Nucleotide Specificity of DNA Binding of the Aryl Hydrocarbon Receptor:ARNT Complex Is Unaffected by Ligand Structure

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that mediates the toxic and biological effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, dioxin) and a wide variety of structurally diverse ligands through its ability to translocate into the nucleus and bind to a specific DNA recognition site (the dioxin-responsive element [DRE]) adjacent to responsive genes. Although the sequence of the DRE is well defined, several reports suggested that the nucleotide specificity of AhR DNA binding may vary depending on the structure of its bound ligand. Given the potential toxicological significance of this hypothesis, an unbiased DNA-selection-and-PCR-amplification approach was utilized to directly determine whether binding and activation of the AhR by structurally diverse agonists alter its nucleotide specificity of DNA binding. Guinea pig hepatic cytosolic AhR activated in vitro by equipotent concentrations of TCDD, 3-methylcholanthrene, -naphthoflavone, indirubin, L-kynurenine, or YH439 was incubated with a pool of DNA oligonucleotides containing a 15-base pair variable region consisting of all possible nucleotides. The AhR-bound oligonucleotides isolated by immunoprecipitation were PCR amplified and used in subsequent rounds of selection. Sequence analysis of a total of 196 isolated oligonucleotides revealed that each ligand-activated AhR:ARNT complex only bound to DRE-containing DNA oligonucleotides; no non-DRE-containing DNA oligonucleotides were identified. These results demonstrate that the binding and activation of the AhR by structurally diverse agonists do not appear to alter its nucleotide specificity of DNA binding and suggest that stimulation of gene expression mediated by direct DNA binding of ligand-activated AhR:ARNT complexes is DRE dependent.