Misregulation of Scm3p/HJURP Causes Chromosome Instability in Saccharomyces cerevisiae and Human Cells

The kinetochore (centromeric DNA and associated proteins) is a key determinant for high fidelity chromosome transmission. Evolutionarily conserved Scm3p is an essential component of centromeric chromatin and is required for assembly and function of kinetochores in humans, fission yeast, and budding yeast. Overexpression of HJURP, the mammalian homolog of budding yeast Scm3p, has been observed in lung and breast cancers and is associated with poor prognosis; however, the physiological relevance of these observations is not well understood. We overexpressed SCM3 and HJURP in Saccharomyces cerevisiae and HJURP in human cells and defined domains within Scm3p that mediate its chromosome loss phenotype. Our results showed that the overexpression of SCM3 (GALSCM3) or HJURP (GALHJURP) caused chromosome loss in a wild-type yeast strain, and overexpression of HJURP led to mitotic defects in human cells. GALSCM3 resulted in reduced viability in kinetochore mutants, premature separation of sister chromatids, and reduction in Cse4p and histone H4 at centromeres. Overexpression of CSE4 or histone H4 suppressed chromosome loss and restored levels of Cse4p at centromeres in GALSCM3 strains. Using mutant alleles of scm3, we identified a domain in the N-terminus of Scm3p that mediates its interaction with CEN DNA and determined that the chromosome loss phenotype of GALSCM3 is due to centromeric association of Scm3p devoid of Cse4p/H4. Furthermore, we determined that similar to other systems the centromeric association of Scm3p is cell cycle regulated. Our results show that altered stoichiometry of Scm3p/HJURP, Cse4p, and histone H4 lead to defects in chromosome segregation. We conclude that stringent regulation of HJURP and SCM3 expression are critical for genome stability.

动粒（kinetochore，即着丝粒DNA及其结合蛋白）是保障染色体高保真传递的关键决定因素。进化保守的Scm3p是着丝粒染色质的必需组分，对人类、裂殖酵母与酿酒酵母中动粒的组装与功能具有不可或缺的作用。酿酒酵母Scm3p的哺乳动物同源蛋白HJURP的过表达现象已在肺癌与乳腺癌中被观测到，且与不良预后相关；然而此类观测结果的生理相关性尚未得到充分阐释。我们分别在酿酒酵母中过表达SCM3与HJURP，并在人类细胞中过表达HJURP，同时鉴定了Scm3p中介导其染色体丢失表型的结构域。研究结果显示，过表达SCM3（GALSCM3）或HJURP（GALHJURP）会使野生型酵母菌株出现染色体丢失现象，而HJURP过表达则会引发人类细胞的有丝分裂缺陷。GALSCM3过表达会降低动粒突变体的存活率，导致姐妹染色单体提前分离，并减少着丝粒处的Cse4p与组蛋白H4的水平。过表达CSE4或组蛋白H4可抑制GALSCM3菌株的染色体丢失，并恢复其着丝粒处Cse4p的水平。通过利用scm3的突变等位基因，我们鉴定出Scm3p的N端结构域可介导其与CEN DNA的相互作用，并明确GALSCM3诱导的染色体丢失表型源于不含Cse4p/H4的Scm3p在着丝粒处的异常结合。此外，我们证实与其他研究系统类似，Scm3p的着丝粒定位受细胞周期调控。我们的研究结果表明，Scm3p/HJURP、Cse4p与组蛋白H4的计量比失衡会引发染色体分离缺陷。综上，我们认为对HJURP与SCM3表达的严格调控对于维持基因组稳定性至关重要。