Global analysis of core histones reveals nucleosomal surfaces required for chromosome bi-orientation

Abstract

The attachment of sister kinetochores to microtubules from opposite spindle poles is essential for faithful chromosome segregation. Kinetochore assembly requires centromere‐specific nucleosomes containing the histone H3 variant CenH3. However, the functional roles of the canonical histones (H2A, H2B, H3, and H4) in chromosome segregation remain elusive. Using a library of histone point mutants in Saccharomyces cerevisiae, 24 histone residues that conferred sensitivity to the microtubule‐depolymerizing drugs thiabendazole (TBZ) and benomyl were identified. Twenty‐three of these mutations were clustered at three spatially separated nucleosomal regions designated TBS‐I, ‐II, and ‐III (TBZ/benomyl‐sensitive regions I–III). Elevation of mono‐polar attachment induced by prior nocodazole treatment was observed in H2A‐I112A (TBS‐I), H2A‐E57A (TBS‐II), and H4‐L97A (TBS‐III) cells. Severe impairment of the centromere localization of Sgo1, a key modulator of chromosome bi‐orientation, occurred in H2A‐I112A and H2A‐E57A cells. In addition, the pericentromeric localization of Htz1, the histone H2A variant, was impaired in H4‐L97A cells. These results suggest that the spatially separated nucleosomal regions, TBS‐I and ‐II, are necessary for Sgo1‐mediated chromosome bi‐orientation and that TBS‐III is required for Htz1 function.

The INO80 chromatin remodeling complex prevents polyploidy and maintains normal chromatin structure at centromeres

“…As described above, H2A.Z contributes to proper chromosome segregation in budding yeast (Krogan et al 2004;Measday et al 2005;Sopko et al 2006;Kawashima et al 2011), raising the possibility that, as in higher eukaryotes, H2A.Z contributes to the conformation of chromatin structure of centromeric regions. Our data show that the pericentric regions of budding yeast chromosomes 4 and 12 have aberrant chromatin structure when Ino80 or Ies6 is missing, and there is an increase in H2A.Z enrichment at these locations in ino80 and ies6 mutant strains.…”

“…We first tested the ability of the mutant strains to survive in the presence of the microtubule-destabilizing drug benomyl and found that, similar to strains with defective centromere or kinetochore structure (Kawashima et al 2011;Storchova et al 2011), both ies6 and ino80 strains are hypersensitive compared with wild type (Fig. 5A).…”

“…In addition, H2A.Z was found enriched in chromatin flanking budding yeast centromeres, and loss of H2A.Z led to chromosome segregation defects (Krogan et al 2004). More recently, the enrichment of H2A.Z flanking the centromeres was found to be impaired in mutants that have defective kinetochore attachment (Kawashima et al 2011), raising the possibility that H2A.Z also contributes to the structure and function of point centromeres.…”

Kinetochore Function and Chromosome Segregation Rely on Critical Residues in Histones H3 and H4 in Budding Yeast

“…Although many screens for histone mutations that affect genomic stability have been reported (Smith et al 1996;Hyland et al 2005;Matsubara et al 2007;Dai et al 2008;Sakamoto et al 2009;Kawashima et al 2011;Yu et al 2011), none have been specifically directed at measuring chromosome segregation frequencies or sensitivity to Ipl1/Aurora downregulation. While this work was in progress, Kawashima et al (2011) reported a systematic screen of all histone mutants for sensitivity to the microtubule-depolymerizing agents benomyl and thiabendazole. There was little overlap of mutants identified in their study with those identified here, with the exception of H3 G44A and H4 Y98A.…”

Roles of common subunits within distinct multisubunit complexes

“…S3A). Thirtyeight phenotypic residues of H2B have been identified by previous screening of the histone-GLibrary (nonfused H2B point mutant library) (23)(24)(25). We individually mutated 3 (H2B-D71, -L109, or -K123) of 38 residues in the H2B-H2A and H2B-Htz1 fusions.…”