ANALYSIS OF THE CHROMATIN-REMODELING COMPLEX RSC

描述（由申请人提供）：SWI/SNF家族复合物是大的、丰富的蛋白质机器，其动员核小体以调节DNA结合因子进入染色质。这些复合物具有一组在整个真核生物中保守的七个“核心”蛋白质，以及为特定功能定制复合物的其他蛋白质。SWI/SNF家族染色质重塑复合物（称为“重塑物”）有助于调节转录、DNA修复和基因组稳定性等过程。除了在正常染色体生物学中的这些关键作用之外，SWI/SNF重塑的失调与许多癌症相关，并且是横纹肌样肿瘤的原因。我们专注于SWI/SNF-家庭重塑RSC，这是一个必不可少的和丰富的重塑从芽殖酵母，已作为原型理解重塑功能。RSC是一种15蛋白复合物，既能载玻片又能激发核小体。我们的工作解决了染色质重塑中的核心问题：重塑者如何重新定位和排出核小体，这些重塑模式如何调节，以及核小体上的共价修饰如何影响RSC靶向和机制。RSC可以被认为是一个蛋白质模块的镶嵌，这些模块一起工作以进行染色质重塑： 1)一个进行DNA易位的催化模块，涉及ATP酶（Sth 1）和一对肌动蛋白相关蛋白（ARP），2）一组具有核小体结合基序（溴结构域）的蛋白质，可能有助于将RSC靶向特定核小体，3）一组真菌特异性蛋白质，调节RSC参与特定染色体过程。目的1应用生物化学，单分子和结构的方法来了解如何DNA易位模块动员和激活核小体，并检查RSC核小体复合物的结构工程。目的2着重于通过ARPs和Sth 1上存在的三个结构域（HSA、post-HSA和Prosth 1 #1）调节Sth 1 ATP酶，这三个结构域有助于将ARPs与ATP酶调节联系起来。目的3着重于理解RSC中的八个溴结构域，特别是必需蛋白Rsc 4中的串联溴结构域，如何参与RSC靶向或活性。RSC的见解应该告知这些类似的蛋白质在哺乳动物SWI/SNF复合物的功能，并大大有助于我们理解染色质重塑过程。

DESCRIPTION (provided by applicant): SWI/SNF-family complexes are large, abundant protein machines that mobilize nucleosomes to regulate the access of DNA-binding factors to chromatin. These complexes have a set of seven 'core' proteins that are conserved throughout eukaryotes, as well as additional proteins that tailor the complex for specialized functions. SWI/SNF-family chromatin remodeling complexes (termed 'remodelers') help regulate transcription, DNA repair and genome stability, among other processes. Beyond these critical roles in normal chromosomal biology, the misregulation of SWI/SNF remodelers is associated with many cancers, and is the cause of rhabdoid tumors. We focus on the SWI/SNF-family remodeler RSC, which is an essential and abundant remodeler from the budding yeast that has served as a prototype for understanding remodeler functions. RSC is a 15-protein complex that both slides and ejects nucleosomes. Our work addresses central issues in chromatin remodeling: how do remodelers reposition and eject nucleosomes, how are these remodeling modes regulated, and how do covalent modifications on nucleosomes affect RSC targeting and mechanism? RSC can be considered a mosaic of protein modules that work together to conduct chromatin remodeling: 1) a catalytic module that conducts DNA translocation, which involves an ATPase (Sth1) and a pair of actin-related proteins (ARPs), 2) a set of proteins with nucleosome-binding motifs (bromodomains) that may help target RSC to particular nucleosomes, and 3) a set of fungalspecific proteins that regulate RSC participation in particular chromosomal processes. Aim 1 applies biochemical, single-molecule, and structural approaches to understand how the DNA translocation module mobilizes and ejects nucleosomes, and also examines the structural engineering of the RSC-nucleosome complex. Aim 2 focuses on the regulation of the Sth1 ATPase by the ARPs and by three domains present on Sth1 (HSA, post-HSA, and Protrusion #1) which help link the ARPs to ATPase regulation. Aim 3 focuses on understanding how the eight bromodomains in RSC, especially the tandem bromodomains in the essential protein Rsc4, are involved in RSC targeting or activity. Insights from RSC should inform the functions of these similar proteins in mammalian SWI/SNF complexes, and contribute considerably to our understanding of the chromatin remodeling process.