Mishra Prashant K, Guo Jiasheng, Dittman Lauren E, Haase Julian, Yeh Elaine, Bloom Kerry, Basrai Munira A
Genetics Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892.
Department of Biology, University of North Carolina, Chapel Hill, NC 27599.
Mol Biol Cell. 2015 Jun 1;26(11):2067-79. doi: 10.1091/mbc.E14-08-1335. Epub 2015 Apr 1.
Evolutionarily conserved histone H3 variant Cse4 and its homologues are essential components of specialized centromere (CEN)-specific nucleosomes and serve as an epigenetic mark for CEN identity and propagation. Cse4 is a critical determinant for the structure and function of the kinetochore and is required to ensure faithful chromosome segregation. The kinetochore protein Pat1 regulates the levels and spatial distribution of Cse4 at centromeres. Deletion of PAT1 results in altered structure of CEN chromatin and chromosome segregation errors. In this study, we show that Pat1 protects CEN-associated Cse4 from ubiquitination in order to maintain proper structure and function of the kinetochore in budding yeast. PAT1-deletion strains exhibit increased ubiquitination of Cse4 and faster turnover of Cse4 at kinetochores. Psh1, a Cse4-specific E3-ubiquitin ligase, interacts with Pat1 in vivo and contributes to the increased ubiquitination of Cse4 in pat1∆ strains. Consistent with a role of Psh1 in ubiquitination of Cse4, transient induction of PSH1 in a wild-type strain resulted in phenotypes similar to a pat1∆ strain, including a reduction in CEN-associated Cse4, increased Cse4 ubiquitination, defects in spatial distribution of Cse4 at kinetochores, and altered structure of CEN chromatin. Pat1 interacts with Scm3 and is required for its maintenance at kinetochores. In conclusion, our studies provide novel insights into mechanisms by which Pat1 affects the structure of CEN chromatin and protects Cse4 from Psh1-mediated ubiquitination for faithful chromosome segregation.
进化上保守的组蛋白H3变体Cse4及其同源物是特殊着丝粒（CEN）特异性核小体的重要组成部分，并作为CEN身份和传播的表观遗传标记。Cse4是动粒结构和功能的关键决定因素，是确保染色体忠实分离所必需的。动粒蛋白Pat1调节着丝粒处Cse4的水平和空间分布。PAT1的缺失导致CEN染色质结构改变和染色体分离错误。在本研究中，我们表明Pat1保护与CEN相关的Cse4不被泛素化，以维持芽殖酵母中动粒的正常结构和功能。PAT1缺失菌株表现出Cse4泛素化增加以及动粒处Cse4的周转加快。Psh1是一种Cse4特异性E3泛素连接酶，在体内与Pat1相互作用，并导致pat1∆菌株中Cse4泛素化增加。与Psh1在Cse4泛素化中的作用一致，在野生型菌株中瞬时诱导PSH1导致了与pat1∆菌株相似的表型，包括与CEN相关的Cse4减少、Cse4泛素化增加、Cse4在动粒处的空间分布缺陷以及CEN染色质结构改变。Pat1与Scm3相互作用，是其在动粒处维持所必需的。总之，我们的研究为Pat1影响CEN染色质结构以及保护Cse4免受Psh1介导的泛素化以实现忠实染色体分离的机制提供了新的见解。
