Binding of transformed Ah receptor complex to a dioxin responsive transcriptional enhancer: evidence for two distinct heteromeric DNA-binding forms.
Guinea pig hepatic Ah receptor (AhR) complex was transformed in vitro to its DNA-binding form by incubation with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, dioxin). Transformed TCDD-AhR was covalently cross-linked by UV-irradiation to a bromodeoxyuridine-substituted oligonucleotide containing its specific DNA recognition site, the dioxin responsive element (DRE). Denaturing gel electrophoresis and autoradiography identified four TCDD-inducible protein-DNA complexes, with molecular masses of approximately 97, 105, and 115 kDa and a somewhat broader complex at 247 kDa. The 247-kDa complex appears to contain two distinct protein-DNA complexes of approximately 232 and 256 kDa and represents two proteins covalently cross-linked to a single DRE oligonucleotide, while the 97, 105, and 115-kDa complexes represent single protein-DRE cross-links. UV cross-linking to DRE oligonucleotides containing variable numbers of BrdU residues revealed that the 105-kDa protein, identified as the AhR ligand-binding subunit by photoaffinity labeling with a radioiodinated AhR agonist, cross-links to the DRE core consensus (5'-GCGTG-3'); the 97- and 115-kDa non-ligand-binding proteins differentially cross-link immediately 5'-ward of the core. Overall, our results not only demonstrate that the critical protein-DNA contacts which occur between the AhR complex and the DRE are made primarily by the ligand-binding subunit but also indicate that the AhR complex exists as two distinct heteromeric DNA-binding forms, containing one 105-kDa ligand-binding subunit and either one 115- or one 97-kDa non-ligand-binding subunit.
Swanson
H I
HI
Department of Biochemistry, Michigan State University, East Lansing 48824.
Tullis
K
K
Denison
M S
MS
D000818:Animals; D001483:Base Sequence; D001973:Bromodeoxyuridine / Q000378:metabolism; D017124:Conserved Sequence; D003432:Cross-Linking Reagents; D003600:Cytosol / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D004147:Dioxins / Q000737:chemistry; D004742:Enhancer Elements, Genetic / Q000187:drug effects; D005786:Gene Expression Regulation; D006168:Guinea Pigs; D008297:Male; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000737:chemistry / Q000378:metabolism; D000072317:Polychlorinated Dibenzodioxins / Q000494:pharmacology*; D011487:Protein Conformation; D018336:Receptors, Aryl Hydrocarbon / Q000737:chemistry / Q000378:metabolism*; D012689:Sequence Homology, Nucleic Acid; D014466:Ultraviolet Rays
D003432:Cross-Linking Reagents; D004268:DNA-Binding Proteins; D004147:Dioxins; D009838:Oligodeoxyribonucleotides; D000072317:Polychlorinated Dibenzodioxins; D018336:Receptors, Aryl Hydrocarbon; C054916:2-azido-3-iodo-7,8-dibromodibenzo-1,4-dioxin; D001973:Bromodeoxyuridine