Li Yinghui, Li Guangyu, Li Chunyi, Zhao Yanyan
Department of Medical Genetics, China Medical University, Shenyang 110001, China.
Acta Biochim Biophys Sin (Shanghai). 2007 Apr;39(4):247-54. doi: 10.1111/j.1745-7270.2007.00280.x.
Transcriptional regulation of the neuronal nitric oxide synthase gene (nNOS) is particularly complex as 12 distinct transcripts derived from different first exons are expressed in a tissue- and cell-specific manner. The exon 1f mRNA is relatively highly expressed in nervous system and relies upon exon 1f-specific promoter activity. Using conventional and real-time reverse transcription-polymerase chain reaction, we found exon 1f mRNA was the major transcript of the nNOS gene in human neuroblastoma SK-N-SH cells. We analyzed a 1090 bp fragment of 1f promoter by TRANSFAC-TESS and Match softwares and luciferase assay, and found an important positive transcriptional regulation region that contained a putative nuclear factor (NF)-kappaB binding site. Subsequently, using electrophoresis mobility shift and chromatin immunoprecipitation assays, we identified this site to be the NF-kappaB responsive element, a crucial positive regulator in the activation of the nNOS 1f promoter. Taken together, our study identified an NF-kappaB responsive element within nNOS 1f promoter and showed that it plays an important role in the transactivation of nNOS 1f mRNA, the major transcript of nNOS in SK-N-SH cells.
神经元型一氧化氮合酶基因（nNOS）的转录调控尤为复杂，因为源自不同首个外显子的12种不同转录本以组织和细胞特异性方式表达。外显子1f mRNA在神经系统中相对高表达，并依赖于外显子1f特异性启动子活性。使用常规和实时逆转录-聚合酶链反应，我们发现外显子1f mRNA是人类神经母细胞瘤SK-N-SH细胞中nNOS基因的主要转录本。我们通过TRANSFAC-TESS和Match软件以及荧光素酶测定分析了1f启动子的1090 bp片段，发现了一个重要的正向转录调控区域，该区域包含一个假定的核因子（NF）-κB结合位点。随后，使用电泳迁移率变动分析和染色质免疫沉淀测定，我们确定该位点为NF-κB反应元件，是nNOS 1f启动子激活中的关键正向调节因子。综上所述，我们的研究在nNOS 1f启动子内鉴定出一个NF-κB反应元件，并表明它在SK-N-SH细胞中nNOS的主要转录本nNOS 1f mRNA的反式激活中起重要作用。
