人RAG2基因5′近端染色质结构定量差异分析及可接近区域转录调控机制研究

Quantitative difference analysis of chromatin structure across the 5′proximal region of human RAG2 and study of its transcriptional regulatory mechanisms on the accessible areas

目的研究人类重组激活基因2(RAG2)5′近端染色质可接近区域调控RAG2表达的分子机制。方法采用染色质可接近性实时聚合酶链反应(CHART-PCR)分析RAG2基因5′近端区域的DNaseⅠ超敏位点(DHS),比较RAG阳性和阴性淋巴细胞以及非淋巴细胞的染色质开放状态变化。采用双荧光素酶报告基因分析DHS的转录调控活性,采用胶迁徙试验和染色质免疫沉淀试验证实GATA3的体外(in vitro)和在体(in vivo)结合,采用PCR定点突变和显性负突变体技术证实GATA3对RAG2基因的调控作用。结果人RAG2基因5′上游近端区域染色质在T与B细胞中处于不同的开放状态,RAG阳性T细胞的核心启动子活性与其特异性DHS相吻合。T细胞特异性转录因子GATA3结合于T细胞特异性DHS的高度保守区域,特异性上调报告基因及在体RAG2 m RNA在T细胞中的表达。结论 RAG2基因5′近端区域通过染色质开放状态变化调控基因表达的特异性。GATA3与高度保守的DHS区域结合,上调内源性人RAG2在T细胞中的表达。

Objective To study molecular mechanism of the regulation of RAG2 expression regulated the human recombination activating gene 2（RAG2） 5′proximal chromatin accessible areas.Methods To compare the open states change of chromatin of RAG positive and negative lymphocytes as well as non lymphocyte chromatin,chromatin accessibility by real-time polymerase chain reaction（CHART-PCR） was used for DNase Ⅰ analysis of RAG2 gene 5′proximal regions of hypersensitive sites（DHS）.Double luciferases reporter assay was used to examine the transcriptional regulation effects of the DHS regions.Electrophoresis mobility shift assay and chromatin immunoprecipitation assays were used to detect the binding of GATA3 in vitro or in vivo.PCR site-directed mutant assay and dominant-negative mutant assay were used to identify the regulatory effects of GATA3 on the reporter gene（in vitro）,as well as on RAG2 gene（in vivo）.Results In T and B cells,the chromatin structures of 5′ proximal region of human RAG2 were in distinct open states.In RAG positive T cells,the activity of RAG2 core promoter corresponded to its specific DHS.GATA3,a T-cell specific transfactor,bound to a highly conserved sequence within the specific DHS,and increased the expression of the reporter genes as well as RAG2 m RNA of the T cells in vivo.Conclusion RAG2 gene 5′proximal area through open states change of chromatin regulate the specificity of the gene expression,GATA3 regulates the expression of endogenous RAG2 in T cells by binding to highly conserved DHS regions.