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

FEBS Letters

Chang, Wen-Chang¹  Chen, Ching-Jiunn¹  Huang, Huei-Sheng¹ 

[1] Department of Pharmacology, College of Medicine, National Cheng Kung University, Tainan 701, Taiwan

关键词: PHGPx;    NF-Y;    A431 cell;    PHGPx;    phospholipid hydroperoxide glutathione peroxidase;    CBFs;    CCAAT-box binding factors;    SECIS;    selenocysteine insertion sequence;    Sec;    selenocysteine;    Se-S;    selenenyl sulfide;    ORE;    oxygen responsive element;    PdBu;    phorbol 12;    13-dibutyrate;   

DOI  :  10.1016/S0014-5793(99)00866-2

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

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.

【 授权许可】

Unknown