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.

A nucleosome is composed of intrinsically disordered histone tails and a structured nucleosome core surrounded by DNA. A variety of modifiable residues on the intrinsically disordered histone tails have been identified in the last decade. Mapping of the functional residues on the structured nucleosome core surface was recently initiated by global analysis of a comprehensive histone point mutant library (histone-GLibrary). It stands to reason that a functional relationship exists between modifiable residues on the intrinsically disordered histone tails and functional residues on the structured nucleosome core; however, this matter has been poorly explored. During transcription elongation, trimethylation of histone H3 at lysine 36 (H3-K36me3) is mediated by histone methyltransferase Set2, which binds to RNA polymerase II. Here, we used a histone-GLibrary that encompasses the nucleosomal DNA entry ⁄ exit site to show that six residues (H2A-G107, H2A-I112, H2A-L117, H3-T45, H3-R49 and H3-R52) form a surface on the structured nucleosome core and regulate H3-K36me3. Trimethylation at H3-K4 introduced by histone methyltransferase Set1 was not affected by the mutation of any of the six residues. Chromatin immunoprecipitation analysis showed that most of these residues are critical for the chromatin association of RNA polymerase II and Set2, suggesting that these components regulate H3-K36me3 through functional interactions with the structured nucleosome core surface.

Significance The same protein is often a subunit of more than one multisubunit protein complex, each of which has a distinct function within cells. Mutating such a protein would cause multiple cellular defects; therefore, it is difficult to distinguish between the function of a protein in one complex from its functions in other complexes. Here, we developed a unique strategy to overcome this problem, which will help to analyze a variety of biological processes by revealing the specific roles played by such proteins within multisubunit protein complexes.

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