In vitro transformation of the human Ah receptor and its binding to a dioxin response element.
Many biological effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, dioxin) are mediated by a soluble intracellular protein, the Ah receptor (AhR). After binding of TCDD to the cytoplasmic AhR there occurs a poorly understood "transformation" step, wherein the TCDD-AhR complex is converted to a form that can bind to DNA with high affinity. The binding of transformed AhR to a specific dioxin-responsive element (DRE) upstream of a given gene stimulates transcriptional activation of that gene. Using a gel retardation assay we examined the interaction of transformed human cytosolic TCDD-AhR complexes with a synthetic DNA oligonucleotide containing a single DRE site. Transformation and DNA binding of human AhR in vitro was ligand dependent and specific for DRE-containing DNA. Unlike rodent hepatic AhR, in vitro transformation of human AhR was completely temperature dependent. Although at 4 degrees AhR binds ligand, no transformation of human TCDD-AhR complex was observed at 4 degrees even after 24 h; however, rapid transformation as measured by DNA binding was detectable as early as 10 min after warming to 22 degrees, with maximal binding by about 60 min. Calf thymus DNA-Sepharose or DRE-Sepharose column chromatography showed that transformed human cytosolic AhR interacts with DNA as a single species. The absolute temperature dependency of human AhR transformation mimics that observed in vivo and provides a useful system to study the mechanism of AhR transformation in detail.
Harper
P A
PA
Division of Clinical Pharmacology and Toxicology, Hospital for Sick Children, Toronto, Ontario, Canada.
Giannone
J V
JV
Okey
A B
AB
Denison
M S
MS
D000818:Animals; D001483:Base Sequence; D002478:Cells, Cultured; D016384:Consensus Sequence; D004268:DNA-Binding Proteins / Q000378:metabolism*; D005786:Gene Expression Regulation; D066298:In Vitro Techniques; D008024:Ligands; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D051381:Rats; D018336:Receptors, Aryl Hydrocarbon; D011955:Receptors, Drug / Q000378:metabolism*; D012045:Regulatory Sequences, Nucleic Acid; D013329:Structure-Activity Relationship; D013696:Temperature
D004268:DNA-Binding Proteins; D008024:Ligands; D009838:Oligodeoxyribonucleotides; D018336:Receptors, Aryl Hydrocarbon; D011955:Receptors, Drug