THE Y-BOX MOTIF MEDIATES REDOX-DEPENDENT TRANSCRIPTIONAL ACTIVATION IN MOUSE CELLS

We show here that the OxyR response element (ORE) in the bacterial oxy R promoter can also function as a redox-dependent enhancer in mammalia n cells. Fusion of ORE to an SV40 basal promoter driving chloramphenic ol acetyltransferase (CAT) expression confers H2O2 inducibility to exp ression of the cat gene in mouse Hepa-1 hepatoma cells. Nuclear extrac ts from these cells contain DNA-binding proteins that specifically int eract with ORE DNA, cannot be competed 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-prot ein interaction, Recombinant cDNA clones that express ORE-binding prot eins were isolated from a mouse hepatoma expression library and found to be representatives of two different members of the murine Y-box fam ily of transcription factors, Canonical Y-box and ORE oligonucleotides compete with each other for binding to Y-box proteins in gel shift as says and antibodies to FRGY2, a Xenopus Y-box protein, supershift both Y-box and ORE DNA-protein complexes, In addition, antisense oligonucl eotides to mouse YB-1 mRNA abolish induction of ORE-mediated cat expre ssion by H2O2, and luciferase reporter constructs containing ORE, or t he Y-box from the human MHC class II HLA DQ gene, exhibit identical do se-dependent H2O2 inducibilities, which can be abolished by addition o f 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.