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. Each of these genomic rearrangements is mediated by a single V(D)J recombinase activity that recognizes sequences common to all antigen receptor gene segments. Despite these shared features, receptor gene assembly is regulated at several levels, including tissue-, stage- and allele-specificity. Correlations between recombination and transcriptional status support the notion that specificity is enforced at the level of gene segment accessibility. The molecular determinants of recombinational accessibility; however, have not been delineated. Aberrations in Ig and TCR rearrangement 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 fundamental aspects of these regulatory mechanisms will be addressed: 1. Efficient rearrangement of chromosomal gene segments in contingent upon the presence of transcriptional promoters and enhancers, in cis. The independent contributions of enhancer activity, promoter activation, and transcriptional readthrough to recombinational accessibility will be dissected with modified versions of TCRbeta miniloci. Subsequent studies will probe the features of chromatin remodeling that confer recombinational accessibility to chromosomal gene segments. 2. The role of endogenous promoter and enhancer elements in directing efficient assembly of the Ig heavy chain locus will be assessed by targeted deletion strategies. In addition, targeted embryonic stem cells will be used to determine whether transcriptional activation initiates recombination at inert antigen receptor loci. 3. Assembly of the Iglambda light chain locus is impaired in precursor B lymphocytes that are blocked for nuclear import NF- kappaB transcription factors. However, previously characterized Iglambda enhancers lack obvious NF-kappaB sites. A series of genetic approaches will be employed to dissect the roles of known Iglambda enhancers, NF-kappaB, and other trans-acting factors in regulating the stage-specific assembly of VlambdaJlambda gene segments. Collectively, these studies will yield fundamental insights into the mechanisms that direct, the re-direct recombinase activity to specific antigen receptor loci, and thereby ensure proper lymphocyte development.
