Transcription Factor Interactions with Nucleosomes

描述(由申请人提供)：染色质结构的调节在所有研究的核过程中起主要作用。染色质的主要构件是核小体。这种颗粒包含四个核心组蛋白的两个拷贝；这些蛋白质形成了一个核心，组成了146个碱基对的DNA。核小体曾经被认为是静态的组成部分，现在已知是动态的。一大类复合体，依赖于ATP的染色质重塑复合体，利用ATP水解的能量来重塑核小体的结构和位置。这项应用的一个主要焦点是探索重塑复合体在功能上的不同程度。要了解染色质结构的调节方式，以及染色质结构的调节如何有助于核过程的调节，就必须从机制上理解这类复合体的全谱活性。这将通过将SWI/SNF家族重塑复合体与其他重塑复合体在三个方面进行比较来完成：目的1：ATPase结构域在重塑中的作用。我们将创造具有‘交换’的ATPase结构域的嵌合蛋白，以确定ATPase结构域在功能中的作用。目的2：研究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与不同组蛋白变体组装的核小体上发挥作用的能力。目的3：ATP依赖的重塑和高阶染色质结构。我们将使用可量化的方法，通过一系列重塑蛋白质来检查连接子长度和紧凑状态的变化对重塑的影响。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.