The CENP-A complex and centromere identity

描述（由申请人提供）：此申请是为一名医学博士研究生提供的个人奖学金，其研究训练计划旨在帮助她成为一名独立的内科科学家。有丝分裂过程中染色体的准确分离对所有真核生物的生存和发育至关重要。染色体分离缺陷导致细胞死亡或非整倍体，从而导致胚胎发育中的先天性缺陷或自然流产，并导致体细胞癌症进展。准确的染色体分离需要有丝分裂纺锤体微管附着在着丝点上，着丝点组装在染色体着丝粒上。着丝粒位置由称为着丝粒蛋白A （CENP-A）的组蛋白H3变体表观遗传指定，该变体直接与另一种必需的着丝粒蛋白CENP-C结合。利用氢-氘交换耦合质谱（H/DX-MS）方法进行的初步研究表明，CENP-C结合改变了CENP-A核小体的结构特性，表明CENP-C有助于定义功能性着丝粒染色质的结构。在Aim 1中，H/DX-MS研究将扩展到测试这种结构改变是否仅针对CENP-A核小体，并且将在细胞中测试CENP-C突变体，以研究CENP-C诱导的改变的消除是否与着丝粒缺陷有关。本建议的第二个重点是确保着丝粒在细胞周期中准确传播的机制。细胞周期蛋白依赖性激酶（Cdk） 1/2调节新表达的CENP-A分子装载到新复制的着丝粒DNA的时间，可能是通过磷酸化CENP-A装载途径中的关键参与者。在Aim 2中，将使用细胞培养中氨基酸稳定同位素标记（SILAC）的方法来鉴定亲和纯化的含CENP-A复合物上的cdk1 /2依赖性磷酸化位点，并在细胞中使用荧光脉冲追踪标记方法来鉴定控制CENP-A加载时间的磷酸化位点。这些实验将揭示着丝粒位置是如何精确指定和随时间传播的，这是维持基因组稳定性的关键过程。

DESCRIPTION (provided by applicant): This application is for an individual fellowship for an MD-PhD student, with a research training plan designed to aid her long-term goal of becoming an independent physician-scientist. The accurate segregation of chromosomes during mitosis is essential for the survival and development of all eukaryotes. Defects in chromosome segregation causes cell death or aneuploidy, which causes congenital defects or spontaneous abortions in embryogenesis, and contributes to cancer progression in somatic cells. Accurate chromosome segregation requires attachment of mitotic spindle microtubules to the kinetochore, which is assembled on the chromosomal centromere. Centromere location is specified epigenetically by the histone H3 variant termed centromere protein A (CENP-A), which directly binds to another essential centromere protein CENP-C. Preliminary studies using the method of hydrogen-deuterium exchange coupled to mass spectrometry (H/DX-MS) suggest that CENP-C binding alters structural properties of CENP-A nucleosomes, suggesting that CENP-C helps define the architecture of functional centromeric chromatin. In Aim 1, H/DX-MS studies will be extended to test whether such structural alterations are specific to CENP-A nucleosomes, and CENP-C mutants will be tested in cells to investigate whether abolishment of CENP-C-induced alterations is linked to centromere defects. The second focus of this proposal is the mechanisms ensuring accurate propagation of centromere location across cell cycles. Cyclin- dependent kinase (Cdk) 1/2 regulates the timing for the loading of newly expressed CENP-A molecules into newly replicated centromeric DNA, possibly by phosphorylating key player(s) in the CENP-A-loading pathway. In Aim 2, the method of stable isotope labeling by amino acids in cell culture (SILAC) will be used to identify Cdk1/2-dependent phosphorylation sites on affinity- purified CENP-A-containing complexes, and a fluorescent pulse-chase labeling approach will be used in cells to identify the phosphorylation sites that control the timing of CENP-A loading. These experiments will shed key insight into how centromere location is accurately specified and propagated over time, which are crucial processes for maintaining genomic stability.