A redox mechanism controls differential DNA binding activities of hypoxia-inducible factor (HIF) 1alpha and the HIF-like factor.
Hypoxia-inducible factor 1alpha (HIF-1alpha) and the HIF-like factor (HLF) are two highly related basic Helix-Loop-Helix/Per-Arnt-Sim (bHLH/PAS) homology transcription factors that undergo dramatically increased function at low oxygen levels. Despite strong similarities in their activation mechanisms (e.g. they both undergo rapid hypoxia-induced protein stabilization, bind identical target DNA sequences, and induce synthetic reporter genes to similar degrees), they are both essential for embryo survival via distinct functions during vascularization (HIF-1alpha) or catecholamine production (HLF). It is currently unknown how such specificity of action is achieved. We report here that DNA binding by HLF, but not by HIF-1alpha, is dependent upon reducing redox conditions. In vitro DNA binding and mammalian two-hybrid assays showed that a unique cysteine in the DNA-binding basic region of HLF is a target for the reducing activity of redox factor Ref-1. Although the N-terminal DNA-binding domain of HIF-1alpha can function in the absence of Ref-1, we found that the C-terminal region containing the transactivation domain requires Ref-1 for full activity. Our data reveal that the hypoxia-inducible factors are subject to complex redox control mechanisms that can target discrete regions of the proteins and are the first to establish a discriminating control mechanism for differential regulation of HIF-1alpha and HLF activity.
Lando
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D
Department of Biochemistry, University of Adelaide, Adelaide 5005, South Australia.
Pongratz
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I
Poellinger
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Whitelaw
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ML
D000595:Amino Acid Sequence; D019757:Carbon-Oxygen Lyases / Q000235:genetics; D003545:Cysteine / Q000235:genetics / Q000378:metabolism; D004247:DNA / Q000378:metabolism*; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D005786:Gene Expression Regulation; D006367:HeLa Cells; D018257:Helix-Loop-Helix Motifs; D006801:Humans; D051793:Hypoxia-Inducible Factor 1; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D016376:Oligonucleotides, Antisense / Q000378:metabolism; D010084:Oxidation-Reduction; D011994:Recombinant Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D012694:Serine / Q000235:genetics / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*
D004268:DNA-Binding Proteins; C497442:HIF1A protein, human; D051793:Hypoxia-Inducible Factor 1; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D009687:Nuclear Proteins; D016376:Oligonucleotides, Antisense; D011994:Recombinant Proteins; D014157:Transcription Factors; D012694:Serine; D004247:DNA; D019757:Carbon-Oxygen Lyases; C085108:APEX1 protein, human; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase; D003545:Cysteine