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

基本信息

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
项目类别：
财政年份：
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

项目摘要

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.

进化保守的CSE4，丝粒组蛋白H3变体（人类中的CENP-A）及其伴侣SCM3（人类中的HJURP）对于染色体分离至关重要，这在许多癌症中已被证明过表达。据报道，HJURP的过表达和误定位在肺癌细胞和Hjurp表达升高的患者中的存活率降低。 Hjurp是否诱发肿瘤发生是否尚不清楚。我们表明，HJURP和SCM3的化学计量分析导致人和酵母细胞中的染色体分离和动力学完整性缺陷，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。现在，基因组宽度屏幕将使我们能够识别抑制或增强与SCM3/HJURP过表达相关的表型的基因/途径，以推断出对癌症的外推。在这些研究的持续过程中，我们表明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和忠实的染色体隔离的结构完整性需要PAT1。与Kerry Bloom合作，我们使用了PAT1 NULL菌株来定义酵母动物学上的CSE4分子的数量。使用PAT1的研究将有助于我们了解丝粒染色质的拓扑结构如何调节染色体分离的研究领域，目前在很大程度上尚未探索。组蛋白的翻译后修饰（PTM）对于包括染色体隔离在内的许多染色质活性至关重要。组蛋白脱纤酶（HDAC）抑制剂用于治疗某些癌症，但是，我们不完全了解这些抑制剂的分子靶标。我们研究了丝粒组蛋白在萌芽酵母中PTM的性质和作用，其长期目标是针对组蛋白进行抗癌治疗的PTM。我们首次表明，发芽的酵母中心粒含有低乙酰化组蛋白H4，并且增加了赖氨酸16（H4K16）上组蛋白H4的乙酰化增加导致染色体错误分离。我们还发现，染色体分离需要H4K16乙酰转移酶，SAS2和H4K16脱乙酰基酶的平衡。值得注意的是，SAS2和SIR2都有人类同源物。现在，我们将确定H4的乙酰化模式是否受细胞周期调节，如果H4乙酰化改变会影响丝粒染色质的结构以及组蛋白脱纤酶（HDAC）在染色体分离中的作用。我们建议将HDAC抑制剂与损害动力学功能的药物相结合，可能对癌症治疗更有效，对正常细胞的影响最小。为了研究CSE4的PTM，我们为CSE4的生化纯化设计了一种创新的方法，这促进了对CSE4的PTM的首次全面分析。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与中心粒的关联，并确定IPL1在体内磷酸化CSE4和体外的忠实染色体分离，我们的研究表明，CSE4的磷酸化和甲基化调节了S. ceremosome in S. ceregregation in S. cereviesiae。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性。我们表明，酿酒酵母SPT4突变体对人类SPT4补充的CSE4和染色体隔离缺陷表现出错误的定位。我们通过表明CSE4错误定位的原因和影响通过表明CSE4的组蛋白剂量和错误定位到非中心位点与染色体损失相关。我们的研究定义了CSE4的N末端在其UB介导的蛋白水解中用于忠实的染色体分离。我们正在与查理·布恩（Charlie Boone）合作，以确定介导CSE4蛋白水解进行忠实染色体隔离的途径。长期目标是确定将特别导致杀死过表达CENP-A的癌细胞的途径。我们的实验室最近报告了基因组稳定性“单倍体”（HI）的基因的鉴定和表征。 HI是一种基因的单个功能副本不足以维持正常活性并导致突变表型。 HI导致肿瘤发生的更高发病率，许多肿瘤表现出非整倍性。我们设计了一个新颖的屏幕，以识别和表征使用几乎所有基因（6500）的Hemizygous酵母缺失库“单倍弹性”（HI）用于基因组稳定性的基因（HI）。我们将BCY1和进化保守的γ微管蛋白复合物的新作用定义为染色体分离的HI。我们的研究定义了γ微管蛋白复合物在纺锤体组织中的新作用。