The Y-box motif mediates redox-dependent transcriptional activation in mouse cells.
We show here that the OxyR response element (ORE) in the bacterial oxyR promoter can also function as a redox-dependent enhancer in mammalian cells. Fusion of ORE to an SV40 basal promoter driving chloramphenicol acetyltransferase (CAT) expression confers H2O2 inducibility to expression of the cat gene in mouse Hepa-1 hepatoma cells. Nuclear extracts from these cells contain DNA-binding proteins that specifically interact with ORE DNA, cannot be completed by cognate oligonucleotides to AP-1 or NF kappa B, and are constitutively expressed, since treatment with H2O2 causes no detectable changes in binding activity or DNA-protein interaction. Recombinant cDNA clones that express ORE-binding proteins were isolated from a mouse hepatoma expression library and found to be representatives of two different members of the murine Y-box family of transcription factors. Canonical Y-box and ORE oligonucleotides compete with each other for binding to Y-box proteins in gel shift assays and antibodies to FRGY2, a Xenopus Y-box protein, supershift both Y-box and ORE DNA-protein complexes. In addition, antisense oligonucleotides to mouse YB-1 mRNA abolish induction of ORE-mediated cat expression by H2O2, and luciferase reporter constructs containing ORE, or the Y-box from the human MHC class II HLA-DQ gene, exhibit identical dose-dependent H2O2 inducibilities, which can be abolished by addition of 2-mercaptoethanol to the culture medium. These results suggest that the Y-box proteins may be an integral component of a eukaryotic redox signaling pathway.
Department of Environmental Health, University of Cincinnati Medical Center, Ohio 45267-0056, USA.
J L
JL
Duh
H
H
Zhu
H G
HG
Shertzer
D W
DW
Nebert
A
A
Puga
D016475:3T3 Cells; D000595:Amino Acid Sequence; D000818:Animals; D001419:Bacteria / Q000235:genetics; D001483:Base Sequence; D022762:CCAAT-Enhancer-Binding Proteins; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D015500:Chloramphenicol O-Acetyltransferase / Q000096:biosynthesis; D003001:Cloning, Molecular; D016384:Consensus Sequence; D004268:DNA-Binding Proteins / Q000096:biosynthesis / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D005802:Genes, MHC Class II; D006683:HLA-DQ Antigens / Q000096:biosynthesis / Q000235:genetics*; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D007700:Kinetics; D008114:Liver Neoplasms, Experimental; D008297:Male; D051379:Mice; D008969:Molecular Sequence Data; D051839:NFI Transcription Factors; D009687:Nuclear Proteins; D009838:Oligodeoxyribonucleotides; D016376:Oligonucleotides, Antisense / Q000494:pharmacology; D010084:Oxidation-Reduction; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000096:biosynthesis; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction; D013539:Simian virus 40 / Q000235:genetics; D013737:Testis / Q000378:metabolism; D013873:Thionucleotides; D014157:Transcription Factors / Q000096:biosynthesis / Q000378:metabolism*; D015533:Transcriptional Activation; D014162:Transfection; D014407:Tumor Cells, Cultured; D014981:Xenopus; D051840:Y-Box-Binding Protein 1
D022762:CCAAT-Enhancer-Binding Proteins; D004268:DNA-Binding Proteins; D006683:HLA-DQ Antigens; D051839:NFI Transcription Factors; D009687:Nuclear Proteins; D009838:Oligodeoxyribonucleotides; D016376:Oligonucleotides, Antisense; D012333:RNA, Messenger; D011994:Recombinant Proteins; D013873:Thionucleotides; D014157:Transcription Factors; D051840:Y-Box-Binding Protein 1; C070856:YBX1 protein, human; D006861:Hydrogen Peroxide; D015500:Chloramphenicol O-Acetyltransferase