Analysis of SWI1, SWI2, amd SWI3 proteins

描述（由申请人提供）：我们研究的总体目标是确定染色体结构如何影响基因表达以及转录机制如何与这种结构竞争。我们的总体策略是关注进化上保守的蛋白质复合物SWI/SNF，它是酵母基因子集表达和几种转录激活因子活性所必需的。酵母的遗传学研究表明，SWI/SNF通过拮抗染色质介导的转录抑制发挥作用，我们最近的研究表明，SWI/SNF和Gcn5p组蛋白乙酰转移酶在有丝分裂后期发挥全球作用。体外研究表明，-2Mda SWI/SNF复合物可以利用ATP水解的能量破坏核小体结构，并且SWI/SNF可以通过与多种转录激活剂的直接相互作用靶向特定的核小体。在下一个预算期内，我们将继续利用酵母中强大的遗传和生化机会来研究SWI/SNF在体内的作用以及SWI/SNF在体外破坏核小体结构的生化机制。本研究的第一个目的是研究SWI/SNF和Gcn5p HAT在有丝分裂后期的作用。这一目的是通过Affymetrix基因芯片表达分析，从同步细胞分离的RNA，染色质免疫沉淀和体内染色质结构分析来解决。第二个目的是研究组蛋白H3丝氨酸10磷酸化在体内和体外调节重塑活动中的作用。目的3将验证Ashi p抑制因子通过阻断SWI/SNF的募集或重塑活性来发挥作用的假设。第四个目标将使用一系列SWI2/SNF2 ATP酶基序突变体和光亲和交联王方法来定义ATP结合和水解在SWI/SNF重塑中的作用。本目的还将使用光亲和交联和荧光方法来验证SWI/SNF作用引起核小体DNA运动的假设。第五个目标将通过电子显微镜方法研究SWI/SNF的结构。该目的还描述了一种条件降解策略，以确定单个SWI/SNF亚基在体内和体外功能中的作用

DESCRIPTION (provided by applicant): The overall objective of our research is to determine how chromosome structure affects gene expression and how the transcription machinery contends with this structure. Our general strategy is to focus on an evolutionarily conserved protein complex, SWI/SNF, which is required for expression of a subset of yeast genes and for the activity of several transcriptional activators. Genetic studies in yeast suggest that SWI/SNF functions by antagonizing chromatin-mediated transcriptional repression, and our recent studies suggest a global role for SWI/SNF and the Gcn5p histone acetyltransferase during late mitosis. In vitro studies indicate that the -2Mda SWI/SNF complex can use the energy of ATP hydrolysis to disrupt nucleosome structure and that SWI/SNF can be targeted to specific nucleosomes through direct interactions with a variety of transcriptional activators. Over the next budget period we will continue to exploit the powerful genetic and biochemical opportunities available in yeast to investigate the role of SWI/SNF in vivo and the biochemical mechanism by which SWI/SNF disrupts nucleosome structure in vitro.The first aim of this proposal will investigate the roles of SWI/SNF and the Gcn5p HAT during late mitosis. This aim is addressed by Affymetrix gene chip expression analyses with RNA isolated from synchronized cells, chromatin immunoprecipitations, and in vivo chromatin structural analyses. The second objective will investigate the role of histone H3 serine 10 phosphorylation in the regulation of remodeling activities in vivo and in vitro. Aim 3 will test the hypothesis that the Ashi p repressor functions by blocking the recruitment or remodeling activity of SWI/SNF. The fourth objective will use a battery of SWI2/SNF2 ATPase motif mutants and a photoaffinity crosslin king method to define the role of ATP binding and hydrolysis in SWI/SNF remodeling. This aim will also use photoaffinity crosslinking and fluorescence methods to test the hypothesis that SWI/SNF action causes movements of nucleosomal DNA. The fifth objective will probe the structure of SWI/SNF by electron microscopy methods. This aim also describes a conditional degron strategy to define the role(s) of individual SWI/SNF subunits for both in vivo and in vitro functions