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).

我们使用酿酒酵母的染色体传递保真度（ctf）突变体和缺失菌株集合来鉴定和表征着丝粒功能和检查点功能所需的基因。对 ctf 突变体的研究导致了 SPT4 和 NUP170 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 Spt4p 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 spt4 菌株与人类 SPT4 互补。最重要的是，我们证明酿酒酵母 SPT4 有助于组蛋白 H3 变体 Cse4p 的正确定位。我们研究了 Cse4p 定位的机制，最近发现 Cse4p 的错误定位和组蛋白化学计量的改变会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 CenH3 染色质的组装/功能。我们最近在酿酒酵母中的 Cse4p 定位和组蛋白剂量的结果与在粟酒裂殖酵母中的结果一致，表明潜在机制的保守性。因此，在酿酒酵母中阐明 Cse4p 定位机制以及染色质修饰在着丝粒功能中的作用的研究可能有助于我们了解人类和其他系统中的类似途径。我们还希望确定 Spt4p 及其相互作用伙伴 Spt5p 和 Spt6p 以及组蛋白在酵母和人类染色质结构、染色体分离和基因沉默中的分子作用。为了证明我们在酿酒酵母中的发现的功能相关性，我们计划将我们的研究扩展到高等真核生物。为此，我们正在与博士合作。 Caplen 和 Roschke 在 RNAi 研究中研究了人类 Spt4p/Spt5p/Spt6p 在染色体分离和 CENP-A 功能中的作用。 我们对核孔复合体 (NPC) 基因 NUP170 的研究使我们能够在酿酒酵母中的 SAC 蛋白 Mad1p 和 Mad2p 与 NPC 之间建立一种新的关系。在我们的工作之后，其他几项研究（包括人类细胞系的研究）报告了 NPC 成分在动粒功能中的作用。我们的研究首次报道了酿酒酵母中 SAC 激活后 Mad1p、Mad2p 和 Bub3p 定位于着丝粒的情况。我们最近定义了染色体传递和检查点功能所需的 Mad1p 结构域。 NPC 在有丝分裂中的作用的进一步相关性基于我们与 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。除了染色体分离之外，DNA 损伤和复制检查点途径还通过响应基因毒性应激而停止细胞周期，从而确保基因组稳定性。我们最近建立了氧化应激基因 SOD1 和 CCS1 与 MEC1 介导的 DNA 损伤和复制停滞检查点通路之间的功能关系。最近的遗传分析结果表明，Sod1p 和 Ccs1p 在 DNA 修复、基因组稳定性和端粒维护中发挥作用。我们对 Sod1p 和 Ccs1p 的研究将揭示酿酒酵母中与氧化应激、氧化还原状态和检查点途径相关的分子机制，这些机制可能适用于其他系统。 我们对酿酒酵母中忠实染色体传递的分子决定因素的研究将帮助我们了解人类的类似过程及其对人类疾病的影响。我们的实验室具有独特的优势，可以利用传统的遗传、生化和细胞生物学方法以及高通量基因组分析来开展我们的研究项目。我们使用一系列基因缺失菌株和菌落挑选机器人来识别可能的癌症药物靶标，并通过查理布恩（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。