The somatic multi-step recombination process that assembles variable regions of immunoglobulin and T-cell receptor (TCP) genes generates the enormous diversity of antigen receptor molecules required for mammalian immunity and is pivotal to normal lymphopoeisis. All recombination events are mediated by a single V(D)J recombinase activity that recognizes substrate sequences common to all antigen receptor gene segments. In spite of these shared features, receptor gene assembly is regulated at several levels, including tissue-, stage- and allele-specificity. Observed correlations between locus recombination and transcriptional status support the notion that specificity of recombinational events is imposed by modulations in accessibility of chromosomal gene segments to the recombinase complex. The molecular determinants of accessibility, however, have not been delineated. Aberrations in recombinational targeting can have severe consequences; e.g., impaired lymphocyte development or chromosomal translocations that result in lymphoid tumors. The objective of the experiments described in this proposal is to elucidate the molecular mechanisms that properly target recombinase activity to distinct genetic loci during lymphocyte development. Three distinct facets of this problem will be addressed: 1. Efficient recombination of TCRbeta minilocus substrates and endogenous antigen receptor loci critically depends on the presence of transcriptional enhancers, in cis. The independent contributions of transcription and enhancer activity to recombinational accessibility will be dissected with modified versions of TCRbeta recombination substrates. A novel recombinase-inducible cell system (DR37), that supersedes existing systems for analysis of recombinational control, will be employed. Guided by initial results, subsequent substrates will probe the detailed molecular featrues of transcription, enhancer activity, or enhancer-dependent transcription that confer recombinational accessibility. 2. Recombinational activation of an endogenous antigen receptor locus will be studied in a non-lymphoid cell line engineered to express recombinase activity. Cre-lox mediated gene targeting will be employed to replace inactive transcriptional control elements at the JH locus with analogous, active versions. 3. The second, Vbeta-to-DJbeta stage of TCRbeta gene assembly will be studied in the context of tissue-specific recombination. Transgenic TCRbeta minilocus substrates rearrange D and Jbeta gene segments in all lymphocytes, but rearrange Vbeta (to DJbeta) segments only in pre-T cells. Modified versions of this substrate will be introduced into RAG-2 -/- blastocysts (via ES cells), to define the sequences and activities that direct these most stringently regulated recombinational events. Collectively, these studies will yield fundamental insights into the molecular elements that direct recombinase activity to initiate assembly of particular antigen receptor loci and subsequently, redirect recombination in order to ensure lymphocyte mono-specificity. Such insights are expected to provide a greater understanding of mechanisms that confer differential, long-range regulation to complex genetic loci.
