title: 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.
abstract: Sterol-regulated transcription of the farnesyl diphosphate (FPP) synthase gene is dependent on two cis elements in the proximal promoter. These elements, an inverted CCAAT box and sterol regulatory element 3 (SRE-3), bind NF-Y and sterol regulatory element-binding protein 1 (SREBP-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 sites for both NF-Y and SREBP-1 in gel mobility shift assays, we demonstrate that the addition of NF-Y increases the binding of SREBP-1 to SRE-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 base pairs (bp) are inserted between the inverted CCAAT box and SRE-3. Promoter-reporter genes, containing either the wild-type FPP synthase promoter sequence or containing the 4-bp insertion between the inverted CCAAT box and SRE-3, were transiently transfected into cells. The activity 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 the 4-bp insert, consistent with defective binding of SREBP to the promoter in vivo. These studies suggest that the binding of SREBP-1 to SRE-3 in the FPP synthase promoter, and subsequent stimulation of transcription, is dependent on synergistic binding and a functional interaction between SREBP-1 and NF-Y.
authors.affiliation: Departments of Biological Chemistry and Medicine and the Molecular Biology Institute, UCLA, Los Angeles, California 90095, USA.
authors.forename: J
authors.initials: J
authors.lastname: Ericsson
authors.forename: S M
authors.initials: SM
authors.lastname: Jackson
authors.forename: P A
authors.initials: PA
authors.lastname: Edwards
mesh_terms: D019883:Alkyl and Aryl Transferases; D000818:Animals; D022762:CCAAT-Enhancer-Binding Proteins; D016466:CHO Cells; D002460:Cell Line; D006224:Cricetinae; D004268:DNA-Binding Proteins / Q000378:metabolism*; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology*; D051228:Geranyltranstransferase; D006367:HeLa Cells; D006801:Humans; D009687:Nuclear Proteins / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D011994:Recombinant Proteins / Q000378:metabolism; D051780:Sterol Regulatory Element Binding Protein 1; D013261:Sterols / Q000378:metabolism*; D014157:Transcription Factors; D014158:Transcription, Genetic; D014166:Transferases / Q000235:genetics* / Q000378:metabolism
chemical_list: D022762:CCAAT-Enhancer-Binding Proteins; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D011994:Recombinant Proteins; C497387:SREBF1 protein, human; D051780:Sterol Regulatory Element Binding Protein 1; D013261:Sterols; D014157:Transcription Factors; D014166:Transferases; D019883:Alkyl and Aryl Transferases; D051228:Geranyltranstransferase