Characterization of hypoxia-inducible factor 1 and regulation of DNA binding activity by hypoxia.
Hypoxia-inducible factor 1 (HIF-1) is a DNA binding activity detected in nuclear extracts from Hep3B cells cultured in 1% O2 but not in extracts from cells cultured in 20% O2. HIF-1 binds to a sequence within the human erythropoietin gene enhancer that is required for hypoxic activation of transcription. Induction of HIF-1 is inhibited by cycloheximide, which also inhibits induction of erythropoietin RNA. We now demonstrate that induction of both HIF-1 and erythropoietin RNA is inhibited by the protein kinase inhibitor 2-aminopurine. HIF-1 binding to DNA was eliminated by phosphatase treatment of nuclear extracts. Actinomycin D also inhibited HIF-1 induction, suggesting that de novo transcription is required. The kinetics of HIF-1 induction by hypoxia paralleled the kinetics of erythropoietin gene transcriptional induction. HIF-1 DNA binding activity decayed rapidly when hypoxic cells were exposed to increased oxygen tension. In vitro, the kinetics of HIF-1 association with, and dissociation from, its binding site were extremely rapid, with a t1/2 for both processes of < 1 min. These findings are consistent with the proposed function of HIF-1 as a physiologic regulator of gene expression that responds to changes in cellular oxygen tension. Methylation interference analysis indicated that HIF-1 makes specific contacts with DNA in the major groove.
Wang
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GL
Department of Pediatrics, Johns Hopkins University School of Medicine, Baltimore, Maryland 21287-3914.
Semenza
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GL
D015075:2-Aminopurine / Q000494:pharmacology; D001483:Base Sequence; D001665:Binding Sites; D015687:Cell Hypoxia; D002478:Cells, Cultured; D004247:DNA / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D003609:Dactinomycin / Q000494:pharmacology; D004921:Erythropoietin / Q000235:genetics; D051793:Hypoxia-Inducible Factor 1; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D008745:Methylation; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D009838:Oligodeoxyribonucleotides; D010100:Oxygen / Q000378:metabolism*; D010744:Phosphoric Monoester Hydrolases; D011485:Protein Binding; D012333:RNA, Messenger / Q000096:biosynthesis; D014157:Transcription Factors; D014158:Transcription, Genetic
D004268:DNA-Binding Proteins; D051793:Hypoxia-Inducible Factor 1; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D009687:Nuclear Proteins; D009838:Oligodeoxyribonucleotides; D012333:RNA, Messenger; D014157:Transcription Factors; D004921:Erythropoietin; D003609:Dactinomycin; D015075:2-Aminopurine; D004247:DNA; D010744:Phosphoric Monoester Hydrolases; D010100:Oxygen