Functional analysis of the promoter of human sterol 27-hydroxylase gene in HepG2 cells.
Human sterol 27-hydroxylase catalyses the first step in the alternative pathway of bile acids biosynthesis in hepatocytes. However the gene encoding this enzyme (CYP27 gene) is expressed in every tissue and some evidence suggests that this enzyme plays a role in cholesterol homeostasis. Although modulation of CYP27 expression has been reported, the mechanisms underlying the regulation of this gene in human tissues is still poorly understood. To elucidate the mechanism governing CYP27 expression we cloned a 4.3 kb fragment of the 5' flanking region of the human CYP27 gene and constructed deletion mutants which were transfected into HepG2 cells. Functional assays showed that the -217/-10 nucleotide region from the translation start site (minimal promoter), devoid of TATA and CAAT boxes, contains all the elements for basal transcription. Foot-printing analysis of minimal promoter showed four protected regions (A-D). Regions A, B and D each contain one Sp1 binding site, and region C contains a HNF4 site. Electrophoretic mobility shift assays demonstrated that Sp1, Sp3 and HNF4 transcription factors bind these sites. Mutagenesis of any of these sites resulted in the loss of promoter activity. Co-transfection of the minimal promoter with Sp1 and Sp3 expression vectors transactivated CYP27 gene promoter in Drosophila SL2 cells, which lack endogenous Sp proteins. Transactivation of the minimal promoter was also observed in HeLa cells co-transfected with HNF4 expression vector. Therefore, Sp1, Sp3 and HNF4 co-operate in the expression of the human CYP27 gene in HepG2 cells.
Garuti
Rita
R
Dipartimento di Scienze Biomediche, Università di Modena e Reggio Emilia, Via Campi 287, I-41100, Modena, Italy.
Croce
Maria Antonietta
MA
Piccinini
Luana
L
Tiozzo
Roberta
R
Bertolini
Stefano
S
Calandra
Sebastiano
S
D024506:5' Flanking Region / Q000235:genetics; D000818:Animals; D001483:Base Sequence; D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; D001665:Binding Sites / Q000235:genetics; D006528:Carcinoma, Hepatocellular / Q000235:genetics / Q000473:pathology; D002460:Cell Line; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics / Q000378:metabolism; D053493:Cholestanetriol 26-Monooxygenase; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics*; D004247:DNA / Q000235:genetics / Q000378:metabolism; D018983:DNA Footprinting; D004268:DNA-Binding Proteins; D003850:Deoxyribonuclease I / Q000378:metabolism; D024202:Electrophoretic Mobility Shift Assay; D006367:HeLa Cells; D051557:Hepatocyte Nuclear Factor 4; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D017384:Sequence Deletion; D016329:Sp1 Transcription Factor / Q000235:genetics / Q000378:metabolism; D013250:Steroid Hydroxylases / Q000235:genetics*; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism; D014407:Tumor Cells, Cultured
D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; D004268:DNA-Binding Proteins; D051557:Hepatocyte Nuclear Factor 4; C403128:MLX protein, human; D010750:Phosphoproteins; D011993:Recombinant Fusion Proteins; D016329:Sp1 Transcription Factor; D014157:Transcription Factors; D004247:DNA; D003577:Cytochrome P-450 Enzyme System; D013250:Steroid Hydroxylases; C506831:CYP27A1 protein, human; D053493:Cholestanetriol 26-Monooxygenase; D015500:Chloramphenicol O-Acetyltransferase; D003850:Deoxyribonuclease I