DNA-BINDING AND TRANSCRIPTIONAL ENHANCEMENT BY PURIFIED TCDD-CENTER-DOT-AH RECEPTOR COMPLEX

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcriptio nal enhancer which mediates the biochemical and toxic effects of 2,3,7 ,8-tetrachlorodibenzo-p-dioxin (TCDD) and structurally related chemica ls 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 dioxi n-responsive enhancer sequence (DRE) and that it comprises the AhR lig and-binding monomer and its dimerization partner, ARNT. The present st udies 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 itse lf (in the absence of additional cytosolic or nuclear factors) is capa ble of enhancing transcription in an in vitro system. The purified AhR retained in vitro DRE binding activity in the presence of carrier pro tein and dithiothreitol, and its affinity for the DRE oligonucleotide was equivalent to that of the other receptor preparations (crude and p artially purified cytosolic and crude nuclear). When the ligand recept or 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 Ah R monomer and ARNT protein. All receptor preparations also gave a simi lar footprint of interaction with G-residues within the DRE consensus sequence, as assessed by methylation interference, Furthermore, purifi ed and partially purified receptors were able to stimulate transcripti on from a DRE-containing template in a cell-free system in the presenc e of HeLa cell nuclear extract, Transcriptional enhancement was recept or dose-dependent, TCDD dependent, and specific for the DRE sequence u pstream of the promotor in our template construct, These data document for the first time that a purified TCDD All receptor complex retains both specific DNA binding and transcriptional activities. This observa tion constitutes an important step toward understanding the mechanism of gene regulation by TCDD since it implies that the transformed recep tor ligand complex itself is competent as a transcriptional enhancer w ithout a requirement for other factors. (C) 1997 Academic Press.