Distinct Factors Regulate the Murine RAG-2 Promoter in B- and T-Cell Lines

Abstract

The recombination activating genes RAG-1 and RAG-2 are expressed in a lymphoid-cell-specific and developmentally regulated fashion. To understand the transcriptional basis for this regulation, we have cloned and characterized the murine RAG-2 promoter. The promoter was lymphoid cell specific, showing activity in various B- and T-cell lines but little activity in nonlymphoid cells. To our surprise, however, the promoter was regulated differently in B and T cells. Using nuclear extracts from B-cell lines, we found that the B-cell-specific transcription factor BSAP (Pax-5) could bind to a conserved sequence critical for promoter activity. BSAP activated the promoter in transfected cells, and the BSAP site was occupied in a tissue-specific manner in vivo. An overlapping DNA sequence binding to a distinct factor was necessary for promoter activity in T cells. Full promoter activity in T cells was also dependent on a more distal DNA sequence whose disruption had no effect on B-cell activity. The unexpected finding that a B-cell-specific factor regulates the RAG-2 promoter may explain some of the recently observed differences in the regulation of RAGtranscription between B and T cells.

ACKNOWLEDGMENTS

We thank Eugenia Spanopoulou for providing us with 293T cells and the pEF-BOS expression vector, Meinrad Busslinger for sharing with us anti-BSAP antisera, and Steven Desiderio and Patty Zwollo for providing the BSAP cDNA. We thank Cynthia Guidos, Antony Rosen, David Schatz, Robert Siliciano, and Stephen Smale for gifts of various cell lines. We also thank Chi Dang for access to his luminometer and Qian-Fei Wang for performing gel mobility shift analyses using recombinant BSAP. This paper was improved by criticisms offered by Chi Dang and Kees Murre, by various members of the Schlissel lab, and by anonymous reviewers.

M.S.S. acknowledges the support of NIH grant HL48702, the W. W. Smith Foundation, and the Leukemia Society of America. J.L. is a Howard Hughes Medical Institute predoctoral fellow and a member of the Biochemistry, Cellular and Molecular Biology Training Program at Johns Hopkins.