Negative regulation of gene expression at the transcriptional level is a pivotal control mechanism in many biological processes, including cell growth regulation, tissue formation, development and differentiation. Defects in genes that encode transcriptional repressors may result in developmental abnormalities and tumorigenesis. Despite the important roles repressor proteins are likely to play, very few of them have been identified and studied in eukaryotic cells. The intent of this investigation is to study transcriptional repression and its alteration by viral oncoproteins. It is expected that the proposed studies will provide insights into the mechanisms of transcriptional repression in eukaryotic cells as well as modulation of activities of transcriptional factors by viral oncoproteins. We have recently cloned a eukaryotic repressor YY1 (yin yang 1) and have demonstrated that both the endogenous and the recombinant YY1 proteins are able to repress transcription directed by a basal promoter or the SV40 enhancer/promoter. In addition, YY1 also activates transcription in response to the adenovirus E1A protein, suggesting that YY1 has dual functionality in regulating transcription. Our goal is to understand how YY1 represses transcription and how it can be converted into an activator in the presence of E1A. We will begin by performing an extensive structure/function study of YY1 to define the repressor domain in YY1 and to identify amino acid residues important for transcriptional repression, to determine potential functions of specific sequence motifs in other parts of the YY1 proteins, and to identify domains in YY1 involved in its response to E1A. To understand the activating activity of YY1 in the presence of E1A, we will delineate the domains in the E1A protein that are responsible for relieving repression and for activating transcription through YY1 using available deletion and point mutants of E1A. We will investigate the possibilities that E1A may change the transcriptional activities of YY1 by either affecting posttranslational modifications of YY1 and/or by altering its interactions with other cellular proteins. We will also study the direct interaction of YY1 with E1A in greater detail and map the interacting domains in both proteins. Finally, as an important step towards understanding the molecular mechanisms of transcriptional repression mediated by YY1, we will identify and clone target proteins for the repressor domain of YY1.
