INTERACTION OF THE REGULATORY DOMAINS OF THE MURINE CYP1A1 GENE WITH 2 DNA-BINDING PROTEINS IN ADDITION TO THE AH RECEPTOR AND THE AH RECEPTOR NUCLEAR TRANSLOCATOR (ARNT)

The aromatic hydrocarbon (Ah) receptor complex is a ligand-activated t ranscriptional activator consisting of at least two protein components . The ligand-binding component is the AhR protein, a cytosolic recepto r encoded by the Ahr gene, which, upon ligand binding, translocates to the nucleus in a heterodimeric complex with the ARNT (Ah receptor nuc lear translocator) component. The complex binds to several discrete DN A domains containing aromatic hydrocarbon responsive elements (AhRE) p resent in the regulatory region of the murine cytochrome P(1)450 Cyp1a 1 gene and of the other genes in the [Ah] gene battery. As a consequen ce of binding, a transcriptional complex is formed that activates the expression of these genes by as yet unidentified mechanisms. We have a nalyzed DNA-protein interactions in four of these domains, specificall y, the AhREs located between -1085 and -482 (sites A, C, E, and D) of the upstream regulatory region of the murine Cyp1a1 gene. We found tha t two DNA-binding proteins, present in cytosolic and nuclear extracts of mouse Hepa-1 cells, showed overlapping DNA-binding specificities to those of the Ah receptor. One of these proteins had an apparent molec ular mass of 35-40 kDa, bound only to AhRE3 (site D), and has been ide ntified tentatively as a member of the C/EBP family of transcription f actors. The second protein, purified by DNA-affinity chromatography, h ad an apparent molecular mass of 95 kDa and bound to a larger DNA moti f that included the AhRE sequence, in AhRE3 and AhRE5 (sites D and A), but not in AhRE1 or AhRE2 (sites C and E). This protein was not AhR n or was it ARNT, since it was found in receptorless (Ahr(-)) and in nuc lear translocation-defective (Arnt(-)) cells, as well as in cells that had not been exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD; di oxin), a potent inducer of Cyp1a1 expression. Evidence from in vivo me thylation protection indicated that two G residues flanking AhRE3, one of which is required for binding of the 95-kDa protein, may be protec ted from methylation in uninduced cells and become exposed upon dioxin treatment, suggesting that the 95-kDa protein may be constitutively b ound to AhRE3, and be displaced by binding of the Ah receptor complex. These results lend support to the concept that the transcriptional re gulation of the [Ah] battery genes could be modulated by combinatorial interactions of the Ah receptor complex with other transcription fact ors.