ANALYSIS OF THE CHROMATIN-REMODELING COMPLEX RSC

描述（由申请人提供）：我们的目标是了解重塑和修改染色体结构的因素如何参与基因调控。染色质在调节转录中起着重要作用，主要是通过限制或促进转录因子进入基因。染色质结构的改变被用来正确调节许多对细胞分化和增殖重要的基因，而这一过程的错误调节是许多癌症的基础。染色质结构的形成及其相互转化涉及到大型复合物的协同作用，这些复合物修饰和重新定位/重塑染色质结构的基本单位，称为核小体。我们的研究重点是酵母重塑复合体RSC，它与哺乳动物的SWl/SNF复合体高度相似。因此，RSC的研究将与人类SWI/SNF功能直接相关。我们的具体目标集中在了解核小体如何重新定位，核小体修饰如何影响这一过程，以及修饰和重新定位/重塑如何协调：1)确定DNA易位如何用于染色质重塑。DNA易位已成为重塑者的统一特性。我们将使用预稳态动力学和特殊的核小体底物来了解易位如何应用于核小体的重塑。2)确定含溴域和含bah模块在体内RSC靶向和体外RSC重塑中的作用。RSC复合物含有必要的溴结构域和两个必要的BAH结构域，这可能有助于RSC识别修饰的核小体和/或与修饰剂协调。我们将识别识别的修饰，并确定它们对RSC结合和重塑的影响。3)明确肌动蛋白和肌动蛋白相关蛋白在染色质调节复合体中的作用。肌动蛋白相关蛋白和肌动蛋白存在于许多重塑剂和修饰剂中。我们将研究它们作为异聚模的功能，调控RSC和NuA4（修饰剂）功能。

DESCRIPTION (provided by applicant): Our objectives are to understand how factors that remodel and modify chromosome structure contribute to gene regulation. Chromatin plays a major role in regulating trascription, primarily by restricting or promoting the access of transcription factors to genes. Chromatin structural changes are utilized to properly regulate many genes important for cell differentiation and proliferation, and misregulation of this process underlies many cancers. The formation of chromatin structures and their interconversion involves the concerted action of large complexes that modify and reposition/remodel the basic unit of chromatin structure, termed the nucleosome. Our studies focus on the essential yeast remodeling complex RSC, which is highly similar to mammalian SWl/SNF complexes. Thus, studies on RSC will have direct relevance to human SWI/SNF function. Our specific aims focus on understanding how nucleosomes are repositioned, how nucleosome modifications affect this process, and how modification and repositioning/remodeling are coordinated:1) Determine how DNA translocation is utilized for chromatin remodeling. DNA translocation has emerged as the unifying property of remodelers. We will use pre-steady state kinetics and special nucleosome substrates to understand how translocation is applied to remodel nucleosomes.2) Determine the role of RSC bromodomain- and BAH-containing modules in RSC targeting in vivo and in RSC remodeling in vitro. RSC complex contains essential bromodomains, and two essential BAH domains, which likely assist in RSC recognition of modified nucleosomes and/or coordination with modifiers. We will identify the modifications recognized, and determine their affect on RSC binding and remodeling.3) Define the role of actin and actin-related proteins in chromatin regulating complexes. Actin-related proteins and actin are present in many remodelers and modifiers. We will investigate their functions as hetermeric modules, in regulating RSC and NuA4 (modifier) function.