Deposition of Centromeric Histone H3 Variant CENP-A/Cse4 into Chromatin Is Facilitated by Its C-Terminal Sumoylation.

Centromeric localization of CENP-A (Cse4 in Saccharomyces cerevisiae, CID in flies, CENP-A in humans) is essential for faithful chromosome segregation. Mislocalization of overexpressed CENP-A contributes to aneuploidy in yeast, flies, and humans, and is proposed to promote tumorigenesis in human cancers. Hence, defining molecular mechanisms that promote or prevent mislocalization of CENP-A is an area of active investigation. In budding yeast, evolutionarily conserved histone chaperones Scm3 and chromatin assembly factor-1 (CAF-1) promote localization of Cse4 to centromeric and noncentromeric regions, respectively. Ubiquitin ligases, such as Psh1 and Slx5, and histone chaperones (HIR complex) regulate proteolysis of overexpressed Cse4 and prevent its mislocalization to noncentromeric regions. In this study, we have identified sumoylation sites lysine (K) 215/216 in the C terminus of Cse4, and shown that sumoylation of Cse4 K215/216 facilitates its genome-wide deposition into chromatin when overexpressed. Our results showed reduced levels of sumoylation of mutant Cse4 K215/216R/A [K changed to arginine (R) or alanine (A)] and reduced interaction of mutant Cse4 K215/216R/A with Scm3 and CAF-1 when compared to wild-type Cse4 Consistent with these results, levels of Cse4 K215/216R/A in the chromatin fraction and localization to centromeric and noncentromeric regions were reduced. Furthermore, in contrast to GAL-CSE4, which exhibits Synthetic Dosage Lethality (SDL) in psh1∆, slx5∆, and hir2∆ strains, GAL-cse4K215/216R does not exhibit SDL in these strains. Taken together, our results show that deposition of Cse4 into chromatin is facilitated by its C-terminal sumoylation.

Skp, Cullin, F-box (SCF)-Met30 and SCF-Cdc4-Mediated Proteolysis of CENP-A Prevents Mislocalization of CENP-A for Chromosomal Stability in Budding Yeast.

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.

A Genome-Wide Screen Reveals a Role for the HIR Histone Chaperone Complex in Preventing Mislocalization of Budding Yeast CENP-A.

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.

N-terminal Sumoylation of Centromeric Histone H3 Variant Cse4 Regulates Its Proteolysis To Prevent Mislocalization to Non-centromeric Chromatin.

Stringent regulation of cellular levels of evolutionarily conserved centromeric histone H3 variant (CENP-A in humans, CID in flies, Cse4 in yeast) prevents its mislocalization to non-centromeric chromatin. Overexpression and mislocalization of CENP-A has been observed in cancers and leads to aneuploidy in yeast, flies, and human cells. Ubiquitin-mediated proteolysis of Cse4 by E3 ligases such as Psh1 and Sumo-Targeted Ubiquitin Ligase (STUbL) Slx5 prevent mislocalization of Cse4. Previously, we identified Siz1 and Siz2 as the major E3 ligases for sumoylation of Cse4. In this study, we have identified lysine 65 (K65) in Cse4 as a site that regulates sumoylation and ubiquitin-mediated proteolysis of Cse4 by Slx5. Strains expressing cse4 K65R exhibit reduced levels of sumoylated and ubiquitinated Cse4 in vivo Furthermore, co-immunoprecipitation experiments reveal reduced interaction of cse4 K65R with Slx5, leading to increased stability and mislocalization of cse4 K65R under normal physiological conditions. Based on the increased stability of cse4 K65R in psh1∆ strains but not in slx5∆ strains, we conclude that Slx5 targets sumoylated Cse4 K65 for ubiquitination-mediated proteolysis independent of Psh1. In summary, we have identified and characterized the physiological role of Cse4 K65 in sumoylation, ubiquitin-mediated proteolysis, and localization of Cse4 for genome stability.

Polo kinase Cdc5 associates with centromeres to facilitate the removal of centromeric cohesin during mitosis.

Sister chromatid cohesion is essential for tension-sensing mechanisms that monitor bipolar attachment of replicated chromatids in metaphase. Cohesion is mediated by the association of cohesins along the length of sister chromatid arms. In contrast, centromeric cohesin generates intrastrand cohesion and sister centromeres, while highly cohesin enriched, are separated by >800 nm at metaphase in yeast. Removal of cohesin is necessary for sister chromatid separation during anaphase, and this is regulated by evolutionarily conserved polo-like kinase (Cdc5 in yeast, Plk1 in humans). Here we address how high levels of cohesins at centromeric chromatin are removed. Cdc5 associates with centromeric chromatin and cohesin-associated regions. Maximum enrichment of Cdc5 in centromeric chromatin occurs during the metaphase-to-anaphase transition and coincides with the removal of chromosome-associated cohesin. Cdc5 interacts with cohesin in vivo, and cohesin is required for association of Cdc5 at centromeric chromatin. Cohesin removal from centromeric chromatin requires Cdc5 but removal at distal chromosomal arm sites does not. Our results define a novel role for Cdc5 in regulating removal of centromeric cohesins and faithful chromosome segregation.

SUMO-Targeted Ubiquitin Ligase (STUbL) Slx5 regulates proteolysis of centromeric histone H3 variant Cse4 and prevents its mislocalization to euchromatin.

Centromeric histone H3, CENP-ACse4, is essential for faithful chromosome segregation. Stringent regulation of cellular levels of CENP-ACse4 restricts its localization to centromeres. Mislocalization of CENP-ACse4 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.

Nbs1 ChIP-Seq Identifies Off-Target DNA Double-Strand Breaks Induced by AID in Activated Splenic B Cells.

Activation-induced cytidine deaminase (AID) is required for initiation of Ig class switch recombination (CSR) and somatic hypermutation (SHM) of antibody genes during immune responses. AID has also been shown to induce chromosomal translocations, mutations, and DNA double-strand breaks (DSBs) involving non-Ig genes in activated B cells. To determine what makes a DNA site a target for AID-induced DSBs, we identify off-target DSBs induced by AID by performing chromatin immunoprecipitation (ChIP) for Nbs1, a protein that binds DSBs, followed by deep sequencing (ChIP-Seq). We detect and characterize hundreds of off-target AID-dependent DSBs. Two types of tandem repeats are highly enriched within the Nbs1-binding sites: long CA repeats, which can form Z-DNA, and tandem pentamers containing the AID target hotspot WGCW. These tandem repeats are not nearly as enriched at AID-independent DSBs, which we also identified. Msh2, a component of the mismatch repair pathway and important for genome stability, increases off-target DSBs, similar to its effect on Ig switch region DSBs, which are required intermediates during CSR. Most of the off-target DSBs are two-ended, consistent with generation during G1 phase, similar to DSBs in Ig switch regions. However, a minority are one-ended, presumably due to conversion of single-strand breaks to DSBs during replication. One-ended DSBs are repaired by processes involving homologous recombination, including break-induced replication repair, which can lead to genome instability. Off-target DSBs, especially those present during S phase, can lead to chromosomal translocations, deletions and gene amplifications, resulting in the high frequency of B cell lymphomas derived from cells that express or have expressed AID.

Misregulation of Scm3p/HJURP causes chromosome instability in Saccharomyces cerevisiae and human cells.

The kinetochore (centromeric DNA and associated proteins) is a key determinant for high fidelity chromosome transmission. Evolutionarily conserved Scm3p is an essential component of centromeric chromatin and is required for assembly and function of kinetochores in humans, fission yeast, and budding yeast. Overexpression of HJURP, the mammalian homolog of budding yeast Scm3p, has been observed in lung and breast cancers and is associated with poor prognosis; however, the physiological relevance of these observations is not well understood. We overexpressed SCM3 and HJURP in Saccharomyces cerevisiae and HJURP in human cells and defined domains within Scm3p that mediate its chromosome loss phenotype. Our results showed that the overexpression of SCM3 (GALSCM3) or HJURP (GALHJURP) caused chromosome loss in a wild-type yeast strain, and overexpression of HJURP led to mitotic defects in human cells. GALSCM3 resulted in reduced viability in kinetochore mutants, premature separation of sister chromatids, and reduction in Cse4p and histone H4 at centromeres. Overexpression of CSE4 or histone H4 suppressed chromosome loss and restored levels of Cse4p at centromeres in GALSCM3 strains. Using mutant alleles of scm3, we identified a domain in the N-terminus of Scm3p that mediates its interaction with CEN DNA and determined that the chromosome loss phenotype of GALSCM3 is due to centromeric association of Scm3p devoid of Cse4p/H4. Furthermore, we determined that similar to other systems the centromeric association of Scm3p is cell cycle regulated. Our results show that altered stoichiometry of Scm3p/HJURP, Cse4p, and histone H4 lead to defects in chromosome segregation. We conclude that stringent regulation of HJURP and SCM3 expression are critical for genome stability.

Activation-induced cytidine deaminase induces reproducible DNA breaks at many non-Ig Loci in activated B cells.

After immunization or infection, activation-induced cytidine deaminase (AID) initiates diversification of immunoglobulin (Ig) genes in B cells, introducing mutations within the antigen-binding V regions (somatic hypermutation, SHM) and double-strand DNA breaks (DSBs) into switch (S) regions, leading to antibody class switch recombination (CSR). We asked if, during B cell activation, AID also induces DNA breaks at genes other than IgH genes. Using a nonbiased genome-wide approach, we have identified hundreds of reproducible, AID-dependent DSBs in mouse splenic B cells shortly after induction of CSR in culture. Most interestingly, AID induces DSBs at sites syntenic with sites of translocations, deletions, and amplifications found in human B cell lymphomas, including within the oncogene B cell lymphoma11a (bcl11a)/evi9. Unlike AID-induced DSBs in Ig genes, genome-wide AID-dependent DSBs are not restricted to transcribed regions and frequently occur within repeated sequence elements, including CA repeats, non-CA tandem repeats, and SINEs.