Genetics Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, United States of America.
PLoS Genet. 2011 Sep;7(9):e1002303. doi: 10.1371/journal.pgen.1002303. Epub 2011 Sep 29.
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.
着丝粒（着丝粒 DNA 和相关蛋白）是高保真染色体传递的关键决定因素。进化上保守的 Scm3p 是着丝粒染色质的重要组成部分，是人类、裂殖酵母和 budding yeast 中着丝粒组装和功能所必需的。在肺癌和乳腺癌中观察到 budding yeast Scm3p 的哺乳动物同源物 HJURP 的过表达，并与预后不良相关；然而，这些观察结果的生理相关性尚不清楚。我们在酿酒酵母中过表达 SCM3 和 HJURP，在人细胞中过表达 HJURP，并定义了介导 Scm3p 染色体丢失表型的结构域。我们的结果表明，SCM3（GALSCM3）或 HJURP（GALHJURP）的过表达导致野生型酵母菌株中的染色体丢失，并且 HJURP 的过表达导致人细胞中的有丝分裂缺陷。GALSCM3 导致动粒突变体的活力降低、姐妹染色单体过早分离以及着丝粒处 Cse4p 和组蛋白 H4 的减少。CSE4 或组蛋白 H4 的过表达抑制染色体丢失并恢复 GALSCM3 菌株中着丝粒处 Cse4p 的水平。使用 scm3 的突变等位基因，我们鉴定了 Scm3p 中 N 端的一个结构域，该结构域介导其与 CEN DNA 的相互作用，并确定 GALSCM3 的染色体丢失表型是由于缺乏 Cse4p/H4 的 Scm3p 与着丝粒的关联。此外，我们确定与其他系统类似，Scm3p 的着丝粒关联是细胞周期调节的。我们的结果表明，Scm3p/HJURP、Cse4p 和组蛋白 H4 的比例改变导致染色体分离缺陷。我们得出结论，严格调节 HJURP 和 SCM3 的表达对于基因组稳定性至关重要。
