Differential activation of dual promoters alters Dβ2 germline transcription during thymocyte development

Ag receptor genes are assembled through somatic rearrangements of V, D, and J gene segments. This process is directed in part by transcriptional enhancers and promoters positioned within each gene locus. Whereas enhancers coordinate reorganization of large chromatin stretches, promoters are predicted to facilitate the accessibility of proximal downstream gene segments. In TCR beta locus, rearrangement initiates at two D-J cassettes, each of which exhibits transcriptional activity coincident with DJ rearrangement in CD4/CD8 double-negative pro-T cells. Consistent with a model of promoter-facilitated recombination, assembly of the DJ beta 1 cassette is dependent on a D beta 1 promoter (PD beta 1) positioned immediately 5' of the D. Assembly of DJ beta 2 proceeds independent from that of DJ beta 1, albeit with less efficiency. To gain insight into the mechanisms that selectively alter D usage, we have defined transcriptional regulation at D beta 2. We find that both DJ beta cassettes generate germline messages in murine CD44(+)CD25(-) double-negative 1 cells. However, transcription of unrearranged DJ beta 2 initiates at multiple sites 400-550 bp downstream of the D beta 2. Unexpectedly, loci from which germline promoter activity has been deleted by DJ rearrangement redirect transcription to sites immediately 5' of the new DJ beta 2 joint. Our analyses suggest that 3'-PD beta 2 activity is largely controlled by NF-kappa B RelA, whereas 5'-PD beta 2 activity directs germline transcription of DJ beta 2 joints from initiator elements 76 bp upstream of the D beta 2 5' recombination signal sequence. The unique organization and timing of D beta 2 promoter activity are consistent with a model in which promoter placement selectively regulates the rearrangement potential of D beta 2 during TCR beta locus assembly.