Molecular Determinants of Chromosome Transmission and Cell Cycle Regulation

我们使用染色体传递保真度（ctf）突变体和酿酒酵母的缺失菌株收集来鉴定和表征着丝点功能和检查点功能所需的基因。通过对ctf突变体的研究，鉴定并鉴定了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。我们确定了Spt4p在异色沉默中的新作用。通过跨种方法，我们发现酵母spt4菌株与人spt4是互补的。最重要的是，我们发现酿酒葡萄球菌的SPT4有助于组蛋白H3变体Cse4p的适当定位。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导致染色体传播缺陷。我们希望研究染色质修饰剂和着丝点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关于Cse4p定位和组蛋白剂量的研究结果与pombe中的结果一致，表明其潜在机制是守恒的。因此，在酿酒酵母中的研究阐明了Cse4p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。我们还希望在酵母和人类中确定Spt4p及其相互作用伙伴Spt5p和Spt6p以及组蛋白在染色质结构，染色体分离和基因沉默中的分子作用。为了证明我们的发现在酿酒葡萄球菌中的功能相关性，我们计划将我们的研究扩展到高等真核生物。为此目的，我们正在与博士合作。Caplen和Roschke在RNAi研究中研究人类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葡萄球菌的SAC蛋白Mad1p和Mad2p与NPC之间建立新的关系。在我们的工作之后，包括人类细胞系在内的其他几项研究报道了NPC成分在着丝点功能中的作用。我们的研究首次报道了s.s erevisiae在SAC激活时Mad1p、Mad2p和Bub3p定位于着丝点。我们最近定义了Mad1p的一个区域，它是染色体传递和检查点功能所必需的。我们与Belanger博士的合作表明纺锤体极体（SPB）和有丝分裂退出网络突变体之间存在遗传相互作用，进一步证实了NPC在有丝分裂中的作用。除了染色体分离外，DNA损伤和复制检查点途径通过停止细胞周期以响应基因毒性应激来确保基因组的稳定性。我们最近建立了氧化应激基因SOD1和ccs1与MEC1介导的DNA损伤和复制停止检查点途径之间的功能关系。最近的遗传分析结果表明，Sod1p和Ccs1p在DNA修复、基因组稳定和端粒维持中起作用。我们对Sod1p和Ccs1p的研究将揭示酿酒酵母中氧化应激、氧化还原状态和检查点途径相关的分子机制，这可能适用于其他系统。我们对酿酒酵母忠实染色体传播的分子决定因素的研究将有助于我们了解人类的类似过程及其对人类疾病的影响。我们的实验室是独一无二的，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识别可能的癌症药物靶点，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。

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).