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).

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 Î±-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

Project description:The ring-shaped cohesin complex links sister chromatids until their timely segregation during mitosis. Cohesin is enriched at centromeres, where it provides the cohesive counter-force to bi-polar tension produced by the mitotic spindle. As a consequence of spindle tension, centromeric sequences transiently split in pre-anaphase cells, in some organisms up to several micrometeres. This M-bM-^@M-^Xcentromere breathingM-bM-^@M-^Y presents a paradox, how sister sequences separate where cohesin is most enriched. We now show that in the budding yeast S. cerevisiae, cohesin binding diminishes over centromeric sequences that split during breathing. We see no evidence for cohesin translocation to surrounding sequences, suggesting that cohesin is removed from centromeres during breathing. Two pools of cohesin can be distinguished. Cohesin loaded before DNA replication, that has established sister chromatid cohesion, disappears during breathing. In contrast, cohesin loaded after DNA replication is partly retained. As sister centromeres re-associate after transient separation, cohesin is re-loaded in a manner independent of the canonical cohesin loader Scc2/Scc4. Efficient centromere re-association requires the cohesion establishment factor Eco1, suggesting that re-establishment of sister chromatid cohesion contributes to the dynamic behaviour of centromeres in mitosis. These findings provide new insights into cohesin behaviour at centromeres. Keywords: ChIP-chip SAMPLES Origin of each biological sample: Saccharomyces cerevisiae (W303background) Growth conditions: Cells containing the MET3-CDC20 allele were grown at 25M-BM-0C in SC medium lacking methionine with 2% raffinose or 2% glucose as the carbon source (Uhlmann et al. 2000). Cultures were synchronized in G1 with M-NM-1-factor (5 M-NM-<g/ml) and released into medium containing 100 mM hydroxyurea (HU) for arrest in early S-phase. For arrest in metaphase, cells were released from M-NM-1-factor or HU arrest by filtration and re-suspension in YP medium supplemented with 5 mM methionine to deplete Cdc20. To inactivate cohesin loading, cells harbouring the temperature sensitive scc2-4 allele (Ciosk et al. 2000) were shifted to the restrictive temperature of 35M-BM-0C for 1 hour before release. To prevent metaphase spindle assembly, or to disassemble already formed spindles, 7.5 M-NM-<g/ml nocodazole was added to the medium for 1 hour. To wash out nocodazole, cells were filtered again, washed, and re-suspended in fresh medium lacking nocodazole for 1 hour. Expression of Scc1 to induce differentially epitope-tagged cohesin under control of the GAL1 promoter, or of Cdc14, was achieved by addition of 2% galactose for 1 hour to cultures grown in raffinose-containing medium. EXPERIMENTAL DESIGN Experiment type: ChIP-chip = Chromatin immunoprecipitation (ChIP) followed by hybridization to a high-density oligonucleotide microarray (chip). Number of hybridizations performed: 13 ChIP-chip samples; 2 SUPernatant samples Analysis: ChIP samples were normalized against SUPernatant fractions Quality control: Confirmation of results by duplication of experiments, by usage of different epitope tags for the same protein, and by usage of different subunits of the same protein complex. Also, whole cell extract, SUPernatant, and ChIP fractions were checked by Western blotting. Protocols: ChIP-chip and hybridization protocols were performed as previously described (Katou et al. 2003; Lengronne et al. 2004; Lengronne et al. 2006). A summary is provided below: ChIP. Cells were grown under the conditions described above and cross-linked with 1% formaldehyde. Cells were disrupted using a Multi-Beads Shocker, and whole cell extracts were sonicated to obtain 400-600 bp genomic DNA fragments. Chromatin immunoprecipitation (ChIP) was performed with anti-HA antibody or anti-PK monoclonal antibody coupled to protein A Dynabeads. Immunprecipates were eluted and incubated overnight at 65M-BM-:C to reverse the cross-link. The immunoprecipitated genomic DNA was then incubated with proteinase K, extracted twice with phenol/chloroform/isoamylalcohol, precipitated, resuspended in TE and incubated with RNaseA. The DNA was purified, concentrated by ethanol precipitation and amplified by PCR after random priming. 10ug of the amplified DNA was digested with DNase I and end-labelled using TdT Terminal Transferase and Biotin-N6-ddATP. Hybridization and scanning. Samples were hybridized in buffer containing SSPE, TritonX-100, denatured salmon sperm DNA and biotin-labelled control oligonucleotide, oligo B2. Samples were denatured at 95M-BM-:C for 10 minutes before being hybridized to the arrays for 16 hours at 42M-BM-:C in a GeneChip Hybridization Oven 640. The washing and scanning protocol (FlexMidi_euk2v3_450), provided by Affymetrix, was performed automatically on a GeneChip Fluidics Station 450. Arrays were scanned using the GeneChip Scanner 3000 7G, and primary analysis of tiling chip data was performed using the Affymetrix GeneChip Operating Software (GCOS). ARRAY DESIGN General array design: in situ synthesized arrays from Affymetrix Location and ID of each spot on arrays: available upon request from Affymetrix Probe type: oligonucleotide Availability of arrays: commercially available from Affymetrix S. cerevisiae (Chromosome VI) rikDACF, P/N# 510636 S. cerevisiae T iling Array (Forward), P/N# 520286 REFERENCES Ciosk R, Shirayama M, Shevchenko A, Tanaka T, Toth A, Shevchenko A, Nasmyth K (2000) Cohesin's binding to chromosomes depends on a separate complex consisting of Scc2 and Scc4 proteins. Mol Cell 5: 1-20 Katou Y, Kanoh Y, Bandoh M, Noguchi H, Tanaka H, Ashikari T, Sugimoto K, Shirahige K (2003) S-phase checkpoint proteins Tof1 and Mrc1 form a stable replication-pausing complex. Nature 424: 1078-1083 Lengronne A, Katou Y, Mori S, Yokobayashi S, Kelly GP, Itoh T, Watanabe Y, Shirahige K, Uhlmann F (2004) Cohesin relocation from sites of chromosomal loading to places of convergent transcription. Nature 430: 573-578 Lengronne A, McIntyre J, Katou Y, Kanoh Y, Hopfner K-P, Shirahige K, Uhlmann F (2006) Establishment of sister chromatid cohesion at the S. cerevisiae replication fork. Mol Cell 23: 787-799 Uhlmann F, Wernic D, Poupart M-A, Koonin EV, Nasmyth K (2000) Cleavage of cohesin by the CD clan protease separin triggers anaphase in yeast. Cell 103: 375-386

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Galanthamine hydrobromide 72h

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