摘要
目的鉴定二甲基精氨酸二甲胺水解酶2（DDAH2）基因启动子区的一个NF-κB反应元件，证明其在DDAH2转录激活中的作用。方法应用多种软件分析DDAH2启动子区，寻找潜在的转录因子结合位点；构建系列截短的DDAH2基因启动子荧光素酶报告基因载体，转染人胚肾HEK293细胞，通过荧光素酶活性检测分析各段启动子的活性；应用电泳泳动迁移实验（electrophoresis mobility shift assay，EMSA）和染色质免疫沉淀（chromatin immunoprecipitation，CHIP）方法分别在体外及体内鉴定NF-κB反应元件；构建NF-κB位点突变的DDAH2基因启动子荧光素酶报告基因载体，转染细胞，比较其与野生型启动子活性差异。结果DDAH2基因启动子区存在多种潜在的转录因子结合位点；DDAH2的各段启动子具有高转录活性，其中-530～-437区域为一个正调控区域；DDAH2基因启动子-476～469区为NF-κB反应元件，NF-κB可特异性结合于该位点；该NF-κB元件突变使DDAH2基因启动子活性显著下降。结论DDAH2基因启动子-476～469区为NF-κB反应元件，该元件在DDAH2转录激活中具有重要的作用。
Objective To identify a NF-κB responsive element within the dimethylarginine dimethylaminohydrolase 2 gene （DDAH） promoter and demonstrate its role in DDAH2 transactivation. Methods DDAH2 promoter was analyzed with software to identify potential binding sites of transcription factors. A series of truncated DDAH2 promoter luciferase reporter plasmids were constructed and transfected into human embryonic kidney derived HEK293 cells. Luciferase assays were carried out to analyze the activity of the promoter. Electrophoresis mobility shift assay （EMSA） and chromatin immunoprecipitation （CHIP） were used to identify the NF-κB responsive element in vitro and in vivo. DDAH2 promoter luciferase reporter plasmid with mutated NF-κB site was constructed and transfected into cells, and its activity was compared with that of the wild-type plasmid. Results Potential bindings sites of many transcription factors were found within the DDAH2 promoter. The transcription activity of the DDAH2 promoter was high, and -530 to -437 was a positive regulating region. -476 to --469 of the DDAH2 promoter was a NF-κB responsive element, to which NF-κB can specifically bind. Mutation of the NF-κB element could significantly decrease the DDAH2 promoter activity. Conclusion - 476 to - 469 of the DDAH2 promoter was a NF-κB responsive element and is important for the transactivation of DDAH2.
出处
《中华医学遗传学杂志》
CAS
CSCD
北大核心
2014年第3期322-326,共5页
Chinese Journal of Medical Genetics
基金
国家自然科学基金（30900807）
