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集中在以下：1）进化保守的PAT1（与拓扑异构酶II相关的蛋白质）和polo激酶CDC5调节忠实的染色体隔离的centromeric关联。在许多癌症中，CSE4及其伴侣SCM3（人类的Hjurp）对于染色体隔离至关重要，在许多癌症中都过表达和定位。 Hjurp表达升高的患者的存活率降低。 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的分子机制，以进行忠实的染色体分离。我们已经表明，HjURP和SCM3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，2011）。未来的研究将利用全基因组筛查来识别显示出具有HJURP过表达的致死性的基因/途径，以便对HjURP表达失调的癌症可能治疗。 SCM3与PAT1相互作用（与拓扑异构酶II相关的蛋白质），并且我们发现了PAT1在丝粒染色质和染色体分离的拓扑中的作用（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）。我们的结果表明，PAT1调节着丝粒染色质的结构完整性和CSE4的定位用于忠实的染色体分离。正在进行的研究旨在了解centromeric染色质的拓扑如何影响染色体分离的研究领域，目前在很大程度上尚未探索。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。我们的研究表明，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）。未来的研究将使我们能够了解CDC5调节丝粒粘着素的去除的机制。 2）丝粒组蛋白的翻译后修饰（PTMS）会影响染色体分离。以前在其他系统中已经报道了丝粒组蛋白H4的独特乙酰化模式，但是，这种模式的生理作用尚未完全了解。使用单个核小体的发芽酵母，我们确定丝粒组蛋白H4的乙酰化模式会影响染色体分离。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脱位（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）。未来的研究将检查HDACI与影响染色体分离的药物是否对癌症治疗更有效，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对CSE4的PTM进行首次全面分析（Boeckmann等，2013）。鉴定了CSE4中的乙酰化，甲基化和磷酸化的保守位点。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。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是否在人CENP-A中保守。 3）严格调节CSE4的细胞水平可防止其基因组稳定性的错误定位。我们先前表明，酿酒酵母SPT4突变体表明CSE4和染色体隔离缺陷的错误定位是由人SPT4补充的（Basrai等，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异染色质错误定位（Ohkuni等，2016）。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网中鉴定出的酵母基因，以了解防止CSE4错误定位基因组稳定性的分子机制。 4）CENP-A的错误定位会导致人类细胞中的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法，以解决一个长期的问题，即过表达过表达的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，并为CENP-A过表达如何有助于CENP-A过表达的CENP-A过表达癌症提供机械洞察力。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。