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Cell Cycle Progression and Chromatin Remodeling in Yeast

酵母细胞周期进展和染色质重塑

基本信息

批准号：
7209776
负责人：
BREHON C LAURENT
金额：
$ 28.13万
依托单位：
ICAHN SCHOOL OF MEDICINE AT MOUNT SINAI
依托单位国家：
美国
项目类别：
财政年份：
1998
资助国家：
美国
起止时间：
1998-08-01 至 2009-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7209776
关键词：
Affinity Binding Biological Assay Bromodomain Cell Cycle Cell Cycle Progression Cell Cycle Stage Cell physiology Cells Centromere Chromatin Chromatin Remodeling Factor Chromosome Cohesion Chromosome Segregation Chromosomes Complex Coupled DNA DNA Damage DNA Repair DNA Repair Pathway Drug resistance Environment Event Genes Genetic Genetic Screening Genetic Transcription Genome Genomics Goals Grant Hypersensitivity In Vitro Kinetochores Lead Life Link Maintenance Malignant Neoplasms Mass Spectrum Analysis Microtubules Molecular Mutagens Mutate Pathway interactions Phosphorylation Phosphorylation Site Phosphotransferases Physical condensation Process Protein Kinase Protein Overexpression Proteins Recruitment Activity Resistance Role Sfh1 Signal Pathway Sister Chromatid Site Structure Testing Yeasts chromatin immunoprecipitation chromatin remodeling chromosome movement cohesin cohesion condensin hSWI/SNF human disease hydroxyurea in vivo interest mutant polypeptide repaired research study segregation transmission process

项目摘要

DESCRIPTION (provided by applicant): The long-term objective of this project is to understand the impact of chromatin dynamics on cell cycle progression. Chromatin is remodeled locally during transcription, replication, recombination, and DNA repair and globally during the cell cycle. This project will focus on the yeast ATP-dependent chromatin remodeler, RSC, in regulating the chromosomal structural events that occur during chromosome segregation, including the cohesion and condensation of sister chromatids and the maintenance of genome integrity. The metazoan counterparts, PBAF and hSWI/SNF, function in related cellular pathways and perturbations are tightly linked to human disease and cancers, underscoring the critical importance of remodeling factors in normal cellular physiology. In the first specific aim, roles for RSC in the cohesion and condensation of sister chromatids for chromosome segregation will be assessed using a combination of in vivo chromatin immunoprecipitation (CHIP) assays, live-cell analysis of chromosome movement, chromosome spread, and genetic assays. The recent discovery that RSC cycles on and off chromatin in a cell cycle-dependent manner will be exploited to identify polypeptides that physically associate with the chromatin-bound RSC complex, directly linking RSC to cellular functions. In the second specific aim, cell cycle-dependent targets of RSC will be identified in a genome-wide localization analysis and the chromatin environment of these sites examined to test the hypothesis that RSC binding is correlated to specific structure. In addition, the rsc mutant hypersensitivities to several genotoxic agents, suggesting a direct or indirect role for RSC in maintaining genome integrity, will be pursued by assaying for completion of DNA repair pathway functions in rsc mutants, and for interactions with mutants defective in distinct repair mechanisms. The hypothesis that RSC is specifically recruited to sites of DNA breaks will also be tested. The experiments proposed in the third aim will examine the intracellular signaling pathways that regulate RSC in genomic transmission. Sfhlp phosphorylation sites determined by mass spectrometry will be mutated and the cellular consequences for chromosome segregation tested. The G1-specific kinase that phosphorylates Sfh1 p will be sought in a kinase chip assay. A coupled genetic screen will identify proteins that mechanistically link the PKC1 pathway and RSC in genomic transmission.

描述（由申请人提供）：本项目的长期目标是了解染色质动力学对细胞周期进程的影响。染色质在转录、复制、重组和DNA修复过程中局部重塑，在细胞周期过程中全局重塑。本项目将重点研究酵母ATP依赖性染色质重塑因子RSC在染色体分离过程中对染色体结构事件的调控，包括姐妹染色单体的凝聚和凝聚以及基因组完整性的维持。后生动物对应物PBAF和hSWI/SNF在相关细胞途径和扰动中的功能与人类疾病和癌症紧密相关，强调了重塑因子在正常细胞生理学中的至关重要性。在第一个具体目标中，将使用体内染色质免疫沉淀（CHIP）测定、染色体运动的活细胞分析、染色体扩散和遗传测定的组合来评估RSC在姐妹染色单体的凝聚和凝聚中的作用。最近的发现，RSC周期和关闭染色质在细胞周期依赖性的方式将被利用，以确定物理上与染色质结合的RSC复合物，直接连接RSC的细胞功能的多肽。在第二个具体目标中，将在全基因组定位分析中鉴定RSC的细胞周期依赖性靶标，并检查这些位点的染色质环境，以检验RSC结合与特定结构相关的假设。此外，rsc突变体对几种遗传毒性药物的超敏反应，表明RSC在维持基因组完整性方面的直接或间接作用，将通过测定rsc突变体中DNA修复途径功能的完成以及与不同修复机制缺陷突变体的相互作用来进行研究。也将检验RSC特异性募集至DNA断裂位点的假设。在第三个目标中提出的实验将检查在基因组传递中调节RSC的细胞内信号传导途径。通过质谱法确定的Sfhlp磷酸化位点将被突变，并测试染色体分离的细胞后果。将在激酶芯片测定中寻找磷酸化Sfh 1 p的G1特异性激酶。一个耦合的遗传筛选将确定蛋白质，机械连接的PKC 1途径和RSC的基因组传输。