DNA Binding and Transcriptional Enhancement by Purified TCDD·Ah Receptor Complex

Abstract

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcriptional enhancer which mediates the biochemical and toxic effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and structurally related chemicals and which may have a role in the normal development of some tissues. We have previously reported the purification of the transformed TCDD·receptor complex from rat liver cytosol based on binding to its dioxin-responsive enhancer sequence (DRE) and that it comprises the AhR ligand-binding monomer and its dimerization partner, ARNT. The present studies were designed to compare the DRE-binding characteristics of the purified receptor with the cruder preparations that are commonly used and ultimately to determine whether the purified receptor complex itself (in the absence of additional cytosolic or nuclear factors) is capable of enhancing transcription in anin vitrosystem. The purified AhR retainedin vitroDRE binding activity in the presence of carrier protein and dithiothreitol, and its affinity for the DRE oligonucleotide was equivalent to that of the other receptor preparations (crude and partially purified cytosolic and crude nuclear). When the ligand·receptor complex was bound to a DRE oligonucleotide containing BrdU and then UV-irradiated, two proteins in each of the receptor preparations were found to crosslink to BrdU–DRE, and we concluded that they are the AhR monomer and ARNT protein. All receptor preparations also gave a similar footprint of interaction with G-residues within the DRE consensus sequence, as assessed by methylation interference. Furthermore, purified and partially purified receptors were able to stimulate transcription from a DRE-containing template in a cell-free system in the presence of HeLa cell nuclear extract. Transcriptional enhancement was receptor dose-dependent, TCDD-dependent, and specific for the DRE sequence upstream of the promotor in our template construct. These data document for the first time that a purified TCDD·Ah receptor complex retains both specific DNA binding and transcriptional activities. This observation constitutes an important step toward understanding the mechanism of gene regulation by TCDD since it implies that the transformed receptor·ligand complex itself is competent as a transcriptional enhancer without a requirement for other factors.