Molecular Basis of Centromere Specification and Inheritance

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质复合体，在染色体分离过程中将纺锤体微管连接到染色体上。着丝粒的错误调节会对染色体分离产生不利影响，导致非整倍体或染色体内容异常，这种情况在90%以上的癌症中都存在。着丝粒普遍受着丝粒特异的组蛋白H3变异体CENP-A控制。CENP-A在着丝粒上取代了典型的组蛋白H3，为动粒组装提供了平台。在着丝粒DNA复制过程中，染色质在复制分叉之前被分解。值得注意的是，在DNA复制后，CENP-A被忠实地重组到子着丝粒的核小体中，但在其他地方却没有。亲本CENP-A如何被忠实地招募，即遗传到着丝粒 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有毁灭性的影响。在正常细胞中，非着丝粒区域如何防止CENP-A误掺入也在很大程度上仍未被探索。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(裂殖酵母)，这是一种简单的、遗传上容易处理的真核模式生物。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。我们最近已经证明，参与DNA复制的蛋白质是将CENP-A忠实地装载到着丝粒上所必需的。在目标1中，我们将检验这一假设，即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传播新复制的DNA上的着丝粒组装。此外，我们的初步结果表明，像在多细胞生物体中一样，CENP-A在S.pombe中的过表达导致染色体错误分离和CENP-A在非着丝粒染色质上的组装。利用这个系统，我们证明了CENP-A的N-末端结构域在阻止CENP-A在非着丝粒区域掺入方面起着关键作用。在目标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的假设，以保护非着丝粒染色质免受错误组装CENP-A的影响。在目标3中，我们将进行两个新的互补全基因组遗传筛选，以确定参与1)促进内源性CENP-A在着丝粒组装和2)保护非着丝粒染色质不组装CENP-A的因素。这些因素的进一步表征将为CENP-A如何准确定位于着丝粒染色质提供新的见解。这项拟议的研究将阐明人类细胞中染色体分离的过程，并有望更好地了解癌症的进展和新癌症治疗方法的开发。

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.