Interplay between Chromatin and Co-activator Complexes

描述（由申请人提供）：染色质修饰/重塑机器和共激活因子之间的相互作用是真核细胞中差异基因激活的基础。关于这些大的大分子复合物如何协调它们的活动以在染色质上组装前起始复合物（PIC），人们知之甚少。我们最近开始研究两种主要的共激活剂，TFIID/TFIIA（DA）和30-亚基介体复合物（Med）如何结合染色质以支持GAL 4-VP 16在体外激活转录。使用纯化的p300和STAGA组蛋白乙酰转移酶，我们已经确定了p300和Med之间的直接相互作用，以及STAGA和Med。有趣的是，p300的相互作用被废除后乙酰化。这将作为一个模型，蛋白质-蛋白质相互作用是如何重新排列的催化事件在PIC组装。我们的建议的基石是固定化染色质模板测定，这使我们能够关联招聘的共激活因子和染色质酶与转录激活。 在目标#1中，我们将描述Med和p300之间的相互作用，以了解它们如何对接以及乙酰化如何改变这种相互作用。在目标#2中，我们将利用纯化的染色质重塑机器和共激活剂来研究这些复合物如何协作以在核小体模板上组装PIC。目的#3利用我们的分析和PIC组装的知识来研究抑制性组蛋白甲基化和HP 1的结合如何改变转录中的特定步骤，从而导致基因沉默。 我们的研究旨在阐明染色质上PIC组装和转录的生化原理。对转录调控机制的详细了解对于将研究基因表达的概念应用于疾病导向的问题是必要的。

DESCRIPTION (provided by applicant): The interplay between chromatin modification/remodeling machines and co-activators underlies differential gene activation in eukaryotic cells. Little is known about how these large, macromolecular complexes coordinate their activities to assemble a pre-initiation complex (PIC) on chromatin. We recently began to examine how two major co-activators, TFIID/TFIIA (DA) and the 30-subunit Mediator complex (Med), bind chromatin to support GAL4-VP16 activated transcription in vitro. Using purified p300 and STAGA histone acetyltransferases, we have identified direct interactions between p300 and Med, as well as STAGA and Med. Intriguingly, the p300 interaction is abolished upon acetylation. This will serve as a model for how protein-protein interactions are rearranged by catalytic events during PIC assembly. The cornerstone of our proposal is the immobilized chromatin template assay, which allows us to correlate recruitment of co-activators and chromatin enzymes with transcriptional activation. In Aim #1 we will characterize the interactions between Med and p300 to understand how they dock and how acetylation alters this interaction. In Aim #2 we will utilize purified chromatin remodeling machines and co-activators to study how these complexes collaborate to assemble a PIC on nucleosomal templates. Aim #3 employs our assays and knowledge of PIC assembly to examine how repressive histone methylation and binding of HP1 alter specific steps in transcription resulting in gene silencing. Our study is designed to elucidate the biochemical principles underlying PIC assembly and transcription on chromatin. A detailed understanding of mechanisms of transcriptional control is necessary to apply concepts derived from studying gene expression to disease-oriented problems.