Sterol regulation of human fatty acid synthase promoter I requires nuclear factor-Y- and Sp-1-binding sites.
To understand cholesterol-mediated regulation of human fatty acid synthase promoter I, we tested various 5'-deletion constructs of promoter I-luciferase reporter gene constructs in HepG2 cells. The reporter gene constructs that contained only the Sp-1-binding site (nucleotides -82 to -74) and the two tandem sterol regulatory elements (SREs; nucleotides -63 to -46) did not respond to cholesterol. Only the reporter gene constructs containing a nuclear factor-Y (NF-Y) sequence, the CCAAT sequence (nucleotides -90 to -86), an Sp-1 sequence, and the two tandem SREs responded to cholesterol. The NF-Y-binding site, therefore, is essential for cholesterol response. Mutating the SREs or the NF-Y site and inserting 4 bp between the Sp-1- and NF-Y-binding sites both resulted in a minimal cholesterol response of the reporter genes. Electrophoretic mobility-shift assays using anti-SRE-binding protein (SREBP) and anti-NF-Ya antibodies confirmed that these SREs and the NF-Y site bind the respective factors. We also identified a second Sp-1 site located between nucleotides -40 and -30 that can substitute for the mutated Sp-1 site located between nucleotides -82 and -74. The reporter gene expression of the wild-type promoter and the Sp-1 site (nucleotides -82 to -74) mutant promoter was similar when SREBP1a [the N-terminal domain of SREBP (amino acids 1-520)] was constitutively overexpressed, suggesting that Sp-1 recruits SREBP to the SREs. Under the same conditions, an NF-Y site mutation resulted in significant loss of reporter gene expression, suggesting that NF-Y is required to activate the cholesterol response.
Xiong
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Verna and Marrs McLean Department of Biochemistry and Molecular Biology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA.
Chirala
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Wakil
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D001483:Base Sequence; D001665:Binding Sites; D023081:CCAAT-Binding Factor; D022762:CCAAT-Enhancer-Binding Proteins; D002784:Cholesterol / Q000502:physiology*; D017931:DNA Primers; D004268:DNA-Binding Proteins / Q000235:genetics; D064429:Fatty Acid Synthases / Q000235:genetics / Q000378:metabolism; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology*; D006801:Humans; D009687:Nuclear Proteins / Q000235:genetics; D011401:Promoter Regions, Genetic; D016329:Sp1 Transcription Factor / Q000378:metabolism*; D051780:Sterol Regulatory Element Binding Protein 1; D014157:Transcription Factors / Q000378:metabolism*
D023081:CCAAT-Binding Factor; D022762:CCAAT-Enhancer-Binding Proteins; D017931:DNA Primers; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; C497387:SREBF1 protein, human; D016329:Sp1 Transcription Factor; D051780:Sterol Regulatory Element Binding Protein 1; D014157:Transcription Factors; C402244:nuclear factor Y; D002784:Cholesterol; D064429:Fatty Acid Synthases