Molecular Determinants of Chromosome Transmission and Cell Cycle Regulation

我们使用多生物（酵母，小鼠和人类细胞）和多学科（遗传学，细胞生物学，生化和全基因组）方法来研究忠实的染色体分离，这是每个活细胞的基本过程。遗传筛选是一个起点，深入的机制研究为着丝粒基因的新作用和新着丝粒基因的鉴定提供了证据。我们已经确定并定义了Cse4在染色体分离中的翻译后修饰（乙酰化、甲基化、磷酸化、聚合化和泛素化）的作用。我们的研究重点是了解Cse4相关蛋白在染色体分离中的作用，并确定防止Cse4错定位到非着丝粒区域的途径。在第一个项目中，我们定义了Scm3、Pat1、Cdc5和Sgo1在着丝粒染色质组装中的作用，并表征了着丝粒组蛋白翻译后修饰在染色体忠实分离中的作用。我们确定Cse4伴侣蛋白Scm3（人类中的HJURP）的化学计量失衡导致人类和酵母细胞中的染色体错分离，从而提供了HJURP过表达与癌症中有丝分裂缺陷之间的联系（Mishra等人，2011）。Scm3与Pat1（与拓扑异构酶II相关的蛋白质）相互作用，Pat1调节着丝染色质的拓扑结构（Mishra et al., 2013）。我们使用pat1缺失菌株来确定酵母着丝点上Cse4分子的数量（Hasse, Mishra 2013, Mishra et al., 2015），并为pat1在着丝粒染色质的结构完整性和Cse4的定位中发挥结构作用提供了证据，以实现忠实的染色体分离。除着丝点蛋白外，内聚蛋白与着丝粒的结合以及沿着染色体的长度确保有丝分裂过程中姐妹染色单体的忠实分离。我们报道了进化上保守的polo激酶，Cdc5与着丝粒染色质结合，促进着丝粒内聚蛋白的去除（Mishra等人，2016），Cdc5介导的Cse4磷酸化调节忠实的染色体分离（Mishra等人，2019）。此外，进化上保守的Sgo1保护着着丝粒内聚，它与Cse4相互作用，这是忠实的染色体分离所必需的（Mishra等人，2018）。我们最近报道，进化上保守的Hpr1阻止着丝粒染色质上r环的积累，影响着丝粒的组装并导致染色体不稳定（Mishra et al., 2021）。我们对Cse4的翻译后修饰（PTMs）进行了全面分析，并确定了乙酰化、甲基化和磷酸化的保守位点（Boeckmann et al., 2013）。我们确定进化上保守的Aurora B/Ipl1激酶在体内和体外磷酸化Cse4以实现可靠的染色体分离（Boeckmann等人，2013），并且细胞周期调节的Cse4甲基化可阻止CIN （Mishra等人，2023）。利用单核小体的出芽酵母，我们首次提供了证据，证明酵母着丝粒含有低乙酰化的组蛋白H4，赖氨酸16 （H4K16）上组蛋白H4乙酰化的增加导致染色体错误分离（Choy等，2011）。尽管HDAC抑制剂（HDACi）用于临床试验，但我们并不完全了解它们的作用模式。使用HDACi进行全基因组筛选，以确定易受组蛋白乙酰化改变的途径。我们的研究结果表明，染色体分离突变体对HDACi更敏感（Choy et al., 2015）。未来的研究将使我们能够了解Cse4的PTMs在染色体分离中的分子作用，并确定这些PTMs是否在人类CENP-A中保守。在第二个项目中，我们专注于识别防止Cse4和CIN错误定位的途径。我们之前的研究表明，酿酒葡萄球菌的spt4突变体表现出Cse4的错位和染色体分离缺陷，这些缺陷与人类的spt4互补（Basrai et al ., 1996; Crotti and Basrai 2004）。我们通过证明组蛋白剂量改变和Cse4错定位于非着丝粒染色质与染色体丢失相关，确定了Cse4错定位的原因和影响（Au et al., 2008）。我们发现了Cse4的N端在泛素（Ub）介导的忠实染色体分离蛋白水解中的新作用（Au等，2013），并表明Cse4被sumyylation， Slx5对sumy化的Cse4的泛素化调节其蛋白水解，以防止错定位到常染色质（Ohkuni等，2016,2018,2020）。全基因组方法已用于鉴定防止Cse4错定位到常染色质的调节因子，这些研究揭示了组蛋白伴侣的作用（Ciftci-Yilmaz et al., 2018）。F-box蛋白Cdc4和Met30在Cse4蛋白水解（Au等，2020），Dbf4依赖激酶（DDK） （Eisenstatt等，2020）和Cdc48 （Ohkuni等，2022）。此外，减少组蛋白H4的剂量可以防止Cse4的错误定位（Eisenstatt et al., 2021）。在第三个项目中，我们重点研究了人类细胞和异种移植小鼠模型中CENP-A错定位的原因和后果。在许多癌症中观察到CENP-A的错误定位，这与预后不良有关。因此，了解CENP-A过表达如何促进肿瘤发生以及是否可以利用CENP-A过表达癌症的预后、诊断和靶向治疗是至关重要的。我们建立了细胞系并优化了基于细胞生物学的检测方法，以解决一个长期存在的问题，即过度表达的CENP-A的错误定位是否会导致CIN。我们确定了在HeLa和稳定的二倍体RPE1细胞中组成或诱导表达CENP-A会导致CENP-A错定位到非着丝粒区域。有丝分裂效应的综合分析表明，过表达的CENP-A对染色体分离缺陷和微核发生率的影响呈剂量依赖性。在过表达CENP-A的细胞中，着丝粒蛋白定位的改变导致了原生着丝粒的减弱。在过表达CENP-A的细胞中，组蛋白伴侣DAXX的缺失可防止CENP-A错定位并挽救CIN表型。这些结果表明，CENP-A的错位是CENP-A过表达细胞发生CIN的主要原因之一。我们的研究提供了第一个证据，证明了CENP-A错定位于非着丝粒染色质如何导致人类细胞中的CIN，并提供了关于CENP-A过表达如何导致CENP-A过表达的癌症中的非整倍体的机制见解（Shrestha等，2017）。我们最近报道了假二倍体DLD1细胞系和异种移植小鼠模型中过表达的CENP-A的错误定位导致CIN、非整倍体和核型异质性（Shrestha等，2021）。我们正在研究在全基因组筛选中鉴定的酵母基因的人类同源物，并使用全基因组方法鉴定和表征防止CENP-A和CIN错定位的途径。我们最近报道了组蛋白H3伴侣CHAF1B可以阻止人类细胞中CENP-A和CIN的错定位（Shrestha等，2023）。总之，我们使用多有机体和多学科方法的研究为着丝点功能缺陷如何导致人类癌症的非整倍性提供了机制见解。我们乐观地认为，我们的研究将有助于将基础科学研究转化为临床，并有助于诊断、预后和治疗显示CENP-A过表达的癌症。

We use multi-organismal (yeast, mouse and human cells) and multi-disciplinary (genetic, cell biology, biochemical and genome-wide) approaches to study faithful chromosome segregation, a fundamental process of every living cell. Genetic screens served as a starting point and in-depth mechanistic studies have provided evidence for new roles for kinetochore genes and the identification of new kinetochore genes. We have identified and defined roles for post-translational modifications (acetylation, methylation, phosphorylation, sumoylation and ubiquitination) of Cse4 in chromosome segregation. Our research is focused on understanding the role of Cse4-associated proteins in chromosome segregation and defining pathways that prevent mislocalization of Cse4 to non-centromeric regions. In the first project we defined roles for Scm3, Pat1, Cdc5 and Sgo1 for the assembly of centromeric chromatin and characterized role of post-translational modifications of centromeric histones in faithful chromosome segregation. We determined that imbalanced stoichiometry of a Cse4 chaperone, Scm3 (HJURP in humans) leads 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). Scm3 interacts with Pat1 (Protein associated with topoisomerase II) and Pat1 regulates the topology of centromeric chromatin (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) and provided evidence for a structural role for Pat1 in the structural integrity of centromeric chromatin and localization of Cse4 for faithful chromosome segregation. 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. We reported that evolutionarily conserved polo kinase, Cdc5 associates with centromeric chromatin to facilitate the removal of centromeric cohesins (Mishra et al., 2016) and Cdc5-mediated phosphorylation of Cse4 regulates faithful chromosome segregation (Mishra et al., 2019). Furthermore, evolutionarily conserved Sgo1 which protects centromeric cohesion interacts with Cse4 and this is required for faithful chromosome segregation (Mishra et al., 2018). We recently reported that evolutionarily conserved Hpr1 prevents the accumulation of R-loops at centromeric chromatin affects the assembly of kinetochore and leads to chromosomal instability (Mishra et al., 2021). We have done a comprehensive analysis of Post-translational modifications (PTMs) of Cse4 and identified conserved sites for acetylation, methylation, and phosphorylation (Boeckmann et al., 2013). We determined that evolutionarily conserved Aurora B/Ipl1 kinase phosphorylates Cse4 in vivo and in vitro for faithful chromosome segregation (Boeckmann et al., 2013) and that cell cycle regulated methylation of Cse4 prevents CIN (Mishra et al., 2023). Using budding yeast with a single nucleosome we provided 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. A genome-wide screen with an HDACi was used 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 allow us to understand the molecular role of PTMs of Cse4 in chromosome segregation and determine if these PTMs are conserved in human CENP-A. In the second project we have focused on the identification of pathways that prevent mislocalization of Cse4 and CIN. 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). We identified a novel role for the N terminus of Cse4 in ubiquitin (Ub)-mediated proteolysis for faithful chromosome segregation (Au et al., 2013) and showed that Cse4 is sumoylated and ubiquitination of sumoylated Cse4 by Slx5 regulates its proteolysis to prevent mislocalization to euchromatin (Ohkuni et al., 2016, 2018, 2020). Genome-wide approaches have been used to identify regulators that prevent mislocalization of Cse4 to euchromatin and these studies revealed a role for histone chaperones (Ciftci-Yilmaz et al., 2018). F-box proteins Cdc4 and Met30 in Cse4 proteolysis (Au et al., 2020), Dbf4 dependent kinase (DDK) (Eisenstatt et al., 2020) and Cdc48 (Ohkuni et al., 2022). Furthermore, reduced dosage of histone H4 prevents mislocalization of Cse4 (Eisenstatt et al., 2021). In the third project, we have focused on causes and consequences of mislocalization of CENP-A in human cells and xenograft mouse model. Mislocalization of CENP-A has been observed in many cancers and this correlates with poor prognosis. Hence, 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 (Shrestha et al., 2017). We recently reported that mislocalization of overexpressed CENP-A in pseudodiploid DLD1 cell line and xenograft mouse model contribute to CIN, aneuploidy with karyotypic heterogeneity (Shrestha et al., 2021). We are pursuing studies with human homologs of the yeast genes identified in genome wide screens and using genome-wide approaches to identify and characterize pathways that prevent mislocalization of CENP-A and CIN. We recently reported that histone H3 chaperone, CHAF1B prevents mislocalization of CENP-A and CIN in human cells (Shrestha et al., 2023). n summary, our studies using multi-organismal and multi-disciplinary approaches have provided mechanistic insights for how defects in kinetochore function contribute to aneuploidy in human cancers. We are optimistic that our studies will help translate basic science research to the clinic and aid in the diagnosis, prognosis and treatment of cancers that show overexpression of CENP-A.