Molecular Determinants of Chromosome Transmission and Cell Cycle Regulation

我们使用了一个大的染色体传递保真度突变体的参考集，ctf突变体< 1 bb0 S。cerevisiae</ i>在染色体丢失的菌落颜色试验中。我们鉴定的第一个基因是<I>S。cerevisiae SPT </ i>基因。我们已经确定Spt4p是着丝粒和异色染色质的一个组成部分，在着丝粒功能和基因沉默中起作用。SPT4的人类同源物HsSPT4能够在功能上补充<I>S的表型。酵母spt4 < / I >突变体。这些研究代表了酵母突变体<I>(spt4)</I>沉默缺陷被人类基因（HsSPT4）补充的第一个例子之一，以及人蛋白在体内<I> </I>与出芽酵母的着丝点关联。我们还确定spt4突变体将着丝粒组蛋白变体Cse4p错误定位到非着丝粒区域。Cse4p的人类同源基因CENP-A在结直肠癌中有错误定位的报道。我们最近证实组蛋白H3和Cse4p的剂量影响染色体传递保真度。我们的初步研究表明，除着丝粒蛋白外，着丝粒染色质的状态对染色体传递保真度至关重要。未来的研究将确定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，我们正在与Natasha Caplen博士的实验室合作进行RNAi研究，研究人类Spt4p/Spt5p/Spt6p在染色体分离和CENP-A功能中的作用。对第二个着丝粒突变体的研究表明，核孔蛋白基因<I b> NUP170</I>是<I b> S染色体传递保真度所必需的。酵母< / I >。我们在Nup170p蛋白复合体成员和有丝分裂纺锤体检查点蛋白Mad1p和Mad2p之间建立了重要的物理和功能联系。纺锤体检查点蛋白（Mad1p, Mad2p, Mad3p, Bub1p， Bub3p和Mps1p）监测纺锤体与着丝点的相互作用，如果单染色体的微管附着发生改变，则停止后期。正在进行的研究开始揭示Mad1p中不同的亚结构域，这些亚结构域是核孔或检查点/染色体分离功能所需的。对第三种纺锤体检查点蛋白Bub3p的研究首次证明，这种纺锤体检查点蛋白可以在体内与单个有缺陷的着丝点结合。我们的实验系统将允许我们建立检查点蛋白复合物的组装顺序，并帮助阐明染色体分离所需的检查点蛋白结构域，以及酵母和人类的检查点和核孔功能。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<I>SOD1</I>和<I>LYS7</I>和<I>MEC1</I>介导的DNA损伤和复制停止检查点途径之间的功能关系。我们将继续对Sod1p和Lys7p进行研究，以揭示< 1 >S中氧化应激、氧化还原状态和检查点途径相关的分子机制研究。</I>，可能适用于其他系统。< 1 b> S染色体忠实传递的分子决定因素研究。cerevisiae</I>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一个集落挑选机器人来识别可能的癌症药物靶点，并通过合成基因组（SGA）分析进行基因筛选，该分析由查理·布恩（多伦多大学）的实验室开发。

We have used a large reference set of chromosome transmission fidelity mutants, the ctf mutants of <I>S. cerevisiae</I>in a colony color assay for chromosome loss. The first gene we characterized was <I>S. cerevisiae SPT </I> gene. We have established that Spt4p is a component of centromeric and heterochromatic chromatin with roles in kinetochore function and gene silencing. A human homolog of SPT4, HsSPT4, is able to functionally complement the phenotypes of <I>S. cerevisiae spt4</I>mutants. These studies represent one of the first examples of the silencing defects of a yeast mutant <I>(spt4)</I>being complemented by a human gene (HsSPT4) and the <I>in vivo</I>association of a human protein to the kinetochores of budding yeast. We also determined that spt4 mutants mis-localize the centromeric histone variant Cse4p to non-centromeric regions. Mis-localization of CENP-A, the human homolog of Cse4p has been reported in colorectal cancers. We have recently established that dosage of histone H3 and Cse4p affect chromosome transmission fidelity. Our preliminary studies show that in addition to kinetochore proteins, the state of centromeric chromatin is crucial for chromosome transmission fidelity. Future studies will 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 the laboratory of Dr. Natasha Caplen in RNAi studies to investigate the role of human Spt4p/Spt5p/Spt6p in chromosome segregation and function of CENP-A. Studies of the second kinetochore mutant showed that the nucleoporin gene <I>NUP170</I> is required for chromosome transmission fidelity in <I>S. cerevisiae</I>. We established an important physical and functional link between members of the Nup170p protein complex and mitotic spindle checkpoint proteins Mad1p and Mad2p. Spindle checkpoint proteins (Mad1p, Mad2p, Mad3p, Bub1p, Bub3p and Mps1p) monitor the interaction of the spindle apparatus with the kinetochores and halt anaphase if the microtubule attachment of even a single chromosome is altered. Ongoing studies are beginning to shed light led on the distinct sub-domains within Mad1p that are required for nucleopore or checkpoint/chromosome segregation functions. Studies with a third spindle checkpoint protein, Bub3p, have demonstrated for the first time that this spindle checkpoint protein can associate in vivo with a single defective kinetochore. Our experimental system will allow us to establish the order of assembly of checkpoint protein complexes and help elucidate the domains of checkpoint proteins required for chromosome segregation and checkpoint and nucleopore functions in both yeast and humans. 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 <I>SOD1</I> and <I>LYS7</I>and the <I>MEC1</I> mediated checkpoint pathway for DNA damage and replication arrest. We will continue our studies with Sod1p and Lys7p to unravel molecular mechanisms studies that correlate oxidative stress, redox state and checkpoint pathways in <I>S. cerevisiae</I>that may be applicable to other systems. Our research on the molecular determinants of faithful chromosome transmission in <I>S. cerevisiae</I>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)