The assembly of immunoglobulin (Ig) and T-cell receptor (TCR) genes by V(D)J recombination generates the enormous diversity of antigen receptors required for mammalian immunity. Proper targeting of these recombination events is critical for lymphocyte development and to avoid chromosomal translocations that result in lymphoid tumors. Although each V(D)J rearrangement is mediated by a single recombinase that recognizes a set of common DNA sequences, receptor gene assembly is tightly regulated by tissue-, stage-, and allele-specific mechanisms. We have discovered that efficient rearrangement of minilocus substrates relies on the activity of germline promoter elements, which impart accessibility to neighboring gene segments. We now propose to build on this recent discovery and dissect the molecular mechanisms by which transcriptional promoters target chromosomal gene segments for efficient recombination. Three fundamental aspects of these regulatory mechanisms will be studied in vitro and in vivo: (1) The precise features of promoter activation that control accessibility will be dissected using cell lines that inducibly express V(D)J recombinase. The proposed experiments will define the independent contributions of transcriptional initiation, read through, transcriptional polarity, factor binding, RNA Polymerase II transcription, and chromatin modifiers to minilocus accessibility. (2) Regulatory models emerging from these in vitro studies will guide crc/lox targeting strategies to determine the transcriptional requirements for activating an inert antigen receptor locus in non-lymphoid cells. (3) We will use targeted mutagenesis to test the in vivo role of a germline promoter, and its synergy with a neighboring enhancer, in conferring accessibility to IgH gene segments. Together, these studies will resolve a fundamental question in immunology; how do developing lymphocytes regulate choices amongst large arrays of gene segments, any of which could serve as sequence-specific targets for the V(D)J recombinase complex?
