Purification of the DNA binding form of dioxin receptor. Role of the Arnt cofactor in regulation of dioxin receptor function.

Abstract

The basic region/helix-loop-helix dioxin receptor mediates signal transduction by dioxin (2,3,7,8-tetrachlorodibenzo-p-dioxin). Upon ligand binding the dioxin receptor is converted from a latent, non-DNA binding form to a form that directly interacts with target genes by binding to dioxin-responsive transcriptional control elements. We have purified by conventional and DNA affinity chromatographic procedures the ligand-activated, DNA binding form of dioxin receptor to examine its architecture and functional properties. We observed that the DNA binding activity of the receptor was labile. Most notably, this activity was lost following DNA affinity purification. In complementation experiments we have identified an auxiliary factor(s) that exhibited very poor, if any, intrinsic affinity for the DNA target sequence in vitro but strongly increased the DNA binding activity of the purified receptor-containing material identified by immunoblot analysis. In a similar fashion the in vitro expressed basic region/helix-loop-helix factor Arnt (that has been postulated to modulate the nuclear translocation function of the receptor) reconstituted the DNA binding function of the purified receptor, and the purified auxiliary factor reconstituted receptor activity upon addition to an extract from mutant, Arnt-deficient hepatoma cells. Conversely, purified dioxin receptor reconstituted DNA binding activity in extracts from receptor-deficient hepatoma cells which express bona fide levels of Arnt. Interestingly, UV cross-linking studies using a BrdU-substituted DNA target sequence indicated that primarily the receptor protein was bound to DNA. Moreover, we demonstrate that purified receptor or Arnt exhibited virtually no detectable affinity for the target sequence individually but, in the presence of one another, showed a strong synergy in DNA binding activity in vitro. Importantly, simultaneous expression of the receptor and Arnt resulted in synergistic induction of gene expression in vivo. These data demonstrate that Arnt plays a central role in control of dioxin receptor function by cooperatively modulating the DNA binding activity of the receptor in vitro and dioxin-dependent transactivation in vivo.