Interplay between Chromatin and Co-activator Complexes

描述(申请人提供)：在染色质上形成RNA聚合酶II(POL II)预起始复合体(PIC)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体内PIC在染色质上的组装提供了大量的见解。尽管有大量的文献，但对实际的生化机制了解很少。很少有实验室使用系统来研究这个问题，这些系统允许在体外分析染色质修饰对PIC的影响。组蛋白H3K4和H3K27甲基化的相反现象分别由三胸(MLL/Set1)和多梳(PRC2)复合体介导，由于它们在发育和分化过程中发挥着重要作用，因此特别令人感兴趣。要对它们的机制有一个基本的了解，就需要在一个确定的转录系统中重建和分析这些事件。关于PrC1、PrC2和MLL/Set1复合体的基础酶学已经做了大量的工作。我建议显著扩展这些研究，以解决这些复合体如何影响我们的GAL4-VP16体外模型系统中的PIC组装以及由Sox2、Oct4、Myc、Klf4和Nanog控制的天然干细胞启动子的重要方面。形成该提案基石的一项特别强大的技术是固定化模板分析。该分析使用生物素化模板与NAVE和化学甲基化的组蛋白组装成染色质。这些模板被附着在包被链霉亲和素的珠子上，以从提取物中捕捉图片，并通过免疫印迹分析其组成。组蛋白修饰/重塑实验和体外转录实验研究了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将研究MLL/Set1和H3K4甲基化如何影响PIC的组装。目的#2将研究PrC1和PrC2对PIC沉默的具体机制，以及同时含有三甲基化H3K4和H3K27的二价区的机制。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Sox2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作和测试假设，即PIC如何在核小体上组装，染色质的共价修饰以及绑定这些修饰的机器如何调节这一过程。这些知识将提供适用于许多生物体转录调控的基本信息。与公共健康相关：基因调控最重要的方面之一是理解染色质--保护真核生物基因组的核蛋白结构--如何控制基因何时启动，何时基因关闭。这一步骤对所有生物体来说都是基本的，了解这一过程是理解人类疾病、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一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.