Abstract
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.
Publication types
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Research Support, U.S. Gov't, P.H.S.
MeSH terms
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3T3 Cells
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Amino Acid Sequence
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Animals
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Bacteria / genetics
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Base Sequence
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CCAAT-Enhancer-Binding Proteins*
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Cell Line
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Cell Nucleus / metabolism
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Chloramphenicol O-Acetyltransferase / biosynthesis
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Cloning, Molecular
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Consensus Sequence
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DNA-Binding Proteins / biosynthesis
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DNA-Binding Proteins / metabolism*
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Escherichia coli / genetics
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Escherichia coli / metabolism
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Gene Expression Regulation, Enzymologic / drug effects
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Genes, MHC Class II*
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HLA-DQ Antigens / biosynthesis
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HLA-DQ Antigens / genetics*
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Humans
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Hydrogen Peroxide / pharmacology
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Kinetics
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Liver Neoplasms, Experimental
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Male
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Mice
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Molecular Sequence Data
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NFI Transcription Factors
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Nuclear Proteins
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Oligodeoxyribonucleotides
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Oligonucleotides, Antisense / pharmacology
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Oxidation-Reduction
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Promoter Regions, Genetic
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RNA, Messenger / biosynthesis
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Rats
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Recombinant Proteins / biosynthesis
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Sequence Homology, Amino Acid
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Signal Transduction
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Simian virus 40 / genetics
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Testis / metabolism
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Thionucleotides
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Transcription Factors / biosynthesis
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Transcription Factors / metabolism*
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Transcriptional Activation*
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Transfection
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Tumor Cells, Cultured
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Xenopus
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Y-Box-Binding Protein 1
Substances
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CCAAT-Enhancer-Binding Proteins
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DNA-Binding Proteins
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HLA-DQ Antigens
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NFI Transcription Factors
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Nuclear Proteins
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Oligodeoxyribonucleotides
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Oligonucleotides, Antisense
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RNA, Messenger
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Recombinant Proteins
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Thionucleotides
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Transcription Factors
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Y-Box-Binding Protein 1
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YBX1 protein, human
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Hydrogen Peroxide
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Chloramphenicol O-Acetyltransferase
Associated data
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GENBANK/L35549
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GENBANK/M24069
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GENBANK/M24070
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GENBANK/M60419
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GENBANK/M83888
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GENBANK/X57621