The repression of nuclear factor I/CCAAT transcription factor (NFI/CTF) transactivating domain by oxidative stress is mediated by a critical cysteine (Cys-427).
The activity of the nuclear factor I/CCAAT transcription factor (NFI/CTF) is negatively regulated by oxidative stress. The addition of relatively high (millimolar) H(2)O(2) concentrations inactivates cellular NFI DNA-binding activity whereas lower concentrations can repress NFI/CTF transactivating function. We have investigated the mechanism of this regulation using Gal4 fusion proteins and transfection assays. We show that micromolar H(2)O(2) concentrations repress the transactivating domain of NFI/CTF in a dose-dependent manner and are less or not active on other transcription factors' transactivating domains. Studies using deletions and point mutations pointed to the critical role of Cys-427. Indeed, when this cysteine is mutated into a serine, the repression by H(2)O(2) is totally blunted. Mutation of other cysteine, serine and tyrosine residues within the transactivating domain had no clear effect on the repression by H(2)O(2). Finally, treatment of cells with the thiol-alkylating reagent N-ethylmaleimide leads to a decrease in the transactivating function, which is dependent on Cys-427. This study shows that transactivating domains of transcription factors can constitute very sensitive targets of oxidative stress and highlights the critical role of these domains.
Morel
Y
Y
INSERM U490, Université Paris V-René Descartes, Centre Universitaire des Saints-Pères, 45, rue des Saints-Pères, 75006 Paris, France.
Barouki
R
R
D001665:Binding Sites; D022762:CCAAT-Enhancer-Binding Proteins; D003545:Cysteine / Q000187:drug effects / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D005033:Ethylmaleimide / Q000494:pharmacology; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology*; D051839:NFI Transcription Factors; D009687:Nuclear Proteins; D016877:Oxidants / Q000494:pharmacology; D018384:Oxidative Stress / Q000187:drug effects / Q000235:genetics; D011487:Protein Conformation; D013439:Sulfhydryl Reagents / Q000494:pharmacology; D014157:Transcription Factors; D015533:Transcriptional Activation / Q000187:drug effects; D014407:Tumor Cells, Cultured; D051840:Y-Box-Binding Protein 1
D022762:CCAAT-Enhancer-Binding Proteins; D004268:DNA-Binding Proteins; D051839:NFI Transcription Factors; C497647:NFIC protein, human; D009687:Nuclear Proteins; D016877:Oxidants; D013439:Sulfhydryl Reagents; D014157:Transcription Factors; D051840:Y-Box-Binding Protein 1; C070856:YBX1 protein, human; D006861:Hydrogen Peroxide; D003545:Cysteine; D005033:Ethylmaleimide