Cloning And Identification Of The Promoter Of The Murine Stimulator Of Interferon Gene

Backgroud:The innate immune system is the first line of defense against viral infections.STING is an important adapter protein in the signaling pathway of anti-DNA viral infection.It can also recognize cAMP and cGMP directly as pattern recognition receptor when involved in anti-intracellular bacterial infection.It is also participate in the anti-RNA virus immune.More studies revealed that STING plays an important role in anti-tumor immunity and autoimmune diseases.The current researches on STING are almost focused on its molecular mechanism of activation of downstream kinase TBK1 and transcription factor IRF3 and its function in the process of innate immune response induced by different pathogen-associated molecular models.STING is highly conserved in the evolution,and STING in human and murine has quiet high homology.The mechanism of expression of murine STING is known little.In this study,we focused on the characterization and regulation mechanism of murine STING promoter.Objective:The objective of this study was to clone the promoter sequences of murine STING and identify its core promoter region,and then to investigate the transcripation factors involved in the promoter activity,then clarify the regulatory mechanism of murine STING gene expression.Methods:Based on bioinformatics database,the location of murine STING promoter was predicted.The genomic DNA extracted from NIH3T3 cells was used as a template for PCR amplification to obtain the promoter region sequence of STING.Then the sequence was directly subcloned into the multiple cloning sites of poromoterless pGL3-Basic vector to construct recombinant reporter plasmid.Transfected into NIH3T3 and HEK 293 cells,the luciferase activity was measured by Dual-Luciferase reporter assay system to identify its promoter activity.Then,six promoter fragments with different length were obtained by walking deletion and cloned into pGL3-basic vector.The promoter activities of those plasmids were determined by relative luciferase activity and to find the minimal functional area of STING promoter.Bioinformatics methods were used to predict the potential transcriptional factor bingding sites.Point mutagenesis,si-RNA interference,gene over-expression and chromatin immunoprecipitation(ChIP)experiments were used to analyze the influence of transcription factors to the promoter activity of murine STING gene.Results:It was confirmed that by double digestion and sequencing that the recommbined reporter plasmid containing murine STING promoter region was constructed successfully.Compared with the basic vector pGL3-Basic,the promoter of STING showed significant activity in NIH3T3 cells and HEK 293 cells.Analysis of the promoter activities of the truncated fragments found that the minimal promoter region was located between-77～+177 nt to TSS(Transcription Start Site).Bioinformatics analysis showed that the minimal STING promoter region contained some potential transcription factor binding sites including GATA-1、Sp1/Sp3、STAT and IK2.Point mutation results showed that the binding sites of transcription factors GATA1 and Sp1/Sp3 maintained STING basic transcriptional activity.si-RNA interference and gene overexpression experiments showed that GATA1 and Sp3 can positively regulate STING promoter activity.ChIP experimental results showed that the transcription factors GATA1 and Sp3 can bind directly to the murine STING promoter region.Conclusion:The murine STING gene promoter recommbined reporter plasmids were constructed successfully and showed strong promoter activities.And the core promoter region was located at the-77 to +177 nt toTSS.The transcriptional factors GATA1 and Sp3 had positive regulation to the murine STING promoter activity.