SYNERGISTIC BINDING OF STEROL REGULATORY ELEMENT-BINDING PROTEIN AND NF-Y TO THE FARNESYL DIPHOSPHATE SYNTHASE PROMOTER IS CRITICAL FOR STEROL-REGULATED EXPRESSION OF THE GENE

Sterol-regulated transcription of the farnesyl diphosphate (FPP) synth ase gene is dependent on two cis elements in the proximal promoter. Th ese elements, an inverted CCAAT box and sterol regulatory element 3 (S RE-3), bind NF-Y and sterol regulatory element-binding protein 1 (SREB P-1), respectively. We now demonstrate that the binding of recombinant SREBP-1 to its cognate site (SRE-3) within the FPP synthase promoter in vitro is enhanced by binding of NF-Y to the upstream inverted CCAAT box, Using an FPP synthase promoter fragment containing the binding s ites for both NF-Y and SREBP-1 in gel mobility shift assays, we demons trate that the addition of NF-Y increases the binding of SREBP-1 to SR E-3 over 20-fold. In contrast, NF-Y does not stimulate the binding of SREBP-1 to SRE-3 when the inverted CCAAT box is either mutated or 4 ba se pairs (bp) are inserted between the inverted CCAAT box and SRE-3. P romoter-reporter genes, containing either the wildtype FPP synthase pr omoter sequence or containing the 4-bp insertion between the inverted CCAAT box and SRE-3, were transiently transfected into cells. The acti vity of the wild-type promoter-reporter gene increased when the cells were either incubated in sterol-depleted medium or were co-transfected with an expression vector encoding transcriptionally active SREBP-1. This increase in activity was attenuated when the promoter contained t he 4-bp insert, consistent with defective binding of SREBP to the prom oter in vivo. These studies suggest that the binding of SREBP-1 to SRE -3 in the FPP synthase promoter, and subsequent stimulation of transcr iption, is dependent on synergistic binding and a functional interacti on between SREBP-1 and NF-Y.