Characterization Of Transcription Regulation Of Mouse DsbA-L Gene

Objective：Disulfide-bond-A oxidoreductase-like protein （DsbA-L）is a recentlyidentified adiponectin interactive protein，it plays an important role in the regulation ofadiponectin multimerization and insulin sensitivity. Overexpression of DsbA-L suppressesthe inhibitory effect of ER stress on adiponectin multimerization and stability.Thefat-specific DsbA-L transgenic mice displayed resistance to diet-induced obesity, insulinresistance, and hepatic steatosis. Recent study showed that DsbA-L is highly expressed inadipose tissus and the protein level increased dramatically during3T3-L1differentiation.The level of DsbA-L is negatively correlated with obesity in mice and humans. However,there is only limited information regarding the regulation of DsbA-L gene expression. Thisstudy focuses on the mouse DsbA-L gene promoter activity analysis, to explore theDsbA-L gene promoter and transcription factors involved in the expression of DsbA-L andthe possible mechanism.Methods：1. The luciferase reporter gene plasmid construction of DsbA-L gene promoter and5′or3′end of the series of deletion，were transfected into HEK293and3T3-L1cells，through the Dual luciferase assay system to detect the activity of different parts of thepromoter in order to determine the minimal promoter and cis-regulatory element essentialfor the transcriptional regulation．2. In view of the potential cis-regulatory element，construction of DsbA-L genemutant luciferase reporter gene plasmid．Dual luciferase assay system was used to detectthe activity changes with or without mutation in HEK293cells and3T3-L1cells to test thefunctional importance．3. Electrophoretic mobility shift assay（EMSA）and Chromatin immunoprecipitation assay（ChIP） were carried out to identify the Sp1-binding site．4. Dual luciferase assay system was used to test whether knock down of theendogenous Sp1protein by siRNA or overexpression of Sp1by transient transfection ofSp1vector could affect the DsbA-L promoter activity．5. The effect of high fat diet and3T3-L1differentiation on the transcription activityof DsbA-L were analyzed by electrophoretic mobility shift assay（EMSA）and real-timePCR.Results：1. The proximal promoter of DsbA-L located between-186to-34relative to TSS.2. A search for transcription factor binding sites using the software MAPPERrevealed the presence of a putative Sp1-binding site in the first intron region from+391to+432. EMSA and ChIP assays confirmed that Sp1bound specifically to this region.Overexpression and RNA interference experiments confirmed that DsbA-L promotertranscriptional activity might be negatively regulated by Sp1though the putativeSp1-binding sites.3. A gradual reduction in the binding activity of Sp1to the DsbA-L intronicsequence，as well as the protein level of Sp1，which coincided with the increases inDsbA-L mRNA levels in the course of3T3-L1adipogenesis.4. DsbA-L mRNA was significantly decreased in adipose tissues of HFD-fed miceand the binding activity of Sp1was markedly increased in adipose tissues of diet-inducedobesity mice as compared to those of ND-fed mice.Conclusion：Sp1acts as an inhibitor of the DsbA-L gene transcription via binding toDsbA-L intronic sequence.Part2. The mechanism of resveratrol on the regulation ofDsbA-L gene expressionObjective:To explore the potential mechanism of resveratrol-induced upregulation of theDsbA-L gene.Methods：1. The DsbA-L mRNA and protein levels were detected by real time quantitative PCR and western blot, respectively.2. The promoter activity of DsbA-L gene was detected by Dual Luciferase ReporterAssay System.3. The effect of resveratrol on the binding activity of transcription factor Sp1to theDsbA-L intronic sequence was analyzed by electrophoretic mobility shift assay（EMSA）.4. The effect of resveratrol on the expression of Sp1was determined by Westernblot.Results：1. Resveratrol increased the expression of mRNA and protein of DsbA-L in bothHepG2cells and3T3-L1cells.2. The luciferase activity of p(-186to+432)Luc, the plasmid containing DsbA-Lpromoter and intron1region encompassing a Sp1-binding site, was significantly increasedby treated with resveratrol, whereas resveratrol had no effect on the luciferase activity ofthe reporter plasmids, p(-186to+432)Lucmut and p(-186to+391)Luc, which did notcontain the Sp1site.3. Resveratrol reduced the binding of transcription factor Sp1to the Sp1-binding siteof DsbA-L, as well as the protein level of Sp1.Conclusions: Resveratrol may increase the expression of DsbA-L gene via repressionthe expression of Sp1.