RAG2is regulated differentially in B and T cells by elements 5′ of the promoter

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Abstract
To studyRAG2gene regulationin vivo, we developed a blastocyst complementation method in whichRAG2-deficient embryonic stem cells were transfected with genomic clones containingRAG2and then assessed for their ability to generate lymphocytes. ARAG2genomic clone that contained only theRAG2promoter sequences rescued V(D)J recombination inRAG2-deficient pro-B cell lines, but did not rescue development ofRAG2-deficient lymphocytesin vivo. However, inclusion of varying lengths of sequences 5′ of theRAG2promoter generated constructs capable of rescuing onlyin vivoB 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 forRAGexpression sufficient to generate normal B or T cell numbers, implying redundancy among 5′ elements. We conclude thatRAG2expressionin vivorequires 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.