Molecular Determinants of Chromosome Transmission and Cell Cycle Regulation

我们正在进行的研究主要集中在以下方面：1) 进化上保守的 Pat1（与拓扑异构酶 II 相关的蛋白质）和 polo 激酶 Cdc5 的着丝粒关联调节忠实的染色体分离。 Cse4 及其伴侣 Scm3（人类中的 HJURP）对于染色体分离至关重要，在许多癌症中过度表达且错误定位。 HJURP 表达升高的患者生存率降低。 HJURP 过度表达在肿瘤发生中的作用尚不清楚。我们正在研究调节 Cse4/CENP-A 及其相互作用蛋白 Scm3/HJURP 表达和定位的分子机制，以实现忠实的染色体分离。我们已经证明，HJURP 和 Scm3 的化学计量不平衡会导致人类和酵母细胞中的染色体错误分离，从而提供 HJURP 过度表达与癌症有丝分裂缺陷之间的联系（Mishra 等人，2011）。未来的研究将利用全基因组筛选来鉴定显示 HJURP 过度表达致死性的基因/通路，以可能治疗 HJURP 表达失调的癌症。 Scm3 与 Pat1（与拓扑异构酶 II 相关的蛋白质）相互作用，我们发现 Pat1 在着丝粒染色质拓扑和染色体分离中的作用 (Mishra et al., 2013)。我们使用 pat1 缺失菌株来定义酵母着丝粒处 Cse4 分子的数量 (Hasse, Mishra 2013, Mishra et al., 2015)。我们的结果表明，Pat1 调节着丝粒染色质的结构完整性和 Cse4 的定位，以实现忠实的染色体分离。正在进行的研究旨在了解着丝粒染色质的拓扑结构如何影响染色体分离，这是目前很大程度上尚未探索的研究领域。除了着丝粒蛋白之外，粘连蛋白与着丝粒以及沿着染色体长度的结合确保了有丝分裂期间姐妹染色单体的忠实分离。我们的研究表明，进化上保守的 polo 激酶 Cdc5 与着丝粒染色质结合，以促进着丝粒粘连蛋白的去除 (Mishra et al., 2016)。未来的研究将使我们能够了解 Cdc5 调节着丝粒粘连蛋白去除的机制。 2）着丝粒组蛋白的翻译后修饰（PTM）影响染色体分离。着丝粒组蛋白 H4 的独特乙酰化模式先前已在其他系统中报道过，然而，这种模式的生理作用尚未完全了解。使用具有单个核小体的芽殖酵母，我们确定着丝粒组蛋白 H4 的乙酰化模式影响染色体分离。我们提供了第一个证据，证明酵母着丝粒含有低乙酰化组蛋白 H4，并且组蛋白 H4 在赖氨酸 16 (H4K16) 上的乙酰化增加会导致染色体错误分离 (Choy et al., 2011)。尽管 HDAC 抑制剂 (HDACi) 用于临床试验，但我们仍不完全了解其作用方式。因此，我们使用 HDACi 进行了全基因组筛选，以确定易受组蛋白乙酰化改变影响的途径。我们的结果表明染色体分离突变体对 HDACi 更敏感 (Choy et al., 2015)。未来的研究将检验 HDACi 与影响染色体分离的药物相结合是否能更有效地治疗癌症，同时对正常细胞的影响最小。 Cse4 生化纯化的创新方法使我们能够首次对 Cse4 的 PTM 进行全面分析（Boeckmann 等人，2013）。鉴定了 Cse4 中乙酰化、甲基化和磷酸化的保守位点。我们生成了磷酸化特异性抗体，并显示了磷酸化 Cse4 与着丝粒的关联，并确定进化上保守的 Aurora B/Ipl1 激酶在体内和体外磷酸化 Cse4 以实现忠实的染色体分离。未来的研究将使我们能够了解 Cse4 磷酸化和甲基化在染色体分离中的分子作用，并确定这些 PTM 在人类 CENP-A 中是否保守。 3) Cse4细胞水平的严格调节可防止其错误定位以保证基因组稳定性。我们之前表明，酿酒酵母 spt4 突变体表现出 Cse4 的错误定位和染色体分离缺陷，这些缺陷由人类 SPT4 补充（Basrai 等人，1996 年以及 Crotti 和 Basrai 2004 年）。我们通过证明组蛋白剂量的改变和 Cse4 向非着丝粒染色质的错误定位与染色体丢失相关来确定 Cse4 错误定位的原因和影响 (Au et al., 2008)。防止 Cse4 错误定位的一种机制是 E3 连接酶 Psh1 泛素介导的 Cse4 蛋白水解。我们发现了 Cse4 N 末端在泛素 (Ub) 介导的蛋白质水解中的新作用，以实现忠实的染色体分离 (Au et al., 2013)。我们最近报道，Cse4 被苏酰化，Slx5 对苏酰化 Cse4 的泛素化调节其蛋白水解，以防止错误定位到常染色质 (Okhuni et al., 2016)。我们采用全基因组方法来识别防止 Cse4 错误定位到常染色质的调节因子。我们的研究揭示了组蛋白伴侣和其他 E3 Ub 连接酶在 Cse4 蛋白水解中的作用。我们正在进行的研究旨在深入分析筛选中确定的酵母基因，以了解防止 Cse4 错误定位以实现基因组稳定性的分子机制。 4) CENP-A 的错误定位导致人类细胞中的 CIN。鉴于CENP-A高表达的临床意义及其与癌症的相关性，了解CENP-A过表达如何促进肿瘤发生以及CENP-A表达是否可用于CENP-A过表达癌症的预后、诊断和靶向治疗至关重要。我们建立了细胞系并优化了基于细胞生物学的检测方法，以解决过度表达的 CENP-A 的错误定位是否会导致 CI​​N 这一长期存在的问题。我们确定 CENP-A 在 HeLa 和稳定二倍体 RPE1 细胞中的组成型或诱导型表达会导致 CENP-A 错误定位到非着丝粒区域。有丝分裂效应的综合分析表明，CENP-A 过表达对染色体分离缺陷和微核发生率较高的影响具有剂量依赖性。着丝粒蛋白定位的改变导致 CENP-A 过表达细胞中天然着丝粒的减弱。组蛋白伴侣 DAXX 的耗尽可防止 CENP-A 错误定位并挽救 CENP-A 过表达细胞中的 CIN 表型。这些结果表明，CENP-A 的错误定位是 CENP-A 过表达细胞中 CIN 的主要原因之一。我们的研究为 CENP-A 错位到非着丝粒染色质如何导致人类细胞中的 CIN 提供了第一个证据，并为 CENP-A 过度表达如何导致 CENP-A 过度表达癌症中的非整倍性提供了机制见解。我们正在对在全基因组筛选中鉴定的酵母基因的人类同源物进行研究，并使用其他方法来识别和表征防止 CENP-A 错误定位的途径，以实现基因组稳定性。

Our ongoing research is focused on the following: 1) Centromeric association of evolutionarily conserved Pat1 (Protein associated with topoisomerase II) and polo kinase Cdc5 regulate faithful chromosome segregation. Cse4 and its chaperone Scm3 (HJURP in humans), both of which are essential for chromosome segregation, are overexpressed and mis-localized in many cancers. Patients with elevated HJURP expression show a reduced survival rate. The role of HJURP overexpression in tumorigenesis is not yet understood. We are investigating the molecular mechanisms that regulate expression and localization of Cse4/CENP-A and its interacting proteins Scm3/HJURP for faithful chromosome segregation. We have shown that the imbalanced stoichiometry of HJURP and Scm3 lead to chromosome mis-segregation in both human and yeast cells thereby providing a link between HJURP overexpression and mitotic defects in cancers (Mishra et al., 2011). Future studies will utilize genome-wide screens to identify genes/pathways that show lethality with overexpression of HJURP for possible treatment of cancers with deregulated HJURP expression. Scm3 interacts with Pat1 (Protein associated with topoisomerase II) and we have uncovered a role for Pat1 in the topology of centromeric chromatin and chromosome segregation (Mishra et al., 2013). We used a pat1 deletion strain to define the number of Cse4 molecules at the yeast kinetochore (Hasse, Mishra 2013, Mishra et al., 2015). Our results show that Pat1 regulates the structural integrity of centromeric chromatin and localization of Cse4 for faithful chromosome segregation. Ongoing research is aimed at understanding how topology of centromeric chromatin affects chromosome segregation an area of research that is largely unexplored at the present time. In addition to kinetochore proteins, association of cohesins with centromeres and along the length of the chromosomes ensures faithful segregation of sister chromatids during mitosis. Our studies have shown that evolutionarily conserved polo kinase, Cdc5 associates with centromeric chromatin to facilitate the removal of centromeric cohesins (Mishra et al., 2016). Future studies will allow us to understand the mechanism by which Cdc5 regulates removal of centromeric cohesins. 2) Post-translational modifications (PTMs) of centromeric histones affect chromosome segregation. Distinctive acetylation pattern of centromeric histone H4 has been previously reported in other systems, however, the physiological role for this pattern is not fully understood. Using budding yeast with a single nucleosome we determined that the acetylation pattern of centromeric histone H4 affects chromosome segregation. We provide the first evidence that yeast centromeres contain hypoacetylated histone H4 and that increased acetylation of histone H4 on lysine 16 (H4K16) leads to chromosome mis-segregation (Choy et al., 2011). Even though HDAC inhibitors (HDACi) are used in clinical trials we do not fully understand their mode of action. Hence, we performed a genome-wide screen with an HDACi to identify pathways that are vulnerable to altered histone acetylation. Our results showed that chromosome segregation mutants are more sensitive to HDACi (Choy et al., 2015). Future studies will examine if combining HDACi with drugs that affect chromosome segregation are more effective for cancer treatment with a minimal effect on normal cells. An innovative approach for the biochemical purification of Cse4, allowed us to provide the first comprehensive analysis of PTMs of Cse4 (Boeckmann et al., 2013). Conserved sites for acetylation, methylation, and phosphorylation in Cse4 were identified. We generated a phospho-specific antibody and showed the association of phosphorylated Cse4 with centromeres and determined that evolutionarily conserved Aurora B/Ipl1 kinase phosphorylates Cse4 in vivo and in vitro for faithful chromosome segregation. Future studies will allow us to understand the molecular role of Cse4 phosphorylation and methylation in chromosome segregation and determine if these PTMs are conserved in human CENP-A. 3) Stringent regulation of cellular levels of Cse4 prevents its mislocalization for genome stability. We showed previously that S. cerevisiae spt4 mutants show mislocalization of Cse4 and chromosome segregation defects that are complemented by human SPT4 (Basrai et al, 1996 and Crotti and Basrai 2004). We established the cause and effect of Cse4 mislocalization by showing that altered histone dosage and mislocalization of Cse4 to non-centromeric chromatin correlate with chromosome loss (Au et al., 2008). One mechanism that prevents mislocalization of Cse4 is ubiquitin-mediated proteolysis of Cse4 by E3 ligase Psh1. We identified a novel role for the N terminus of Cse4 in ubiquitin (Ub)-mediated proteolysis for faithful chromosome segregation (Au et al., 2013). We recently reported that Cse4 is sumoylated and ubiquitination of sumoylated Cse4 by Slx5 regulates its proteolysis to prevent mislocalization to euchromatin (Ohkuni et al., 2016). We have undertaken genome-wide approaches to identify regulators that prevent mislocalization of Cse4 to euchromatin. Our studies have revealed a role for histone chaperones and other E3 Ub ligases in Cse4 proteolysis. Our ongoing studies are aimed at in-depth analysis of the yeast genes identified in the screen to understand the molecular mechanisms that prevent mislocalization of Cse4 for genome stability. 4) Mislocalization of CENP-A contributes to CIN in human cells. Given the clinical significance of high CENP-A expression and its correlation with cancer, it is critical to understand how CENP-A overexpression contributes to tumorigenesis and whether CENP-A expression can be exploited for prognosis, diagnosis and targeted treatment of CENP-A overexpressing cancers. We established cell lines and optimized cell biology based assays to address a long-standing question of whether mislocalization of overexpressed CENP-A contributes to CIN. We determined that constitutive or inducible expression of CENP-A in HeLa and stable diploid RPE1 cells results in mislocalization of CENP-A to non-centromeric regions. Comprehensive analysis for mitotic effects showed a dose-dependent effect of CENP-A overexpression on chromosome segregation defects and higher incidence of micronuclei. Altered localization of kinetochore proteins contributes to a weakening of the native kinetochore in CENP-A overexpressing cells. Depletion of the histone chaperone DAXX prevents CENP-A mislocalization and rescues the CIN phenotype in CENP-A overexpressing cells. These results show that mislocalization of CENP-A is one of the major contributors for CIN in CENP-A overexpressing cells. Our studies provide the first evidence for how mislocalization of CENP-A to non-centromeric chromatin contributes to CIN in human cells and provide mechanistic insights into how CENP-A overexpression may contribute to aneuploidy in CENP-A overexpressing cancers. We are pursuing studies with human homologs of the yeast genes identified in genome wide screens and using other approaches to identify and characterize pathways that prevent mislocalization of CENP-A for genome stability.