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）： 项目摘要/摘要 在细胞分裂过程中，染色体被复制并平等地分离到每个子细胞中。着丝粒是一种特殊的染色体结构，负责染色体的正确分离。着丝粒引导着丝粒的组装，着丝粒是一种多蛋白复合物，在染色体分离过程中将纺锤体微管与染色体连接起来。着丝粒的错误调控会对染色体分离产生不利影响，导致非整倍体或染色体含量异常，这种情况存在于 90% 以上的所有癌症中。着丝粒普遍受着丝粒特异性组蛋白 H3 变体 CENP-A 控制。 CENP-A 取代了着丝粒处的典型组蛋白 H3，并为着丝粒的组装提供了平台。在着丝粒 DNA 复制过程中，染色质在复制叉之前被分解。值得注意的是，DNA 复制后，CENP-A 忠实地重新组装成子着丝粒的核小体，但其他地方则不然。亲本 CENP-A 如何忠实地招募（即遗传）至着丝粒 DNA复制后的核小体是完全未知的。此外，CENP-A错误定位到非着丝粒区域对染色体分离具有破坏性影响。如何保护非着丝粒区域免受正常细胞中 CENP-A 错误掺入的影响也很大程度上尚未得到探索。我们的长期目标是了解着丝粒遗传和规范的分子基础。为了实现这一目标，我们建议使用裂殖酵母（Schizosaccharomyces pombe），这是一种简单的、遗传上易于处理的真核模型生物。在裂殖酵母中，着丝粒调控的许多方面在进化上对于人类来说是保守的。我们最近表明，参与 DNA 复制的蛋白质是将 CENP-A 忠实加载到着丝粒所必需的。在目标 1 中，我们将测试以下假设：DNA 复制组件与 CENP-A 蛋白相互作用，在准备细胞分裂的细胞中在新复制的 DNA 上传播着丝粒组装。此外，我们的初步结果表明，与多细胞生物一样，CENP-A在粟酒裂殖酵母中的过度表达会导致染色体错误分离以及CENP-A在非着丝粒染色质处的组装。使用该系统，我们证明了 CENP-A 的 N 末端结构域在阻止 CENP-A 在非着丝粒区域的掺入中发挥着关键作用。在目标 2 中，我们将提出这样一个假设：CENP-A 的 N 末端结构域与染色质重塑因子相互作用，以保护非着丝粒染色质免于错误组装 CENP-A。在目标 3 中，我们将进行两项新颖的互补全基因组遗传筛选，以确定涉及 1) 促进内源性 CENP-A 在着丝粒处组装和 2) 保护非着丝粒染色质免于组装 CENP-A 的因素。对这些因素的进一步表征将为 CENP-A 如何精确靶向着丝粒染色质提供新的见解。拟议的研究将揭示人类细胞中染色体分离的控制过程，并有望更好地了解癌症进展和开发新的癌症治疗方法。