Interplay Between Chromatin and Co-Activator Complexes

描述（由申请人提供）：我们的实验室对RNA聚合酶II前起始复合物（PIC）与染色质转录所需的组蛋白修饰和重塑酶协调其功能的生化机制感兴趣。在之前的资助周期中，我们已经建立了关于Mediator和TFIID辅激活因子如何与p300、Chd 1和佐贺协调的模型，以及沉默蛋白如HP 1和PRC 1如何影响这一过程的模型。 我们最近的实验集中在真核启动子上发现的H2 AZ-H3.3变体染色质如何调节转录。我们首次在体外重建了乙酰化染色质变体的转录刺激，表明它增强了p400-Tip 60和Ino 80的结合，现在将继续研究其机制。我们采用固定化模板测定，这使我们能够捕获的PIC从HeLa和小鼠胚胎干细胞提取物和分析不同的PIC因子在体外的功能。体外研究伴随着基于模型细胞的系统中的ChIP，以及基因表达综合数据库中的全基因组数据集的分析。 在目标1中，我们将获得增强的p400-Tip 60与变异染色质的结合对基因激活的作用的知识。目的2：分析Ino 80在不同染色质上的结合和功能，以及Mediator在此过程中的作用。我们的重点是Ino 80如何应对含有和缺乏H2 AZ和H3.3的核小体障碍。目的3检查指示中介体与TFIID相互作用的特定亚基，其形成控制一般因子、Pol II和起始所需的众多染色质因子的结合的核心结构。我们的研究结果将提供一个详细的机制，染色质上的基因激活使用最先进的生化方法。

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.