ANALYSIS OF THE CHROMATIN-REMODELING COMPLEX RSC

我们的目标是了解ATP依赖的染色质重塑复合物（CRC）如何有助于转录和其他重要的细胞过程的调节。 染色质抑制转录，所有真核生物都利用一组相关的CRC来消除这种抑制。 酵母S.酿酒酵母含有两种相关的CRCs，SWI/SNF和RSC，它们在组成和体外活性上与它们的后生动物对应物高度相似。 酵母SWI/SNF是一种罕见的、非必需的复合物，其在许多pol II启动子处重塑染色质。 相比之下，RSC是丰富的和必需的生存能力，但其功能是未知的。 人SWI/SNF是丰富的和必需的生存能力，突变可导致侵袭性横纹肌样肿瘤。 我们的具体目标是确定RSC如何靶向特定的基因组基因座并鉴定RSC相互作用蛋白，目的是更多地了解这些保守的染色质重塑复合物如何有助于控制基因表达：1）鉴定RSC作用的基因组靶标。 RSC的Rsc 3组分与已知的序列特异性DNA结合转录激活因子高度相似。我们将通过对rsc 3 Ts-突变体的DNA微阵列分析来鉴定Rsc 3（和RSC）靶标。 Rsc 3结合位点将通过位点选择研究来确定，并且染色质免疫沉淀实验将用于测试候选启动子在体内的Rsc 3占有率。 2)确定Rsc 1、Rsc 2和溴结构域（BD）的功能。 Rsc 1和Rsc 2成员包含BD和BAH结构域，这两个结构域在许多重要的转录调节因子中发现，但功能未知。 我们将使用随机诱变对Rsc 2 BD进行结构/功能分析，分离条件突变，并利用条件突变体的遗传抑制来分离BD相互作用蛋白。 3)检查BAH区域的结构/功能。 我们将使用随机诱变对Rsc 2 BAH区域进行结构/功能分析，并利用条件突变体的抑制来分离BAH相互作用蛋白。 4)确定RSC中肌动蛋白相关蛋白（阿普）的功能。我们将纯化缺少阿普蛋白的RSC并测试其功能，用我们的阿普Ts-突变体进行DNA微阵列分析，并获得阿普突变体的遗传抑制子以鉴定需要阿普功能的细胞过程。

Our objective is to understand how ATP-dependent chromatin remodeling complexes (CRCs) contribute to the regulation of transcription and other vital cellular processes. Chromatin represses transcription, and all eukaryotes utilize a related set of CRCs to remove this repression. The yeast S. cerevisiae contains two related CRCs, SWI/SNF and RSC, which are highly similar in composition and in vitro activities to their metazoan counterparts. Yeast SWI/SNF is a rare, non-essential complex that remodels chromatin at many pol II promoters. In contrast, RSC is both abundant and essential for viability, but its functions are unknown. Human SWI/SNF is both abundant and essential for viability, and mutations can cause aggressive rhabdoid tumors. Our Specific Aims are to determine how RSC is targeted to particular genomic loci and to identify RSC- interacting proteins, with the goal of understanding more about how these conserved chromatin remodeling complexes contribute to the control of gene expression: 1) Identify the genomic targets for RSC action. The Rsc3 component of RSC is highly similar to known sequence-specific DNA-binding transcriptional activators. We will identify Rsc3 (and RSC) targets through DNA microarray analysis of rsc3 Ts- mutants. The Rsc3 binding site will be determined by site-selection studies, and chromatin immunoprecipitation experiments will be utilized to test candidate promoters for Rsc3 occupancy in vivo. 2) Determine the function of Rsc1, Rsc2, and the bromodomain (BD). Rsc1 and Rsc2 members that contain BDs and a BAH domain, two motifs of unknown function found in many important transcriptional regulators. We will perform a structure/function analysis of the Rsc2 BD using random mutagenesis, isolate conditional mutations, and utilize genetic suppression of conditional mutants to isolate BD- interactions proteins. 3) Examine the structure/function of a BAH region. We will perform a structure/function analysis of the Rsc2 BAH region using random mutagenesis, and utilize suppression of conditional mutants to isolate BAH-interacting proteins. 4) Ascertain the function of actin-related proteins (Arp) in RSC. We will purify RSC lacking Arp proteins and test for function, perform DNA microarray analysis with our arp Ts- mutants, and acquire genetic suppressors of arp mutants to identify cellular processes that require Arp function.