Cohesin spreads bi-directionally to form chromatin jets in C. elegans
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ABSTRACT:
ORGANISM(S): Caenorhabditis elegans
PROVIDER: GSE237663 | GEO | 2023/09/30
REPOSITORIES: GEO
Similar Datasets
Project description:cohesin spreads bi-directionally to form chromatin jets in C. elegans
| PRJNA996120 | ENA
Project description:The cohesin complex holds together newly-replicated chromatids and is involved in diverse pathways that preserve genome integrity. We show that in budding yeast, cohesin is transiently recruited to active replication origins and it spreads along DNA as forks progress. When DNA synthesis is impeded, cohesin accumulates at replication sites and is critical for the recovery of stalled forks. Cohesin enrichment at replication forks does not depend on H2A(X) formation, which differs from its loading requirements at DNA double-strand breaks (DSBs). However, cohesin localization is largely reduced in rad50delta mutants and cells lacking both Mec1 and Tel1 checkpoint kinases. Interestingly, cohesin loading at replication sites depends on the structural features of Rad50 that are important for bridging sister chromatids, including the CXXC hook domain and the length of the coiled-coil extensions. Together, these data reveal a novel function for cohesin in the maintenance of genome integrity during S phase. Scc1 ChIP, Rad50 ChIP and BrdU IP in wild type and mutants at different stages of the cell cycle.
2012-10-01 | E-GEOD-39078 | biostudies-arrayexpress
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: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 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.
2008-12-31 | GSE13517 | GEO
Project description:The cohesin complex holds together newly-replicated chromatids and is involved in diverse pathways that preserve genome integrity. We show that in budding yeast, cohesin is transiently recruited to active replication origins and it spreads along DNA as forks progress. When DNA synthesis is impeded, cohesin accumulates at replication sites and is critical for the recovery of stalled forks. Cohesin enrichment at replication forks does not depend on H2A(X) formation, which differs from its loading requirements at DNA double-strand breaks (DSBs). However, cohesin localization is largely reduced in rad50delta mutants and cells lacking both Mec1 and Tel1 checkpoint kinases. Interestingly, cohesin loading at replication sites depends on the structural features of Rad50 that are important for bridging sister chromatids, including the CXXC hook domain and the length of the coiled-coil extensions. Together, these data reveal a novel function for cohesin in the maintenance of genome integrity during S phase.
2012-10-01 | GSE39078 | GEO
Project description:Condensins are molecular motors that compact DNA for chromosome segregation and gene regulation. In vitro experiments have begun to elucidate the mechanics of condensin function but how condensin loading and translocation along DNA controls eukaryotic chromosome structure in vivo remains poorly understood. To address this question, we took advantage of a specialized condensin, which organizes the 3D conformation of X chromosomes to mediate dosage compensation (DC) in C. elegans. Condensin DC is recruited and spreads from a small number of recruitment elements on the X chromosome (rex). We found that ectopic insertion of rex sites on an autosome leads to bidirectional spreading of the complex over hundreds of kilobases. On the X chromosome, strong rex sites contain multiple copies of a 12-bp sequence motif and act as TAD borders. Inserting a strong rex and ectopically recruiting the complex on the X chromosome or an autosome creates a loop-anchored TAD. Unlike the CTCF system, which controls TAD formation by cohesin, direction of the 12-bp motif does not control the specificity of loops. In an X;V fusion chromosome, condensin DC linearly spreads into V and increases 3D DNA contacts, but fails to form TADs in the absence of rex sites. Finally, we provide in vivo evidence for the loop extrusion hypothesis by targeting multiple dCas9-Suntag complexes to an X chromosome repeat region. Consistent with linear translocation along DNA, condensin DC accumulates at the block site. Together, our results support a model whereby strong rex sites act as insulation elements through recruitment and bidirectional spreading of condensin DC molecules and form loop-anchored TADs.
2021-03-24 | GSE169250 | GEO
Project description:Condensins are molecular motors that compact DNA for chromosome segregation and gene regulation. In vitro experiments have begun to elucidate the mechanics of condensin function but how condensin loading and translocation along DNA controls eukaryotic chromosome structure in vivo remains poorly understood. To address this question, we took advantage of a specialized condensin, which organizes the 3D conformation of X chromosomes to mediate dosage compensation (DC) in C. elegans. Condensin DC is recruited and spreads from a small number of recruitment elements on the X chromosome (rex). We found that ectopic insertion of rex sites on an autosome leads to bidirectional spreading of the complex over hundreds of kilobases. On the X chromosome, strong rex sites contain multiple copies of a 12-bp sequence motif and act as TAD borders. Inserting a strong rex and ectopically recruiting the complex on the X chromosome or an autosome creates a loop-anchored TAD. Unlike the CTCF system, which controls TAD formation by cohesin, direction of the 12-bp motif does not control the specificity of loops. In an X;V fusion chromosome, condensin DC linearly spreads into V and increases 3D DNA contacts, but fails to form TADs in the absence of rex sites. Finally, we provide in vivo evidence for the loop extrusion hypothesis by targeting multiple dCas9-Suntag complexes to an X chromosome repeat region. Consistent with linear translocation along DNA, condensin DC accumulates at the block site. Together, our results support a model whereby strong rex sites act as insulation elements through recruitment and bidirectional spreading of condensin DC molecules and form loop-anchored TADs.
2021-03-24 | GSE168802 | GEO
Project description:Condensins are molecular motors that compact DNA for chromosome segregation and gene regulation. In vitro experiments have begun to elucidate the mechanics of condensin function but how condensin loading and translocation along DNA controls eukaryotic chromosome structure in vivo remains poorly understood. To address this question, we took advantage of a specialized condensin, which organizes the 3D conformation of X chromosomes to mediate dosage compensation (DC) in C. elegans. Condensin DC is recruited and spreads from a small number of recruitment elements on the X chromosome (rex). We found that ectopic insertion of rex sites on an autosome leads to bidirectional spreading of the complex over hundreds of kilobases. On the X chromosome, strong rex sites contain multiple copies of a 12-bp sequence motif and act as TAD borders. Inserting a strong rex and ectopically recruiting the complex on the X chromosome or an autosome creates a loop-anchored TAD. Unlike the CTCF system, which controls TAD formation by cohesin, direction of the 12-bp motif does not control the specificity of loops. In an X;V fusion chromosome, condensin DC linearly spreads into V and increases 3D DNA contacts, but fails to form TADs in the absence of rex sites. Finally, we provide in vivo evidence for the loop extrusion hypothesis by targeting multiple dCas9-Suntag complexes to an X chromosome repeat region. Consistent with linear translocation along DNA, condensin DC accumulates at the block site. Together, our results support a model whereby strong rex sites act as insulation elements through recruitment and bidirectional spreading of condensin DC molecules and form loop-anchored TADs.
2021-03-24 | GSE169468 | 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