DIOXIN RECEPTOR AND C/EBP REGULATE THE FUNCTION OF THE GLUTATHIONE-S-TRANSFERASE YA GENE XENOBIOTIC RESPONSE ELEMENT

The rat glutathione S-transferase Va gene xenobiotic response element (XRE) has both constitutive and xenobiotic-inducible activity. We present evidence that the XRE is regulated by both the constitutive C/EBP transcription factor and the xenobiotic-activated dioxin receptor. A ligand-activated XRE-binding protein was shown to be dioxin receptor by specific antibody immunodepletion and binding of highly purified receptor. Identification of C/EBPalpha as the constitutive binding protein was demonstrated by competition with a C/EBP binding site, protein-DNA cross-linking to determine the molecular weight of the constitutive protein(s), specific antibody immunodepletion, and binding of purified bacterially expressed C/EBPalpha. Mutational analysis of the XRE revealed that the constitutive factor (C/EBPalpha) shares a nearly identical overlapping binding site with the dioxin receptor. In functional testing of the putative C/EBP-XRE interaction, cotransfected C/EBPalpha activated an XRE test promoter in the non-xenobiotic-responsive HeLa cell line. Unexpectedly, cotransfected C/EBPalpha had no effect on basal activity but significantly increased the xenobiotic response of the XRE test promoter in the xenobiotic-responsive, C/EBP-positive HepG2 cell line. Furthermore, inhibition of C/EBP-binding protein(s) in HepG2 cells by transfection of C/EBP oligonucleotides suppressed the xenobiotic response. These results suggest that C/EBPalpha and dioxin receptor recognize the same DNA sequence element and that transcriptional regulation can occur by cooperative interactions between these two transcription factors.