Clone And Identification Of The Promoter Of The Stimulator Of Interferon Genes

Objective:To clone STING gene promoter and identify its core promoter region and then clarify the regulatory mechanism for STING gene expression.Methods:The genomic DNA extracted from HEK 293 cells was as a template for PCR amplification to obtain the promoter region sequence of STING.Then the promomoter region sequence was directly subcloned into the multiple cloning sites of pGL3-Basic vector to construct recombinant reporter plasmid.Transfected HEK 293 cells,the luciferase activity measured using Dual-Luciferase reporter assay system was calculated by relative activity units(RLU)to identify its promoter activity.Piecewise missing was conducted at STING gene 5' end using deletion analysis method,multiple truncated fragment was inserted into the basic vector pGL3-Basic,detected the double luciferase activity of each truncated fragments to find the core functional areas of STING promoter.Using point mutagenesis,RNA interference,gene over-expression and chromatin immunoprecipitation(ChIP)experiments to analyze the influence of transcription factors to the promoter activity of STING promoter region.Results:It was confirmed that by double digestion and sequencing the double reporter plasmid containing STING promoter region sequence was constructed successfully.After analysis of luciferase activity,compared with the basic vector pGL3-Basic,the promoter of STING showed very significant activity in HEK 293 cells.Analysis of the promoter activity of the truncated fragments found,the promoter activity of truncated fragments at-124?+ 1 bp was close to the maximum,suggesting the core promoter region of STING is located between-124?+ 1 bp.STING promoter region contains some binding sites including SRY?Sp1?E2F?HOX?CREB?c-Myc and GATA-1 so on by using bioinformatics analysis.Point mutation results showed that the binding sites of transcription factors CREB and c-Myc maintained STING basic transcriptional activity.ChIP experimental results showed that the binding of CREB and c-Myc and STING promoter region.RNA interference and gene overexpression experiments also showed that CREB and c-Myc can promote STING promoter activity.Conclusion:The reporter plasmids constructed successfully of STING gene promoter showed strong promoter activity,and the core promoter region was found at the-124 to +1 bp region of the upstream of the transcription start site.The transcription factors CREB and c-Myc have positive regulation to STING promoter activity.