Promoter Nucleosome Disassembly from a Metazoan Model System A long-standing pursuit in modern biology is to understand how eukaryotic genes, tightly compacted into chromatin, are transformed into highly active transcription units. Due to the complexity of genome packaging and organization, we have a very limited understanding of the molecular mechanisms that control gene expression at the level of chromatin architecture. Central to the transformation of a silenced gene into a highly active transcription unit is the mobilization of nucleosomes. In recent years, a number of studies have established the existence of nucleosome-free regions within the promoters of eukaryotes genes. These regions represent a common "signature" of transcriptionally active genes, where the degree of promoter DNA accessibility may directly correlate with gene activity. Unfortunately, little is known about the molecular steps involved in the formation of these regions, or the relevant proteins. We recently developed a biochemically-defined and biologically relevant model system for studying nucleosome disassembly using a human retroviral (HTLV-I) promoter. In this purely recombinant chromatin system, DNA-bound activators recruit the cellular coactivator p300 to the promoter. Following p300 recruitment, the histones become highly acetylated and the nucleosomes are disassembled from the promoter by the histone chaperone Nap1. Nucleosome disassembly is strictly acetyl-CoA dependent, uncoupled from transcription, and independent of ATP. These observations define a novel role for the acetyltransferase p300 and the histone chaperone Nap1 in the facilitation of acetylation-dependent nucleosome eviction. The Nyborg Project proposes to biochemically characterize nucleosome eviction with respect to histone acetylation, Nap1-mediated disassembly, and the impact of histone HI. Our studies will be enhanced by the highly complementary research proposed by our collaborators in the Luger and Stargell Projects, the efforts of our co-investigator Dr. Jeff Hansen, and the essential expertise and infrastructure provided by the Core facilities. This independent, yet highly interdependent, research effort will advance our understanding of the molecular mechanism underlying eukaryotic gene activation.
This proposal seeks to better understand the step-wise events required for the activation of genes in higher eukaryotes. Inappropriate gene expression is linked to a significant fraction of human disease states, including genetic disorders and cancer.
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