p53-dependent suppression of uridine phosphorylase gene expression through direct promoter interaction.
Uridine phosphorylase (UPase) is a key enzyme in the pyrimidine salvage pathway. It reversibly catalyzes the catabolism of uridine to uracil; controls the homeostatic regulation of uridine concentration in plasma and tissues; and plays a role in the intracellular activation of 5-fluorouracil. We cloned the murine UPase gene promoter, a 1703-bp fragment, and determined the transcription initiation sites located at +1 and +92 bp of the cDNA sequence. Through transient expression analysis of the 5'-flanking region of UPase gene, we have evaluated the promoter activity for a series of fragments with 5'- to 3'-deletion in murine breast cancer EMT-6 cells and immortalized murine fibroblast NIH 3T3 cells. Cotransfection of the UPase promoter constructs (from -1619 to -445) containing p53 binding motif with the wild-type p53 construct resulted in a significant reduction of luciferase activity; however, this effect disappeared with the additional deletion of the -445 to -274 sequence to suggest the existence in this promoter region of a putative p53 recognition element. Similar cotransfection in murine embryo fibroblasts p53-/- confirmed the inhibitory role of p53 on the UPase promoter activity. The specificity of the interaction is demonstrated by nuclear protein-specific binding to the putative p53 recognition sequence using gel mobility shift assay and DNase I footprinting analysis. These data indicate the UPase gene is a novel target of p53, and its expression is down-regulated by p53 at the promoter level.
Zhang
D
D
Department of Internal Medicine (Oncology), Yale University School of Medicine, New Haven, Connecticut 06520, USA.
Cao
D
D
Russell
R
R
Pizzorno
G
G
D016475:3T3 Cells; D000818:Animals; D001483:Base Sequence; D002874:Chromosome Mapping; D015972:Gene Expression Regulation, Neoplastic / Q000502:physiology; D020868:Gene Silencing / Q000502:physiology*; D051379:Mice; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic / Q000235:genetics / Q000502:physiology*; D011485:Protein Binding; D011743:Pyrimidines / Q000378:metabolism; D012333:RNA, Messenger / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000235:genetics; D012265:Ribonucleotides / Q000378:metabolism; D014158:Transcription, Genetic / Q000235:genetics / Q000502:physiology; D014407:Tumor Cells, Cultured; D016159:Tumor Suppressor Protein p53 / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D014543:Uridine Phosphorylase / Q000037:antagonists & inhibitors* / Q000096:biosynthesis / Q000235:genetics
D011743:Pyrimidines; D012333:RNA, Messenger; D012265:Ribonucleotides; D016159:Tumor Suppressor Protein p53; D014543:Uridine Phosphorylase; C030986:pyrimidine