Analysis of the yeast SWI1, SWI2, and 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。我们的一般策略是集中在一个进化上保守的蛋白质复合物，SWI/SNF，这是必需的酵母基因的一个子集的表达和转录激活因子的活性。在酵母中的遗传研究表明SWI/SNF通过拮抗染色质介导的转录抑制而促进转录，并且我们的体外研究表明~1Mda SWI/SNF复合物可以利用ATP水解产生的能量来移动核小体并破坏核小体结构。SWI/SNF复合物对于哺乳动物的发育是必不可少的，SWI/SNF亚基的失活导致人类癌症。该建议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。该建议的第一个目的是检验SWI/SNF和ISWI重塑复合物在许多诱导基因中拮抗作用的假设。这一目标是解决染色质免疫沉淀分析和核小体作图方法。第二个目标是检验SWI/SNF通过破坏染色质的高阶折叠来促进转录的假设。这些研究将涉及重塑核小体的沉降速度分析以及Sin-组蛋白的生化分析。目的3描述了单分子测定和SWI/SNF-DNA光亲和交联研究，以剖析ATP水解在染色质重塑活性中的作用。第四个目标将解决的结构SWI/SNF绑定到一个monolithome使用单粒子重建cryo-EM图像。这个目的还调查了组装SWI/SNF所需的亚基-亚基相互作用。这项提案描述了研究，重点是如何染色体结构影响基因表达，以及正常的细胞机制如何与这种结构竞争。具体来说，我们建议继续研究一种新的蛋白质机器，SWI/SNF复合物，通过重塑染色体结构促进基因激活。这种机制对于哺乳动物的发育是必不可少的，人类SWI/SNF的失活会导致多种癌症。

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 transcriptional activators. Genetic studies in yeast indicate that SWI/SNF facilitates transcription by antagonizing chromatin-mediated transcriptional repression, and our in vitro studies indicate that the ~1Mda SWI/SNF complex can use the energy derived from ATP hydrolysis to mobilize nucleosomes and disrupt nucleosome structure. SWI/SNF complexes are essential for mammalian development and inactivation SWI/SNF subunits lead to cancers in humans. This proposal continues 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 test the hypothesis that the SWI/SNF and ISWI remodeling complexes function antagonistically at many inducible genes. This aim is addressed by chromatin immunoprecipitation assays and nucleosome mapping methods. The second objective will test the hypothesis that SWI/SNF facilitates transcription by disrupting the higher order folding of chromatin. These studies will involve sedimentation velocity analyses of remodeled nucleosomes as well as biochemical analyses of Sin-histones. Aim 3 describes single molecule assays and SWI/SNF-DNA photo-affinity crosslinking studies to dissect the role of ATP hydrolysis in chromatin remodeling activity. The fourth aim will address the structure of SWI/SNF bound to a mononucleosome using single particle reconstruction of cryo-EM images. This aim also investigates the subunit-subunit interactions required for assembly of SWI/SNF. This proposal describes research that is focused on how chromosome structure affects gene expression and how the normal cellular machinery contends with this structure. Specifically, we propose continued studies on a novel protein machine, the SWI/SNF complex that facilitates gene activation by remodeling chromosome structure. This machine is essential for mammalian development and inactivation of human SWI/SNF leads to a variety of cancers.