Molecular Determinants of Chromosome Transmission and Cell Cycle Regulation

我们使用了大量的染色体传递保真度突变体，<i>酿酒酵母</i>的CTF突变体进行了染色体丢失的菌落显色试验。我们鉴定的第一个基因是酿酒酵母spt基因。我们已经确定Spt4p是着丝粒和异染色质的一种成分，在着丝粒功能和基因沉默中发挥作用。SPT4的人类同源物HsSPT4能够在功能上补充酿酒酵母spt4突变体的表型。这些研究代表了酵母突变体(Spt4)的沉默缺陷被人类基因(HsSPT4)补充的第一个例子，以及人类蛋白质在体内与萌芽酵母的动点的关联。我们还确定spt4突变体将着丝粒的组蛋白变异体Cse4p错位定位到非着丝粒区域。CENP-A是人类Cse4p的同源物，已有报道在结直肠癌中定位错误。我们最近证实，组蛋白H3和Cse4p的剂量影响染色体传递的保真度。我们的初步研究表明，除了动粒蛋白外，着丝粒染色质的状态对染色体传递的保真度也是至关重要的。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Natasha Caplen博士的实验室合作进行RNAi研究，以研究人类Spt4p/Spt5p/Spt6p在染色体分离和CENP-A功能中的作用。对第二个动粒突变体的研究表明，在酿酒酵母中，核孔蛋白基因NUP170是染色体传递保真度所必需的。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)监控纺锤体与着丝点的相互作用，如果单个染色体的微管附着发生变化，就会在后期停止。正在进行的研究开始阐明Mad1p中核孔或检查点/染色体分离功能所需的不同亚域。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在体内可以与单个有缺陷的着丝粒联系在一起。我们的实验系统将使我们能够建立检查点蛋白复合体的组装顺序，并帮助阐明酵母和人类中染色体分离以及检查点和核孔功能所需的检查点蛋白结构域。除了染色体分离外，DNA损伤和复制检查点途径还通过停止细胞周期来响应遗传毒性压力，从而确保基因组的稳定性。我们最近建立了氧化应激基因SOD1LYS7和Mec1介导的DNA损伤和复制抑制的检查点通路之间的功能关系。我们将继续我们对Sod1p和Lys7p的研究，以揭示与酿酒酵母氧化应激、氧化还原状态和检查点途径相关的分子机制研究，这些研究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有助于我们理解人类中类似的过程及其在人类疾病中的意义。我们的实验室独一无二地准备好利用传统的遗传、生化和细胞生物学方法，以及高通量基因组分析来进行我们的研究项目。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抗癌药物靶点，并通过合成基因组(SGA)分析进行基因筛选，该分析是在查理·布恩(Charlie Boone)的实验室开发的。多伦多)

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)