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: 9556375
• 负责人: Munira Basrai
• 金额: $ 169.42万
• 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/9556375
• 关键词: Acetylation; Address; Affect; Amyloid Beta Precursor Protein-Binding Protein 2; Aneuploidy; Area; Biochemical; Biological Assay; Biological Models; Cell Cycle Regulation; Cells; Cellular biology; Centromere; Chromatin; Chromosomal Instability; Chromosomal Stability; Chromosome Segregation; Chromosomes; Clinical Trials; Complement; DAXX gene; DNA; DNA Sequence; Defect; Diagnosis; Diploidy; Dose; Drosophila polo protein; Ensure; Euchromatin; Excision; Future; Gene Dosage; Genes; Genome; Genome Stability; Hela Cells; Histone Acetylation; Histone Deacetylase Inhibitor; Histone H3; Histone H4; Histones; Human; In Vitro; Incidence; Kinetochores; Lead; Length; Link; Lysine; Malignant Neoplasms; Methylation; Microtubules; Mitosis; Mitotic; Molecular; Molecular Chaperones; Normal Cell; Nucleosomes; Pathway interactions; Patients; Pattern; Pharmaceutical Preparations; Phenotype; Phospho-Specific Antibodies; Phosphorylation; Phosphotransferases; Physiological; Post Translational Modification Analysis; Post-Translational Protein Processing; Proteins; Proteolysis; Regulation; Reporting; Research; Role; Saccharomyces cerevisiae; Saccharomycetales; Sister Chromatid; Site; Solid Neoplasm; Survival Rate; System; Time; Topoisomerase II; Ubiquitin-mediated Proteolysis Pathway; Ubiquitination; Variant; Yeasts; base; cancer therapy; cell growth regulation; centromere protein A; chromosome loss; clinically significant; cohesin; daughter cell; dosage; established cell line; fly; genome-wide; genome-wide analysis; in vivo; inducible gene expression; innovation; insight; mutant; novel; outcome forecast; overexpression; prevent; segregation; stoichiometry; targeted treatment; transmission process; tumorigenesis; ubiquitin-protein ligase

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.

我们正在进行的研究主要集中在以下方面：1)进化保守的Pat1（与拓扑异构酶II相关的蛋白）和polo激酶Cdc5的着丝粒关联调节忠实的染色体分离。Cse4和它的伴侣Scm3（人类中的HJURP）都是染色体分离所必需的，在许多癌症中过度表达和错误定位。HJURP表达升高的患者生存率降低。HJURP过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的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的不平衡化学计量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）。未来的研究将利用全基因组筛选来确定HJURP过表达的致命性基因/途径，从而可能治疗HJURP表达不受控制的癌症。Scm3与Pat1（与拓扑异构酶II相关的蛋白质）相互作用，我们已经发现了Pat1在着丝染色质拓扑结构和染色体分离中的作用（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）。我们的研究结果表明，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，这是目前尚未开发的研究领域。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。我们的研究表明，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 et al., 2016）。未来的研究将使我们能够了解Cdc5调节着丝粒内聚蛋白去除的机制。着丝粒组蛋白的翻译后修饰（PTMs）影响染色体分离。着丝粒组蛋白H4的独特乙酰化模式先前在其他系统中有报道，然而，这种模式的生理作用尚未完全了解。利用单核小体芽殖酵母，我们确定了着丝粒组蛋白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。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）。未来的研究将检验HDACi与影响染色体分离的药物是否在对正常细胞影响最小的情况下更有效地治疗癌症。一种创新的Cse4生化纯化方法使我们能够首次对Cse4的PTMs进行全面分析（Boeckmann et al., 2013）。确定了Cse4中乙酰化、甲基化和磷酸化的保守位点。我们产生了磷酸化特异性抗体，并证明了磷酸化的Cse4与着丝粒的关联，并确定了进化上保守的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。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PTMs是否在人类CENP-A中保守。3)严格调控Cse4的细胞水平可以防止其错定位，从而保证基因组的稳定性。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）。防止Cse4错定位的一种机制是E3连接酶Psh1介导的泛素介导的Cse4蛋白水解。我们发现了Cse4的N端在泛素（Ub）介导的蛋白质水解中对忠实染色体分离的新作用（Au et al., 2013）。我们最近报道了Cse4被sumylated， Slx5对sumylated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 et al., 2016）。我们已经采取了全基因组方法来鉴定防止Cse4错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到的酵母基因，以了解防止Cse4错定位的分子机制，从而保持基因组的稳定性。4)人细胞中CENP-A的错位导致了CIN的发生。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是否可以利用CENP-A过表达的癌症的预后、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。我们的研究首次证明了CENP-A在非着丝粒染色质上的错误定位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过表达癌症中的非整倍体的机制见解。我们正在研究在基因组宽筛选中鉴定的酵母基因的人类同源物，并使用其他方法鉴定和表征防止CENP-A错定位的途径，以保持基因组稳定性。