The developmentally-regulated appearance of important T cell subpopulations represents an excellent model system for studying the roles of specialized transcription factors that affect the phenotype of the immune system. A critical determinant of T cell subtype and function is the T cell receptor (TCR), an alpha beta (or gamma delta) heterodimer that forms a surface-bound complex with CD3 and CD4 (or CD8) antigens to recognize cellular MHC-bound antigens. The expression of distinct TCR heterodimers is controlled by powerful enhancers located downstream of the rearranged genes. These enhancers also play a role in a number of T-cell leukemias, by deregulating the expression of errantly translocated proto-oncogenes that fall within their control during somatic gene rearrangement. The short 115 bp C-alpha enhancer, containing three essential protein binding sites (CREB, TCF-1 and TCF-2), is a novel T cell-specific enhancer that activates the V-alpha promoter across an extensive genomic distance of >30kB. We have purified the T cell-specific enhancer-binding protein, TCF-1, and shown that it is critical for TCRalpha enhancer activity in vivo. We propose to isolate the gene encoding TCF-1 and develop reagents necessary to characterize the expression and function of this protein in T and pre-T cell lines, as well as in primary thymocyte cultures isolated at different stages of development. We have found that the activity of TCF-I is dependent upon the adjacent location of two other proteins, CREB and TCF-2. To study the mechanism of this context-dependent enhancer activity in greater detail we suggest an in vitro approach, using active Jurkat transcription extracts and purified TCF-1 and TCF-2 proteins, and an in vivo analysis of altered synthetic enhancers. Interestingly, the minimal C-alpha-enhancer is active in gamma delta T cell lines that normally do not transcribe the TCR C-alpha gene. This lineage-specific restriction of T lymphocyte receptors requires "silencer" elements that lie upstream of the minimal enhancer. Thus, TCR gamma delta+ cell lineages silence the TCRalpha enhancer whereas alpha beta+ cell lineages are thought to silence the TCR gamma enhancer. In this proposal, we describe a combined in vivo and in vitro approach to study the mechanism of silencer function and the possible involvement of alpha beta lineage-restricted anti-silencer proteins. Taken together, these studies will significantly advance our understanding of the development of individual T cell subtypes and the mechanisms underlying lineage-restricted transcriptional activation and repression.
NIH ExPorter
NIH ExPorter