PMC:4608092 / 26449-29120 JSONTXT

{"project":"2_test","denotations":[{"id":"26126547-8594589-55294633","span":{"begin":387,"end":389},"obj":"8594589"},{"id":"26126547-22615451-55294634","span":{"begin":521,"end":523},"obj":"22615451"},{"id":"26126547-12671680-55294635","span":{"begin":524,"end":526},"obj":"12671680"},{"id":"26126547-10072525-55294636","span":{"begin":829,"end":831},"obj":"10072525"}],"text":"Induction of RNF213 by IFN-β Is Mediated by STATx\nWe examined the putative binding sites for transcriptional factors in the promoter region up to 3 kb from the transcriptional start site of the RNF213 gene. We performed a computer search for potential regulatory elements in this region using MatInspector V2.2 at the TRANSFAC website (http://www.gene-regulation.com/pub/databases.html).24 We found a single STATx-binding site at the −514 position (Figure6A). STAT1 is a signaling molecule in the IFN-β-signaling pathway.25,26 Therefore, we conducted promoter assays using a fusion plasmid containing the 3-kb RNF213 promoter region and a luciferase reporter gene. The promoter significantly increased luciferase activity after treatment with IFN-β (Figure6B), whereas disruption of the STATx-binding site by missense mutagenesis27 abrogated promoter activity (Figure6C). Therefore, we conclude that IFN-β upregulates RNF213 through the STATx-binding site in its promoter region.\nFigure 6 Activation of RNF213 promoter activity in HUVECs after IFN-β treatment. A, Constructs for the RNF213 gene promoter-luciferase fusion plasmids and the sequences of the STATx-binding site (potential STATx1-binding site; WT or mutant). Mutated nucleotides are underlined. B, Dose-response effects of IFN-β on RNF213 gene promoter activity. Each column with a bar represents mean±SD of 3 independent experiments. Blue columns represent pGL4.14 vector. Red columns represent RNF213 WT promoter pGL4.14 vector. Fold represents relative mean luciferase activity of RNF213 WT promoter pGL4.14 to PGL4.14. *P\u003c0.05, luciferase activity at 0.1, 1, or 10 ng/mL of IFN-β were compared with 0 ng/mL of IFN-β in cells transfected with RNF213 WT promoter pGL4.14 (red) by Student t test. #P\u003c0.05 luciferase activity in cells transfected with RNF213 WT promoter pGL4.14 (red) was compared with cells transfected with pGL4.14 (blue) at the same IFN-β dose by Student t test. C, Mutation analysis of RNF213 gene promoter activity in HUVECs. HUVECs were transiently transfected with the RNF213 WT promoter pGL4.14 plasmid or RNF213 STATx mutation promoter pGL4.14 plasmid, and luciferase activities were measured after 24 hours of IFN-β treatment. Fold inductions by 10 ng/mL of IFN-β (red) were compared with the activities of IFN-β 0 ng/mL (blue). Each column with a bar represents mean±SD of 3 independent experiments. *P\u003c0.05, by Student t test compared with 0 ng/mL of IFN-β with the same promoter. #P\u003c0.05 by Student t test compared between RNF213 WT promoter and mutation promoter at 10 ng/mL of IFN-β. HUVECs indicates human umbilical vein endothelial cells; IFN, interferon; WT, wild type.\n\nR"}