Pat1 protects centromere-specific histone H3 variant Cse4 from Psh1-mediated ubiquitination.
ABSTRACT: Evolutionarily conserved histone H3 variant Cse4 and its homologues are essential components of specialized centromere (CEN)-specific nucleosomes and serve as an epigenetic mark for CEN identity and propagation. Cse4 is a critical determinant for the structure and function of the kinetochore and is required to ensure faithful chromosome segregation. The kinetochore protein Pat1 regulates the levels and spatial distribution of Cse4 at centromeres. Deletion of PAT1 results in altered structure of CEN chromatin and chromosome segregation errors. In this study, we show that Pat1 protects CEN-associated Cse4 from ubiquitination in order to maintain proper structure and function of the kinetochore in budding yeast. PAT1-deletion strains exhibit increased ubiquitination of Cse4 and faster turnover of Cse4 at kinetochores. Psh1, a Cse4-specific E3-ubiquitin ligase, interacts with Pat1 in vivo and contributes to the increased ubiquitination of Cse4 in pat1? strains. Consistent with a role of Psh1 in ubiquitination of Cse4, transient induction of PSH1 in a wild-type strain resulted in phenotypes similar to a pat1? strain, including a reduction in CEN-associated Cse4, increased Cse4 ubiquitination, defects in spatial distribution of Cse4 at kinetochores, and altered structure of CEN chromatin. Pat1 interacts with Scm3 and is required for its maintenance at kinetochores. In conclusion, our studies provide novel insights into mechanisms by which Pat1 affects the structure of CEN chromatin and protects Cse4 from Psh1-mediated ubiquitination for faithful chromosome segregation.
Project description:Regulating levels of centromeric histone H3 (CenH3) variant is crucial for genome stability. Interaction of Psh1, an E3 ligase, with the C terminus of Cse4 has been shown to contribute to its proteolysis. Here, we demonstrate a role for ubiquitination of the N terminus of Cse4 in regulating Cse4 proteolysis for faithful chromosome segregation and a role for Doa1 in ubiquitination of Cse4.
Project description:Centromeric histone H3, CENP-A<sup>Cse4</sup>, is essential for faithful chromosome segregation. Stringent regulation of cellular levels of CENP-A<sup>Cse4</sup> restricts its localization to centromeres. Mislocalization of CENP-A<sup>Cse4</sup> is associated with aneuploidy in yeast, flies and tumorigenesis in human cells; thus, defining pathways that regulate CENP-A levels is critical for understanding how mislocalization of CENP-A contributes to aneuploidy in human cancers. Previous work in budding yeast has shown that ubiquitination of overexpressed Cse4 by Psh1, an E3 ligase, partially contributes to proteolysis of Cse4. Here, we provide the first evidence that Cse4 is sumoylated by E3 ligases Siz1 and Siz2 in vivo and in vitro. Ubiquitination of Cse4 by Small Ubiquitin-related Modifier (SUMO)-Targeted Ubiquitin Ligase (STUbL) Slx5 plays a critical role in proteolysis of Cse4 and prevents mislocalization of Cse4 to euchromatin under normal physiological conditions. Accumulation of sumoylated Cse4 species and increased stability of Cse4 in slx5? strains suggest that sumoylation precedes ubiquitin-mediated proteolysis of Cse4. Slx5-mediated Cse4 proteolysis is independent of Psh1 since slx5? psh1? strains exhibit higher levels of Cse4 stability and mislocalization compared to either slx5? or psh1? strains. Our results demonstrate a role for Slx5 in ubiquitin-mediated proteolysis of Cse4 to prevent its mislocalization and maintain genome stability.
Project description:Shugoshin is an evolutionarily conserved protein, which is involved in tension sensing on mitotic chromosomes, kinetochore biorientation, and protection of centromeric (CEN) cohesin for faithful chromosome segregation. Interaction of the C-terminus of Sgo1 with phosphorylated histone H2A regulates its association with CEN and pericentromeric (peri-CEN) chromatin, whereas mutations in histone H3 selectively compromise the association of Sgo1 with peri-CEN but not CEN chromatin. Given that histone H3 is absent from CEN and is replaced by a histone H3 variant CENP-A<sup>Cse4</sup>, we investigated if CENP-A<sup>Cse4</sup> interacts with Sgo1 and promotes its association with the CEN chromatin. In this study, we found that Sgo1 interacts with CENP-A<sup>Cse4</sup> in vivo and in vitro. The N-terminus coiled-coil domain of Sgo1 without the C-terminus (sgo1-NT) is sufficient for its interaction with CENP-A<sup>Cse4</sup>, association with CEN but not the peri-CEN, and this CEN association is cell cycle dependent with maximum enrichment in mitosis. In agreement with the role of CENP-A<sup>Cse4</sup> in CEN maintenance of Sgo1, depletion of CENP-A<sup>Cse4</sup> results in the loss of Sgo1 and sgo1-NT from the CEN chromatin. The N-terminus of Sgo1 is required for genome stability as a mutant lacking the N-terminus (sgo1-CT) exhibits increased chromosome missegregation when compared to a sgo1-NT mutant. In summary, our results define a novel role for the N-terminus of Sgo1 in CENP-A<sup>Cse4</sup> mediated recruitment of Sgo1 to CEN chromatin for faithful chromosome segregation.
Project description:The kinetochore (centromeric DNA and associated protein complex) is essential for faithful chromosome segregation and maintenance of genome stability. Here we report that an evolutionarily conserved protein Pat1 is a structural component of Saccharomyces cerevisiae kinetochore and associates with centromeres in a NDC10-dependent manner. Consistent with a role for Pat1 in kinetochore structure and function, a deletion of PAT1 results in delay in sister chromatid separation, errors in chromosome segregation, and defects in structural integrity of centromeric chromatin. Pat1 is involved in topological regulation of minichromosomes as altered patterns of DNA supercoiling were observed in pat1? cells. Studies with pat1 alleles uncovered an evolutionarily conserved region within the central domain of Pat1 that is required for its association with centromeres, sister chromatid separation, and faithful chromosome segregation. Taken together, our data have uncovered a novel role for Pat1 in maintaining the structural integrity of centromeric chromatin to facilitate faithful chromosome segregation and proper kinetochore function.
Project description:The budding yeast kinetochore is ~68 nm in length with a diameter slightly larger than a 25 nm microtubule. The kinetochores from the 16 chromosomes are organized in a stereotypic cluster encircling central spindle microtubules. Quantitative analysis of the inner kinetochore cluster (Cse4, COMA) reveals structural features not apparent in singly attached kinetochores. The cluster of Cse4-containing kinetochores is physically larger perpendicular to the spindle axis relative to the cluster of Ndc80 molecules. If there was a single Cse4 (molecule or nucleosome) at the kinetochore attached to each microtubule plus end, the cluster of Cse4 would appear geometrically identical to Ndc80. Thus, the structure of the inner kinetochore at the surface of the chromosomes remains unsolved. We have used point fluorescence microscopy and statistical probability maps to deduce the two-dimensional mean position of representative components of the yeast kinetochore relative to the mitotic spindle in metaphase. Comparison of the experimental images to three-dimensional architectures from convolution of mathematical models reveals a pool of Cse4 radially displaced from Cse4 at the kinetochore and kinetochore microtubule plus ends. The pool of displaced Cse4 can be experimentally depleted in mRNA processing pat1? or xrn1? mutants. The peripheral Cse4 molecules do not template outer kinetochore components. This study suggests an inner kinetochore plate at the centromere-microtubule interface in budding yeast and yields information on the number of Ndc80 molecules at the microtubule attachment site.
Project description:The histone H3 variant Cse4 specifies centromere identity in Saccharomyces cerevisiae by its incorporation into a special nucleosome positioned at CEN DNA and promotes the assembly of the kinetochore complex, which is required for faithful chromosome segregation. Our previous work showed that Cse4 is also associated with the partitioning locus STB of the 2?m circle--a multicopy plasmid that resides in the yeast nucleus and propagates itself stably. Cse4 is essential for the functional assembly of the plasmid partitioning complex, including the recruitment of the yeast cohesin complex at STB. We have located Cse4 association strictly at the origin-proximal subregion of STB. Three of the five directly repeated tandem copies of a 62-bp consensus sequence element constituting this region are necessary and sufficient for the recruitment of Cse4. The association of Cse4 with STB is dependent on Scm3, the loading factor responsible for the incorporation of Cse4 into the CEN nucleosome. A chromosomally integrated copy of STB confers on the integration site the capacity for Cse4 association as well as cohesin assembly. The localization of Cse4 in chromatin digested by micrococcal nuclease is consistent with the potential assembly of one Cse4-containing nucleosome, but not more than two, at STB. The remarkable ability of STB to acquire a very specialized, and strictly regulated, chromosome segregation factor suggests its plausible evolutionary kinship with CEN.
Project description:The centromeric histone H3 variant (CenH3) is essential for chromosome segregation in eukaryotes. We identify posttranslational modifications of Saccharomyces cerevisiae CenH3, Cse4. Functional characterization of cse4 phosphorylation mutants shows growth and chromosome segregation defects when combined with kinetochore mutants okp1 and ame1. Using a phosphoserine-specific antibody, we show that the association of phosphorylated Cse4 with centromeres increases in response to defective microtubule attachment or reduced cohesion. We determine that evolutionarily conserved Ipl1/Aurora B contributes to phosphorylation of Cse4, as levels of phosphorylated Cse4 are reduced at centromeres in ipl1 strains in vivo, and in vitro assays show phosphorylation of Cse4 by Ipl1. Consistent with these results, we observe that a phosphomimetic cse4-4SD mutant suppresses the temperature-sensitive growth of ipl1-2 and Ipl1 substrate mutants dam1 spc34 and ndc80, which are defective for chromosome biorientation. Furthermore, cell biology approaches using a green fluorescent protein-labeled chromosome show that cse4-4SD suppresses chromosome segregation defects in dam1 spc34 strains. On the basis of these results, we propose that phosphorylation of Cse4 destabilizes defective kinetochores to promote biorientation and ensure faithful chromosome segregation. Taken together, our results provide a detailed analysis, in vivo and in vitro, of Cse4 phosphorylation and its role in promoting faithful chromosome segregation.
Project description:Restricting the localization of the histone H3 variant CENP-A (Cse4 in yeast, CID in flies) to centromeres is essential for faithful chromosome segregation. Mislocalization of CENP-A leads to chromosomal instability (CIN) in yeast, fly and human cells. Overexpression and mislocalization of CENP-A has been observed in many cancers and this correlates with increased invasiveness and poor prognosis. Yet genes that regulate CENP-A levels and localization under physiological conditions have not been defined. In this study we used a genome-wide genetic screen to identify essential genes required for Cse4 homeostasis to prevent its mislocalization for chromosomal stability. We show that two Skp, Cullin, F-box (SCF) ubiquitin ligases with the evolutionarily conserved F-box proteins Met30 and Cdc4 interact and cooperatively regulate proteolysis of endogenous Cse4 and prevent its mislocalization for faithful chromosome segregation under physiological conditions. The interaction of Met30 with Cdc4 is independent of the D domain, which is essential for their homodimerization and ubiquitination of other substrates. The requirement for both Cdc4 and Met30 for ubiquitination is specifc for Cse4; and a common substrate for Cdc4 and Met30 has not previously been described. Met30 is necessary for the interaction between Cdc4 and Cse4, and defects in this interaction lead to stabilization and mislocalization of Cse4, which in turn contributes to CIN. We provide the first direct link between Cse4 mislocalization to defects in kinetochore structure and show that SCF-mediated proteolysis of Cse4 is a major mechanism that prevents stable maintenance of Cse4 at non-centromeric regions, thus ensuring faithful chromosome segregation. In summary, we have identified essential pathways that regulate cellular levels of endogenous Cse4 and shown that proteolysis of Cse4 by SCF-Met30/Cdc4 prevents mislocalization and CIN in unperturbed cells.
Project description:Centromeric localization of the evolutionarily conserved centromere-specific histone H3 variant CENP-A (Cse4 in yeast) is essential for faithful chromosome segregation. Overexpression and mislocalization of CENP-A lead to chromosome segregation defects in yeast, flies, and human cells. Overexpression of CENP-A has been observed in human cancers; however, the molecular mechanisms preventing CENP-A mislocalization are not fully understood. Here, we used a genome-wide synthetic genetic array (SGA) to identify gene deletions that exhibit synthetic dosage lethality (SDL) when Cse4 is overexpressed. Deletion for genes encoding the replication-independent histone chaperone HIR complex (HIR1, HIR2, HIR3, HPC2) and a Cse4-specific E3 ubiquitin ligase, PSH1, showed highest SDL. We defined a role for Hir2 in proteolysis of Cse4 that prevents mislocalization of Cse4 to noncentromeric regions for genome stability. Hir2 interacts with Cse4 in vivo, and hir2? strains exhibit defects in Cse4 proteolysis and stabilization of chromatin-bound Cse4 Mislocalization of Cse4 to noncentromeric regions with a preferential enrichment at promoter regions was observed in hir2? strains. We determined that Hir2 facilitates the interaction of Cse4 with Psh1, and that defects in Psh1-mediated proteolysis contribute to increased Cse4 stability and mislocalization of Cse4 in the hir2? strain. In summary, our genome-wide screen provides insights into pathways that regulate proteolysis of Cse4 and defines a novel role for the HIR complex in preventing mislocalization of Cse4 by facilitating proteolysis of Cse4, thereby promoting genome stability.
Project description:During cell division, segregation of sister chromatids to daughter cells is achieved by the poleward pulling force of microtubules, which attach to the chromatids by means of a multiprotein complex, the kinetochore. Kinetochores assemble at the centromeric DNA organized by specialized centromeric nucleosomes. In contrast to other eukaryotes, which typically have large repetitive centromeric regions, budding yeast CEN DNA is defined by a 125 bp sequence and assembles a single centromeric nucleosome. In budding yeast, as well as in other eukaryotes, the Cse4 histone variant (known in vertebrates as CENP-A) is believed to substitute for histone H3 at the centromeric nucleosome. However, the exact composition of the CEN nucleosome remains a subject of debate. We report the use of a novel ChIP approach to reveal the composition of the centromeric nucleosome and its localization on CEN DNA in budding yeast. Surprisingly, we observed a strong interaction of H3, as well as Cse4, H4, H2A, and H2B, but not histone chaperone Scm3 (HJURP in human) with the centromeric DNA. H3 localizes to centromeric DNA at all stages of the cell cycle. Using a sequential ChIP approach, we could demonstrate the co-occupancy of H3 and Cse4 at the CEN DNA. Our results favor a H3-Cse4 heterotypic octamer at the budding yeast centromere. Whether or not our model is correct, any future model will have to account for the stable association of histone H3 with the centromeric DNA.