Characterization of the proximal enhancer element and transcriptional regulatory factors for murine recombination activating gene‐2
Characterization of the proximal enhancer element and transcriptional regulatory factors for murine recombination activating gene‐2
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DOI：
10.1002/eji.200425185
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发表时间：
2005-02
影响因子：
5.4
通讯作者：
Xing-Cheng Wei;Jun-ichi Dohkan;H. Kishi;Chun-xiao Wu;Sachiko Kondo;A. Muraguchi
中科院分区：
文献类型：
--
作者：
Xing-Cheng Wei;Jun-ichi Dohkan;H. Kishi;Chun-xiao Wu;Sachiko Kondo;A. Muraguchi
增殖激活基因（RAG）-1和RAG-2是V（D）J重组所必需的，并在淋巴细胞中特异性表达。我们先前鉴定了两个推定的增强子元件，近端和远端增强子，分别位于小鼠RAG-2的5′上游的-2.6和-8 kb处，并详细表征了远端增强子元件。在这项研究中，为了在体外和体内表征近端增强子，我们首先在近端增强子（Ep）中定义了一个170 bp的核心增强子元件，并确定了它在各种细胞中的活性。EP仅在B淋巴细胞系中赋予增强子活性，但在T淋巴细胞系或非淋巴细胞系中不赋予增强子活性。对携带与Ep相连的EGFP报告基因的转基因小鼠的分析表明，Ep激活了骨髓和脾脏中报告基因的转录，但在胸腺或非淋巴组织中没有。Ep在骨髓中的B220+IgM-和B220+IgM+亚群以及脾脏中的B220+亚群中均具有活性。使用电泳迁移率变动分析和突变分析，我们发现Ikaros和CCAAT/增强子结合蛋白协同结合Ep，并作为负责B细胞特异性增强子活性的转录因子发挥作用。这些结果证明了Ep作为B-淋巴谱系中RAG-2特异性表达的顺式调节增强子元件的作用。
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′ 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.