DNA sequence determinants for binding of transformed Ah receptor to a dioxin-responsive enhancer

• Published in: Biochemistry (Easton) Vol. 31; no. 21; pp. 5060 - 5067
• Main Authors: Yao, Eveline F., Denison, Michael S.
• Format: Journal Article
• Language: English
• Published: Washington, DC American Chemical Society 01.06.1992
• Subjects: Animals; Base Sequence; Biological and medical sciences; Chemical and industrial products toxicology. Toxic occupational diseases; DNA - metabolism; DNA-Binding Proteins - genetics; DNA-Binding Proteins - metabolism; Enhancer Elements, Genetic; Guinea Pigs; Liver - metabolism; Medical sciences; Molecular Sequence Data; Mutagenesis; Polychlorinated Dibenzodioxins - pharmacology; Receptors, Aryl Hydrocarbon; Receptors, Drug - genetics; Receptors, Drug - metabolism; Sequence Alignment; Substrate Specificity; Toxicology; Various organic compounds; Dioxin derivatives; Toxicity; Cytochrome P450; Rodentia; Polycyclic aromatic compound; In vitro; Pig; Vertebrata; Biological fixation; Enhancer sequence; Mammalia; Guinea pig; Gene; Animal; DNA; Artiodactyla; Ungulata; Biological receptor
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi00136a019

We have utilized gel retardation analysis and DNA mutagenesis to examine the specific interaction of transformed guinea pig hepatic cytosolic TCDD.AhR complex with a dioxin-responsive element (DRE). Sequence alignment of the mouse CYPIA1 upstream DREs has identified a common invariant "core" consensus sequence of TNGCGTG flanked by several variable nucleotides. Competitive gel retardation analysis using a series of DRE oligonucleotides containing single or multiple base substitutions has allowed identification of those nucleotides important for TCDD.AhR.DRE complex formation. A putative TCDD.AhR DNA-binding consensus sequence of GCGTGNNA/TNNNC/G has been derived. The four core nucleotides, CGTG, appear to be critical for TCDD-inducible protein-DNA complex formation since their substitution decreased AhR binding affinity by 100-800-fold; the remaining conserved bases are also important, albeit to a lesser degree (3-5-fold). The 5'-ward thymine, present in the invariant core sequence of all the DREs identified to date, appears not to be involved in DNA binding of the AhR. The results obtained here indicate that although the primary interaction of the TCDD.AhR complex with the DRE occurs with the conserved "core" sequence, nucleotides flanking the core also contribute to the specificity of DRE binding.