ORIENTATION OF THE HETERODIMERIC ARYL-HYDROCARBON (DIOXIN) RECEPTOR COMPLEX ON ITS ASYMMETRIC DNA RECOGNITION SEQUENCE

The 2,3,7,8-tetrachlorodibenzo-p-dioxin-tranformed aryl hydrocarbon re ceptor (AHR) complex binds to xenobiotic-responsive element (XRE) sequ ences in the 5' flanking region of the CYP1A1 gene, resulting in initi ation of transcription. Both components of the transformed AHR complex [the ligand-binding AHR monomer and the AHR nuclear translocator (ARN T)] directly contact the XRE. These proteins belong to a novel subclas s of basic helix-loop-helix transcription factors. The binding sites o f AHR and ARNT on the asymmetric XRE were determined using nuclear ext racts of 2,3,7,8-tetrachiorodibenzo-p-dioxin-treated Hepa-1c1c7 cells and a panel of double-stranded oligonucleotides containing XRE1 of the CYP1A1 gene (5'-TTGCGTGAGAA-3') in which all combinations of three, t wo,or one of the thymines indicated were substituted by the photoreact ive thymine analog 5-bromodeoxyuracil. Covalent cross-linking analysis and immunoprecipitation with antibodies specific for AHR or ARNT demo nstrated that ARNT directly contacts the 3'-most thymine position, tha t AHR directly contacts the second thymine position, and that neither protein contacts the 5'-most thymine position. The thymine position co ntacted by ARNT lies within a three-nucleotide sequence (5'-GTG-3') id entical to a half-site of an E-box element (5'-CACGTG-3') that is reco gnized by a number of other basic helix-loop-helix transcription facto rs. AHR binds to a portion of the XRE that does not resemble an E-box. Additional experiments demonstrated that neither protein loops over t o contact residues located beyond the other's binding site.