Cell Cycle Progression and Chromatin Remodeling in Yeast

描述（由申请人提供）：该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修复过程中进行局部重塑，在细胞周期中进行全局重塑。本项目将重点研究酵母atp依赖性染色质重塑酶（RSC）对染色体分离过程中发生的染色体结构事件的调节作用，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。后生动物对应体PBAF和hSWI/SNF在相关细胞通路和扰动中的功能与人类疾病和癌症密切相关，强调了重塑因子在正常细胞生理学中的关键重要性。在第一个特定目标中，RSC在姐妹染色单体的内聚和凝聚中对染色体分离的作用将通过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。最近发现RSC以细胞周期依赖的方式在染色质上和染色质外循环，这将被用来鉴定与染色质结合的RSC复合物物理相关的多肽，直接将RSC与细胞功能联系起来。在第二个特定目标中，将在全基因组定位分析中确定RSC的细胞周期依赖靶点，并检查这些位点的染色质环境，以验证RSC结合与特定结构相关的假设。此外，rsc突变体对几种基因毒性药物的超敏感性表明rsc在维持基因组完整性方面具有直接或间接的作用，将通过分析rsc突变体中DNA修复途径功能的完成情况以及与不同修复机制缺陷的突变体的相互作用来进行研究。RSC被专门招募到DNA断裂位点的假设也将被验证。在第三个目标中提出的实验将检查在基因组传播中调节RSC的细胞内信号通路。质谱测定的Sfhlp磷酸化位点将发生突变，并对染色体分离的细胞后果进行测试。磷酸化Sfh1 p的g1特异性激酶将在激酶芯片试验中寻找。偶联遗传筛选将确定在基因组传播中机制连接PKC1途径和RSC的蛋白质。

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.