Cell Cycle Progression and Chromatin Remodeling in Yeast

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，在细胞周期中全局重塑。这个项目将集中在酵母ATP依赖的染色质重构体RSC，在调节染色体分离过程中发生的染色体结构事件，包括姐妹染色单体的凝聚和凝聚和维持基因组的完整性。后生动物对应的PBAF和hSWI/SNF在相关的细胞通路和扰动中发挥作用，与人类疾病和癌症密切相关，这突显了重塑因子在正常细胞生理中的关键作用。在第一个具体目标中，将结合体内染色质免疫沉淀(CHIP)分析、染色体运动的活细胞分析、染色体传播和遗传分析来评估RSC在姐妹染色单体凝聚和凝聚中的作用。最近发现，RSC以细胞周期依赖的方式开启和关闭染色质，这将被用来识别与染色质结合的RSC复合体物理上相关的多肽，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，并检查这些位置的染色质环境，以验证RSC结合与特定结构相关的假设。此外，RSC突变体对几种遗传毒性物质的超敏反应，表明RSC在维持基因组完整性方面的直接或间接作用，将通过检测RSC突变体中DNA修复途径功能的完成，以及与修复机制不同的突变体的相互作用来寻求。RSC被特异性地招募到DNA断裂部位的假设也将得到检验。第三个目标中提出的实验将检查在基因组传输中调节RSC的细胞内信号通路。质谱学确定的Sfhlp磷酸化位点将发生突变，并测试染色体分离的细胞后果。使Sfh1p磷酸化的G1特异性激酶将在激酶芯片分析中寻找。一个耦合的遗传筛选将识别在基因组传输中机械地连接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.