Transcriptional repression of human cad gene by hypoxia inducible factor-1alpha.
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-1alpha 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-1alpha but abolished by overexpression of dominant-negative HIF-1alpha. A chromatin immunoprecipitation assay provides further evidence that HIF-1alpha binds to the cad promoter in vivo. These data demonstrate that the cad-gene expression is repressed by HIF-1alpha, which represents a functional link between hypoxia and cell-cycle arrest.
Chen
Ko-Fan
KF
Department of Physiology, College of Medicine, National Cheng Kung University, Tainan 70101, Taiwan.
Lai
Yen-Yu
YY
Sun
H Sunny
HS
Tsai
Shaw-Jenq
SJ
D001221:Aspartate Carbamoyltransferase / Q000096:biosynthesis / Q000235:genetics*; D001665:Binding Sites; D002223:Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing) / Q000096:biosynthesis / Q000235:genetics*; D002453:Cell Cycle; D015687:Cell Hypoxia; D002460:Cell Line; D002478:Cells, Cultured; D003676:Deferoxamine / Q000494:pharmacology; D004080:Dihydroorotase / Q000096:biosynthesis / Q000235:genetics*; D020868:Gene Silencing; D006801:Humans; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D007502:Iron Chelating Agents / Q000494:pharmacology; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000378:metabolism; D012097:Repressor Proteins / Q000378:metabolism*; D020218:Response Elements; D014157:Transcription Factors / Q000378:metabolism*; D014158:Transcription, Genetic
C033626:CAD trifunctional enzyme; C497442:HIF1A protein, human; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D007502:Iron Chelating Agents; D012333:RNA, Messenger; D012097:Repressor Proteins; D014157:Transcription Factors; D001221:Aspartate Carbamoyltransferase; D004080:Dihydroorotase; D002223:Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing); D003676:Deferoxamine