Interplay between Chromatin and Co-activator Complexes

描述（申请人提供）：染色质上RNA聚合酶II（Pol II）前起始复合物（PIC）的形成是真核基因调控中最基本的过程之一。遗传学、染色质免疫沉淀和哺乳动物细胞中的分子研究为PIC在体内染色质上的组装提供了许多见解。尽管有大量的文献，但对实际的生化机制了解甚少。很少有实验室使用允许分析染色质修饰对体外PIC的影响的系统来研究这个问题。组蛋白H3 K4和H3 K27甲基化分别由三胸（MLL/Set 1）和多梳（PRC 2）复合物介导的相反现象，由于它们在发育和分化过程中发挥的重要作用而特别令人感兴趣。对它们的机制的初步理解需要在定义的转录系统中重新创建和分析这些事件。在PRC 1、PRC 2和MLL/Set 1复合物的基础酶学方面已经做了大量的工作。我建议显着扩展这些研究，以解决这些复合物如何影响PIC组装在我们的模型GAL 4-VP 16在体外系统和天然干细胞启动子控制的Sox 2，Oct 4，Myc，Klf 4和Nanog的重要方面。一个特别强大的技术，将形成基石的建议是固定化模板测定。该测定采用生物素化的模板，其与天然的和化学甲基化的组蛋白组装成染色质。将模板连接到链霉亲和素包被的珠上，以从提取物中捕获PIC，并通过免疫印迹分析其组成。使用组蛋白修饰/重塑测定和体外转录研究PIC的功能。固定化模板方法将用于解决以下三个目标：目标#1将检查MLL/Set 1和H3 K4甲基化如何影响PIC的组装。目的#2将检查PRC 1和PRC 2对PIC的沉默的具体机制以及含有三甲基化H3 K4和H3 K27的二价结构域的机制。目的#3将研究干细胞激活剂如何组装成增强体并在体外激活染色质化的Sox 2和Nanog基因的转录。我们的研究将利用哺乳动物基因调控的大量知识来制定和测试有关PIC如何在核小体上组装以及染色质的共价修饰以及结合这些修饰的机器如何调节这一过程的假设。这些知识将提供适用于许多生物体转录调控的基本信息。公共卫生相关性：基因调控最重要的方面之一是了解染色质（保护真核基因组的核蛋白结构）如何控制基因何时开启以及基因关闭时的替换。这一步骤对所有生物体都是至关重要的，对这一过程的了解是理解人类疾病、分化和发育过程中基因调控的关键。我们的建议将使用一个模型系统来了解这个过程的生化细节和机制。

DESCRIPTION (provided by applicant): The formation of RNA polymerase II (Pol II) preinitiation complexes (PICs) on chromatin is one of the most fundamental processes in eukaryotic gene regulation. Genetics, chromatin immunoprecipitation and molecular studies in mammalian cells have provided numerous insights into PIC assembly on chromatin in vivo. Despite the extensive literature, there is a poor understanding of the actual biochemical mechanisms. Few labs have studied the problem using systems that allow analysis of the effect of chromatin modifications on PICs in vitro. The opposing phenomena of histone H3K4 and H3K27 methylation mediated by the trithorax (MLL/Set1) and polycomb (PRC2) complexes, respectively, are of particular interest due to the important role they play during development and differentiation. A rudimentary understanding of their mechanism requires that these events be recreated and analyzed in a defined transcription system. There has been much work done on the basic enzymology of the PRC1, PRC2 and MLL/Set1 complexes. I propose to significantly extend these studies to address important aspects of how these complexes affect PIC assembly in both our model GAL4-VP16 in vitro system and on natural stem cell promoters controlled by Sox2, Oct4, Myc, Klf4 and Nanog. A particularly powerful technique that will form the cornerstone of the proposal is the immobilized template assay. This assay employs biotinylated templates assembled into chromatin with naove and chemically methylated histones. The templates are attached to streptavidin-coated beads to capture the PICs from extracts and analyze their composition by immunoblotting. The functions of the PIC are studied using histone modification/remodeling assays and in vitro transcription. The immobilized template approach will be used to address the following three aims: Aim #1 will examine the how MLL/Set1 and H3K4 methylation affect the assembly of a PIC. Aim #2 will examine the specific mechanism of silencing by PRC1 and PRC2 on the PIC and the mechanism of bivalent domains containing both trimethylated H3K4 and H3K27. Aim #3 will examine how stem cell activators assemble into enhanceosomes and activate transcription of chromatinized Sox2 and Nanog genes in vitro. Our study will leverage the vast body of knowledge from mammalian gene regulation to craft and test hypotheses for how PICs assemble on nucleosomes and how covalent modifications of chromatin, and the machines that bind those modifications, regulate this process. The knowledge will provide fundamental information applicable to transcription regulation in many organisms. PUBLIC HEALTH RELEVANCE: One of the most important aspects of gene regulation is understanding how chromatin, the nucleoprotein structure that protects eukaryotic genome, controls when genes are turned on and replaced when genes are turned off. This step is fundamental to all organisms and knowledge of the process is key to understanding gene regulation during disease, differentiation and development in humans. Our proposal will use a model system to understand the biochemical details and mechanism of this process.