Protein-DNA interactions at a dioxin-responsive enhancer. Analysis of six bona fide DNA-binding sites for the liganded Ah receptor.

Abstract

The DNA upstream of the dioxin-inducible CYP1A1 gene contains six distinct sites to which the liganded Ah receptor binds in intact mouse hepatoma cells. Here, we have analyzed these six bona fide receptor-binding sites in order to study the relationships between DNA sequence, receptor binding, and dioxin responsiveness. Gel retardation studies reveal that the sites vary by about 7-fold in their relative affinities for the liganded receptor. Within this range, there is no obvious association between the strength of receptor binding and the degree of dioxin responsiveness, as measured in transfection experiments. In fact, one site binds the receptor well but fails to respond to dioxin. This observation implies that the receptor-DNA binding event per se is not sufficient to confer dioxin responsiveness upon a linked gene. Comparison of the DNA sequences of the six receptor-binding sites permits the derivation of a "functional consensus" recognition sequence, which is more extended in length than the "core"-binding sequence previously described. In corroboration of these results, protein-DNA cross-linking studies indicate that the liganded receptor contacts base pairs beyond the core sequence. Our observations also indicate that the liganded receptor can tolerate limited sequence heterogeneity at its DNA-binding site and still elicit a response to dioxin. This finding might reflect corresponding heterogeneity in the amino acid sequence of the liganded receptor's DNA-binding domain.