Molecular cloning and characterization of the human dihydropyrimidine dehydrogenase promoter

Several studies have demonstrated that dihydropyrimidine dehydrogenase (EC 1.3.1.2) has a critical role in the pharmacokinetics of the anticancer agen t 5-fluorouracil. We previously reported the structural organization of the human DPYD gene. In this article, we describe the molecular cloning and fu nctional characterization of 1.2 kb of the 5' flanking region of the DPYD g ene. Sequence analysis demonstrated that this region of the DPYD gene lacks the typical TATA or CCAAT boxes with several GC-rich regions containing po tential cis-regulatory elements. Progressive 5' deletions of the 5' flankin g region were fused to the luciferase reporter gene and transient expressio n measured following transfection into HeLa and 293 cells. Comparative anal ysis of luciferase activity revealed that a 208 bp region of the DPYD gene (-121/+86) contained equivalent transcriptional activity to the complete 1. 2 kb 5' flanking region of the DPYD gene. Site-directed mutagenesis of the luciferase reporter constructs demonstrated that the -72/-23 sequence conta ined two regulatory regions (designated elements I and II) essential for pr omoter activity. Gel shift experiments demonstrated that both regulatory el ements specifically bind with protein(s) from nuclear extracts of 293 cells . Competitive binding experiments with 293 nuclear extracts and radiolabele d oligonucleotides (corresponding to elements I and II) suggest that the sa me protein(s) bind to both regulatory elements. We conclude that constituti ve expression of the DPYD gene involves a limited GC-rich region of the 5' flanking sequence of the DPYD gene which contains two regulatory elements. (C) 2000 Elsevier Science B.V. All rights reserved.