Functional identification of the proximal promoter region of human pyridoxine 5′-phosphate oxidase gene

Abstract

Pyridoxine 5′-phosphate oxidase (PNPO) is a key enzyme for the biosynthesis of pyridoxal 5′-phosphate. Pyridoxal 5′-phosphate is the catalytically active form of vitamin B6, and acts as a cofactor involved in a high diversity of biochemical reactions. The expression and regulation of PNPO are implicated in numerous physiological and pathological processes. The genomic organization of human PNPO has been reported previously whereas the promoter functional identification is not there yet. In this study, we identified the proximal promoter region of human PNPO gene and analyzed its function. Bioinformatics analysis showed that the transcription start site is at 153 bp upstream of the translation initiation site ATG. Progressive truncation analysis of the 5′-flanking region of PNPO gene demonstrated that two important regulatory regions are located at −996/−852 and −412/+85 bp relative to the transcription start site. From the regulatory regions one USF site, two E2F1 site and multiple Sp1 sites were found. Deletion and mutation experiments indicated that these cis-regulatory elements contribute to the basic promoter activity on different degrees. Downregulation of the transcription factors further indicated that E2F1 plays a dominant role in the transcriptional regulation of PNPO through the binding site at −867/−857, which was confirmed by ChIP assay. Our present study should facilitate further studies on the mechanism regulating the expression of this important gene.