鸡miR-17-92基因簇上游A/T富集区启动子活性鉴定与分析

Identification and analysis of promoter activity of A/T-rich region upstream of chicken miR-17-92 cluster

文章采用基因定点突变和报告基因技术作A/T富集区启动子鉴定和转录调控分析,研究鸡miR-17-92基因簇上游A/T富集区是否具有启动子活性。PromPredict预测分析提示miR-17-92基因簇上游-388^-444 bp处可能是启动子区域。转录因子结合位点分析发现,A/T富集区存在3个E2F1潜在结合位点,分别位于miR-17-92基因簇上游-1 273 bp(结合位点1)、-1 186 bp(结合位点2)和-753 bp(结合位点3)处。报告基因活性分析发现,鸡miR-17-92基因簇上游A/T富集区具有启动子活性,其中miR-17-92基因簇上游-440/-1区域启动子活性最强。共转染分析显示,转录因子E2F1极显著抑制该A/T富集区启动子活性(P<0.01);进一步定点突变分析表明E2F1通过E2F1结合位点1和2抑制A/T富集区启动子活性。报告基因分析发现,与对A/T富集区启动子作用不同,E2F1促进miR-17-92基因簇宿主基因(MIR17HG)启动子活性。文章首次发现鸡miR-17-92基因簇上游存在一个新的转录调控区,对揭示鸡miR-17-92基因簇转录调控具有重要意义。

The A/T- rich genomic region upstream of miR- 17- 92 cluster was analyzed by online bioinformatics software. The promoter of the A/T- rich region and its transcriptional regulation was investigated by reporter gene assay and the site- directed mutagenesis. PromPredict analysis showed that a putative promoter region was located from -388 to -444 bp upstream of chicken miR-17-92 cluster. Transcription factor binding site analysis showed that there were three potential E2F1 binding sites located within the A/T- rich region:- 1 273 bp (binding site 1),- 1 186 bp (binding site 2) and - 753 bp (binding site 3) upstream of the miR- 17- 92 cluster. The reporter gene assay showed that the A/T- rich region had promoter activity, and the -440 bp region (-440 to -1 bp) upstream of the miR-17-92 cluster had the strongest promoter activity. Cotransfection analysis showed that the transcription factor E2F1 significantly inhibited the promoter activity of the A/T- rich region (P<0.01). Further site- directed mutagenesis showed that E2F1 inhibited the promoter activity via E2F1 binding sites 1 and 2. Reporter gene analysis showed that, unlike its effect on the promoter activity of the A/T- rich region, E2F1 enhanced the promoter activity of miR- 17- 92 cluster host gene (MIR17HG). The present study first identified a new transcription regulatory region upstream of the chicken miR-17-92 cluster. The outcome of the present study pave the way for elucidating the transcriptional regulation mechanisms of chicken miR-17-92 cluster.