TRANSCRIPTIONAL REGULATION OF T-CELL RECEPTOR ALPHA GENE

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Tissue-specific transcriptional regulation of the T cell receptor alpha chain gene requires the productive rearrangement of gene segments within the TCR alpha/delta chromosomal locus, the generation of an active or "accessible" chromatin structure, and the recruitment of a distal 3' TCR alpha enhancer. This enhancer can regulate its cognate Valpha promoter from distances as great as 69 kb, depending upon the precise combination of variable, joining, and constant regions during rearrangement. In addition, enhancer function appears to be controlled, in part, by nearby silencer elements that restrict its activity to the appropriate stage of T lymphocyte development. The minimal TCR alpha enhancer has been mapped to approximately 116 base pairs of DNA and is regulated by four proteins: T cell-restricted Lef-1, Ets-1, and CBF (Core Binding Factor) and a member of the ubiquitous CREB family of proteins that interacts with cAMP response elements. The importance of the TCR alpha enhancer in other critical aspects of TCR alpha gene activation has been demonstrated by showing that a deletion of this element from the alpha/delta chromosomal locus prevents productive gene rearrangement in T cells. The implication is that the enhancer is required for transcription of the unrearranged locus, which occurs prior to gene rearrangement, and/or to modulate chromatin structure within the locus so that it becomes accessible to V(D)J recombinases. In this proposal, we plan to study the mechanism of alpha enhancer function using in vitro transcription and chromatin assembly systems that accurately reproduce tissue-specific and enhancer- dependent TCR alpha gene expression. First, experiments are designed to characterize the DNA topology and nucleosomal structure required to generate a functional enhancer since this does not occur using naked DNA templates in vitro. Second, the mechanism by which specific proteins regulate enhancer activity will be analyzed by examining whether they can modulate promoter structure at a distance. Third, a repressor protein that inactivates TCR alpha gene expression in the absence of an active enhancer will be purified and characterized for its site of DNA binding and ability to interact with other transcription factors in vitro. Fourth, since very little is known about Valpha promoter regulation, we plan to identify the basal factors that compose the transcription initiation complex on this TATA-less sequence and to analyze the relation of these proteins to promoter and enhancer function. Finally, we will assess the role of the TCR alpha silencer in regulating lineage-specific transcription by analyzing whether this element can modulate TCR alpha promoter or enhancer activity in vitro. The information gained from an analysis of the TCR alpha enhancer in these in vitro experiments should be particularly relevant to health-related issues since the TCR alpha/delta chromosomal locus is the site of many aberrant translocations that are associated with a variety of leukemias and lymphomas. The activation of oncogenes in these cells is thought to result from their translocation into the vicinity of an active TCR alpha enhancer.