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 pres ent evidence that the XRE is regulated by both the constitutive C/EBP transcription factor and the xenobiotic-activated dioxin receptor. A l igand-activated XRE-binding protein was shown to be dioxin receptor by specific antibody immunodepletion and binding of highly purified rece ptor. Identification of C/EBPalpha as the constitutive binding protein was demonstrated by competition with a C/EBP binding site, protein-DN A 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 reve aled that the constitutive factor (C/EBPalpha) shares a nearly identic al overlapping binding site with the dioxin receptor. In functional te sting of the putative C/EBP-XRE interaction, cotransfected C/EBPalpha activated an XRE test promoter in the non-xenobiotic-responsive HeLa c ell line. Unexpectedly, cotransfected C/EBPalpha had no effect on basa l activity but significantly increased the xenobiotic response of the XRE test promoter in the xenobiotic-responsive, C/EBP-positive HepG2 c ell line. Furthermore, inhibition of C/EBP-binding protein(s) in HepG2 cells by transfection of C/EBP oligonucleotides suppressed the xenobi otic response. These results suggest that C/EBPalpha and dioxin recept or recognize the same DNA sequence element and that transcriptional re gulation can occur by cooperative interactions between these two trans cription factors.