9631067 Seto These studies involve biochemical approaches to study the mechanism of YY1 action with particular emphasis on determining how YY1 represses transcription. YY1 is a mammalian sequence-specific DNA-binding protein with complex transcriptional properties. In some genes YY1 activates transcription, in others it represses transcription and in at least one case it functions as part of the basal transcription complex. Previously, a number of proteins have been identified that interact with YY1. Of particular interest is a protein that possesses sequence homology with the yeast global transcriptional regulated RPD3. The mammalian RPD3 protein is a potent transcriptional repressor when directed to a promoter. These studies involve the functional interaction of YY1 and the mammalian RPD3 protein. The hypothesis to be tested is that the negative regulation by YY1 is achieved through its ability to selectively interact with the conserved cellular protein, RPD3. Specifically, the idea that YY1 regulates transcription by tethering mammalian RPD3 to DNA as a co-repressor and that this transcriptional mechanism is conserved from yeast to human will be tested. With recombinant DNA and in vitro mutagenesis techniques, the mammalian RPD3-YY1 interaction will be analyzed in detail. A number of YY1 mutants will be constructed and tested for their ability to associate with mammalian RPD3 and for their ability to modulate transcription in the presence or absence of the RPD3 protein. Conversely, different mammalian RPD3 mutants will be tested for their ability to associate with YY1 and for their ability to modulate transcription in vivo and in vitro. These studies will help answer some of the questions that are critical for a thorough understanding of the intricate mechanisms operating to orchestrate gene expression in eukaryotic cells. ***
