c-Myb binds to a sequence in the proximal region of the RAG-2 promoter and is essential for promoter activity in T-lineage cells.
The RAG-2 gene encodes a component of the V(D)J recombinase which is essential for the assembly of antigen receptor genes in B and T lymphocytes. Previously, we reported that the transcription factor BSAP (PAX-5) regulates the murine RAG-2 promoter in B-cell lines. A partially overlapping but distinct region of the proximal RAG-2 promoter was also identified as an important element for promoter activity in T cells; however, the responsible factor was unknown. In this report, we present data demonstrating that c-Myb binds 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 transactivation depends on the proximal promoter Myb consensus site. By using a chromatin immunoprecipitation (ChIP) strategy, fractionation of chromatin with anti-c-Myb antibody specifically enriched endogenous RAG-2 promoter DNA sequences. DNase I genomic footprinting revealed that the c-Myb site is occupied in a tissue-specific fashion in vivo. Furthermore, an integrated RAG-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 enriched. Finally, this lack of binding of c-Myb to a chromosomally integrated mutant RAG-2 promoter construct in vivo was associated with a striking decrease in promoter activity. We conclude that c-Myb regulates the RAG-2 promoter in T cells by binding to this consensus c-Myb binding site.
Wang
Q F
QF
Department of Molecular and Cell Biology, University of California, Berkeley, California 94720-3200, USA.
Lauring
J
J
Schlissel
M S
MS
D000818:Animals; D002460:Cell Line; D019070:Cell Lineage; D004247:DNA / Q000378:metabolism; D018983:DNA Footprinting; D004254:DNA Nucleotidyltransferases / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics*; D004591:Electrophoresis, Polyacrylamide Gel; D017930:Genes, Reporter; D006801:Humans; D051379:Mice; D008810:Mice, Inbred C57BL; D009687:Nuclear Proteins; D011233:Precipitin Tests; D011401:Promoter Regions, Genetic / Q000235:genetics*; D020598:Proto-Oncogene Proteins c-myb / Q000235:genetics / Q000378:metabolism*; D013601:T-Lymphocytes / Q000502:physiology*; D014158:Transcription, Genetic; D014162:Transfection; D019895:Transposases / Q000235:genetics*; D045567:VDJ Recombinases
D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D020598:Proto-Oncogene Proteins c-myb; C499311:RAG2 protein, human; C499308:Rag2 protein, mouse; C064655:V(D)J recombination activating protein 2; D004247:DNA; D004254:DNA Nucleotidyltransferases; D019895:Transposases; D045567:VDJ Recombinases