Molecular Mechanisms of Chromosome Segregation in Yeast

描述(申请人提供)：所有生物体都有机制确保有丝分裂和减数分裂产生的细胞含有适当数量的染色体。该细胞监测染色体只复制一次，然后正确地分配给子细胞。这一点至关重要，因为错误会导致染色体的不完全补充，这与癌症高度相关，并会导致人类自发流产、唐氏症和其他发育障碍。我们的长期目标是了解影响染色体分布保真度的分子机制。许多染色体分离机制从萌芽酵母到人类都是保守的。由于在萌芽酵母中易于进行遗传操作，我们使用酿酒酵母作为我们的模式生物。特别是，这项建议将探索染色体分离的分子机制，特别关注着丝粒和着丝粒。着丝粒是染色体上的顺式作用序列，是正确分离所必需的。尽管着丝粒序列在不同生物体之间差异很大，但着丝粒普遍由组蛋白H3变异体标记，称为Cse4/CENP-A。这种组蛋白变体与着丝粒序列结合到核小体中，着丝粒序列对着丝粒的形成至关重要，着丝粒是一种对微管附着至关重要的多蛋白质结构，因此对染色体分离至关重要。这项建议的目标是从分子上详细了解含有Cse4/CENP-A的核小体是如何在基因组中建立和维持的。特别是，我们将表征一种新的Cse4相关因子在Cse4沉积、动粒功能和染色体分离中的作用。我们的最终目标是在体外重建着丝粒染色质和内部着丝粒。这些类型的实验将帮助我们建立第一个关于着丝粒染色质和任何有机体内部动粒形成的详细分子理解。我们的研究将帮助我们评估着丝粒和着丝点在染色体分离中的作用的现有模型，并可能有助于阐明癌症的病因和新的治疗途径。与公共健康相关人体内的每个细胞都必须保持正确的染色体数目。当染色体不能在细胞间准确分割时，就会产生非整倍体，即染色体过多或过少的状态。非整倍体与癌症高度相关，并会导致自发流产和唐斯综合征等发育障碍。特别是，这项提案的主题Cse4/CENP-A是一种对着丝粒和着丝点的形成至关重要的蛋白质，着丝粒和着丝点是染色体准确分离所需的特征。CSE4/CENP-A已被证明在人类原发结直肠癌中过度表达和错误定位[1]。一种相关蛋白CENP-H在人类细胞中过度表达时会导致非整倍体[2]。过度表达CID，CENP-A的苍蝇同源物，促进异位着丝粒和多中心染色体的形成，从而导致染色体错误分离、非整倍体和生长缺陷[3]。这些结果表明，Cse4/CENP-A对基因组的维持有显著的贡献。更好地理解Cse4/CENP-A定位于着丝粒及其参与着丝粒功能的分子要求，对于我们理解有助于精确染色体分离的基本机制是至关重要的。1.Tomonaga，T.等人，着丝粒蛋白-A在人类原发性结直肠癌中的过度表达和错误靶向。《癌症资源》，2003年。63：第3511-6页。2.Tomonaga，T.等，着丝粒蛋白H在人原发结直肠癌中表达上调，其过度表达导致非整倍体。癌症资源，2005年。65：第4683-9页。3.Heun，P.等人，果蝇着丝粒特异性组蛋白CID的错误定位促进了功能异位动点的形成。Dev Cell，2006。10：第303-15页。

DESCRIPTION (provided by applicant): All organisms have mechanisms to ensure that cells produced from mitotic and meiotic divisions contain the proper number of chromosomes. The cell monitors that chromosomes are copied exactly once and then distributed correctly to daughter cells. This is critical since errors result in an incomplete chromosome complement which is highly correlated with cancer and causes spontaneous miscarriage, Downs, and other developmental disorders in humans. Our long term goal is to understand the molecular mechanisms that contribute to the fidelity of chromosome distribution. Many chromosome segregation mechanisms are conserved from budding yeast to man. Due to the ease of genetic manipulations in budding yeast, we use S. cerevisiae as our model organism. In particular, this proposal will explore the molecular mechanisms of chromosome segregation with particular attention to centromeres and kinetochores. Centromeres are cis acting sequences on chromosomes that are required for their correct segregation. Although centromere sequence is highly variable between organisms, centromeres are universally marked by a histone H3 variant, known as Cse4/CENP-A. This histone variant is incorporated into nucleosomes with centromere sequence which is critical to direct the formation of the kinetochore, a multi-protein structure essential for microtubule attachment and therefore chromosome segregation. The goal of this proposal is to understand in molecular detail how Cse4/CENP-A- containing nucleosomes are established and maintained in the genome. In particular we will characterize the role of a novel Cse4-associated factor on Cse4-deposition, kinetochore function and chromosome segregation. Our ultimate goal is to reconstruct centromeric chromatin and the inner kinetochore in vitro. These types of experiments will help us build the first detailed molecular understanding of centromeric chromatin and inner kinetochore formation in any organism. Our studies will help us evaluate current models for the function of centromeres and kinetochores in chromosome segregation, and may help elucidate the etiology of cancer and new avenues for therapy. PUBLIC HEALTH RELEVANCE Each cell in the body must maintain the correct number of chromosomes. When chromosomes are not accurately divided between cells, aneuploidy results, which is the state of having too many or too few chromosomes. Aneuploidy is highly correlated with cancer and causes spontaneous miscarriage and developmental disorders such as Downs syndrome. In particular, the topic of this proposal, Cse4/CENP-A, is a protein that is essential for the formation of centromeres and kinetochores, features of chromosomes that are required for their accurate segregation. Cse4/CENP-A has been shown to be overexpressed and mistargeted in human primary colorectal cancers [1]. An associated protein, CENP-H, induces aneuploidy when overexpressed in human cells [2]. Overexpression of CID, the fly homolog of CENP-A, promotes formation of ectopic centromeres and multicentric chromosomes which causes chromosome missegregation, aneuploidy, and growth defects [3]. These results demonstrate that Cse4/CENP-A significantly contributes to genome maintenance. A better understanding of the molecular requirements for localization of Cse4/CENP-A to centromeres and its participation in kinetochore function is crucial to our understanding of basic mechanisms that contribute to accurate chromosome segregation. 1. Tomonaga, T., et al., Overexpression and mistargeting of centromere protein-A in human primary colorectal cancer. Cancer Res, 2003. 63: p. 3511-6. 2. Tomonaga, T., et al., Centromere protein H is up-regulated in primary human colorectal cancer and its overexpression induces aneuploidy. Cancer Res, 2005. 65: p. 4683-9. 3. Heun, P., et al., Mislocalization of the Drosophila centromere-specific histone CID promotes formation of functional ectopic kinetochores. Dev Cell, 2006. 10: p. 303-15.