Genetic Analysis of Cell Cycle Dependent Transcription

描述（由申请人提供）：长期目标是了解 染色质动力学在转录和细胞周期中的作用。染色质 在细胞周期中经历折叠的全局变化和 在转录、DNA修复和重组过程中折叠。这些变化是 与正常细胞生理学密切相关，因为多项观察结果表明， 将染色质重塑因子的改变与人类疾病联系起来， 癌拟议中的研究将检验以下假设： 细胞周期调节转录辅抑制子Hir 1 p和Hir 2 p以及 泛素化的组蛋白H2 B影响转录，因为它们 在染色质折叠方面的直接作用。在第一个具体目标中，酵母 通过结合DNA，将鉴定由Hir 1 p和Hir 2 p直接调控的基因 用染色质免疫沉淀进行微阵列杂交分析。的 第二个具体目标将区分染色质依赖性和 非染色质依赖的Hir抑制机制 在RNA聚合酶II缺陷的突变体中， 全酶组分或组蛋白脱乙酰酶。Hir蛋白的能力， 在体外组装核小体，并将DNA组织成拓扑上不同的 将测试体内结构域。第三个具体目标将审查 调节Hir蛋白在细胞周期中的功能。染色质 免疫沉淀将用于鉴定细胞周期信号， 控制Hir-dependent募集Swi/Snf核小体重塑 因子的HTA 1基因，和非变性染色质制备将被使用 以了解Hir蛋白和Swi/Snf是否在染色质上和染色质外循环。的假设 Swi/Snf在G1晚期将有丝分裂Clb细胞周期蛋白募集到HTA 1启动子， 通过染色质免疫沉淀进行检测。在第四个具体目标中， 泛素化H2 B（uH 2B）的转录作用将通过检测 假设uH 2B招募核小体重塑因子， 基因. uH 2B调控的基因及其基因组定位 将通过与染色质偶联的DNA微阵列杂交来鉴定 免疫沉淀。

DESCRIPTION (Provided by applicant): The long-term goal is to understand the role of chromatin dynamics in transcription and the cell cycle. Chromatin undergoes global changes in folding during the cell cycle and local changes in folding during transcription, DNA repair, and recombination. These changes are intimately linked to normal cell physiology because multiple observations have linked alterations in chromatin remodeling factors to human diseases and cancer. The proposed studies will test the hypothesis that two factors - the cell cycle-regulated transcriptional corepressors Hir1p and Hir2p and a ubiquitinated species of histone H2B - affect transcription because of their direct roles in aspects of chromatin folding. In the first specific aim, yeast genes directly regulated by Hir1p and Hir2p will be identified by combining DNA microarray hybridization analysis with chromatin immunoprecipitation. The second specific aim will distinguish between chromatin-dependent and chromatin-independent mechanisms of Hir repression by analyzing transcription of the histone HTA I gene in mutants that are defective in RNA polymerase II holoenzyme components or histone deacetylases. The ability of Hir proteins to assemble nucleosomes in vitro and to organize DNA into topologically distinct domains in vivo will be tested. The third specific aim will examine the regulation of Hir protein function in the cell cycle. Chromatin immunoprecipitation will be used to identify the cell cycle signals that control the Hir-dependent recruitment of the Swi/Snf nucleosome remodeling factor to the HTA 1 gene, and nondenaturing chromatin preparations will be used to ask if Hir proteins and Swi/Snf cycle on and off chromatin. The hypothesis that Swi/Snf recruits mitotic Clb cyclins to the HTA1 promoter in late G 1 will be tested by chromatin immunoprecipitation. In the fourth specific aim, the transcriptional role of ubiquitinated H2B (uH2B) will be examined by testing the hypothesis that uH2B recruits nucleosome remodeling factors to specific genes. Genes that are regulated by uH2B and the genomic localization of uH2B will be identified by DNA microarray hybridization coupled with chromatin immunoprecipitation.