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: 8763235
• 负责人: Munira Basrai
• 金额: $ 121.62万
• 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/8763235
• 关键词: APPBP2 gene; 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; Complement; Complex; Congenital Abnormality; DNA; Deacetylase; Defect; Deposition; Disease; Drosophila genus; Ensure; Equilibrium; Eukaryota; Failure; Genes; Genetic Materials; Genome Stability; Histone H3; Histone H4; Histones; Homologous Gene; Human; In Vitro; Incidence; Kinetochores; Laboratories; Lead; Life; Link; Lysine; Maintenance; Malignant Neoplasms; Malignant neoplasm of lung; Mediating; Methylation; Mitotic; Mitotic Checkpoint; Molecular; Molecular Chaperones; Molecular Target; Monitor; Nature; Normal Cell; Organism; Orthologous Gene; Pathway interactions; Patients; Pattern; Pharmaceutical Preparations; Phenotype; Phospho-Specific Antibodies; Phosphorylation; Physiological; Play; Post-Translational Protein Processing; Process; Proteins; Proteolysis; Recovery; Reporting; Research; Role; Saccharomyces cerevisiae; 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 association study; in vivo; inhibitor/antagonist; innovation; killings; mutant; novel; overexpression; prevent; repaired; response; segregation; stoichiometry; transmission process; tumor; tumorigenesis

Evolutionarily conserved Cse4, the centromeric histone H3 variant (CENP-A in humans) 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 will help us understand how topological structure of centromeric chromatin regulates chromosome segregation an area of research that is largely unexplored at the present time. Post-translational modifications (PTM) of histones are critical for many chromatin activities including chromosome segregation. Histone deactylase (HDAC) inhibitors are used for treatment of certain cancers, however, we do not fully understand the molecular targets of these inhibitors. We investigated the nature and role of PTM of centromeric histones 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 H4K16 deacetylase, Sir2, is required for chromosome segregation. Notably, both Sas2 and Sir2 have human homologs. We will now determine if the acetylation pattern of H4 is cell cycle regulated, if altered H4 acetylation affect the structure of centromeric chromatin and the role of histone deactylases (HDAC) in chromosome segregation. 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. To study 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 for faithful chromosome segregation Our studies have shown that phosphorylation and methylation of Cse4 regulate chromosome segregation in S. cerevisiae. Overexpression and mis-localization of CENP-A is observed in colorectal cancers and leads to aneuploidy in flies. We showed that S. cerevisiae spt4 mutants show mis-localization of Cse4 and chromosome segregation defects that are complemented by human SPT4. 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. Our studies have defined a novel role for the N-terminus of Cse4 in its Ub mediated proteolysis for faithful chromosome segregation. We are collaborating with Charlie Boone to identify pathways that mediate the proteolysis of Cse4 for faithful chromosome segregation. The long-term objective is to identify pathways that will specifically lead to killing of cancer cells overexpressing CENP-A. Our laboratory recently reported on the identification and characterization of genes that are "haploinsufficient" (HI) for genome stability. 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 screen to identify and characterize genes that are "haploinsufficient" (HI) for genome stability using the hemizygous yeast deletion library representing nearly all genes (6500). We defined novel roles for BCY1 and the evolutionarily conserved Gamma tubulin complex as HI for chromosome segregation. Our studies defined a novel role for the gamma tubulin complex in spindle organization.

进化上保守的Cse 4、着丝粒组蛋白H3变体（人类中的CENP-A）及其伴侣蛋白Scm 3（人类中的HJURP）对于染色体分离是必需的，已经显示在许多癌症中过表达。据报道，HJURP在肺癌和乳腺癌细胞中过表达和错误定位，HJURP表达升高的患者显示存活率降低。HJURP过表达是否诱导肿瘤发生尚不清楚。我们发现，HJURP和SCM 3的不平衡化学计量导致人类和酵母细胞中染色体分离和动粒完整性的缺陷，从而提供了HJURP过表达和癌症中有丝分裂缺陷之间的联系。全基因组筛选现在将允许我们鉴定抑制或增强与SCM 3/HJURP过表达相关的表型的基因/途径，以便可能外推到癌症。在这些研究的继续中，我们已经表明Pat 1（拓扑异构酶II相关蛋白）与Scm 3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帕特1。在与Kerry Bloom的合作中，我们使用pat 1无效菌株来定义酵母动粒处的Cse 4分子的数量。对PAT 1的研究将有助于我们理解着丝粒染色质的拓扑结构如何调节染色体分离，这是一个目前尚未探索的研究领域。组蛋白的翻译后修饰（PTM）对于包括染色体分离在内的许多染色质活动是至关重要的。组蛋白去乙酰化酶（HDAC）抑制剂用于治疗某些癌症，然而，我们并不完全了解这些抑制剂的分子靶点。我们研究了芽殖酵母中着丝粒组蛋白PTM的性质和作用，长期目标是靶向组蛋白PTM用于抗癌治疗。我们首次发现芽殖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（H4 K16）上的乙酰化增加导致染色体错误分离。我们还发现H4 K16乙酰转移酶Sas 2和H4 K16脱乙酰酶Sir 2的平衡是染色体分离所必需的。值得注意的是，Sas 2和Sir 2都有人类同源物。我们现在将确定H4的乙酰化模式是否受细胞周期调节，改变的H4乙酰化是否影响着丝粒染色质的结构和组蛋白脱乙酰酶（HDAC）在染色体分离中的作用。我们认为，HDAC抑制剂与损害动粒功能的药物相结合可能对癌症治疗更有效，对正常细胞的影响最小。为了研究Cse 4的PTM，我们设计了一种用于Cse 4的生物化学纯化的创新方法，这促进了Cse 4的PTM的首次全面分析。Cse 4中乙酰化、甲基化和磷酸化的保守位点被鉴定。我们制备了磷酸化特异性抗体，并显示了磷酸化的Cse 4与着丝粒的结合，并确定了Ipl 1在体内和体外磷酸化Cse 4以实现可靠的染色体分离。啤酒。在结直肠癌中观察到CENP-A的过表达和错误定位，并导致果蝇的非整倍体。我们证明了S.酿酒酵母spt 4突变体显示Cse 4的错误定位和染色体分离缺陷，其由人SPT 4补充。我们通过显示改变的组蛋白剂量和Cse 4到非着丝粒位点的错误定位与染色体丢失相关来确定Cse 4错误定位的原因和影响。我们的研究已经确定了Cse 4的N-末端在其Ub介导的蛋白水解中的新作用，以实现忠实的染色体分离。我们正在与Charlie Boone合作，以确定介导Cse 4蛋白水解的途径，以实现忠实的染色体分离。长期目标是确定特异性杀死过表达CENP-A的癌细胞的途径。我们的实验室最近报道了对基因组稳定性“单倍不足”（HI）的基因的鉴定和表征。HI是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病症。HI导致更高的肿瘤发生率，并且许多肿瘤显示非整倍体。我们设计了一个新的屏幕，以确定和表征基因组稳定性的“单倍不足”（HI）的基因，使用半合子酵母缺失文库代表几乎所有的基因（6500）。我们定义了BCY 1和进化上保守的γ微管蛋白复合物作为染色体分离的HI的新角色。我们的研究定义了一个新的作用，γ微管蛋白复合体在纺锤体组织。