Analysis of the yeast SWI1, SWI2, and SWI3 proteins

描述(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相抗衡。我们的总体策略是专注于进化上保守的蛋白质复合体SWI/SNF，这是酵母基因子集表达和转录激活剂活性所必需的。在酵母中的遗传学研究表明，SWI/SNF通过拮抗染色质介导的转录抑制而促进转录，我们的体外研究表明，~1Mda SWI/SNF复合体可以利用ATP水解产生的能量来动员核小体，破坏核小体结构。SWI/SNF复合体对哺乳动物的发育是必不可少的，而SWI/SNF亚单位失活会导致人类癌症。这一建议继续利用酵母中可用的强大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。这项提议的第一个目的将检验这样的假设，即SWI/SNF和ISWI重塑复合体在许多诱导基因上起拮抗作用。这一目标是通过染色质免疫沉淀分析和核小体定位方法来实现的。第二个目标将检验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的核小体的沉积速度分析以及Sin-组蛋白的生化分析。目的3通过单分子分析和SWI/SNF-DNA光亲和交联法研究ATP水解酶在染色质重塑中的作用。第四个目标将利用冷冻-EM图像的单粒子重建来解决与单核小体结合的SWI/SNF的结构。这一目的也研究了组装SWI/SNF所需的亚基-亚基相互作用。这项建议描述了专注于染色体结构如何影响基因表达以及正常细胞机制如何与这种结构竞争的研究。具体地说，我们建议继续研究一种新的蛋白质机器，SWI/SNF复合体，它通过重塑染色体结构来促进基因激活。这台机器对哺乳动物的发育是必不可少的，而人类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.