesin in chromatin and identified transcription factors, RNA-binding proteins, and chromatin regulators associated with Cohesin. Acute protein degradation followed by time-series genomic binding quantitation and BAT Hi-C analysis were conducted, and the results showed that the transcription factor ZBTB21 contributes to Cohesin chromatin binding, 3D chromatin interactions and transcriptional repression. Strikingly, multiomic analyses revealed that the other four ZBTB factors interacted with Cohesin, and double degradation of ZBTB21 and ZBTB7B led to a further decrease in Cohesin chromatin occupancy. We propose that multiple ZBTB transcription factors orchestrate the chromatin binding of Cohesin to regulate chromatin interactions, and we provide a catalog of many additional proteins associated with Cohesin that warrant further investigation.
2023-06-05 | PXD036004 | Pride
Project description:One bottleneck in understanding the principles of 3D chromatin structures is caused by the paucity of known regulators. Cohesin is essential for 3D chromatin organization, and its interacting partners are candidate regulators. Here, we performed proteomic profiling of the Cohesin in chromatin and identified transcription factors, RNA-binding proteins, and chromatin regulators associated with Cohesin. Acute protein degradation followed by time-series genomic binding quantitation and BAT Hi-C analysis were conducted, and the results showed that the transcription factor ZBTB21 contributes to Cohesin chromatin binding, 3D chromatin interactions and transcriptional repression. Strikingly, multiomic analyses revealed that the other four ZBTB factors interacted with Cohesin, and double degradation of ZBTB21 and ZBTB7B led to a further decrease in Cohesin chromatin occupancy. We propose that multiple ZBTB transcription factors orchestrate the chromatin binding of Cohesin to regulate chromatin interactions, and we provide a catalog of many additional proteins associated with Cohesin that warrant further investigation.
2023-06-05 | PXD028860 | Pride