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Molecular and Cellular Biology, December 2000, p. 9203-9211, Vol. 20, No. 24
0270-7306/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

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

Qian-Fei Wang,dagger Josh Lauring,Dagger and Mark S. Schlissel*

Department of Molecular and Cell Biology, University of California, Berkeley, California 94720-3200

Received 10 August 2000/Returned for modification 9 September 2000/Accepted 19 September 2000

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.

* Corresponding author. Mailing address: Department of Molecular and Cell Biology, 439 LSA (#3200), University of California, Berkeley, CA 94720-3200. Phone: (510) 643-2462. Fax: (510) 642-6845. E-mail: mss{at}uclink4.berkeley.edu.

dagger Present address: Graduate Program in Cellular and Molecular Medicine, Johns Hopkins University School of Medicine, Baltimore, MD 21205.

Dagger Present address: Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, MD 21205.

Molecular and Cellular Biology, December 2000, p. 9203-9211, Vol. 20, No. 24
0270-7306/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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