Sg. Bacsi et al., ORIENTATION OF THE HETERODIMERIC ARYL-HYDROCARBON (DIOXIN) RECEPTOR COMPLEX ON ITS ASYMMETRIC DNA RECOGNITION SEQUENCE, Molecular pharmacology, 47(3), 1995, pp. 432-438
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.