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: 7592969
• 负责人: Munira Basrai
• 金额: $ 112.05万
• 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/7592969
• 关键词: Affect; Aging; Anaphase; Antineoplastic Agents; Biochemical Genetics; Biological Assay; Biological Models; Cell Cycle; Cell Cycle Checkpoint; Cell Cycle Regulation; Cellular biology; Centromere; Chromatin; Chromatin Structure; Chromosome Segregation; Chromosomes; Color; Colorectal Cancer; Complement; Complex; Congenital Abnormality; DNA; DNA Replication Damage; DNA biosynthesis; Defect; Disease; Drug Delivery Systems; Ensure; Etiology; Eukaryota; Eukaryotic Cell; Failure; Future; Gene Deletion; Gene Silencing; Genes; Genetic Materials; Genetic Screening; Genome; Genome Stability; Genomics; Genotoxic Stress; Histone H3; Histones; Homologous Gene; Human; In Vitro; Kinetochores; Laboratories; Life; Light; Link; Localized; Maintenance; Malignant Neoplasms; Mediating; Medical Surveillance; Microtubules; Mitosis; Mitotic; Mitotic/Spindle Checkpoint; Molecular; Monitor; Nature; Nucleoporin Gene; Oxidation-Reduction; Oxidative Stress; Pathway interactions; Phenotype; Process; Proteins; RNA Interference; Recovery; Regulation; Reporting; Research; Research Project Grants; Robot; Role; Saccharomyces cerevisiae; Saccharomycetales; Structure; System; Time; Variant; Yeasts; centromere protein A; chromosome loss; dosage; human disease; in vivo; interest; member; mutant; novel; prevent; reconstitution; repaired; response; transmission process

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)

我们使用了染色体传递保真度突变体的大型参考集，即 <I>S 的 ctf 突变体。酿酒酵母染色体丢失的菌落颜色测定。我们表征的第一个基因是<I>S。酿酒酵母SPT</I>基因。我们已经确定 Spt4p 是着丝粒和异染色质的组成部分，在动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 S 的表型。酿酒酵母spt4突变体。这些研究代表了酵母突变体<I>(spt4)</I>的沉默缺陷被人类基因(HsSPT4)补充以及人类<I>体内</I>关联的第一个例子。蛋白质到芽殖酵母的着丝粒。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。据报道，Cse4p 的人类同源物 CENP-A 在结直肠癌中存在错误定位。我们最近确定组蛋白 H3 和 Cse4p 的剂量会影响染色体传递保真度。我们的初步研究表明，除了着丝粒蛋白之外，着丝粒染色质的状态对于染色体传输保真度也至关重要。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Natasha Caplen博士实验室合作进行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。对第二个动粒突变体的研究表明，核孔蛋白基因<I>NUP170</I>是<I>S中染色体传递保真度所必需的。酿酒酵母</I>。我们在 Nup170p 蛋白复合体成员与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）监控纺锤体与动粒的相互作用，如果单条染色体的微管附着发生改变，就会停止后期。 正在进行的研究开始揭示 Mad1p 内核孔或检查点/染色体分离功能所需的不同子结构域。对第三纺锤体检查点蛋白 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粒相关联。我们的实验系统将使我们能够建立检查点蛋白复合物的组装顺序，并帮助阐明酵母和人类染色体分离以及检查点和核孔功能所需的检查点蛋白的结构域。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<I>SOD1</I>和<I>LYS7</I>与<I>MEC1</I>介导的DNA损伤和复制停滞检查点通路之间的功能关系。我们将继续对 Sod1p 和 Lys7p 进行研究，以阐明与 <I>S 中氧化应激、氧化还原状态和检查点通路相关的分子机制研究。 cerevisiae 可能适用于其他系统。我们对 <I>S 中染色体忠实传递的分子决定因素的研究。酿酒酵母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