Dissecting the Epigenetic Regulation of V-beta Recombination

Abstract

V(D)J recombination in developing lymphocytes is essential for producing a diverse repertoire of antigen receptors (TCR and Ig). During recombination, the proteins encoded by the recombinase activating genes (RAG1 and RAG2) bind specific DNA sequences flanking individual V, D, and J coding segments within each antigen receptor gene, and introduce double strand DNA breaks at the coding sequence⁄targeting sequence boundaries. These double strand breaks are then repaired by ubiquitous DNA repair machinery to generate novel coding segment joints. The ability of each developing lymphocyte to independently assemble unique V(D)J joints results in the enormous diversity of antigen receptors expressed by our immune system. Despite a conserved enzymatic activity in both B and T lymphocytes, the assembly of T cell receptors (TCRs) and Immunoglobulins (Igs) in T and B cells respectively follows a highly orchestrated program in which the accessibility of individual targeting sequences varies during lymphocyte development. For example, when the TCRb locus is rearranged, it initially assembles joints between D and J elements. Only after DJ joining do upstream V sequences become accessible and rearrange with the preassembled DJ's. We have previously shown that DJ rearrangement requires modification of the chromatin structure surrounding individual D and J segments via the coordinated actions of D-associated promoters and a single downstream enhancer. Like the D elements, each V element in TCRb is associated with a transcriptional promoter. But the role these V promoters play in V-to-DJ recombination remains unknown. Similarly, because enhancer deletion ablates D-to-J assembly, the potential role of enhancer activity in V recombination has not been directly tested. We hypothesize that V-to-DJ rearrangement requires both enhancer and promoter-dependent changes in the chromatin surrounding the V RAG binding site, as well as that surrounding the D 5' binding site. To test this hypothesis, I have constructed a panel of recombination substrates which harbor unrearranged or prerearranged DJ elements downstream from a single V element. These "miniloci" were stably transfected into the chromatin of a recombinase-inducible T cell line, and the chromatin status, expression and recombination potential of each was assessed.