title: Characterization of chromatin structure and enhancer elements for murine recombination activating gene-2.
abstract: Recombination-activating genes (RAGs) play a critical role in V(D)J recombination machinery and their expression is specifically regulated during lymphocyte ontogeny. To elucidate the molecular mechanisms regulating murine RAG-2 expression, we examined a chromatin structure of 25-kb DNA segment adjacent to murine RAG-2 by analyzing DNase I hypersensitive (HS) sites. In a RAG-2-expressing murine pre-B cell line, three lymphoid cell-specific HS sites (HS1, HS2, and HS3) were identified. Among these HS sites, one HS site (HS3) that locates in the RAG-2 promoter was associated only with RAG-2-expressing cell lines. Using the transient enhanced green fluorescence protein reporter gene assays, we identified two enhancer elements in the 5'-upstream region of RAG-2 that corresponded to HS1 and HS2. One of the enhancer elements (D3) exhibited enhancer activity only in the lymphoid cell lines. Analysis of the transgenic mice carrying the enhanced green fluorescence protein-reporter gene linked with D3 revealed that D3 activated the reporter gene-expression in the primary lymphoid tissues, but not in the secondary lymphoid tissues or nonlymphoid tissues. D3 was active in CD4(-)CD8(-), but not in CD4(+)CD8(+) or CD4(+)CD8(-) thymocytes in the thymus, and also active in B220(+)IgM(-), but not in B220(+)IgM(+), cells in the bone marrow. Finally, our data suggested that C/EBP may bind to the D3 enhancer and function as one of the transcription factor(s) responsible for the enhancer activity. These results show that the tissue- and stage-specific expression of murine RAG-2 is regulated by alteration of the chromatin structure as well as cis-regulatory enhancer elements.
authors.affiliation: Department of Immunology, Faculty of Medicine, Toyama Medical and Pharmaceutical University, Sugitani, Toyama, Japan.
authors.forename: Xing-Cheng
authors.initials: XC
authors.lastname: Wei
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Kishi
authors.forename: Zhe-Xiong
authors.initials: ZX
authors.lastname: Jin
authors.forename: Wen-Pu
authors.initials: WP
authors.lastname: Zhao
authors.forename: Sachiko
authors.initials: S
authors.lastname: Kondo
authors.forename: Tadashi
authors.initials: T
authors.lastname: Matsuda
authors.forename: Shigeru
authors.initials: S
authors.lastname: Saito
authors.forename: Atsushi
authors.initials: A
authors.lastname: Muraguchi
mesh_terms: D016475:3T3 Cells; D020121:5' Untranslated Regions / Q000276:immunology / Q000378:metabolism; D000818:Animals; D001483:Base Sequence; D019070:Cell Lineage / Q000235:genetics / Q000276:immunology; D002843:Chromatin / Q000737:chemistry* / Q000235:genetics*; D053938:DNA Fragmentation / Q000276:immunology; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics*; D004742:Enhancer Elements, Genetic / Q000276:immunology*; D018507:Gene Expression Regulation, Developmental / Q000276:immunology; D017930:Genes, Reporter / Q000276:immunology; D049452:Green Fluorescent Proteins; D007739:L Cells; D008164:Luminescent Proteins / Q000235:genetics; D008214:Lymphocytes / Q000276:immunology / Q000378:metabolism; D051379:Mice; D008822:Mice, Transgenic; D008969:Molecular Sequence Data; D009928:Organ Specificity / Q000235:genetics / Q000276:immunology; D011401:Promoter Regions, Genetic / Q000276:immunology; D011485:Protein Binding / Q000235:genetics / Q000276:immunology; D011995:Recombination, Genetic / Q000276:immunology*; D014407:Tumor Cells, Cultured
chemical_list: D020121:5' Untranslated Regions; D002843:Chromatin; D004268:DNA-Binding Proteins; D008164:Luminescent Proteins; C499308:Rag2 protein, mouse; C064655:V(D)J recombination activating protein 2; D049452:Green Fluorescent Proteins