The long-term goal of the proposed studies is to understand the molecular mechanisms that mediate allelic exclusion at the T cell receptor (TCR) b locus. Allelic exclusion ensures that individual lymphocytes express a unique antigen receptor and therefore is essential for the clonal activation of lymphocytes during the immune response. Evidence suggests that allelic exclusion is controlled in part through changes in chromatin structure that block the accessibility of substrate gene segments. To identify the underlying cis-regulatory elements, eleven Dnase 1 hypersensitive sites have been identified in a contiguous 95 kb region, starting 20 kb upstream of Db1 and ending 50 kb downstream of Vb14, in DN and DP thymocytes. This proposal aims to systematically test and characterize these putative cis- regulatory elements and their associated factors in allelic exclusion. (1) The role of the two major induced sites in allelic exclusion will be determined by targeted deletion of the sites individually from the endogenous locus. (2) The role of all the identified sites in allelic exclusion will be explored using recombination substrates. The two major induced sites will be deleted simultaneously and the effect of the deletion will be determined in ES cell-derived thymocytes and mature T cells. The remaining nine sites will also be analyzed using recombination substrates by deletion simultaneously and in combinations with the induced sites. Critical sequence motifs within identified sites will be determined by mutations. (3) Trans-acting factors that associate with this identified cis-regulatory elements will be characterized and identified by electrophoretic mobility shift assays and molecular cloning. (4) The presence of additional cis-regulatory elements in the remaining 300 kb region from the endogenous locus and targeted insertion of a Vb13 gene segment upstream of D-b segments. Disregulation of allelic exclusion may block lymphocyte development and/or produce lymphocytes that express more than one type of antigen receptor per cell. An understanding of the control of allelic exclusion at the molecular level may help to elucidate mechanisms that underlie immunodeficiencies and autoimmune diseases.
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