Department of Molecular and Cell Biology, University of California, Berkeley, California 947202, USA.
Genetics. 2011 Jan;187(1):9-19. doi: 10.1534/genetics.110.123117. Epub 2010 Oct 13.
Accurate chromosome segregation is dependent on the centromere-specific histone H3 isoform known generally as CenH3, or as Cse4 in budding yeast. Cytological experiments have shown that Cse4 appears at extracentromeric loci in yeast cells deficient for both the CAF-1 and HIR histone H3/H4 deposition complexes, consistent with increased nondisjunction in these double mutant cells. Here, we examined molecular aspects of this Cse4 mislocalization. Genome-scale chromatin immunoprecipitation analyses demonstrated broader distribution of Cse4 outside of centromeres in cac1Δ hir1Δ double mutant cells that lack both CAF-1 and HIR complexes than in either single mutant. However, cytological localization showed that the essential inner kinetochore component Mif2 (CENP-C) was not recruited to extracentromeric Cse4 in cac1Δ hir1Δ double mutant cells. We also observed that rpb1-1 mutants displayed a modestly increased Cse4 half-life at nonpermissive temperatures, suggesting that turnover of Cse4 is partially dependent on Pol II transcription. We used genome-scale assays to demonstrate that the CAF-1 and HIR complexes independently stimulate replication-independent histone H3 turnover rates. We discuss ways in which altered histone exchange kinetics may affect eviction of Cse4 from noncentromeric loci.
准确的染色体分离依赖于着丝粒特异性组蛋白 H3 异构体，通常称为 CenH3，或在出芽酵母中称为 Cse4。细胞学实验表明，在缺乏 CAF-1 和 HIR 组蛋白 H3/H4 沉积复合物的酵母细胞中，Cse4 出现在着丝粒外的额外位置，这与这些双突变体细胞中非分离的增加一致。在这里，我们检查了这种 Cse4 定位错误的分子方面。全基因组染色质免疫沉淀分析表明，在缺乏 CAF-1 和 HIR 复合物的 cac1Δhir1Δ双突变体细胞中，Cse4 在着丝粒外的分布比在单个突变体中更广泛。然而，细胞学定位表明，必需的内着丝粒成分 Mif2（CENP-C）没有被招募到 cac1Δhir1Δ双突变体细胞中的额外着丝粒 Cse4 上。我们还观察到 rpb1-1 突变体在非许可温度下显示出适度增加的 Cse4 半衰期，这表明 Cse4 的周转率部分依赖于 Pol II 转录。我们使用全基因组测定来证明 CAF-1 和 HIR 复合物独立地刺激复制非依赖性组蛋白 H3 周转率。我们讨论了改变组蛋白交换动力学如何影响 Cse4 从非着丝粒位置的驱逐。
