A second Wpl1 anti-cohesion pathway requires dephosphorylation of fission yeast kleisin Rad21 by PP4.

Cohesin mediates sister chromatid cohesion which is essential for chromosome segregation and repair. Sister chromatid cohesion requires an acetyl-transferase (Eso1 in fission yeast) counteracting Wpl1, promoting cohesin release from DNA We report here that Wpl1 anti-cohesion function includes an additional mechanism. A genetic screen uncovered that Protein Phosphatase 4 (PP4) mutants allowed cell survival in the complete absence of Eso1. PP4 co-immunoprecipitated Wpl1 and cohesin and Wpl1 triggered Rad21 de-phosphorylation in a PP4-dependent manner. Relevant residues were identified and mapped within the central domain of Rad21. Phospho-mimicking alleles dampened Wpl1 anti-cohesion activity, while alanine mutants were neutral indicating that Rad21 phosphorylation would shelter cohesin from Wpl1 unless erased by PP4. Experiments in post-replicative cells lacking Eso1 revealed two cohesin populations. Type 1 was released from DNA by Wpl1 in a PP4-independent manner. Type 2 cohesin, however, remained DNA-bound and lost its cohesiveness in a manner depending on Wpl1- and PP4-mediated Rad21 de-phosphorylation. These results reveal that Wpl1 antagonizes sister chromatid cohesion by a novel pathway regulated by the phosphorylation status of the cohesin kleisin subunit.

The Fun30 chromatin remodeler Fft3 controls nuclear organization and chromatin structure of insulators and subtelomeres in fission yeast.

In eukaryotic cells, local chromatin structure and chromatin organization in the nucleus both influence transcriptional regulation. At the local level, the Fun30 chromatin remodeler Fft3 is essential for maintaining proper chromatin structure at centromeres and subtelomeres in fission yeast. Using genome-wide mapping and live cell imaging, we show that this role is linked to controlling nuclear organization of its targets. In fft3∆ cells, subtelomeres lose their association with the LEM domain protein Man1 at the nuclear periphery and move to the interior of the nucleus. Furthermore, genes in these domains are upregulated and active chromatin marks increase. Fft3 is also enriched at retrotransposon-derived long terminal repeat (LTR) elements and at tRNA genes. In cells lacking Fft3, these sites lose their peripheral positioning and show reduced nucleosome occupancy. We propose that Fft3 has a global role in mediating association between specific chromatin domains and the nuclear envelope.

Pds5 promotes cohesin acetylation and stable cohesin-chromosome interaction.

Pds5 and Wpl1 act as anti-establishment factors preventing sister-chromatid cohesion until counteracted in S-phase by the cohesin acetyl-transferase Eso1. However, Pds5 is also required to maintain sister-chromatid cohesion in G2. Here, we show that Pds5 is essential for cohesin acetylation by Eso1 and ensures the maintenance of cohesion by promoting a stable cohesin interaction with replicated chromosomes. The latter requires Eso1 only in the presence of Wapl, indicating that cohesin stabilization relies on Eso1 only to neutralize the anti-establishment activity. We suggest that Eso1 requires Pds5 to counteract anti-establishment. This allows both cohesion establishment and Pds5-dependent stable cohesin binding to chromosomes.

Cell-cycle regulation of cohesin stability along fission yeast chromosomes.

Sister chromatid cohesion is mediated by cohesin, but the process of cohesion establishment during S-phase is still enigmatic. In mammalian cells, cohesin binding to chromatin is dynamic in G1, but becomes stabilized during S-phase. Whether the regulation of cohesin stability is integral to the process of cohesion establishment is unknown. Here, we provide evidence that fission yeast cohesin also displays dynamic behavior. Cohesin association with G1 chromosomes requires continued activity of the cohesin loader Mis4/Ssl3, suggesting that repeated loading cycles maintain cohesin binding. Cohesin instability in G1 depends on wpl1, the fission yeast ortholog of mammalian Wapl, suggestive of a conserved mechanism that controls cohesin stability on chromosomes. wpl1 is nonessential, indicating that a change in wpl1-dependent cohesin dynamics is dispensable for cohesion establishment. Instead, we find that cohesin stability increases at the time of S-phase in a reaction that can be uncoupled from DNA replication. Hence, cohesin stabilization might be a pre-requisite for cohesion establishment rather than its consequence.

The CDK Pef1 and protein phosphatase 4 oppose each other for regulating cohesin binding to fission yeast chromosomes.

Cohesin has essential roles in chromosome structure, segregation and repair. Cohesin binding to chromosomes is catalyzed by the cohesin loader, Mis4 in fission yeast. How cells fine tune cohesin deposition is largely unknown. Here, we provide evidence that Mis4 activity is regulated by phosphorylation of its cohesin substrate. A genetic screen for negative regulators of Mis4 yielded a CDK called Pef1, whose closest human homologue is CDK5. Inhibition of Pef1 kinase activity rescued cohesin loader deficiencies. In an otherwise wild-type background, Pef1 ablation stimulated cohesin binding to its regular sites along chromosomes while ablating Protein Phosphatase 4 had the opposite effect. Pef1 and PP4 control the phosphorylation state of the cohesin kleisin Rad21. The CDK phosphorylates Rad21 on Threonine 262. Pef1 ablation, non-phosphorylatable Rad21-T262 or mutations within a Rad21 binding domain of Mis4 alleviated the effect of PP4 deficiency. Such a CDK/PP4-based regulation of cohesin loader activity could provide an efficient mechanism for translating cellular cues into a fast and accurate cohesin response.

A screen for cohesion mutants uncovers Ssl3, the fission yeast counterpart of the cohesin loading factor Scc4.

Sister-chromatid cohesion is mediated by cohesin, a ring-shape complex made of four core subunits called Scc1, Scc3, Smc1, and Smc3 in Saccharomyces cerevisiae (Rad21, Psc3, Psm1, and Psm3 in Schizosaccharomyces pombe). How cohesin ensures cohesion is unknown, although its ring shape suggests that it may tether sister DNA strands by encircling them . Cohesion establishment is a two-step process. Cohesin is loaded on chromosomes before replication and cohesion is subsequently established during S phase. In S. cerevisiae, cohesin loading requires a separate complex containing the Scc2 and Scc4 proteins. Cohesin rings fail to associate with chromatin and cohesion can not establish when Scc2 is impaired . The mechanism of loading is unknown, although some data suggest that hydrolysis of ATP bound to Smc1/3 is required . Scc2 homologs exist in fission yeast (Mis4), Drosophila, Xenopus, and human . By contrast, no homolog of Scc4 has been identified so far. We report here on the identification of fission yeast Ssl3 as a Scc4-like factor. Ssl3 is in complex with Mis4 and, as a bona fide loading factor, Ssl3 is required in G1 for cohesin binding to chromosomes but dispensable in G2 when cohesion is established. The discovery of a functional homolog of Scc4 indicates that the machinery of cohesin loading is conserved among eukaryotes.

Kinetochore recruitment of two nucleolar proteins is required for homolog segregation in meiosis I.

Halving of the chromosome number during meiosis I depends on the segregation of maternal and paternal centromeres. This process relies on the attachment of sister centromeres to microtubules emanating from the same spindle pole. We describe here the identification of a protein complex, Csm1/Lrs4, that is essential for monoorientation of sister kinetochores in Saccharomyces cerevisiae. Both proteins are present in vegetative cells, where they reside in the nucleolus. Only shortly before meiosis I do they leave the nucleolus and form a "monopolin" complex with the meiosis-specific Mam1 protein, which binds to kinetochores. Surprisingly, Csm1's homolog in Schizosaccharomyces pombe, Pcs1, is essential for accurate chromosome segregation during mitosis and meiosis II. Csm1 and Pcs1 might clamp together microtubule binding sites on the same (Pcs1) or sister (Csm1) kinetochores.

Control of Shugoshin function during fission-yeast meiosis.

Meiosis consists of a single round of DNA replication followed by two consecutive nuclear divisions. During the first division (MI), sister kinetochores must orient toward the same pole to favor reductional segregation. Correct chromosome segregation during the second division (MII) requires the retention of centromeric cohesion until anaphase II. The spindle checkpoint protein Bub1 is essential for both processes in fission yeast . When bub1 is deleted, the Shugoshin protein Sgo1 is not recruited to centromeres, cohesin Rec8 does not persist at centromeres, and sister-chromatid cohesion is lost by the end of MI. Deletion of bub1 also affects kinetochore orientation because sister centromeres can move to opposite spindle poles in approximately 30% of MI divisions. We show here that these two functions are separable within the Bub1 protein. The N terminus of Bub1 is necessary and sufficient for Sgo1 targeting to centromeres and the protection of cohesion, whereas the C-terminal kinase domain acts together with Sgo2, the second fission-yeast Shugoshin protein, to promote sister-kinetochore co-orientation during MI. Additional analyses suggest that the protection of centromeric cohesion does not operate when sister kinetochores attach to opposite spindle poles during MI. Sgo1-mediated protection of centromere cohesion might therefore be regulated by the mode of kinetochore attachment.

Two fission yeast homologs of Drosophila Mei-S332 are required for chromosome segregation during meiosis I and II.

BACKGROUND: Meiosis produces haploid gametes from diploid progenitor cells. This reduction is achieved by two successive nuclear divisions after one round of DNA replication. Correct chromosome segregation during the first division depends on sister kinetochores being oriented toward the same spindle pole while homologous kinetochores must face opposite poles. Segregation during the second division depends on retention of sister chromatid cohesion between centromeres until the onset of anaphase II, which in Drosophila melanogaster depends on a protein called Mei-S332 that binds to centromeres. RESULTS: We report the identification of two homologs of Mei-S332 in fission yeast using a knockout screen. Together with their fly ortholog they define a protein family conserved from fungi to mammals. The two identified genes, sgo1 and sgo2, are required for retention of sister centromere cohesion between meiotic divisions and kinetochore orientation during meiosis I, respectively. The amount of meiotic cohesin's Rec8 subunit retained at centromeres after meiosis I is reduced in Deltasgo1, but not in Deltasgo2, cells, and Sgo1 appears to regulate cleavage of Rec8 by separase. Both Sgo1 and Sgo2 proteins localize to centromere regions. The abundance of Sgo1 protein normally declines after the first meiotic division, but extending its expression by altering its 3'UTR sequences does not greatly affect meiosis II. Its mere presence within the cell might therefore be insufficient to protect centromeric cohesion. CONCLUSIONS: A conserved protein family based on Mei-S332 has been identified. The two fission yeast homologs are implicated in meiosis I kinetochore orientation and retention of centromeric sister chromatid cohesion until meiosis II.

Kinetochore targeting of fission yeast Mad and Bub proteins is essential for spindle checkpoint function but not for all chromosome segregation roles of Bub1p.

Several lines of evidence suggest that kinetochores are organizing centers for the spindle checkpoint response and the synthesis of a "wait anaphase" signal in cases of incomplete or improper kinetochore-microtubule attachment. Here we characterize Schizosaccharomyces pombe Bub3p and study the recruitment of spindle checkpoint components to kinetochores. We demonstrate by chromatin immunoprecipitation that they all interact with the central domain of centromeres, consistent with their role in monitoring kinetochore-microtubule interactions. Bub1p and Bub3p are dependent upon one another, but independent of the Mad proteins, for their kinetochore localization. We demonstrate a clear role for the highly conserved N-terminal domain of Bub1p in the robust targeting of Bub1p, Bub3p, and Mad3p to kinetochores and show that this is crucial for an efficient checkpoint response. Surprisingly, neither this domain nor kinetochore localization is required for other functions of Bub1p in chromosome segregation.

Psm3 acetylation on conserved lysine residues is dispensable for viability in fission yeast but contributes to Eso1-mediated sister chromatid cohesion by antagonizing Wpl1.

In budding yeast and humans, cohesion establishment during S phase requires the acetyltransferase Eco1/Esco1-2, which acetylates the cohesin subunit Smc3 on two conserved lysine residues. Whether Smc3 is the sole Eco1/Esco1-2 effector and how Smc3 acetylation promotes cohesion are unknown. In fission yeast (Schizosaccharomyces pombe), as in humans, cohesin binding to G(1) chromosomes is dynamic and the unloading reaction is stimulated by Wpl1 (human ortholog, Wapl). During S phase, a subpopulation of cohesin becomes stably bound to chromatin in an Eso1 (fission yeast Eco1/Esco1-2)-dependent manner. Cohesin stabilization occurs unevenly along chromosomes. Cohesin remains largely labile at the rDNA repeats but binds mostly in the stable mode to pericentromere regions. This pattern is largely unchanged in eso1Δ wpl1Δ cells, and cohesion is unaffected, indicating that the main Eso1 role is counteracting Wpl1. A mutant of Psm3 (fission yeast Smc3) that mimics its acetylated state renders cohesin less sensitive to Wpl1-dependent unloading and partially bypasses the Eso1 requirement but cannot generate the stable mode of cohesin binding in the absence of Eso1. Conversely, nonacetylatable Psm3 reduces the stable cohesin fraction and affects cohesion in a Wpl1-dependent manner, but cells are viable. We propose that Psm3 acetylation contributes to Eso1 counteracting of Wpl1 to secure stable cohesin interaction with postreplicative chromosomes but that it is not the sole molecular event by which this occurs.

Role for cohesin in the formation of a heterochromatic domain at fission yeast subtelomeres.

Increasing evidence implicates cohesin in the control of gene expression. Here we report the first analysis of cohesin-dependent gene regulation in fission yeast. Global expression profiling of the mis4-367 cohesin loader mutant identified a small number of upregulated and downregulated genes within subtelomeric domains (SD). These 20- to 40-kb regions between chromosome arm euchromatin and telomere-proximal heterochromatin are characterized by a combination of euchromatin (methylated lysine 4 on histone H3/methylated Tysine 9 on histone H3 [H3K4me]) and heterochromatin (H3K9me) marks. We focused our analysis on the chromosome 1 right SD, which contains several upregulated genes and is bordered on the telomere-distal side by a pair of downregulated genes. We find that the expression changes in the SD also occur in a mutant of the cohesin core component Rad21. Remarkably, mutation of Rad21 results in the depletion of Swi6 binding in the SD. In fact, the Rad21 mutation phenocopied Swi6 loss of function: both mutations led to reduced cohesin binding, reduced H3K9me, and similar gene expression changes in the SD. In particular, expression of the gene pair bordering the SD was dependent both on cohesin and on Swi6. Our data indicate that cohesin participates in the setup of a subtelomeric heterochromatin domain and controls the expression of the genes residing in that domain.

Splicing factor Spf30 assists exosome-mediated gene silencing in fission yeast.

Heterochromatin assembly in fission yeast relies on the processing of cognate noncoding RNAs by both the RNA interference and the exosome degradation pathways. Recent evidence indicates that splicing factors facilitate the cotranscriptional processing of centromeric transcripts into small interfering RNAs (siRNAs). In contrast, how the exosome contributes to heterochromatin assembly and whether it also relies upon splicing factors were unknown. We provide here evidence that fission yeast Spf30 is a splicing factor involved in the exosome pathway of heterochromatin silencing. Spf30 and Dis3, the main exosome RNase, colocalize at centromeric heterochromatin and euchromatic genes. At the centromeres, Dis3 helps recruiting Spf30, whose deficiency phenocopies the dis3-54 mutant: heterochromatin is impaired, as evidenced by reduced silencing and the accumulation of polyadenylated centromeric transcripts, but the production of siRNAs appears to be unaffected. Consistent with a direct role, Spf30 binds centromeric transcripts and locates at the centromeres in an RNA-dependent manner. We propose that Spf30, bound to nascent centromeric transcripts, perhaps with other splicing factors, assists their processing by the exosome. Splicing factor intercession may thus be a common feature of gene silencing pathways.