Molecular Basis of Centromere Specification and Inheritance

着丝粒规格和遗传的分子基础

• 批准号: 8842664
• 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/8842664
• 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; 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）： 项目概要/摘要在细胞分裂过程中，染色体复制并均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。着丝粒引导动粒的组装，动粒是在染色体分离期间将纺锤体微管连接到染色体的多蛋白质复合物。着丝粒的错误调节不利地影响染色体分离，导致非整倍性或异常染色体含量，这是在超过90%的所有癌症中发现的状况。着丝粒普遍受着丝粒特异性组蛋白H3变体CENP-A控制。CENP-A在着丝粒上取代了典型的组蛋白H3，并为着丝粒的组装提供了平台。在着丝粒DNA的复制过程中，染色质在复制叉之前被分解。值得注意的是，在DNA复制后，CENP-A忠实地重新组装到子着丝粒的核小体中，而不是其他地方。如何忠实地招募亲本CENP-A，即，遗传的，到着丝粒的 DNA复制后的核小体完全未知。此外，CENP-A到非着丝粒区域的错误定位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错误掺入的影响也在很大程度上尚未探索。我们的长期目标是了解着丝粒遗传和特化的分子基础。为了实现这一目标，我们建议使用裂殖酵母（裂殖酵母），一个简单的，遗传学上易于处理的真核生物模式生物。在分裂酵母中，着丝粒调控的许多方面在进化上与人类保守。我们最近发现，参与DNA复制的蛋白质是CENP-A忠实装载到着丝粒所必需的。在目的1中，我们将测试DNA复制组件与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上繁殖着丝粒组装的假设。此外，我们的初步结果表明，像在多细胞生物中一样，CENP-A在S.粟酒裂殖酵母导致染色体错误分离和CENP-A在非着丝粒染色质上的组装。使用这个系统，我们已经证明了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起着关键作用。在目的2中，我们将解决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的假设。在目标3中，我们将进行两种新的互补全基因组遗传筛选，以鉴定参与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组装CENP-A的因素。这些因子的进一步表征将为CENP-A如何精确靶向着丝粒染色质提供新的见解。这项研究将揭示人类细胞中染色体分离的过程，并有望更好地了解癌症进展和开发新的癌症治疗方法。