Wee1 and Cdc25 are controlled by conserved PP2A-dependent mechanisms in fission yeast.
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ABSTRACT:
SUBMITTER: Lucena R
PROVIDER: S-EPMC5351916 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Proper cell size is essential for cellular function. Nonetheless, despite more than 100 years of work on the subject, the mechanisms that maintain cell-size homeostasis are largely mysterious [1]. Cells in growing populations maintain cell size within a narrow range by coordinating growth and division. Bacterial and eukaryotic cells both demonstrate homeostatic size control, which maintains population-level variation in cell size within a certain range and returns the population average to that range if it is perturbed [1, 2]. Recent work has proposed two different strategies for size control: budding yeast has been proposed to use an inhibitor-dilution strategy to regulate size at the G1/S transition [3], whereas bacteria appear to use an adder strategy, in which a fixed amount of growth each generation causes cell size to converge on a stable average [4-6]. Here we present evidence that cell size in the fission yeast Schizosaccharomyces pombe is regulated by a third strategy: the size-dependent expression of the mitotic activator Cdc25. cdc25 transcript levels are regulated such that smaller cells express less Cdc25 and larger cells express more Cdc25, creating an increasing concentration of Cdc25 as cells grow and providing a mechanism for cells to trigger cell division when they reach a threshold concentration of Cdc25. Because regulation of mitotic entry by Cdc25 is well conserved, this mechanism may provide a widespread solution to the problem of size control in eukaryotes.
| S-EPMC5479637 | biostudies-literature
Project description:In eukaryotes, the cyclin-dependent kinase Cdk1p (Cdc2p) plays a central role in entry into and progression through nuclear division during mitosis and meiosis. Cdk1p is activated during meiotic nuclear divisions by dephosphorylation of its tyrosine-15 residue. The phosphorylation status of this residue is largely determined by the Wee1p kinase and the Cdc25p phosphatase. In fission yeast, the forkhead-type transcription factor Mei4p is essential for entry into the first meiotic nuclear division. We recently identified cdc25(+) as an essential target of Mei4p in the control of entry into meiosis I. Here, we show that wee1(+) is another important target of Mei4p in the control of entry into meiosis I. Mei4p bound to the upstream region of wee1(+) in vivo and in vitro and inhibited expression of wee1(+), whereas Mei4p positively regulated expression of the adjacent pseudogene. Overexpression of Mei4p inhibited expression of wee1(+) and induced that of the pseudogene. Conversely, deletion of Mei4p did not decrease expression of wee1(+) but inhibited that of the pseudogene. In addition, deletion of Mei4p-binding regions delayed repression of wee1(+) expression as well as induction of expression of the pseudogene. These results suggest that repression of wee1(+) expression is primarily owing to Mei4p-mediated transcriptional interference.
| S-EPMC4612672 | biostudies-literature
Project description:Cell polarization is key for the function of most eukaryotic cells, and regulates cell shape, migration and tissue architecture. Fission yeast, Schizosaccharomyces pombe cells are cylindrical and polarize cell growth to one or both cell tips dependent on the cell cycle stage. Whereas microtubule cytoskeleton contributes to the positioning of the growth sites by delivering polarity factors to the cell ends, the Cdc42 GTPase polarizes secretion via actin-dependent delivery and tethering of secretory vesicles to plasma membrane. How growth is restricted to cell tips and how re-initiation of tip growth is regulated in the cell cycle remains poorly understood. In this work we investigated the function of protein phosphatase type 2A (PP2A) in S. pombe morphogenesis by deleting the evolutionary conserved PTPA-type regulatory subunit that we named pta2. pta2-deleted cells showed morphological defects and altered growth pattern. Consistent with this, actin patches and active Cdc42 were mislocalized in the pta2 deletion. These defects were additive to the lack of Cdc42-GAP Rga4. pta2Δ cells show upregulated Cdc42 activity and pta2 interacts genetically with polarisome components Tea1, Tea4 and For3 leading to complete loss of cell polarity and rounded morphology. Thus, regulation of polarity by PP2A requires the polarisome and involves Pta2-dependent control of Cdc42 activity.
| S-EPMC3293916 | biostudies-literature
Project description:The kinase Cdc2p is a central regulator of entry into and progression through nuclear division during mitosis and meiosis in eukaryotes. Cdc2p is activated at the onset of mitosis by dephosphorylation on tyrosine-15, the phosphorylation status of which is determined mainly by the kinase Wee1p and the phosphatase Cdc25p. In fission yeast, the forkhead-type transcription factor Mei4p is required for expression of many genes during meiosis, with mei4 mutant cells arresting before meiosis I. The mechanism of cell cycle arrest in mei4 cells has remained unknown, however. We now show that cdc25(+) is an important target of Mei4p in control of entry into meiosis I. Forced dephosphorylation of Cdc2p on tyrosine-15 thus induced meiosis I in mei4 mutant cells without a delay, although no spores were formed. We propose that Mei4p acts as a rate-limiting regulator of meiosis I by activating cdc25(+) transcription in coordination with other meiotic events.
| S-EPMC1976235 | biostudies-literature
Project description:This experiment aimed at understanding the correlation between RNA polymerase II occupancy and cell size. For this, we used three strains of increasing average size (wee1-50 < wt < cdc25-22) and performed ChIP-seq experiments using anti-CTD antibodies.
2020-01-01 | E-MTAB-8522 | 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.
2008-12-31 | GSE13517 | 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:Connections between the protein kinases that function within complex cell polarity networks are poorly understood. Rod-shaped fission yeast cells grow in a highly polarized manner, and genetic screens have identified many protein kinases, including the CaMKK-like Ssp1 and the MARK/PAR-1 family kinase Kin1, that are required for polarized growth and cell shape, but their functional mechanisms and connections have been unknown [1-5]. We found that Ssp1 promotes cell polarity by phosphorylating the activation loop of Kin1. Kin1 regulates cell polarity and cytokinesis through unknown mechanisms [4-7]. We performed a large-scale phosphoproteomic screen and found that Kin1 phosphorylates itself and Pal1 to promote growth at cell tips, and these proteins are interdependent for localization to growing cell tips. Additional Kin1 substrates for cell polarity and cytokinesis (Tea4, Mod5, Cdc15, and Cyk3) were also phosphorylated by a second kinase, the DYRK family member Pom1 [8]. Kin1 and Pom1 were enriched at opposite ends of growing cells, and they phosphorylated largely non-overlapping sites on shared substrates. Combined inhibition of both Kin1and Pom1 led to synthetic defects in their shared substrates Cdc15 and Cyk3, confirming a non-redundant functional connection through shared substrates. These findings uncover a new Ssp1-Kin1 signaling pathway, and define its functional and mechanistic connection with Pom1 signaling for cell polarity and cytokinesis. These kinases are conserved in many eukaryotes including humans, suggesting that similar connections and mechanisms might operate in a broad range of cells.
| S-EPMC5760279 | biostudies-literature
Project description:BackgroundCohesin 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.ResultsHere, we present the high-resolution analysis of cohesin localization along fission yeast chromosomes. This reveals that several determinants, thought specific for different organisms, come together to shape the overall distribution. Cohesin is detected at chromosomal loading sites, characterized 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. We furthermore observe that centromeric cohesin spreads out onto chromosome arms during mitosis, dependent on Aurora B kinase activity, emphasizing the plasticity of cohesin behavior.ConclusionsOur findings suggest that features that were thought to differentiate cohesin between organisms collectively define the overall behavior of fission yeast cohesin. Apparent differences between organisms might reflect an emphasis on different aspects, rather than different principles, of cohesin action.
| S-EPMC2718518 | biostudies-literature
Project description:Cytokinesis involves temporally and spatially coordinated action of the cell cycle, cytoskeletal and membrane systems to achieve separation of daughter cells. The septation initiation network (SIN) and mitotic exit network (MEN) signaling pathways regulate cytokinesis and mitotic exit in the yeasts Schizosaccharomyces pombe and Saccharomyces cerevisiae, respectively. Previously, we have shown that in fission yeast, the nucleolar protein Dnt1 negatively regulates the SIN pathway in a manner that is independent of the Cdc14-family phosphatase Clp1/Flp1, but how Dnt1 modulates this pathway has remained elusive. By contrast, it is clear that its budding yeast relative, Net1/Cfi1, regulates the homologous MEN signaling pathway by sequestering Cdc14 phosphatase in the nucleolus before mitotic exit. In this study, we show that dnt1(+) positively regulates G2/M transition during the cell cycle. By conducting epistasis analyses to measure cell length at septation in double mutant (for dnt1 and genes involved in G2/M control) cells, we found a link between dnt1(+) and wee1(+). Furthermore, we showed that elevated protein levels of the mitotic inhibitor Wee1 kinase and the corresponding attenuation in Cdk1 activity is responsible for the rescuing effect of dnt1Δ on SIN mutants. Finally, our data also suggest that Dnt1 modulates Wee1 activity in parallel with SCF-mediated Wee1 degradation. Therefore, this study reveals an unexpected missing link between the nucleolar protein Dnt1 and the SIN signaling pathway, which is mediated by the Cdk1 regulator Wee1 kinase. Our findings also define a novel mode of regulation of Wee1 and Cdk1, which is important for integration of the signals controlling the SIN pathway in fission yeast.
| S-EPMC3820244 | biostudies-literature