Developmental regulation of the RAG2 gene and the T cell receptor delta locus

We investigated the regulation of the recombination activating gene 2 (RAG2) and the T cell receptor (TCR) alpha/delta locus during in vivo lymphocyte development. To examine the expression pattern of RAG2 in primary and peripheral lymphoid tissues, we first generated mice in which a functional, RAG2:GFP fusion gene was knocked-in to the endogenous RAG2 locus. Analysis of these mice demonstrated that RAG2:GFP expression occurred in appropriate stages of developing B and T cells, as well as in immature IgM+ B cells in the bone marrow, extending previous observations that RAG expression continues after surface antigen receptor deposition in both developing B and T cells. We also determined that there are substantial numbers of splenic RAG-expressing cells which mostly resemble pre-B cells. This population decreases in size with age, but reappears following immunization of older RAG2:GFP mice, suggesting a novel role for pre-B-like cells in the immune response. To uncover the specific cis-elements that regulate RAG2 gene expression in vivo, we developed a novel genetic assay. Using this assay, we identified genomic sequences in the region 5' of RAG2 that are capable of driving RAG2 expression in developing B and T cell lineages. Deletional analyses of these sequences further demonstrated that RAG2 is regulated differentially in B versus T lineages by elements upstream of the promoter and that the sequences sufficient to direct B cell expression are located more promoter proximal. To elucidate mechanisms involved in the developmental regulation of TCRalpha/delta genes, we deleted the TCRdelta enhancer (Edelta) from the endogenous locus. We determined that Edelta is important, but not absolutely required, for efficient TCRdelta rearrangement and gammadelta T cell development. Simultaneous deletion of both Edelta and the TCRalpha enhancer (Ealpha) demonstrated that residual TCRdelta rearrangements were not driven by Ealpha, implicating additional elements in TCRdelta locus accessibility. Surprisingly, while deletion of Edelta severely impaired germline TCRdelta expression in double-negative thymocytes, absence of Edelta did not affect expression of mature delta transcripts in gammadelta T cells. We conclude that Edelta has an important role in TCRdelta locus regulation at early, but not late, stages of gammadelta T cell development.