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：
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
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/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

项目摘要

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.

进化保守的CSE4及其伴侣SCM3（人类中的Hjurp）对于染色体隔离至关重要，在许多癌症中已显示出过表达的。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的研究提供了有关丝粒染色质的拓扑结构如何调节染色体隔离的重要见解，目前在很大程度上尚未探索的研究领域。忠实的染色体隔离也受centromeric组蛋白和动元蛋白的翻译后修饰（PTM）调节。我们研究了Cenctromeric组蛋白H4和CSE4在萌芽酵母中的PTM的性质和作用，其长期目标是将组蛋白的PTM用于抗癌治疗。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和组蛋白脱乙酰基酶（HDAC）（HDAC）的平衡。值得注意的是，SAS2和SIR2都有人类同源物。现在，我们正在研究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HDAC在染色体隔离中的作用。 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 PTM的作用，我们设计了一种创新的CSE4生化纯化方法，这促进了CSE4 PTM的首次综合分析。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特异性抗体，并显示了磷酸化的CSE4与丝粒的缔合，并确定IPL1在体内和体外磷酸化CSE4以调节染色体分离。在继续使用PTM的研究中，我们表明CSE4的N末端被泛素化以调节其蛋白水解和定位。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。与查理·布恩（Charlie Boone）合作，我们使用了全基因组筛选来识别几个新的CSE4蛋白水解调节剂，这些调节剂在进化上保守。在结直肠癌中观察到人类同源物（CENP-A）的过度表达和错误定位，并导致果蝇的非整倍性。我们研究的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。鉴于基因组稳定途径的进化保守性，我们决定使用萌芽的酵母投资来利用“单倍弥补”的作用（HI）。 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。我们设计了一个新型的全基因组筛选，它使用了几乎所有基因（6500）的半合并酵母缺失库来识别和表征基因组稳定性HI的基因。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。综上所述，我们对萌芽的酵母菌及其人类同源物的研究正在为许多癌症经常观察到染色体隔离的原因和后果的重要见解。