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. ? ? ?
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