Centromere identity and function

描述(申请人提供)：这项建议集中在遗传学中的一个基本问题上：染色体及其携带的基因如何在细胞分裂时适当地分离到子细胞？这一过程中的缺陷会导致基因组的不均匀分割，进而导致非整倍体。如果这发生在配子发生过程中，这种类型的遗传灾难会导致自然流产或由此产生的后代的出生缺陷。突变由于有丝分裂错误和肿瘤抑制基因突变而导致的单个染色体的获得或丢失 基因或癌基因在肿瘤形成和发展的早期事件中起作用，非整倍体是几乎所有晚期肿瘤的标志。控制其准确分离的染色体元件是着丝粒。分子遗传学和人类患者数据不是通过特定的DNA序列来定义着丝粒的身份，因为在大多数正常着丝粒上发现的着丝粒？卫星重复既不是必要的，也不足以确定功能着丝粒的位置。相反，流行的观点是着丝粒的同一性是由表观遗传学定义的，我们的长期目标是理解这种表观遗传学着丝粒标记的基础以及用于建立和维持它的机制。代表表观遗传标记的主要候选者是CENP-A，这是一种组蛋白H3变体，在功能着丝粒处组装成核小体。这项提案中的具体研究将解决以下问题：CENP-A如何在着丝粒产生表观遗传标记？CENP-A并入多核小体阵列对它们的高阶折叠有什么影响？含有CENP-A的核小体是否直接招募一组特定的着丝粒因子？CENP-A对着丝粒？-卫星DNA有选择性亲和力吗？将CENP-A装载到着丝粒DNA上的机制是什么？哪些细胞蛋白通过着丝粒靶向结构域识别CENP-A？ 结合生化重建、生物物理学、分子遗传学和细胞生物学的方法，我们提出了以下具体目标： 1.确定CENP-A掺入着丝粒核小体的后果 2.确定着丝粒上CENP-A染色质组装的要求 总之，这些研究有望为表观遗传机制提供有价值的见解，这些机制指定并维持着丝粒在染色体上的位置。

DESCRIPTION (provided by applicant): This proposal is focused on a fundamental question in genetics: How are chromosomes and the genes they carry appropriately segregated to the daughter cells at cell division? Defects in this process result in uneven partitioning of the genome that, in turn, causes aneuploidy. If this happens during gametogenesis, this type of genetic catastrophe results in spontaneous abortion or birth defects in resulting offspring. The gain or loss of an individual chromosome resulting from errors in mitotic cell division along with mutation of tumor suppressor genes or oncogenes contributes to early events in tumor formation and progression, and aneuploidy is a hallmark of almost all late stage tumors. The chromosomal element that controls its accurate segregation is the centromere. Molecular genetic and human patient data has defined centromere identity not by a particular DNA sequence, since centromeric ?-satellite repeats found at most normal centromeres are neither necessary nor sufficient to specify the location of a functioning centromere. Rather, the prevailing view is that centromere identity is defined epigenetically, and our long-term goal is to understand the basis for this epigenetic centromere mark and the mechanisms used for its establishment and maintenance. The leading candidate to represent the epigenetic mark is CENP-A, a histone H3 variant that assembles into nucleosomes at functional centromeres. Specific research in this proposal will address the following questions: How does CENP-A generate an epigenetic mark at the centromere? What impact does the incorporation of CENP-A into polynucleosome arrays have on their higher-order folding? Does the CENP-A-containing nucleosome directly recruit a specific set of centromere factors? Is there selective affinity of CENP-A for centromeric ?-satellite DNA? What is the mechanism for loading CENP-A onto centromeric DNA? What cellular proteins recognize CENP-A via its centromere targeting domain? Using a combination of biochemical reconstitution, biophysics, molecular genetics, and cell biology approaches we propose the following specific aims: 1. Determine the consequences of incorporation of CENP-A into centromeric nucleosomes 2. Define the requirements for CENP-A chromatin assembly at centromeres Together, these studies promise to provide valuable insight into the epigenetic mechanisms that specify and maintain the location of the centromere on the chromosome.