Interplay between Chromatin and Co-activator Complexes

描述(由申请人提供)：染色质修饰/重塑机制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。关于这些大分子复合体如何协调它们的活性来组装染色质上的预引发复合体(PIC)，人们知之甚少。我们最近开始研究两个主要的共激活子，TFIID/TFIIA(DA)和30亚单位介体复合体(Med)是如何在体外结合染色质以支持GAL4-VP16激活的转录。利用纯化的p300和STAGA组蛋白乙酰转移酶，我们鉴定了p300和Med以及STAGA和Med之间的直接相互作用。有趣的是，p300相互作用在乙酰化时被取消。这将作为PIC组装过程中催化事件如何重排蛋白质-蛋白质相互作用的模型。我们建议的基础是固定化染色质模板分析，它允许我们将共激活因子和染色质酶的招募与转录激活联系起来。 在目标#1中，我们将表征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构机和共激活剂来研究这些复合体如何协作组装核小体模板上的PIC。目的#3利用我们对PIC组装的分析和知识来研究组蛋白甲基化抑制和HP1结合如何改变转录中的特定步骤，导致基因沉默。 我们的研究旨在阐明PIC在染色质上组装和转录的生化原理。对转录调控机制的详细理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.