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

Abstract

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.

ACKNOWLEDGMENTS

We thank Peggy J. Farnham (University of Wisconsin) for sharing the chromatin immunoprecipitation protocol and Kathryn E. Boyd (Yale) for generous technical assistance with this assay. We also thank Stephen T. Smale (UCLA) for his DNase I genomic footprinting protocol. We are grateful to the late Eugenia Spanopoulou for providing us with 293T cells and the pEF-BOS expression vector and Chi Dang (Hopkins) for sharing with us the murine c-Myb cDNA. We also thank Astar Winoto, Alan Friedman, Shau-Ku Huang, Laurent Bentolila, Hans Brightbill, Jamie Geier, and various members of Schlissel laboratory for critical reading of the manuscript.

This research was funded in part by NIH grants RO1 AI40227 and HL48702 to M.S.S., who also acknowledges the support of a Leukemia Society Scholarship.