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: 8157482
• 负责人: Munira Basrai
• 金额: $ 114.84万
• 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/8157482
• 关键词: Cell Cycle Regulation; Chromosomes; Molecular; transmission process

We have used chromosome transmission fidelity (ctf) mutants and the deletion strain collections of S. cerevisiae to identify and characterize genes required for kinetochore function and checkpoint function. Studies with the ctf mutants led to the identification and characterization for a role of SPT4 and NUP170 in chromosome segregation and spindle assembly checkpoint (SAC) function. We established a novel role for Spt4p in heterochromatic silencing. Using cross-species approach we showed that the yeast spt4 strains are complemented by human SPT4. Most importantly, we showed that S. cerevisiae SPT4 contributes to the proper localization of histone H3 variant Cse4p. We investigated the mechanism of Cse4p localization and have recently established that mislocalization of Cse4p and altered histone stoichiometry lead to defects in chromosome transmission. We wish to examine if chromatin modifiers and post-translational modification of kinetochore proteins affect the assembly/function of CenH3 chromatin. Our recent results with Cse4p localization and histone dosage in S. cerevisiae are consistent with those in S. pombe suggesting conservation of the underlying mechanisms. Thus, studies in S. cerevisiae that elucidate a mechanism for Cse4p localization and the role of chromatin modifications in centromere function may help us understand analogous pathways in humans and other systems. We also wish to establish the molecular role of Spt4p and its interacting partners Spt5p and Spt6p as well as histones in chromatin structure, chromosome segregation and gene silencing in both yeast and humans. To demonstrate the functional relevance of our findings in S. cerevisiae, we plan to extend our research to higher eukaryotes. To this end we are collaborating with Drs. Caplen and Roschke in RNAi studies to investigate the role of human Spt4p/Spt5p/Spt6p in chromosome segregation and function of CENP-A. Our studies with the nuclear pore complex (NPC) gene NUP170 allowed us to establish a novel relationship between SAC proteins Mad1p and Mad2p and the NPC in S. cerevisiae. Subsequent to our work, several other studies including ones with human cell lines, have reported roles for NPC components in kinetochore function. Our studies have led to the first report of Mad1p, Mad2p and Bub3p localization to the kinetochore upon SAC activation in S. cerevisiae. We recently defined a domain of Mad1p that is required for chromosome transmission and checkpoint function. Further relevance for a role of NPC in mitosis is based on our collaboration with Dr. Belanger that show genetic interactions between spindle pole body (SPB) and mitotic exit network mutants. In addition to chromosome segregation, the DNA damage and replication checkpoint pathways ensure genome stability by halting the cell cycle in response to genotoxic stress. We have recently established a functional relationship between oxidative stress genes SOD1 and CCS1and the MEC1 mediated checkpoint pathway for DNA damage and replication arrest. Recent results from genetic analysis have shown that Sod1p and Ccs1p have a role in DNA repair, genome stability and telomere maintenance. Our studies with Sod1p and Ccs1p will unravel molecular mechanisms that correlate oxidative stress, redox state and checkpoint pathways in S. cerevisiae that may be applicable to other systems. Our research on the molecular determinants of faithful chromosome transmission in S. cerevisiae will help us understand analogous processes in humans and their implications in human disease. Our laboratory is uniquely poised to utilize the conventional genetic, biochemical, and cell biology approaches, as well as high-throughput genomic analysis for our research projects. We use an array of gene-deletion strains and a colony picking robot for the identification of possible cancer drug targets and also for genetic screens by Synthetic Genome (SGA) analysis, developed in the laboratory of Charlie Boone (Univ. of Toronto).

我们利用染色体传递保真度突变体和缺失菌株收集了S.酿酒酵母以鉴定和表征动粒功能和检查点功能所需的基因。对ctf突变体的研究导致对SPT 4和NUP 170在染色体分离和纺锤体组装检查点（SAC）功能中的作用的鉴定和表征。我们确定了Spt 4p在异染色质沉默中的新作用。使用跨物种方法，我们表明酵母spt 4菌株与人SPT 4互补。最重要的是，我们证明了S。酿酒酵母SPT 4有助于组蛋白H3变体Cse 4p的正确定位。我们研究了Cse 4p定位的机制，最近确定Cse 4p的错误定位和改变组蛋白化学计量导致染色体传递缺陷。我们希望检查染色质修饰剂和着丝粒蛋白的翻译后修饰是否影响CenH 3染色质的组装/功能。我们最近的研究结果表明，Cse 4p在S. cerevisiae与S.粟酒裂殖酵母表明保护的基本机制。因此，S.酿酒酵母，阐明了Cse 4p定位的机制和染色质修饰在着丝粒功能中的作用，可能有助于我们了解人类和其他系统中的类似途径。我们还希望建立Spt 4p及其相互作用伙伴Spt 5 p和Spt 6p以及组蛋白在酵母和人类的染色质结构、染色体分离和基因沉默中的分子作用。为了证明我们的研究结果在S。酿酒酵母，我们计划将我们的研究扩展到高等真核生物。为此，我们正在与Caplen和Roschke博士合作进行RNAi研究，以研究人类Spt 4p/Spt 5 p/Spt 6p在染色体分离和CENP-A功能中的作用。 我们对核孔复合体（nuclear pore complex，NPC）基因NUP 170的研究使我们能够建立SAC蛋白Mad 1 p和Mad 2 p与S.啤酒。在我们的工作之后，其他几项研究，包括人类细胞系的研究，已经报道了NPC成分在动粒功能中的作用。我们的研究导致了第一个报告Mad 1 p，Mad 2 p和Bub 3 p定位到动粒后SAC激活在S。啤酒。我们最近定义了Mad 1 p的一个结构域，它是染色体传递和检查点功能所必需的。NPC在有丝分裂中的作用的进一步相关性是基于我们与Belanger博士的合作，该合作显示了纺锤体极体（SPB）和有丝分裂出口网络突变体之间的遗传相互作用。除了染色体分离，DNA损伤和复制检查点途径通过响应遗传毒性应激而停止细胞周期来确保基因组稳定性。我们最近建立了氧化应激基因SOD 1和CCS 1与MEC 1介导的DNA损伤和复制阻滞检查点通路之间的功能关系。最近的遗传分析结果表明，Sod 1 p和Ccs 1 p在DNA修复、基因组稳定性和端粒维持中发挥作用。我们对Sod 1 p和Ccs 1 p的研究将揭示S.酿酒酵母，可以适用于其他系统。 本研究对S.酿酒酵母将帮助我们了解人类的类似过程及其在人类疾病中的意义。我们的实验室是唯一准备利用传统的遗传，生物化学和细胞生物学方法，以及高通量基因组分析为我们的研究项目。我们使用一系列基因缺失菌株和菌落挑选机器人来鉴定可能的癌症药物靶点，并通过Charlie Boone（多伦多大学）实验室开发的合成基因组（SGA）分析进行遗传筛选。