Molecular Basis of Centromere Specification and Inheritance

着丝粒规格和遗传的分子基础

• 批准号: 9060967
• 负责人: Fei Li
• 金额: $ 29.97万
• 依托单位: NEW YORK UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-05-01 至 2019-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9060967
• 关键词: Address; Affect; Aneuploidy; Animal Model; Binding Proteins; Biochemical; Biochemistry; Cell Cycle; Cell Cycle Stage; Cell division; Cells; Centromere; Chromatin; Chromatin Remodeling Factor; Chromosome Segregation; Chromosome Structures; Chromosomes; Complex; Cytology; DNA; DNA Polymerase II; DNA biosynthesis; DNA replication fork; Data; Daughter; Development; Diagnosis; Disease; Ensure; Epigenetic Process; Eukaryota; Eukaryotic Cell; Fission Yeast; Foundations; G2 Phase; Genetic; Genetic Screening; Genome; Genomics; Goals; Histone H3; Human; In Vitro; Inherited; Kinetochores; Knowledge; Lead; Light; Link; Malignant Neoplasms; Mediating; Meiosis; Microtubules; Mitosis; Molecular; N-terminal; Nature; Normal Cell; Nucleosomes; Organism; Play; Positioning Attribute; Process; Proteins; Recruitment Activity; Regulation; Research; Role; S Phase; Sequence Analysis; Structure; System; Testing; Variant; Work; abstracting; base; cancer therapy; centromere protein A; chromatin immunoprecipitation; daughter cell; deep sequencing; fluorescence imaging; genome-wide; in vivo; insight; interdisciplinary approach; novel; novel diagnostics; novel therapeutics; overexpression; prevent; protein complex; public health relevance; segregation; tool; tumor progression

DESCRIPTION (provided by applicant): PROJECT SUMMARY/ABSTRACT During cell division, chromosomes are duplicated and equally segregated into each daughter cell. The centromere, a specialized chromosomal structure, is responsible for correct segregation of chromosomes. The centromeres guide the assembly of the kinetochore, a multi-protein complex that links spindle microtubules to chromosomes during chromosome segregation. Mis-regulation of centromeres adversely affects chromosome segregation resulting in aneuploidy, or abnormal chromosome content, a condition found in more than 90% of all cancers. Centromeres are universally governed by the centromere-specific histone H3 variant, CENP-A. CENP-A replaces canonical histone H3 at centromeres, and provides the platform for the assembly of kinetochores. During replication of centromeric DNA, chromatin is disassembled ahead of the replication fork. Remarkably, after DNA replication, CENP-A is faithfully reassembled into nucleosomes of daughter centromeres but not elsewhere. How the parental CENP-A is faithfully recruited, i.e., inherited, to centromeric nucleosomes following DNA replication is completely unknown. In addition, mislocalization of CENP-A to non- centromeric regions has a devastating impact on chromosome segregation. How non-centromeric regions are protected from CENP-A mis-incorporation in normal cells also remains largely unexplored. Our long-term goal is to understand the molecular basis of the inheritance and specification of centromeres. Toward this goal, we propose to use fission yeast (Schizosaccharomyces pombe), a simple, genetically-tractable eukaryotic model organism. In fission yeast, many aspects of centromere regulation are evolutionarily conserved with humans. We have recently shown that proteins involved in DNA replication are required for faithful loading of CENP-A to centromeres. In Aim 1, we will test the hypothesis that DNA replication components interact with the CENP-A protein to propagate centromere assembly on newly replicated DNA in cells preparing for cell division. In addition, our preliminary results indicate that, like in multi-cellular organisms, overexpression of CENP-A in S. pombe results in chromosome mis-segregation and the assembly of CENP-A at non-centromeric chromatin. Using this system, we have demonstrated that the N-terminal domain of CENP-A plays a key role in preventing the incorporation of CENP-A at non-centromeric regions. In Aim 2, we will address the hypothesis that the N- terminal domain of CENP-A interacts with chromatin remodeling factors to protect non-centromeric chromatin from erroneously assembling CENP-A. In Aim 3, we will perform two novel complementary genome-wide genetic screens to identify factors involved in 1) promoting the assembly of endogenous CENP-A at centromeres and 2) protecting non-centromeric chromatin from assembling CENP-A. Further characterization of these factors will provide new insights into how CENP-A is precisely targeted to centromeric chromatin. The proposed research will shed light on the processes governing chromosome segregation in human cells, and hold promise for a better understanding of cancer progression and the development of new cancer treatments.

描述（由申请人提供）： 细胞分裂期间的项目摘要/摘要，染色体被复制并均匀地分离为每个子细胞。 Centromere是一种专门的染色体结构，负责正确的染色体分离。 Centromeres指导动力学的组装，这是一种多蛋白质复合物，将纺锤体微管与染色体分离过程中的染色体联系起来。丝粒的错误调节会不利地影响染色体隔离，导致非整倍性或异常的染色体含量，这是所有癌症中90％以上的疾病。中心粒普遍由Centromere特异性组蛋白H3变体CENP-A支配。 CENP-A代替了centromeres的规范组蛋白H3，并为组装组合提供了平台。在丝粒DNA的复制过程中，染色质在复制叉之前被拆卸。值得注意的是，在DNA复制后，CENP-A忠实地重新组装成女儿centromeres的核小体，但并非其他地方。父母CENP-A如何忠实地招募，即继承到centromeric DNA复制后的核小组完全未知。此外，CENP-A向非丝粒区域的错误定位对染色体分离有毁灭性的影响。非中心区域如何受到正常细胞中的CENP-A错误结构的保护，这在很大程度上仍未得到探索。我们的长期目标是了解中心粒的遗传和规范的分子基础。为了实现这一目标，我们建议使用裂变酵母（Schizosacchomyces pombe），这是一种简单，可削弱的真核模型生物。在裂变酵母中，丝粒调节的许多方面在进化上是对人类的保守。我们最近表明，与CENP-A忠实载荷到丝粒需要涉及DNA复制的蛋白质。在AIM 1中，我们将检验以下假设：DNA复制成分与CENP-A蛋白相互作用，以在准备细胞分裂的细胞中，在新复制的DNA上传播丝粒组装。此外，我们的初步结果表明，与多细胞生物一样，猪链球菌中CENP-A的过表达会导致染色体错误分类和在非中心层染色质下CENP-A的组装。使用该系统，我们已经证明了CENP-A的N末端结构域在防止在非中心区域掺入CENP-A在非中心区域中起关键作用。在AIM 2中，我们将解决以下假设：CENP-A的N末端结构域与染色质重塑因子相互作用，以保护非中心染色质免受错误组装CENP-A的影响。在AIM 3中，我们将执行两种新型的互补基因组遗传筛选，以识别涉及的因素1）促进centromeres的内源性CENP-A组装和2）2）保护非中层染色质免受组装CENP-A。这些因素的进一步表征将为CENP-A精确针对丝粒染色质提供新的见解。拟议的研究将阐明人类细胞中染色体隔离的过程，并有望更好地了解癌症进展和新癌症治疗的发展。