We have identified a transcriptional silencer that is required for the appropriate expression of the CD4 gene. This silencer functions to inhibit expression of the CD4 gene in inappropriate mature T cell subclasses as well as immature T cells and non T cells, indicating that this silencer functions in both a developmental stage-specific- and tissue-specific manner. In this grant, we propose to study the factors that bind to this silencer and contribute to its specificity and function. Our study of the CD4 silencer will provide insight into two important biological questions. First, this study will help define the molecular basis for important biological processes in T cell development and activation. Because of the critical role that CD4 plays in T-cell development, the mechanisms that initiate and terminate cD4 expression are believed to be linked to the thymic selection process. A comprehensive understanding of thymic selection will require the knowledge of the nuclear events that control differential gene expression of the important molecules that in turn control the selection process. Thus, an analysis of the control of CD4 gene expression constitutes an important step in defining these events. Because its expression is required for antigen-specific T-cell activation, the analysis of CD4 expression will also help identify factors that control T cell function in the periphery. Second, this study will help characterize the mechanism for transcriptional silencing in mammals. Although other mammalian silencers have been reported, to date little has been done to characterize the factors that contribute directly to transcriptional silencing. Thus, these experiments will provide important information on this basic transcriptional process. Using DNAse footprinting, we have identified four factor-binding sites in the minimal silencer region. We have detected differences in factor binding to one of these sites that correlate to silencer function; using southwestern analysis, we have identified a 42kd protein binding to this region that is expressed in CD4- cells, but not CD4+ cells. Thus, specificity of silencer function may be conveyed by the binding of this 42kd nuclear factor to this site. In this grant, we will use reporter constructs in transgenic mouse experiments to determine which of the four factor binding sites are important for silencer specificity and function. Once the functionally relevant sites are defined, we will clone the factors that bind to them either by screening lambda-gt11 expression libraries with recognition-site probes or by purifying the factors biochemically and determining their protein sequence. Because of its expression pattern, the 42kd protein that we identified using southwestern analysis is a prime candidate for our studies. Once these novel factors are cloned, we will characterize their expression patterns and begin to characterize their mechanism of function. In this manner, we hope to begin to characterize the molecular events that drive T cell development in the thymus, and those that contribute to basic transcriptional silencing.
