Transcriptional regulation is central to growth and development of eukaryotic organisms. Since the template for transcription in eukaryotes is not naked DNA, but chromatin, a full understanding of transcriptional regulation will require learning how factors which promote transcription interact with chromatin in vivo. The ability to manipulate and analyze chromatin structure in yeast makes this organism ideally suited for such studies. Initial studies using GAL4 binding to nucleosomal sites in yeast as a model system have shown that GAL4 is able to compete successfully with histone proteins for occupancy of a binding site in vivo. Previous results have shown that, in contrast, function of some cis-acting DNA elements is inhibited by incorporation into nucleosomes in vivo. This proposal aims to explore further the binding of GAL4 to nucleosomal templates in vivo by determining a) whether GAL4 can bind to a site in a nucleosome in the absence of replication; b) how the ability of GAL4 to bind to a site in a positioned nucleosome, as compared to a non-nucleosomal site, is affected by its affinity for the particular binding site; and c) how the position of the GAL4 binding site within a nucleosome affects the ability of GAL4 to disrupt nucleosome positioning. These experiments will give insight into the mechanism by which GAL4 successfully competes with the histones for occupancy of its binding site in vivo, thereby increasing our understanding of the rules which govern when a transcription factor can and cannot bind to a site in chromatin in living cells. Preliminary studies have also revealed structural changes in chromatin which depend on the GAL4 activation domain an a TATA site, suggesting that GAL4 may function by recruiting components of the preinitiation complex to a distant (hundreds of bp) TATA site in a chromatin template. This hypothesis will be tested, and whether transcriptional initiation is required for these structural changes will be determined. The involvement of the global activator complex including SWI1-3 in GAL4 binding in chromatin and changes in chromatin structure at the TATA site will be tested. Finally, evidence suggests that a hierarchy exists between transcription factors and chromatin, so that some factors can bind to nucleosomal sites whereas function of other cis-acting elements is inhibited by incorporation into nucleosomes. One possible explanation for this is that transcription factors belonging to different classes behave differently towards chromatin templates. To explore this issue, studies on transcription factor binding to nucleosomal sites in vivo will be extended to transcription factors belonging to the bZIP, homeodomain, and b-hlh classes.
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