The CCAAT-box binding factor NF-Y is required for the expression of phospholipid hydroperoxide glutathione peroxidase in human epidermoid carcinoma A431 cells

Huei Sheng Huang, Ching Jiunn Chen, Wen Chang Chang

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23 Citations (Scopus)

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. Copyright (C) 1999 Federation of European Biochemical Societies.

Original languageEnglish
Pages (from-to)111-116
Number of pages6
JournalFEBS Letters
Volume455
Issue number1-2
DOIs
Publication statusPublished - Jul 16 1999
Externally publishedYes

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Keywords

  • A431 cell
  • NF-Y
  • PHGPx

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Molecular Biology

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