Molecular Determinants of Chromosome Transmission and Cell Cycle Regulation

染色体传递和细胞周期调节的分子决定因素

• 批准号: 10014455
• 负责人: Munira Basrai
• 金额: $ 197.67万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10014455
• 关键词: Acetylation; Address; Amyloid Beta Precursor Protein-Binding Protein 2; Aneuploidy; Area; Basic Science; Biochemical; Biological Assay; Cell Cycle Regulation; Cells; Cellular biology; Centromere; Chromatin; Chromosomal Instability; Chromosomal Stability; Chromosome Segregation; Chromosome Structures; Chromosomes; Clinic; Clinical Trials; Complement; DAXX gene; DNA; DNA Sequence; Defect; Diagnosis; Diploidy; Dose; Drosophila polo protein; Ensure; Euchromatin; Excision; Future; Gene Dosage; Genes; Genetic; Genetic Screening; Genome; Genome Stability; Hela Cells; Histone Acetylation; Histone Deacetylase Inhibitor; Histone H3; Histone H4; Histones; Human; In Vitro; Incidence; Kinetochores; Length; Link; Lysine; Malignant Neoplasms; Mediating; Methylation; Microtubules; Mitosis; Mitotic; Molecular; Molecular Chaperones; Mus; Nucleosomes; Pathway interactions; Phenotype; Phospho-Specific Antibodies; Phosphorylation; Phosphotransferases; Post Translational Modification Analysis; Post-Translational Protein Processing; Process; Proteins; Proteolysis; Regulation; Reporting; Research; Role; Saccharomyces cerevisiae; Saccharomycetales; Sister Chromatid; Site; Solid Neoplasm; Structure; Topoisomerase II; Translating; Ubiquitin-mediated Proteolysis Pathway; Ubiquitination; Variant; Yeasts; anticancer research; base; cancer cell; cancer therapy; centromere protein A; chromosome loss; chromosome missegregation; clinically significant; cohesin; cohesion; daughter cell; dosage; established cell line; fly; genome wide screen; genome-wide; in vivo; inducible gene expression; insight; interdisciplinary approach; mouse model; multidisciplinary; mutant; novel; outcome forecast; overexpression; prevent; segregation; stoichiometry; targeted treatment; tool; transmission process; tumorigenesis; ubiquitin-protein ligase

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 also identified and defined roles for post-translational modifications (acetylation, methylation, phosphorylation, sumoylation and ubiquitination) of Cse4 in chromosome segregation. Our current research is aimed at 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 have 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. Our results show 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 we have shown that 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 have shown 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, we have determined that evolutionarily conserved Sgo1 which protects centromeric cohesion interacts with Cse4 and this is required for faithful chromosome segregation. Biochemical approaches have allowed us to provide a comprehensive analysis of Post-translational modifications (PTMs) of Cse4. Conserved sites for acetylation, methylation, and phosphorylation were identified in Cse4 (Boeckmann et al., 2013). 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. 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. 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 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 regulate cellular levels of Cse4 thereby preventing its mislocalization 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 etal, 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, 2018). 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 (Ciftci-Yilmaz et al., 2018) and essential 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 chromosomal stability. 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 (Shrestha et al., 2017). 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. In summary, our studies using multi-organismal and multi-disciplinary approaches will provide mechanistic insights for how defects in kinetochore function contribute to aneuploidy in human cancers. We are optimistic that our in vivo studies with the mouse model 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.

我们使用多生物（酵母，小鼠和人类细胞）以及多学科（遗传，细胞生物学，生化和全基因组）方法来研究忠实的染色体隔离，这是每个活细胞的基本过程。遗传筛选是起点，深入的机械研究为动物学基因的新作用提供了证据和新的动型基因的鉴定。我们还确定了CSE4在染色体分离中的翻译后修饰（乙酰化，甲基化，磷酸化，磷酸化，磷酸化，磷酸化，磷酸化和泛素化）的作用。我们当前的研究旨在了解CSE4相关蛋白在染色体分离中的作用，并定义途径，以防止CSE4错误地定位到非中心粒子区域。在第一个项目中，我们定义了SCM3，PAT1，CDC5和SGO1的作用，用于集中丝粒染色质的组装，并在忠实的染色体分离中表征了丝粒组蛋白的翻译后修饰的作用。我们的结果表明，CSE4伴侣scm3（人类的HJURP）的化学计量不平衡导致人和酵母细胞中的染色体错误分离，从而在癌症中提供了Hjurp过表达和有丝分裂缺陷之间的联系（Mishra等人（Mishra等人，2011年）。 SCM3与PAT1相互作用（与拓扑异构酶II相关的蛋白质），我们已经表明PAT1调节着丝粒染色质的拓扑（Mishra等，2013）。我们使用PAT1缺失菌株来定义酵母动物学上CSE4分子的数量（Hasse，Mishra，2013； Mishra等，2015），并为PAT1在中心粒染色质的结构完整性和CSE4的定位中提供了结构性作用的证据。用于忠实的染色体隔离。除了动力学蛋白外，粘连蛋白与丝粒的缔合以及沿染色体的长度确保了有丝分裂过程中姐妹染色单体的忠实隔离。我们已经表明，进化保守的polo激酶，CDC5与丝粒染色质相关，以促进去除丝粒粘着素（Mishra等，2016）和CDC5介导的CSE4的CSE4磷酸化，CSE4的磷酸化调节了忠实的染色体染色体（Mishra等人）（Mishra等人，2019年）。此外，我们已经确定保护着丝粒凝聚力的进化保守的SGO1与CSE4相互作用，这对于忠实的染色体隔离是必需的。生化方法使我们能够对CSE4的翻译后修饰（PTMS）进行全面分析。在CSE4中鉴定出用于乙酰化，甲基化和磷酸化的保守位点（Boeckmann等，2013）。我们产生了一种磷酸化特异性抗体，并显示了磷酸化的CSE4与丝粒的缔合，并确定进化保守的Aurora B/IPL1激酶在体内和体外磷酸化的CSE4磷酸化，用于忠实的染色体分离。使用单个核小体的出现酵母，我们提供了第一个证据，表明酵母中心粒含有低乙酰化组蛋白H4，并且在赖氨酸16（H4K16）上增加组蛋白H4的乙酰化会导致染色体错误播种（Choy等，2011）。即使HDAC抑制剂（HDACI）用于临床试验中，我们也不完全了解其作用方式。因此，我们进行了一个带有HDACI的全基因组筛选，以识别容易受到组蛋白乙酰化改变的途径。我们的结果表明，染色体分离突变体对HDACI更敏感（Choy等，2015）。未来的研究将使我们能够理解CSE4 PTM在染色体分离中的分子作用，并确定这些PTM是否在人CENP-A中保守。在第二个项目中，我们集中在调节CSE4细胞水平的途径上，从而防止其错误定位和CIN。我们先前表明，酿酒酵母SPT4突变体表现出对人类SPT4补充的CSE4和染色体隔离缺陷的错误定位（Basrai etal，1996以及Crotti和Basrai 2004）。我们通过表明CSE4错误定位的原因和作用，表明CSE4的组蛋白剂量改变和非中心染色质与染色体损失相关（Au等，2008）。阻止CSE4错误定位的一种机制是E3连接酶PSH1对CSE4的泛素介导的蛋白水解。我们确定了CSE4 N末端在泛素（UB）介导的蛋白水解中的N末端的新作用（Au等，2013）。我们最近报道说，通过SLX5对Sumoypated CSE4的CSE4进行了泛素化，调节其蛋白水解以防止将其定位错误地定位到舌状素（Ohkuni等，2016，2016，2018）。我们已经采用了全基因组方法来识别防止CSE4错误定位到白染色质的调节剂。我们的研究揭示了组蛋白伴侣的作用（Ciftci-Yilmaz等，2018）和CSE4蛋白水解中必需的E3 UB连接酶。我们正在进行的研究旨在深入分析筛网中鉴定的酵母基因，以了解防止CSE4错误定位的分子机制用于染色体稳定性。 CENP-A的错误定位导致人类细胞中的CIN。鉴于高CENP-A表达及其与癌症的相关性的临床意义，因此了解CENP-A过表达如何有助于肿瘤发生以及CENP-A表达是否可以利用用于预后，诊断和针对CENP-A过表达的靶向治疗癌症。我们建立了细胞系和优化的基于细胞生物学的测定方法，以解决一个长期的问题，即过表达CENP-A是否有助于CIN。我们确定CENP-A在HELA和稳定的二倍体RPE1细胞中的组成型或诱导表达导致CENP-A将CENP-A错误定位到非中心粒子区域。全面的有丝分裂作用分析表明，CENP-A过表达对染色体隔离缺陷的剂量依赖性作用和较高的微核发生率。动力学蛋白的定位改变改变了CENP-A过表达细胞中天然动力学的弱化。组蛋白伴侣DAXX的耗竭可防止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的主要因素之一。我们的研究提供了第一个证据，证明CENP-A对非中心染色质的错误定位如何有助于人类细胞中的CIN，并提供机械洞察CENP-A过表达如何有助于CENP-A过表达的癌症（Shrestha等，，，，，，，，，，，，地） 2017）。我们正在研究基因组宽筛查中鉴定出的酵母基因的人类同源物，并使用其他方法来识别和表征途径，以防止CENP-A对基因组稳定性的错误定位。总而言之，我们使用多生物和多学科方法的研究将提供机械见解，以了解动物学功能中的缺陷如何有助于人类癌症中的非整倍性。我们乐观的是，我们使用小鼠模型的体内研究将有助于将基础科学研究转化为诊所，并有助于诊断，预后和治疗表现出CENP-A过表达的癌症。