During T cell development, T-cell receptor (TCR) genes rearrange and express in a tissue- and lineage-specific fashion. How this is achieved at the molecular level is not completely understood. The TCR alpha/delta locus is particularly interesting as the delta gene segments are nested inside the alpha segments. Rearrangements and expression of these TCR alpha and delta genes are differentially regulated in an unknown fashion. Chromatin accessibility, tissue specific regulatory elements and their DNA binding proteins are mostly likely to play a major role in this process. The long term goal of this proposed research is to understand the mechanisms underlying these processes and how the choice of alpha-beta versus gamma-delta T cells is made. Recently, a set of T-cell specific regulatory elements 3' of the TCR alpha/delta locus with a locus-control-region. (LCR) activity was identified. These elements confer high level, T cell specific, developmental specific, position independent and gene copy dependent transcription in transgenic mice. In analogy with the beta-globin LCR, these data suggest that the alpha/delta LCR controls the chromatin structure of the TCR alpha/delta locus. Based on the available data and known characteristics of the beta globin LCR, a LCR competition model was proposed to explain how the choice between TCR alpha versus delta is made. Using the competition hypothesis as a framework and to further characterize the alpha/delta LCR, we design a series of experiments. In this application, we propose: 1) to characterize the regulatory elements in the entire J-alpha(J:joining) intron and flanking sequence of the TCR alpha/delta locus using DNase I hypersensitive site assay, footprinting and sequence analysis; 2) to define the minimal region for the alpha/delta LCR function in transgenic mice; 3) to examine the role of the alpha/delta LCR in TCR delta gene expression; 4) to assess the role of alpha/delta LCR in regulation of rearrangements; 5) to test the LCR competition model by examining the importance of gene order (alpha versus delta) in relation to the alpha/delta LCR. The information obtained should yield considerable insight into the molecular mechanisms of how the choice between TCR alpha versus delta is made and how LCR functions during development.
