Transcription Factor Interactions with Nucleosomes

描述（由申请人提供）：染色质结构的调节在细胞核的所有研究过程中起着重要作用。染色质的主要组成部分是核小体。这种颗粒包含四个核心组蛋白的两个副本;这些蛋白质形成一个核心，组织146个碱基对的DNA。核小体，曾经被认为是一个静态的积木，现在被认为是动态的。一大类复合物，ATP依赖性染色质重塑复合物，利用ATP水解的能量来重塑核小体的结构和位置。本申请的一个主要重点是探索重塑复合物在功能上的差异程度。一个机械的理解，这一系列的复合物的活动的全谱是必要的，以了解染色质结构可以被调制的方式，以及染色质结构的调制可能有助于调节核过程。这将通过比较SWI/SNF家族重塑复合物与其他重塑复合物来完成，目的有三个：目的1：ATP酶结构域在重塑中的作用。我们将创建具有“交换”ATP酶结构域的嵌合蛋白，以定义ATP酶结构域在功能中的作用。目的2：表征组蛋白变体对重塑反应的影响。我们将测试几种不同的重塑蛋白在核小体上发挥作用的能力，这些核小体已与不同的组蛋白变体组装在一起。目的3：ATP依赖性重塑和高级染色质结构。我们将使用可量化的方法来检查连接体长度和压缩状态的变化对重塑蛋白质谱的重塑的影响。我们将开发一种组织培养系统，以表征不同重塑活动引起的染色质结构变化的幅度和范围。

DESCRIPTION (provided by applicant): Regulation of chromatin structure plays a major role in all studied processes in the nucleus. The primary building block of chromatin is the nucleosome. This particle contains two copies each of four core histones; these proteins which form a core which organizes 146 base pairs of DNA. The nucleosome, once thought of as a static building block, is now known to be dynamic. A large class of complexes, the ATP-dependent chromatin remodeling complexes, use the energy of ATP hydrolysis to remodel nucleosome structure and position. A major focus of this application is to explore the extent to which remodeling complexes differ in function. A mechanistic understanding of the full spectrum of activities of this family of complexes is necessary to understanding the ways in which chromatin structure can be modulated, and how modulation of chromatin structure might contribute to the regulation of nuclear processes. This will be done by comparing SWI/SNF-family remodeling complexes with other remodeling complexes in three Aims: Aim 1: The role of the ATPase domain in remodeling. We will create chimeric proteins with 'swapped' ATPase domains to define the role of the ATPase domain in function. Aim 2: Characterization of the effects of histone variants on the remodeling reaction. We will test the ability of several different remodeling proteins to function on nucleosomes that have been assembled with different histone variants. Aim 3: ATP-dependent remodeling and higher order chromatin structure. We will use quantifiable approaches to examine the effects of changes in linker length and compaction status on remodeling by a spectrum of remodeling proteins. We will develop a tissue culture system to characterize the magnitude and spread of changes in chromatin structure caused by different remodeling activities.