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: 9153681
• 负责人: Munira Basrai
• 金额: $ 137.18万
• 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/9153681
• 关键词: Acetylation; Acetyltransferase; Affect; Aging; Aneuploidy; Area; Biochemical; Biological Models; Breast Cancer Cell; Cell Cycle; Cell Cycle Checkpoint; Cell Cycle Regulation; Cells; Centromere; Chromatin; Chromosome Segregation; Chromosomes; Collaborations; Colorectal Cancer; Complex; Congenital Abnormality; DNA; Defect; Deposition; Disease; Drosophila genus; Ensure; Equilibrium; Failure; Genes; Genetic Materials; Genome Stability; Histone Deacetylase; Histone Deacetylase Inhibitor; Histone H3; Histone H4; Histones; Homologous Gene; Human; In Vitro; Incidence; Kinetochores; Lead; Life; Link; Lysine; Maintenance; Malignant Neoplasms; Malignant neoplasm of lung; Methylation; Mitotic; Mitotic Checkpoint; Molecular; Molecular Chaperones; Molecular Target; Nature; Normal Cell; Orthologous Gene; Pathway interactions; Patients; Pattern; Pharmaceutical Preparations; Phenotype; Phospho-Specific Antibodies; Phosphorylation; Physiological; Post-Translational Protein Processing; Proteins; Proteolysis; Recovery; Reporting; Research; Role; Saccharomycetales; Site; Structure; Survival Rate; Time; Topoisomerase II; Variant; Yeasts; cancer cell; cancer therapy; centromere protein A; chromosome loss; deletion library; design; dosage; fly; gamma Tubulin; genome-wide analysis; in vivo; inhibitor/antagonist; innovation; insight; killings; mutant; novel; overexpression; prevent; repaired; response; segregation; stoichiometry; therapeutic target; transmission process; tumor; tumorigenesis

Evolutionarily conserved Cse4 and its chaperone Scm3 (HJURP in humans) which are essential for chromosome segregation have been shown to be overexpressed in many cancers. Overexpression and mis-localization of HJURP has been reported in lung and breast cancer cells and patients with elevated HJURP expression show reduced survival rate. Whether HJURP overexpression induces tumorigenesis is not understood. We showed that imbalanced stoichiometry of HJURP and SCM3 lead to defects in chromosome segregation and kinetochore integrity in human and yeast cells thereby providing a link between HJURP overexpression and mitotic defects in cancers. Genome wide screens will now allow us to identify genes/pathways that suppress or enhance phenotypes associated with overexpression of SCM3/HJURP for possible extrapolation to cancers. In continuation of these studies we have shown that Pat1 (Protein associated with topoisomerase II) interacts with Scm3. We determined that structural integrity of centromeric chromatin and faithful chromosome segregation requires Pat1. In collaboration with Kerry Bloom we used a pat1 null strain to define the number of Cse4 molecules at the yeast kinetochore. Studies with Pat1 have provided important insights into how topological structure of centromeric chromatin regulates chromosome segregation an area of research that is largely unexplored at the present time. Faithful chromosome segregation is also regulated by post-translational modifications (PTM) of centromeric histones and kinetochore proteins. We investigated the nature and role of PTM of centromeric histone H4 and Cse4 in budding yeast with the long-term objective of targeting PTM of histones for anti-cancer therapy. We showed for the first time that budding yeast centromeres contain hypoacetylated histone H4 and also that increased acetylation of histone H4 on lysine 16 (H4K16) leads to chromosome mis-segregation. We also discovered that a balance in H4K16 acetyltransferase, Sas2, and Histone Deacetylase (HDAC), Sir2, is required for chromosome segregation. Notably, both Sas2 and Sir2 have human homologs. We are now investigating if acetylation pattern of H4 is cell cycle regulated, if altered H4 acetylation affect the structure of centromeric chromatin and the role of HDAC's in chromosome segregation. HDAC inhibitors are used for treatment of certain cancers, however, we do not fully understand the molecular targets of these inhibitors. We propose that combining HDAC inhibitors with drugs that compromise kinetochore function may be more effective for cancer treatment with minimal effect on normal cells. In order to investigate the role of PTM of Cse4 we devised an innovative approach for biochemical purification of Cse4 and this facilitated the first comprehensive analysis of PTMs of Cse4. 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 Ipl1 phosphorylates Cse4 in vivo and in vitro to regulate chromosome segregation. In continuation of our studies with PTM, we have shown that the N-terminus of Cse4 is ubiquitinated to regulate its proteolysis and localization. We established the cause and effect of Cse4 mis-localization by showing that altered histone dosage and mis-localization of Cse4 to non-centromeric loci correlates with chromosome loss. In collaboration with Charlie Boone we have used genome-wide screens to identify several new regulators for Cse4 proteolysis that are evolutionarily conserved. Overexpression and mis-localization of human homolog of Cse4 (CENP-A) is observed in colorectal cancers and leads to aneuploidy in flies. The long-term objective of our research is to identify pathways that will specifically lead to killing of cancer cells overexpressing CENP-A. Given the evolutionary conservation of pathways for genome stability we decided to use budding yeastinvestigate the role of "haploinsufficiency" (HI). HI is a condition where a single functional copy of a gene is insufficient to sustain normal activity and leads to a mutant phenotype. HI leads to higher incidences of tumorigenesis and many tumors display aneuploidy. We designed a novel genome-wide screen using the hemizygous yeast deletion library representing nearly all genes (6500) to identify and characterize genes that are HI for genome stability. We defined novel roles for BCY1 and the evolutionarily conserved Gamma Tubulin complex as HI for chromosome segregation. Taken together, our studies with budding yeast and its human homologs are providing critical insights into the causes and consequences of errors in chromosome segregation that are frequently observed in many cancers.

进化上保守的Cse4及其伴侣Scm3(人类中的HJURP)是染色体分离所必需的，已被证明在许多癌症中过表达。HJURP在肺癌和乳腺癌细胞中的过度表达和错误定位已有报道，HJURP表达升高的患者存活率降低。目前尚不清楚HJURP过表达是否会导致肿瘤的发生。我们发现HJURP和SCM3的化学计量不平衡导致人类和酵母细胞中染色体分离和动粒完整性的缺陷，从而在HJURP过表达和癌症中的有丝分裂缺陷之间提供了联系。全基因组筛查现在将使我们能够识别抑制或增强与SCM3/HJURP过度表达相关的表型的基因/途径，从而可能推断为癌症。在这些研究的继续中，我们已经证明Pat1(与拓扑异构酶II相关的蛋白质)与ScM3相互作用。我们确定着丝粒染色质的结构完整性和忠实的染色体分离需要Pat1。在与Kerry Bloom的合作中，我们使用了一株pat1缺失菌株来定义酵母动粒上的Cse4分子的数量。对Pat1的研究为着丝粒染色质的拓扑结构如何调节染色体分离提供了重要的见解，这是目前基本上未被探索的研究领域。忠实的染色体分离也受到着丝粒蛋白和着丝粒蛋白的翻译后修饰(PTM)的调节。我们研究了着丝粒组蛋白H4和Cse4的PTM在芽殖酵母中的性质和作用，并以抗癌治疗为靶向组蛋白的PTM为长期目标。我们首次发现发芽酵母着丝粒含有低乙酰化的组蛋白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的错误分离。我们还发现，H4K16乙酰转移酶SAS2和组蛋白脱乙酰酶Sir2的平衡是染色体分离所必需的。值得注意的是，SAS2和Sir2都有人类同源基因。我们正在研究H4的乙酰化模式是否受细胞周期调节，H4乙酰化是否影响着丝粒染色质的结构以及HDAC在染色体分离中的作用。HDAC抑制剂被用于某些癌症的治疗，然而，我们并不完全了解这些抑制剂的分子靶点。我们建议，将HDAC抑制剂与损害动粒功能的药物联合使用可能对癌症治疗更有效，而对正常细胞的影响最小。为了研究Cse4的PTM的作用，我们设计了一种新的生化纯化方法，这有助于首次对Cse4的PTM进行全面的分析。确定了Cse4中乙酰化、甲基化和磷酸化的保守位点。我们产生了一种磷酸化特异性抗体，证明了磷酸化的Cse4与着丝粒的结合，并确定了Ipl1在体内和体外都能磷酸化Cse4来调节染色体分离。在我们对PTM的继续研究中，我们发现Cse4的N-末端是泛素化的，以调节其蛋白分解和定位。我们通过证明组蛋白剂量的改变和Cse4对非着丝粒基因的错误定位与染色体丢失相关，从而确定了Cse4错误定位的原因和影响。在与Charlie Boone的合作中，我们已经使用全基因组筛选来确定Cse4蛋白分解的几个新的调节因子，它们在进化上是保守的。人类Cse4同源基因(CENP-A)在结直肠癌中过度表达和错误定位，并导致果蝇非整倍体。我们研究的长期目标是确定具体导致过度表达CENP-A的癌细胞被杀死的途径。考虑到基因组稳定性途径的进化保守性，我们决定使用芽殖酵母来研究“单倍体不足”(HI)的作用。HI是指基因的单个功能拷贝不足以维持正常活性并导致突变表型的情况。HI可导致较高的肿瘤发生率，许多肿瘤表现为非整倍体。我们设计了一种新的全基因组筛选，使用代表几乎所有基因的半合子酵母缺失文库(6500)来识别和表征对基因组稳定性具有HI的基因。我们将BCY1和进化上保守的Gamma微管蛋白复合体的新角色定义为HI，用于染色体分离。综上所述，我们对萌芽酵母及其人类同源物的研究为许多癌症中经常观察到的染色体分离错误的原因和后果提供了关键的见解。