The CENP-A complex and centromere identity

CENP-A 复合体和着丝粒身份

• 批准号: 8716947
• 负责人: Lucie Yueqi Guo
• 金额: $ 4.61万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-09-01 至 2017-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8716947
• 关键词: Affinity; Amino Acids; Aneuploidy; Architecture; Binding; CDC2 Protein Kinase; Cell Culture Techniques; Cell Cycle; Cell Cycle Regulation; Cell Death; Cell division; Cells; Centromere; Chromatin; Chromosome Segregation; Chromosome abnormality; Chromosomes; Complex; Congenital Abnormality; Coupled; DNA; DNA biosynthesis; Defect; Deuterium; Development; Doctor of Philosophy; Down Syndrome; Embryo; Embryonic Development; Ensure; Epigenetic Process; Eukaryota; Fellowship; Generations; Genetic; Genome Stability; Goals; Histone H3; Histones; Hydrogen; Individual; Inherited; Kinetochores; Label; Light; Link; Location; Maintenance; Mass Spectrum Analysis; Methods; Microtubules; Mitosis; Mitotic spindle; Mutation; Nucleosomes; Pathway interactions; Phosphorylation Site; Physicians; Physiologic pulse; Play; Process; Property; Proteins; Recruitment Activity; Regulation; Research Training; Role; Scientist; Sister; Sister Chromatid; Solutions; Somatic Cell; Specific qualifier value; Spontaneous abortion; Stable Isotope Labeling; Structure; Students; Testing; Time; Variant; centromere protein A; centromere protein C; congenital heart disorder; design; experience; insight; mutant; physical property; premature; prevent; public health relevance; research study; tumor progression

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.

描述（由申请人提供）：本申请是一个MD-PhD学生的个人奖学金，旨在帮助她成为一个独立的医生，科学家的长期目标的研究培训计划。 染色体在有丝分裂过程中的准确分离对所有真核生物的生存和发育至关重要。 染色体分离的缺陷导致细胞死亡或非整倍性，这导致胚胎发生中的先天性缺陷或自发性流产，并有助于体细胞中的癌症进展。 准确的染色体分离需要有丝分裂纺锤体微管附着在着丝粒上的动粒上。 着丝粒位置由称为着丝粒蛋白A（CENP-A）的组蛋白H3变体表观遗传地指定，其直接结合到另一种必需的着丝粒蛋白CENP-C。 使用氢-氘交换偶联质谱（H/DX-MS）方法的初步研究表明，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。 在目标1中，H/DX-MS研究将扩展到测试这种结构改变是否特异于CENP-A核小体，并将在细胞中测试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。 该建议的第二个焦点是确保着丝粒位置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分子加载到新复制的着丝粒DNA中的时机，可能通过磷酸化CENP-A加载途径中的关键参与者。 在目的2中，将使用细胞培养物中氨基酸的稳定同位素标记方法（SILAC）来鉴定亲和纯化的含CENP-A复合物上的Cdk 1/2依赖性磷酸化位点，并将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如何随着时间的推移而准确指定和传播的关键见解，这是维持基因组稳定性的关键过程。