Regulation of Centromeric Chromatin

描述(申请人提供)：细胞分裂的完美执行对所有生物体的产生和生存至关重要。在每个细胞周期中，染色体必须准确地分割到子细胞，以防止基因组不稳定和非整倍体，这是许多肿瘤和出生缺陷的标志。染色体利用着丝点进行分离，着丝点是一种特殊的蛋白质结构，聚集在着丝粒DNA序列上，并介导与微管的附着。所有真核着丝点的基础是一种保守的内部着丝粒结构，其特征是着丝粒染色质及其相关蛋白。着丝粒染色质的一个特征是CENP-A，这是一种重要的组蛋白H3变体，它在表观遗传学上标记着丝粒，是动粒组装所必需的。周围的着丝粒周围的染色质对分离的保真度也有不同的贡献。为了充分了解确保染色体准确分离的机制，阐明着丝粒和着丝粒周围染色质的功能和维持的突出问题是至关重要的。这项提案将使用纯化的动粒，并结合生化、生物物理、蛋白质组和基因组学方法来解决着丝粒染色质对动粒功能的贡献的一些悬而未决的问题。1)着丝粒结合蛋白如何参与着丝粒的不同功能？2)着丝粒周围染色质如何调节染色体分离？3)着丝粒专一性定位的机制是什么？该提案将使用萌芽酵母进行这些研究，因为它们可以进行生化、遗传学和细胞学研究，而酵母动粒是迄今为止最好的特征。综上所述，这些对发芽酵母中动粒和基本染色质基础的研究将有助于理解所有真核生物中分离的基本机制。这项工作不仅将阐明隔离过程的重要方面，而且从长远来看，将有助于设计更好的治疗干预措施。

DESCRIPTION (provided by applicant): The flawless execution of cell division is essential to the generation and survival of all organisms. During every cell cycle, chromosomes must be accurately partitioned to daughter cells to prevent genomic instability and aneuploidy, a hallmark of many tumors and birth defects. Chromosomes segregate using their kinetochores, the specialized protein structures that assemble on centromeric DNA sequences and mediate attachment to microtubules. The foundation of all eukaryotic kinetochores is a conserved, inner centromere structure characterized by centromeric chromatin and its associated proteins. A hallmark of centromeric chromatin is Cenp-A, an essential histone H3 variant that epigenetically marks centromeres and is required for kinetochore assembly. The surrounding pericentromeric chromatin also makes various contributions to the fidelity of segregation. To fully understand the mechanisms that ensure accurate chromosome segregation, it is critical to elucidate outstanding questions about the functions and maintenance of centromeric and pericentromeric chromatin. This proposal will use purified kinetochores and a combination of biochemical, biophysical, proteomic and genomic approaches to address a number of outstanding questions about the contribution of centromeric chromatin to kinetochore function. 1) How do centromere-binding proteins contribute to the diverse functions of kinetochores? 2) How does pericentromeric chromatin regulate chromosome segregation? 3) What are the mechanisms that contribute to the exclusive localization of centromeres? The proposal will use budding yeast for these studies because they are amenable to biochemical, genetic and cytological studies, and the yeast kinetochore is the best characterized to date. Taken together, these studies of kinetochores and the underlying chromatin foundation in budding yeast will lead toward an understanding of the fundamental mechanisms of segregation in all eukaryotes. This work will not only elucidate important aspects about the process of segregation, but will aid in the design of better therapeutic interventions in the long-term.