title: SREBP-1 binds to multiple sites and transactivates the human ApoA-II promoter in vitro : SREBP-1 mutants defective in DNA binding or transcriptional activation repress ApoA-II promoter activity.
abstract: -Screening of an expression human liver cDNA library resulted in the isolation of several cDNA clones homologous to sterol regulatory element-binding protein-1 (SREBP-1) that recognize the regulatory element AIIAB and AIIK of the human apoA-II promoter. DNaseI footprinting of the apoA-II promoter using SREBP-1 (1 to 460) expressed in bacteria identified 5 overall protected regions designated AIIAB (-64 to -48), AIICD (-178 to -154), AIIDE (-352 to -332), AIIHI (-594 to -574), and AIIK (-760 to -743). These regions contain inverted E-box palindromic or direct repeat motifs and bind SREBP-1 with different affinities. Transient cotransfection experiments in HepG2 cells showed that SREBP-1 transactivated the -911/29 apoA-II promoter 3.5-fold as well as truncated apoA-II promoter segments that contain 1, 2, 3, or 4 SREBP binding sites. Mutagenesis analysis showed that transactivation by SREBP was mainly affected by mutations in element AIIAB. Despite the strong transactivation of the apoA-II promoter by SREBP-1 we could not demonstrate significant changes on the endogenous apoA-II mRNA levels of HepG2 cells after cotransfection with SREBP-1 or in the presence or absence of cholesterol and 25-OH-cholesterol. An SREBP-1 mutant lacking the amino-terminal activation domain bound normally to its cognate sites and repressed the apoA-II promoter activity. Repression was also caused by specific amino acid substitutions of Leu, Val, or Gly for Lys359, which affected DNA binding. Repression by the DNA binding-deficient mutants was abolished by deletion of the amino-terminal activation domain (1 to 90) of SREBP-1. Overall, the findings suggest that the wild-type SREBP-1 can bind and transactivate efficiently the apoA-II promoter in cell culture. SREBP-1 mutants lacking the activation domain bind to their cognate sites and directly repress the apoA-II promoter whereas mutants defective in DNA binding indirectly repress the apoA-II promoter activity, possibly by a squelching mechanism.
authors.affiliation: Section of Molecular Genetics, Cardiovascular Institute, Department of Biochemistry, Boston University Medical Center, Boston, MA, USA.
authors.forename: P
authors.initials: P
authors.lastname: Pissios
authors.forename: H Y
authors.initials: HY
authors.lastname: Kan
authors.forename: S
authors.initials: S
authors.lastname: Nagaoka
authors.forename: V I
authors.initials: VI
authors.lastname: Zannis
mesh_terms: D000595:Amino Acid Sequence; D016633:Apolipoprotein A-II / Q000235:genetics*; D053305:Apolipoprotein C-III; D001056:Apolipoproteins C / Q000235:genetics; D001483:Base Sequence; D001665:Binding Sites; D022762:CCAAT-Enhancer-Binding Proteins; D004247:DNA / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000502:physiology*; D006801:Humans; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000502:physiology*; D011401:Promoter Regions, Genetic; D020218:Response Elements; D051780:Sterol Regulatory Element Binding Protein 1; D013329:Structure-Activity Relationship; D014157:Transcription Factors / Q000378:metabolism / Q000502:physiology*; D015533:Transcriptional Activation; D051720:Upstream Stimulatory Factors
chemical_list: D016633:Apolipoprotein A-II; D053305:Apolipoprotein C-III; D001056:Apolipoproteins C; D022762:CCAAT-Enhancer-Binding Proteins; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; C497387:SREBF1 protein, human; D051780:Sterol Regulatory Element Binding Protein 1; D014157:Transcription Factors; D051720:Upstream Stimulatory Factors; D004247:DNA