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.