Ligand-dependent recruitment of the Arnt coregulator determines DNA recognition by the dioxin receptor.

Abstract

The intracellular basic region/helix-loop-helix (bHLH) dioxin receptor mediates signal transduction by dioxin (2,3,7,8-tetrachlorodibenzo-p-dioxin) and functions as a ligand-activated DNA binding protein directly interacting with target genes by binding to dioxin response elements. Here we show that the partially purified, ligand-bound receptor alone could not bind target DNA. In contrast, DNA binding by the receptor could be induced by addition of a cytosolic auxiliary activity which functionally and biochemically corresponded to the bHLH factor Arnt. While Arnt exhibited no detectable affinity for the dioxin response element in the absence of the dioxin receptor, it strongly promoted the DNA binding function of the ligand-activated but not the ligand-free receptor forms. Arnt also functionally reconstituted in vitro the DNA binding activity of a mutant, nuclear translocation-deficient dioxin receptor phenotype in cytosolic extracts from a dioxin-resistant hepatoma cell line. Importantly, coimmunoprecipitation experiments showed that Arnt physically interacted in solution with the ligand-activated dioxin receptor but failed to heterodimerize with the ligand-free, hsp9O-associated receptor form. Mutational analysis suggested that the functional interaction between these two factors occurred via the bHLH motif of Arnt. These data suggest that dioxin receptor activity is governed by a complex pattern of combinatorial regulation involving repression by hsp9O and then by ligand-dependent recruitment of the positive coregulator Arnt. The dioxin receptor system also provides the first example of signal-controlled dimerization of bHLH factors.Signal transduction by dioxins (most notably 2,3,7,8-tetrachlorodibenzo-p-dioxin [TCDD]) is mediated by the intracellular dioxin (or aryl hydrocarbon) receptor. The receptor binds dioxin and its planar aromatic congeners in a saturable manner with high affinity (for reviews, see references 22 and 49). The potent toxicity of dioxins is well established in animal models but is a matter of debate in humans (for a recent review, see reference 19). In animals the toxic effects are typified by thymic wasting and immune suppression, severe epithelial disorders, and tumor promotion (for a review, see reference 51). At the molecular level, dioxins are very potent inducers of transcription of a battery of target genes encoding xenobiotic metabolizing enzymes such as cytochrome P-450IA1, glutathione S-transferase Ya, aldehyde dehydrogenase, and quinone oxidoreductase (for a review, see reference 34). In addition, dioxin appears to transcriptionally regulate the expression of the growth modulatory genes for interleukin-1 and plasminogen activator inhibitor-2 (59). The dioxin induction response is mediated by single or multiple copies of dioxin-inducible transcriptional control elements (xenobiotic response elements [XREs]) in target promoters (16,18,47). In analogy to the current model of action of steroid hormone receptors (for a review, see reference 3), the ligand-activated dioxin recepto...

The basic helix–loop–helix–PAS protein ARNT functions as a potent coactivator of estrogen receptor-dependent transcription

“…These observations demonstrate that the interaction between the ERs and ARNT represents a functionally distinct mechanism compared with ARNT's role as a dimerization partner for the AhR or HIF-1␣. Deletion of the bHLH or PAS domain of ARNT severely compromises AhR and HIF-1␣ activity because of failure of the mutant forms of ARNT to dimerize with the AhR (20) and HIF-1␣ (23). Deletion of the C-terminal transactivation function of ARNT, on the other hand, does not restrict ARNT͞ AhR interaction and has no effect on AhR-mediated transcription (19,24).…”

“…For this purpose we used mutant forms of ARNT where various functional domains of ARNT are deleted. ARNT mutants lacking the bHLH or the PAS domains are unable to interact with AhR, thus obliterating TCDD-dependent transcription (19,20). In contrast, deletion of the C-terminal transactivating region of ARNT does not interfere with its ability to dimerize with AhR, and the resulting complex is still transcriptionally active in response to TCDD (19,21).…”

A Basic Helix-Loop-Helix–Leucine Zipper Transcription Complex in Yeast Functions in a Signaling Pathway from Mitochondria to the Nucleus

“…Two exceptions to the consensus bHLH-E-box binding are the SREB/ADD1 protein, which is involved in transcriptional regulation of the low-density lipoprotein receptor and in adipocyte differentiation (11,39,42), and the aryl hydrocarbon receptor (ARH)-aryl hydrocarbon receptor nuclear translator protein (ARNT) bHLH heterodimeric complex that functions in xenobiotic metabolism (27)(28)(29)41). SREB/ADD1 is a bHLH protein that can recognize both E-box and non-E-box sites.…”

Development and characterization of polyclonal antibodies against the aryl hydrocarbon receptor protein family (AHR1, AHR2, and AHR repressor) of Atlantic killifish Fundulus heteroclitus

“…Some of the most ubiquitous toxic compounds in the environment, including polychlorinated dibenzo-p-dioxins (PCDDs), polychlorinated biphenyls (PCBs), and some polycyclic aromatic hydrocarbons (PAHs), elicit biochemical and toxic responses through the AHR signaling pathway. Following activation by a ligand such as 2, 3,7,, the AHR dimerizes with the aryl hydrocarbon receptor nuclear translocator (ARNT) (Reyes et al 1992;Whitelaw et al 1993), and interacts with AHR response elements (AHREs, also known as XREs or DREs) to regulate expression of genes encoding biotransformation enzymes (Negishi and Nebert 1979;Nebert and Gonzalez 1987;Telakowski-Hopkins et al 1988). The AHR-ARNT complex also regulates numerous other genes involved in a variety of physiological processes (Puga et al 2000).…”