Molecular mechanisms of transcription activation by HLF and HIF1α in response to hypoxia: Their stabilization and redox signal-induced interaction with CBP/p300

Masatsugu Ema, Kiichi Hirota, Junsei Mimura, Hisaku Abe, Junji Yodoi, Kazuhiro Sogawa, Lorenz Poellinger, Yoshiaki Fujii-Kuriyama

    Research output: Contribution to journalArticlepeer-review

    479 Citations (Scopus)

    Abstract

    Hypoxia-inducible factor 1α (HIF1α) and its related factor, HLF, activate expression of a group of genes such as erythropoietin in response to low oxygen. Transfection analysis using fusion genes of GAL4DBD with various fragments of the two factors delineated two transcription activation domains which are inducible in response to hypoxia and are localized in the C-terminal half. Their sequences are conserved between HLF and HIF1α. One is designated NAD (N-terminal activation domain), while the other is CAD (C-terminal activation domain). Immunoblot analysis revealed that NADs, which were rarely detectable at normoxia, became stabilized and accumulated at hypoxia, whereas CADs were constitutively expressed. In the mammalian two-hybrid system, CAD and NAD baits enhanced the luciferase expression from a reporter gene by co-transfection with CREB-binding protein (CBP) prey, whereas CAD, but not NAD, enhanced β-galactosidase expression in yeast by CBP co-expression, suggesting that NAD and CAD interact with CBP/p300 by a different mechanism. Co-transfection experiments revealed that expression of Ref-1 and thioredoxin further enhanced the luciferase activity expressed by CAD, but not by NAD. Amino acid replacement in the sequences of CADs revealed a specific cysteine to be essential for their hypoxia-inducible interaction with CBP. Nuclear translocation of thioredoxin from cytoplasm was observed upon reducing O2 concentrations.

    Original languageEnglish
    Pages (from-to)1905-1914
    Number of pages10
    JournalEMBO Journal
    Volume18
    Issue number7
    Publication statusPublished - 1999 Apr 1

    Keywords

    • Oxygen sensing
    • Protein stabilization
    • Redox regulation
    • Sulfhydryl modification
    • Thioredoxin

    ASJC Scopus subject areas

    • Genetics
    • Cell Biology

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