DNA binding by the heterodimeric Ah receptor. Relationship to dioxin-induced CYP1A1 transcription in vivo.
The environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin induces the microsomal enzyme cytochrome P4501A1 by increasing the transcription rate of the CYP1A1 gene. Induction requires two basic helix-loop-helix proteins, the ligand-binding aromatic hydrocarbon receptor (AhR) and its heterodimerization partner, the AhR nuclear translocator (Arnt). The AhR/Arnt heterodimer induces transcription by binding to dioxin-responsive elements (DREs) within an enhancer upstream of the CYP1A1 gene. The basic regions of AhR and Arnt are crucial for DRE binding. We have mutated these regions in order to analyze the relationship between DRE binding (determined in vitro using an electrophoretic mobility shift assay) and induction of CYP1A1 transcription (determined in vivo by genetic complementation of AhR-defective and Arnt-defective mouse hepatoma cells, using an RNase protection assay to measure mRNA accumulation). Our findings reveal the amino acids in the basic regions of AhR/Arnt that are important for both DRE binding and induction of transcription. This information provides biological background for the interpretation of structural (e.g. crystallographic) studies of the interactions between AhR/Arnt and the DRE. Our findings also indicate that the in vitro behavior of the mutants does not consistently predict their functional activity in vivo. Thus, genetic complementation constitutes an important and stringent test for analyzing the effects of mutations on AhR/Arnt function.
Dong
L
L
Department of Molecular Pharmacology, Stanford University School of Medicine, Stanford, California 94305-5332, USA.
Ma
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Whitlock
J P
JP
D000595:Amino Acid Sequence; D000818:Animals; D051784:Aryl Hydrocarbon Receptor Nuclear Translocator; D001483:Base Sequence; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism; D017931:DNA Primers / Q000737:chemistry; D004268:DNA-Binding Proteins / Q000378:metabolism; D005816:Genetic Complementation Test; D018257:Helix-Loop-Helix Motifs; D008114:Liver Neoplasms, Experimental; D051379:Mice; D008969:Molecular Sequence Data; D018336:Receptors, Aryl Hydrocarbon / Q000378:metabolism*; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship; D014157:Transcription Factors / Q000378:metabolism*; D014158:Transcription, Genetic; D014407:Tumor Cells, Cultured
C497397:Arnt protein, mouse; D017931:DNA Primers; D004268:DNA-Binding Proteins; D018336:Receptors, Aryl Hydrocarbon; D014157:Transcription Factors; D051784:Aryl Hydrocarbon Receptor Nuclear Translocator; D003577:Cytochrome P-450 Enzyme System