Upstream Stimulatory Factor Binding to the E-box at −65 Is Required for Insulin Regulation of the Fatty Acid Synthase Promoter

Abstract

Fatty acid synthase (FAS) plays a central role in de novo lipogenesis in mammals. We have shown that FAS transcription rate is induced dramatically when fasted animals are refed with a high carbohydrate diet or when streptozotocin-diabetic mice are given insulin. We also reported that FAS gene transcription was up-regulated by insulin through the proximal promoter region from -71 to -50 and that upstream stimulatory factors (USFs), including USF1 and USF2, interact with this region in vitro. In the present study, by using site-directed mutagenesis of the -71/-50 region and correlating functional assays of the mutated promoter with USF binding activities, we demonstrate that the -65/-60 E-box motif (5'-CATGTG-3') is functionally required for insulin regulation and that USFs are in vivo components of the insulin response complex. Mutation of the -65/-60 E-box sequence abolished insulin response in both transiently and stably transfected 3T3-L1 adipocytes in the -2. 1 kb promoter context, which contains all the necessary regulatory elements of the promoter based on our previous transgenic mice studies, and in the minimal -67 promoter context. Gel mobility shift assays demonstrated that USFs can no longer bind to the -71/-50 promoter region when the E-box is mutated. Cotransfection of USF1 and USF2 expression vectors with the FAS promoter-luciferase reporter constructs increased insulin-stimulated FAS promoter activity. Moreover, cotransfection of dominant negative USF1 and USF2 mutants lacking the DNA binding domain inhibited the insulin stimulation of the FAS promoter activity. On the other hand, site-directed mutagenesis of the -65/-60 E-box surrounding sequences within the overlapped tandem copies of sterol regulatory element-binding protein (SREBP) binding sites prevented SREBP from binding to -71/-50 promoter region in vitro but had no effect on insulin regulation of the FAS promoter in vivo. When rat liver nuclear extracts were used in gel mobility shift assays, only USF-containing protein-DNA complexes that can be supershifted by specific USF antibodies were observed. These results demonstrate that upstream stimulatory factor binding to the E-box at -65 is required for insulin regulation of the fatty acid synthase promoter.