Interplay Between Chromatin and Co-Activator Complexes

描述(申请人提供)：我们实验室对RNA聚合酶II预起始复合体(PIC)与组蛋白修饰和染色质转录所需的重塑酶协调其功能的生化机制感兴趣。在之前的资金周期中，我们已经建立了模型，研究了Mediator和TFIID共激活子如何与p300、Chd1和SAGA协调，以及HP1和Prc1等沉默蛋白如何影响带有特定组蛋白修饰的设计染色质模板的这一过程。我们最近的实验集中在真核启动子上发现的H2AZ-H3.3变体染色质如何调节转录。我们首次在体外用乙酰化的变体染色质重建了转录刺激，表明它增强了P400-Tip60和INO80的结合，并将探讨其机制。我们使用固定化模板实验，可以从HeLa和小鼠胚胎干细胞提取液中捕获PIC，并在体外分析不同PIC因子的功能。体外研究伴随着模型细胞系统中的芯片，以及基因表达总括中的全基因组数据集的分析。在目标1中，我们将了解P400-Tip60与不同染色质的增强结合在基因激活中的作用。在目标2中，我们将分析INO80与不同染色质的结合和功能，以及介体在这一过程中所起的作用。我们的重点是INO80如何对含有和缺乏H2 AZ和H3.3的核小体障碍做出反应。目的3研究决定Mediator与TFIID相互作用的特定亚基，这些亚基形成控制一般因子、POL II和启动所需的众多染色质因子结合的核心结构。我们的结果将利用最先进的生物化学方法提供染色质上基因激活的详细机制。

DESCRIPTION (provided by applicant): Our lab is interested in the biochemical mechanisms by which the RNA polymerase II preinitiation complex (PIC) coordinates its functions with histone modification and remodeling enzymes necessary for transcription on chromatin. Work in previous funding cycles has established models for how the Mediator and TFIID co-activators coordinate with p300, Chd1, and SAGA, and how silencing proteins like HP1 and PRC1 influence this process on designer chromatin templates bearing specific histone modifications. Our most recent experiments have focused on how the H2AZ-H3.3 variant chromatin found at eukaryotic promoters regulates transcription. We have for the first time recreated transcriptional stimulation by acetylated variant chromatin in vitro, shown that it enhances binding of p400-Tip60 and Ino80, and will now pursue its mechanism. We employ the immobilized template assay, which allows us to capture PICs from HeLa and mouse embryonic stem cell extracts and analyze the functions of different PIC factors in vitro. The in vitro studies are accompanied by ChIP in a model cell-based system, and by analysis of genome wide data sets in the gene expression omnibus. In Aim 1, we will acquire knowledge of the role of enhanced binding of p400-Tip60 to variant chromatin on gene activation. In Aim 2, we will analyze binding and function of Ino80 on variant chromatin and the role Mediator plays in this process. Our emphasis is on how Ino80 responds to nucleosomal obstacles containing and lacking H2AZ and H3.3. Aim 3 examines the specific subunits dictating interaction of Mediator with TFIID, which form the core structure that controls binding of general factors, Pol II and the numerous chromatin factors necessary for initiation. Our results will provide a detailed mechanism of gene activation on chromatin using state of the art biochemical approaches.