Interplay between Chromatin and Co-activator Complexes

描述（由申请人提供）：染色质上RNA聚合酶II （Pol II）预起始复合物（PICs）的形成是真核生物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在体外分析染色质修饰对PICs的影响的系统研究这个问题。三胸（MLL/Set1）和多梳（PRC2）复合物分别介导组蛋白H3K4和H3K27甲基化的相反现象，由于它们在发育和分化过程中发挥重要作用而受到特别关注。对其机制的基本理解需要在一个确定的转录系统中重现和分析这些事件。关于PRC1， PRC2和MLL/Set1复合物的基本酶学已经做了很多工作。我建议进一步扩展这些研究，以解决这些复合物如何影响体外GAL4-VP16模型和Sox2、Oct4、Myc、Klf4和Nanog控制的天然干细胞启动子中的PIC组装的重要方面。一个特别强大的技术，将形成该建议的基石是固定化模板测定。该试验采用生物素化的模板组装成染色质与原始和化学甲基化组蛋白。模板附着在链霉亲和素包被珠上，从提取物中捕获PICs，并通过免疫印迹分析其组成。通过组蛋白修饰/重塑实验和体外转录研究了PIC的功能。固定化模板方法将用于解决以下三个目标：目的#1将检查MLL/Set1和H3K4甲基化如何影响PIC的组装。Aim #2将研究PRC1和PRC2沉默PIC的具体机制，以及含有三甲基化H3K4和H3K27的二价结构域的机制。Aim #3将研究干细胞激活因子如何在体外组装成增强体并激活染色化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PICs如何在核小体上组装的假设，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。这些知识将为许多生物体的转录调控提供基础信息。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（保护真核生物基因组的核蛋白结构）如何控制基因何时开启和何时关闭。这一步骤是所有生物体的基础，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理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.