title: The CCAAT-box binding factor NF-Y is required for the expression of phospholipid hydroperoxide glutathione peroxidase in human epidermoid carcinoma A431 cells.
abstract: Promoter activation in the expression of phospholipid hydroperoxide glutathione peroxidase (PHGPx) gene in human epidermoid carcinoma A431 cells was studied in the present investigation. Luciferase reporter assays with plasmids carrying a 400 bp of the promoter DNA were performed to analyze the regulatory element in the proximal promoter of human PHGPx gene. Transient transfection with a series of 5'-deletion and internal truncation mutants showed that the 5'-flanking region spanning from -212 to -121 bp was important for the basal expression of PHGPx gene in A431 cells. A region from -170 to -140 bp was protected in DNase I footprinting assays and bound the nuclear proteins in electrophoretic mobility shift assays. This region, denoted FP3, contains the consensus recognition sites for AP-2, CCAAT-box and CRE. The oligonucleotide competitor with the mutation at CCAAT-box could not eliminate the nuclear protein binding in gel-shift assay and the site-directed mutagenesis at the CCAAT-box decreased the luciferase activity of PHGPx promoter for approximate 50% in reporter gene assays. Competition experiments indicate that the binding of nuclear factor to the FP3 region was abolished by oligodeoxyribonucleotide corresponding to NF-Y/CP1 binding site to a greater extent than by those corresponding to sites for CTF/NFI and C/EBP. Taken together, the CCAAT-box in the promoter ranging from -156 to -151 bp, bound to NF-Y/CP1, was essential for the basal expression of human PHGPx gene in A431 cells.
authors.affiliation: Department of Pharmacology, College of Medicine, National Cheng Kung University, Tainan, Taiwan.
authors.forename: H S
authors.initials: HS
authors.lastname: Huang
authors.forename: C J
authors.initials: CJ
authors.lastname: Chen
authors.forename: W C
authors.initials: WC
authors.lastname: Chang
mesh_terms: D022762:CCAAT-Enhancer-Binding Proteins; D002294:Carcinoma, Squamous Cell / Q000201:enzymology* / Q000473:pathology; D004247:DNA; D004268:DNA-Binding Proteins / Q000378:metabolism*; D015971:Gene Expression Regulation, Enzymologic; D005979:Glutathione Peroxidase / Q000235:genetics*; D006801:Humans; D008156:Luciferases / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D009687:Nuclear Proteins / Q000378:metabolism; D000080662:Phospholipid Hydroperoxide Glutathione Peroxidase; D011401:Promoter Regions, Genetic; D012045:Regulatory Sequences, Nucleic Acid; D014407:Tumor Cells, Cultured
chemical_list: D022762:CCAAT-Enhancer-Binding Proteins; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D004247:DNA; D000080662:Phospholipid Hydroperoxide Glutathione Peroxidase; D005979:Glutathione Peroxidase; D008156:Luciferases