Kinetochore function and chromosome segregation rely on critical residues in histones H3 and H4 in budding yeast

Abstract

Accurate chromosome segregation requires that sister kinetochores biorient and attach to microtubules from opposite poles. Kinetochore biorientation relies on the underlying centromeric chromatin, which provides a platform to assemble the kinetochore and to recruit the regulatory factors that ensure the high fidelity of this process. To identify the centromeric chromatin determinants that contribute to chromosome segregation, we performed two complementary unbiased genetic screens using a library of budding yeast mutants in every residue of histone H3 and H4. In one screen, we identified mutants that lead to increased loss of a nonessential chromosome. In the second screen, we isolated mutants whose viability depends on a key regulator of biorientation, the Aurora B protein kinase. Nine mutants were common to both screens and exhibited kinetochore biorientation defects. Four of the mutants map near the unstructured nucleosome entry site, and their genetic interaction with reduced IPL1 can be suppressed by increasing the dosage of SGO1, a key regulator of biorientation. In addition, the composition of purified kinetochores was altered in six of the mutants. Together, this work identifies previously unknown histone residues involved in chromosome segregation and lays the foundation for future studies on the role of the underlying chromatin structure in chromosome segregation.

Keywords: biorientation; chromosomal passenger complex (CPC); chromosome segregation; histones; kinetochore.

Figure 1

Identification of H3 and H4 mutants with chromosome segregation defects. (A and B) The frequency of chromosome loss for the indicated histone H3 (A) and H4 (B) mutants identified in the colony-sectoring assay is plotted [all strains are derived from JDY176 (Table S3)]. (C) Fivefold serial dilutions of strains containing the indicated histone mutant in the presence or absence of deg-ipl1 (SBY719, SBY9837–SBY9847, SBY9880–SBY9888) were plated with (+) or without (−) doxycycline. Note that two different parent cassettes (WT H3 and H4) were used to generate mutants in the corresponding histone, and various pairs of images were cropped to assemble the figure. (D) The H3 (blue) and H4 residues (magenta) identified in both screens are highlighted on the nucleosome structure (Luger et al. 1997). The H3 Q5 residue in the unstructured tail and buried residues of H4 V81 and H4 Y98 are not shown. Front and side views are shown.

Figure 2

The histone mutants do not exhibit replication defects. (A) WT (SBY4) and orc2-1 (SBY11682) strains were shifted to 37° and then subjected to FACS analysis. (B) FACS profiles of histone mutants that exhibit segregation defects. Asynchronous cultures of the indicated histone mutants (SBY9119, SBY9120, SBY9625, SBY9660, SBY9664, SBY9665, SBY9673, SBY9724, SBY9725, SBY9786, SBY9818, SBY9832) were processed for FACS analyses.

Figure 3

Analysis of sister-chromatid segregation in the histone mutant strains. (A) Asynchronous cultures of deg-ipl1 strains (SBY9837–SBY9847) were grown in doxycycline for 0 or 6 hr, and ChrIV segregation was monitored in anaphase cells that had DNA masses at opposite poles. (B) deg-ipl1 mad3Δ strains containing WT H3 and H4 or the mutations indicated (SBY9848–SBY9858) were released from G1 in the presence of doxycycline. ChrIV segregation was monitored in anaphase cells with DNA masses at opposite poles.

Figure 4

Analysis of Cse4 levels and localization to the centromere in the H3 and H4 mutants. (A) The indicated deg-ipl1 histone mutant strains (SBY10182–10192) were grown in doxycycline for 6 hr, and crude lysates were immunoblotted with anti-Cse4 and anti-Pgk1 antibodies for quantitative analysis. A representative immunoblot is shown, and the mean quantified Cse4-to-Pgk1 ratio relative to the corresponding WT parent is reported under each lane. (B) Cse4 ChIP was performed on deg-ipl1 H3 (top) and H4 (bottom) mutant strains (SBY10182–10192) containing centromeric minichromosomes grown in doxycycline for 6 hr. Quantitative real-time PCR was carried out using oligos specific to endogenous CEN3, the minichromosome CEN, and a control locus (PHO5).

Figure 5

Analysis of kinetochore composition on minichromosomes purified from H3 and H4 mutant strains. (A) The indicated deg-ipl1 histone mutant strains (SBY10182–10192) were grown asynchronously in doxycycline for 6 hr and immunoblotted with antibodies against the indicated kinetochore proteins. Tubulin or Pgk1 are shown as loading controls. (B) Centromeric minichromosomes were immunoprecipitated from deg-ipl1 H3 (left) and H4 (right) mutant strains (SBY10182–10192) grown in doxycycline for 6 hr. The purifications were immunoblotted for the indicated outer kinetochore proteins (Spc105, Ndc80) and inner (Mif2, Ctf19, Cse4) kinetochore proteins. Ndc10 directly binds to the centromere and was used as a loading control.

Figure 6

Analysis of SGO1 overexpression in the deg-ipl1 histone mutant strains. Fivefold serial dilutions of deg-ipl1 histone mutant strains containing a control (−) or high-copy SGO1 plasmid (+) (SBY9870, SBY9871, SBY9874 SBY9875, and SBY10284–SBY10297) were analyzed for growth in the presence and absence of doxycycline.