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有助于基因调控。染色质主要通过限制或促进转录因子对基因的访问，在转录调控中起着重要作用。染色质结构的改变被用来适当地调节许多对细胞分化和增殖至关重要的基因，而这一过程的错误调节是许多癌症的基础。染色质结构的形成和相互转换涉及大分子复合体的协同作用，这些复合体修饰和重新定位/重塑染色质结构的基本单位，称为核小体。我们的研究主要集中在基本酵母重塑复合体RSC上，它与哺乳动物的SWL/SNF复合体高度相似。因此，对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/重塑是如何协调的：1)确定DNA易位如何用于染色质重塑。DNA易位已经成为重构体的统一属性。我们将使用稳态前动力学和特殊的核小体底物来了解易位是如何应用于重塑核小体的。2)确定RSC溴结构域和BAH-包含模块在RSC体内靶向和体外RSC重塑中的作用。RSC复合体包含必需的溴结构域和两个必需的BAH结构域，这可能有助于RSC识别修饰的核小体和/或与修饰剂配位。我们将确定识别的修饰，并确定它们对RSC结合和重塑的影响。3)确定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构体和修饰物中。我们将研究它们作为异构体模块在调节RSC和NuA4(修饰物)功能中的作用。

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.