Molecular Mechanisms of Chromosome Segregation in Yeast

描述（由申请方提供）：所有生物体都具有确保有丝分裂和减数分裂产生的细胞含有适当数量染色体的机制。细胞监测染色体复制一次，然后正确地分配给子细胞。这是至关重要的，因为错误会导致不完全的染色体互补，这与癌症高度相关，并导致自然流产、唐斯和人类的其他发育障碍。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。从芽殖酵母到人类，许多染色体分离机制是保守的。酿酒酵母作为我们的模式生物。特别是，这个建议将探讨染色体分离的分子机制，特别关注着丝粒和动粒。着丝粒是染色体上的顺式作用序列，其是正确分离所需的。虽然着丝粒序列在生物体之间高度可变，但着丝粒普遍被称为Cse 4/CENP-A的组蛋白H3变体标记。这种组蛋白变体被掺入具有着丝粒序列的核小体中，所述着丝粒序列对于指导着丝粒的形成至关重要，所述着丝粒是微管附着和因此染色体分离所必需的多蛋白质结构。该提案的目标是在分子上详细了解含有Cse 4/CENP-A的核小体如何在基因组中建立和维持。特别是，我们将描述一种新的Cse 4相关因子对Cse 4沉积，动粒功能和染色体分离的作用。我们的最终目标是在体外重建着丝粒染色质和内部动粒。这些类型的实验将帮助我们建立第一个详细的分子理解着丝粒染色质和内部动粒形成在任何生物体。我们的研究将帮助我们评估目前的模型，着丝粒和动粒在染色体分离的功能，并可能有助于阐明癌症的病因和新的治疗途径。人体内的每个细胞都必须保持正确的染色体数量。当染色体在细胞之间没有被准确地划分时，非整倍性结果，这是具有太多或太少染色体的状态。非整倍体与癌症高度相关，并导致自然流产和发育障碍，如唐斯综合征。特别是，该提案的主题Cse 4/CENP-A是一种蛋白质，对于着丝粒和动粒的形成至关重要，这是染色体精确分离所需的染色体特征。Cse 4/CENP-A已被证明在人类原发性结直肠癌中过表达和错误定位[1]。相关蛋白CENP-H在人细胞中过表达时诱导非整倍性[2]。CID（CENP-A的蝇同源物）的过表达促进异位着丝粒和多着丝粒染色体的形成，这导致染色体错误分离、非整倍性和生长缺陷[3]。这些结果表明Cse 4/CENP-A显著有助于基因组维持。更好地了解Cse 4/CENP-A定位于着丝粒的分子要求及其参与动粒功能对于我们理解有助于准确染色体分离的基本机制至关重要。1. Tomonaga，T.，例如，原发性结直肠癌中着丝粒蛋白-A的过度表达和错误定位癌症研究，2003年。63：p.3511 -6. 2. Tomonaga，T.，例如，着丝粒蛋白H在原发性结直肠癌中表达上调，其过表达诱导异倍体癌症研究，2005年。65：p.4683 -9. 3. Heun，P.，例如，果蝇着丝粒特异性组蛋白CID的错误定位促进功能性异位动粒的形成。Dev Cell，2006年。[10]第303- 315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.