Analysis of gene expression by RNA Seq in mutants of Psh1, Cac2, or the double mutant relative to WT upon overexpression of Cse4 in Saccharomyces cerevisiae.

Correct localization of the centromeric histone variant CenH3/CENP-A/Cse4 is an important part of faithful chromosome segregation. Mislocalization of CenH3 could lead to ectopic centromere formation and missegregation, and could affect DNA replication and transcription. CENP-A is often overexpressed and mislocalized in cancer genomes, but the underlying mechanisms are not understood. One major regulator of Cse4 deposition is Psh1, an E3 ubiquitin ligase that controls levels of Cse4 to prevent deposition into noncentromeric regions. We present evidence that Chromatin assembly factor-1 (CAF-1), an evolutionarily conserved histone H3/H4 chaperone shown previously to interact with CenH3 in flies and human cells, regulates Cse4 deposition in budding yeast. Yeast CAF-1 (yCAF-1) is a heterotrimeric protein complex consisting of CAC1, CAC2, and CAC3, which interacts with Cse4, and can assemble Cse4 nucleosomes in vitro. yCAF-1 regulates the stability of both soluble and chromatin associated Cse4. Loss of yCAF-1 can rescue growth defects and changes in gene expression associated with Cse4 deposition that occur in the absence of Psh1-mediated proteolysis. Incorporation of Cse4 into promoter nucleosomes at transcriptionally active genes depends on yCAF-1. Overall our findings suggest CAF-1 can act as a CenH3 chaperone, regulating levels and incorporation of CenH3 in chromatin. Furthermore, the misincorporation of CenH3 at promoter regions may have negative consequences for gene expression.着丝粒组蛋白变体CenH3（CENP-A/Cse4）的正确定位是保障染色体精准分离的核心环节之一。CenH3的定位异常可导致异位着丝粒形成与染色体分离错误，同时还会影响DNA复制与转录过程。CENP-A在癌症基因组中常出现过表达与定位异常，但其背后的分子机制尚未明确。调控Cse4组装的关键因子之一是Psh1——一种E3泛素连接酶，可通过控制Cse4的蛋白水平，阻止其在非着丝粒区域的异常组装。本研究提供证据表明，染色质组装因子1（Chromatin assembly factor-1, CAF-1）作为一类进化保守的组蛋白H3/H4伴侣蛋白，此前已在果蝇与人类细胞中被证实可与CenH3相互作用，而在酿酒酵母中，它可调控Cse4的染色质组装过程。酿酒酵母CAF-1（yCAF-1）是由CAC1、CAC2与CAC3组成的异源三聚体蛋白复合物，可与Cse4结合，并能在体外完成Cse4核小体的组装。yCAF-1可同时调控可溶性与染色质结合型Cse4的蛋白稳定性。当缺失yCAF-1时，可挽救因Psh1介导的蛋白水解通路失活后，由Cse4异常组装引发的生长缺陷与基因表达谱改变。Cse4在转录活跃基因启动子区域核小体中的掺入过程完全依赖于yCAF-1。综上，本研究结果证实CAF-1可作为CenH3的伴侣蛋白，调控其在染色质中的蛋白水平与染色质掺入过程。此外，CenH3在启动子区域的错误掺入可能对基因表达产生不利影响。