TY - JOUR
T1 - Molecular mechanisms of transcription activation by HLF and HIF1α in response to hypoxia
T2 - Their stabilization and redox signal-induced interaction with CBP/p300
AU - Ema, Masatsugu
AU - Hirota, Kiichi
AU - Mimura, Junsei
AU - Abe, Hisaku
AU - Yodoi, Junji
AU - Sogawa, Kazuhiro
AU - Poellinger, Lorenz
AU - Fujii-Kuriyama, Yoshiaki
PY - 1999/4/1
Y1 - 1999/4/1
N2 - 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.
AB - 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.
KW - Oxygen sensing
KW - Protein stabilization
KW - Redox regulation
KW - Sulfhydryl modification
KW - Thioredoxin
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M3 - Article
C2 - 10202154
AN - SCOPUS:0033118983
VL - 18
SP - 1905
EP - 1914
JO - EMBO Journal
JF - EMBO Journal
SN - 0261-4189
IS - 7
ER -