The overall goal of this proposal is to understand how transcriptional activators contend with chromatin, using the yeast Saccharomyces cerevisiae as a model. Transcriptional regulation is essential to growth and development; aberrant regulation can lead to genetic defects and disease, including cancer. A full understanding of transcriptional regulation requires learning how transcriptional activators interact with chromatin in vivo. Yeast is ideally suited for such investigations: components of both chromatin and the transcription machinery are highly conserved between yeast and higher eukaryotes, and powerful genetic and molecular biological tools are available for its study. Previously, we showed that activation domains enhance transcription factor binding to nucleosomal sites in yeast, using the prototypical activator GAL4 as a model. We now propose to investigate the mechanism by which transcriptional activators bind to and remodel chromatin in yeast, using a combination of genetic and molecular biological approaches. Our studies will include determining whether components of the preinitiation complex or of chromatin remodeling complexes are required for activator-mediated chromatin remodeling; whether the highly conserved histone amino termini, which are known targets for post-translational modification, play a role in facilitating or inhibiting activator-mediated chromatin remodeling; whether binding of GAL4 to a nucleosomal site is facilitated by nucleosome sliding; and how binding site affinity influences the ability of GAL4 to remodel chromatin via nucleosomal binding sites. We also propose to investigate the effect of the type and strength of activation domain on binding of activators to nucleosomal compared to non-nucleosomal sites. We have also found that the yeast transcriptional activator GCN4 binds less well than GAL4 to a nucleosomal site, and that GCN4 needs help from another factor, RAP1, to bind to and activate the HIS4 promoter, whereas GAL4 does not. We propose new experiments to explore the mechanism by which RAP1 potentiates transactivation in the context of chromatin, using the HIS4 promoter as a model. Together, these studies will yield new insight into the mechanism by which transcriptional activators gain access to chromatin in vivo, thereby increasing our knowledge of the role of chromatin in gene regulation.
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