RAG2 is regulated differentially in B and T cells by elements 5' of the promoter.
To study RAG2 gene regulation in vivo, we developed a blastocyst complementation method in which RAG2-deficient embryonic stem cells were transfected with genomic clones containing RAG2 and then assessed for their ability to generate lymphocytes. A RAG2 genomic clone that contained only the RAG2 promoter sequences rescued V(D)J recombination in RAG2-deficient pro-B cell lines, but did not rescue development of RAG2-deficient lymphocytes in vivo. However, inclusion of varying lengths of sequences 5' of the RAG2 promoter generated constructs capable of rescuing only in vivo B cell development, as well as other constructs that rescued both B and T cell development. In particular, the 2-kb 5' region starting just upstream of the RAG2 promoter, as well as the region from 2-7 kb 5', could independently drive B cell development, but not efficient T cell development. Deletion of the 2-kb 5' region from the murine germ line demonstrated that this region was not required for RAG expression sufficient to generate normal B or T cell numbers, implying redundancy among 5' elements. We conclude that RAG2 expression in vivo requires elements beyond the core promoter, that such elements contribute to differential regulation in the B vs. T lineages, and that sequences sufficient to direct B cell expression are located in the promoter-proximal 5' region.
Monroe
R J
RJ
Howard Hughes Medical Institute, The Children's Hospital, Center for Blood Research and Department of Genetics, Harvard Medical School, Boston, MA 02115, USA.
Chen
F
F
Ferrini
R
R
Davidson
L
L
Alt
F W
FW
D000818:Animals; D001402:B-Lymphocytes / Q000276:immunology*; D002460:Cell Line; D019070:Cell Lineage; D004268:DNA-Binding Proteins; D005786:Gene Expression Regulation / Q000276:immunology*; D051379:Mice; D009154:Mutation; D013601:T-Lymphocytes / Q000276:immunology*; D018909:src Homology Domains
D004268:DNA-Binding Proteins; C499308:Rag2 protein, mouse; C064655:V(D)J recombination activating protein 2