DNA-BINDING BY THE HETERODIMERIC AH RECEPTOR - RELATIONSHIP TO DIOXIN-INDUCED CYP1A1 TRANSCRIPTION IN-VIVO

The environmental contaminant 2,3,7,8 tetrachloro- dibenzo-p-dioxin in duces the microsomal enzyme cytochrome P4501A1 by increasing the trans cription rate of the CYP1A1 gene, Induction requires two basic helix-l oop-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 d ioxin-responsive elements (DREs) within an enhancer upstream of the CY P1A1 gene. The basic regions of AhR and Arnt are crucial for DRE bindi ng. We have mutated these regions in order to analyze the relationship between DRE binding (determined in vitro using an electrophoretic mob ility shift assay) and induction of CYP1A1 transcription (determined i n vivo by genetic complementation of AhR-defective and Amt-defective m ouse hepatoma cells, using an RNase protection assay to measure mRNA a ccumulation). 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 i nteractions between AhR/Arnt and the DRE. Our findings also indicate t hat the in, vitro behavior of the mutants does not consistently predic t their functional activity in vivo. Thus, genetic complementation con stitutes an important and stringent test for analyzing the effects of mutations on AhR/Arnt function.