Molecular Mechanisms of Chromosome Segregation in Yeast

描述（由申请人提供）：所有生物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。细胞监控着染色体的精确复制一次，然后正确地分配给子细胞。这是至关重要的，因为错误会导致染色体补体不完整，这与癌症高度相关，并导致人类自然流产、唐氏综合症和其他发育障碍。我们的长期目标是了解有助于染色体分布保真度的分子机制。从出芽酵母到人类，许多染色体分离机制都是保守的。由于在芽殖酵母中易于遗传操作，我们使用酿酒酵母作为我们的模式生物。特别地，本提案将探讨染色体分离的分子机制，特别关注着丝粒和着丝点。着丝粒是染色体上的顺式作用序列，是染色体正确分离所必需的。虽然着丝粒序列在生物体之间是高度可变的，但着丝粒普遍由一种组蛋白H3变体（称为Cse4/CENP-A）标记。这种组蛋白变体被整合到具有着丝粒序列的核小体中，这对于指导着丝点的形成至关重要，着丝点是微管附着和染色体分离所必需的多蛋白质结构。本研究的目的是了解含有Cse4/CENP-A的核小体是如何在基因组中建立和维持的。特别地，我们将描述一种新的cse4相关因子在cse4沉积、着丝点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对任何生物体中着丝粒染色质和内部着丝粒形成的第一个详细的分子理解。我们的研究将有助于我们评估着丝粒和着丝点在染色体分离中的功能的现有模型，并可能有助于阐明癌症的病因和新的治疗途径。人体中的每个细胞都必须保持正确的染色体数目。当染色体不能准确地在细胞间分裂时，就会产生非整倍性，即染色体过多或过少的状态。非整倍体与癌症高度相关，并导致自然流产和发育障碍，如唐氏综合症。特别是，本提案的主题Cse4/CENP-A是一种对着丝粒和着丝点形成至关重要的蛋白质，染色体的特征是它们精确分离所必需的。Cse4/CENP-A已被证明在人类原发性结直肠癌bbb中过度表达和错标。相关蛋白CENP-H在人细胞[2]中过度表达时诱导非整倍体。蝇类的CENP-A同源物CID过表达可促进异位着丝粒和多中心染色体的形成，从而导致染色体错分离、非整倍体和生长缺陷[3]。这些结果表明Cse4/CENP-A在基因组维持中起着重要作用。更好地了解Cse4/CENP-A定位到着丝粒及其参与着丝粒功能的分子要求，对于我们理解染色体精确分离的基本机制至关重要。1. Tomonaga， T., et al.，着丝粒蛋白a在人类原发性结直肠癌中的过表达和错误靶向。癌症研究，2003。63：第3511-6页。2. Tomonaga， T.等，着丝粒蛋白H在原发性人类结直肠癌中上调，其过表达诱导非整倍体。癌症研究，2005。65：第4683-9页。3. Heun， P.等，果蝇着丝粒特异性组蛋白CID的错误定位促进了功能性异位着丝点的形成。Dev Cell， 2006年。10: 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.