Characterization of the proximal enhancer element and transcriptional regulatory factors for murine recombination activating gene‐2

Abstract

Recombination-activating gene (RAG)-1 and RAG-2 are essential for V(D)J recombination and are expressed specifically in lymphoid cells. We previously identified two putative enhancer elements, the proximal and distal enhancers, located at -2.6 and -8 kb, respectively, 5 0 upstream of mouse RAG-2, and characterized the distal enhancer element in detail. In this study, to characterize the proximal enhancer in vitro as well as in vivo, we first defined a 170-bp core enhancer element within the proximal enhancer (Ep) and determined its activity in various cells. Ep conferred enhancer activity only in B-lymphoid cell lines, but not in T-or non-lymphoid cell lines. Analysis of the transgenic mice carrying an EGFP reporter gene linked with Ep revealed that Ep activated the transcription of the reporter gene in bone marrow and spleen, but not in thymus or non-lymphoid tissues. Ep was active in both B220 + IgM -and B220 + IgM + subpopulations in the bone marrow and in the B220 + subpopulation in the spleen. Using electrophoretic mobility shift assays and mutational assays, we found that Ikaros and CCAAT/enhancer binding protein cooperatively bind Ep and function as the transcription factors responsible for B cell-specific enhancer activity. These results demonstrate the role of Ep as a cis-regulatory enhancer element for RAG-2-specific expression in B-lymphoid lineages. IntroductionAssembly of Ig and TCR genes by V(D)J recombination is mediated by the products of recombination-activating gene (RAG)-1 and RAG-2 [1, 2]. RAG proteins act together to recognize recombination signal sequences that flank V, D, and J gene segments and introduce the double-stranded DNA breaks between the recombination signal sequences and the gene segments [3]. The expression of RAG-1 and RAG-2 is restricted to lymphoid cells undergoing V(D)J rearrangement [4, 5]. In the absence of either RAG-1 or RAG-2 gene products, the development of mature lymphocytes is completely abrogated and hence results in severe combined immune deficiency in mouse and human [6][7][8]. In human, missense mutations in either RAG-1 or RAG-2 result in the Omenn syndrome, which is characterized by a poor T lymphocyte repertoire [9].There are two waves of RAG-1 and RAG-2 expression in bone marrow B cell precursors. RAG are expressed first in pro-B cell progenitors undergoing Ig heavy-chain gene rearrangement, and the second expression of RAG is seen during Ig j or k light-chain gene rearrangement occurring in pre-B cells [4,10]. In T cell development, RAG areThe first two authors contributed to this work equally. first expressed in pro-T cells when rearrangement of the TCRb chain gene occurs, and secondary expression of RAG occurs when the TCRa chain loci undergo V-J rearrangement to produce mature T cells [11,12]. The transcription of RAG is regulated at different levels. At the chromatin level, Fuller et al. and we reported that a DNase I-hypersensitive (HS) site was identified in the promoter region of mouse and human RAG-1 only in RAG-expressing lymphocytes [13,14]. We a...

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