c-Myb binds to a sequence in the proximal region of the RAG-2 promoter andis essential for promoter activity in T-lineage cells

The RAG-2 gene encodes a component of the V(D)J recombinase which is essent ial for the assembly of antigen receptor genes in B and T lymphocytes. Prev iously, we reported that the transcription factor BSAP (PAX-5) regulates th e murine RAG-2 promoter in B-cell lines. A partially overlapping but distin ct region of the proximal RAG-2 promoter was also identified as an importan t element for promoter activity in T cells; however, the responsible factor was unknown. In this report, we present data demonstrating that c-Myb bind s to a Myb consensus site within the proximal promoter and is critical for its activity in T-lineage cells. We show that c-Myb can transactivate a RAG -2 promoter-reporter construct in cotransfection assays and that this trans activation depends on the proximal promoter Myb consensus site. By using a chromatin immunoprecipitation (ChIP) strategy, fractionation of chromatin w ith anti-c-hylb antibody specifically enriched endogenous RAG-2 promoter DN A sequences. DNase I genomic footprinting revealed that the c-Myb site is o ccupied in a tissue-specific fashion in vivo, Furthermore, an integrated RA G-2 promoter construct with mutations at the c-Myb site was not enriched in the ChIP assay, while a wild-type integrated promoter construct was enrich ed. Finally, this lack of binding of c-Myb to a chromosomally integrated mu tant RAG-2 promoter construct in vivo was associated with a striking decrea se in promoter activity. We conclude that c-Myb regulates the RAG-2 promote r in T cells by binding to this consensus c-Myb binding site.