Interaction of the regulatory domains of the murine Cyp1a1 gene with two DNA-binding proteins in addition to the Ah receptor and the Ah receptor nuclear translocator (ARNT)

Abstract

The aromatic hydrocarbon ( Ah) receptor complex is a ligand-activated transcriptional activator consisting of at least two protein components. The ligand-binding component is the AhR protein, a cytosolic receptor encoded by the Ahr gene, which, upon ligand binding, translocates to the nucleus in a heterodimeric complex with the ARNT ( Ah receptor nuclear translocator) component. The complex binds to several discrete DNA domains containing aromatic hydrocarbon responsive elements (AhRE) present in the regulatory region of the murine cytochrome P 1450 Cyp1a1 gene and of the other genes in the [ Ah] gene battery. As a consequence of binding, a transcriptional complex is formed that activates the expression of these genes by as yet unidentified mechanisms. We have analyzed DNA-protein interactions in four of these domains, specifically, 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 that 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 molecular mass of 35–40 kDa, bound only to AhRE3 (site D), and has been identified tentatively as a member of the C/EBP family of transcription factors. The second protein, purified by DNA-affinity chromatography, had an apparent molecular mass of 95 kDa and bound to a larger DNA motif 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 nor was it ARNT, since it was found in receptorless ( Ahr − ) and in nuclear translocation-defective ( Arnt − ) cells, as well as in cells that had not been exposed to 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD; dioxin), a potent inducer of Cyp1a1 expression. Evidence from in vivo methylation protection indicated that two G residues flanking AhRE3, one of which is required for binding of the 95-kDa protein, may be protected from methylation in uninduced cells and become exposed upon dioxin treatment, suggesting that the 95-kDa protein may be constitutively bound to AhRE3, and be displaced by binding of the Ah receptor complex. These results lend support to the concept that the transcriptional regulation of the [ Ah] battery genes could be modulated by combinatorial interactions of the Ah receptor complex with other transcription factors.