Interplay between Chromatin and Co-activator Complexes

描述（由申请人提供）：真核细胞中染色质修饰/重塑机器和共激活因子之间的相互作用是差异基因激活的基础。对于这些大的大分子复合物如何协调它们在染色质上组装起始前复合物（PIC）的活动知之甚少。我们最近开始研究两种主要的共激活剂，TFIID/TFIIA （DA）和30亚基介质复合物（Med）如何结合染色质以支持GAL4-VP16在体外激活的转录。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相互作用在乙酰化后被消除。这将作为蛋白质-蛋白质相互作用在PIC组装过程中如何通过催化事件重排的模型。我们建议的基础是固定染色质模板测定，这使我们能够将共激活剂和染色质酶的募集与转录激活联系起来。

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.