Transcriptional repression of human cad gene by hypoxia inducible factor-1α

De novo biosynthesis of pyrimidine nucleotides provides essential precursors for DNA synthesis and cell proliferation. The first three steps of de novo pyrimidine biosynthesis are catalyzed by a multifunctional enzyme known as CAD (carbamoyl phosphate synthetase-aspartate carbamoyltransferase-dihydroorotase). In this work, a decrease in CAD expression is detected in numerous cell lines and primary culture human stromal cells incubated under hypoxia or desferrioxamine (DFO)-induced HIF-1 alpha accumulation. A putative hypoxia response element (HRE) binding matrix is identified by analyzing human cad-gene promoter using a bioinformatic approach. Promoter activity assays, using constructs harboring the cad promoter (-710/+122) and the -67/HRE fragment (25-bases), respectively, demonstrate the suppression of reporter-gene expression under hypoxia. Suppression of cad-promoter activity is substantiated by forced expression of wild-type HIF-1 alpha but abolished by overexpression of dominant-negative HIF-1 alpha. A chromatin immunoprecipitation assay provides further evidence that HIF-1 alpha binds to the cad promoter in vivo. These data demonstrate that the cad-gene expression is repressed by HIF-1 alpha, which represents a functional link between hypoxia and cell-cycle arrest.