The intracellular dioxin receptor mediates signal transduction by dioxin and functions as a ligand-activated transcription factor. It contains a basic helix-loop-helix (bHLH) motif contiguous with a Per-Arnt-Sim (PAS) homology region. In extracts from nonstimulated cells the receptor is recovered in an inducible cytoplasmic form associated with the 90-kDa heat shock protein (hsp90), a molecular chaperone. We have reconstituted ligand-dependent activation of the receptor to a DNA-binding form by using the dioxin receptor and its bHLH-PAS partner factor Arnt expressed by in vitro translation in reticulocyte lysate. Deletion of the PAS domain of the receptor resulted in constitutive dimerization with Arnt. In contrast, this receptor mutant showed low levels of xenobiotic response element-binding activity, indicating that the PAS domain may be important for DNA-binding affinity and/or specificity of the receptor. It was not possible to reconstitute dioxin receptor function with proteins expressed in wheat germ lysate. In line with these observations, reticulocyte lysate but not wheat germ lysate promoted the association of de novo synthesized dioxin receptor with hsp90. At least two distinct domains of the receptor mediated interaction with hsp90: the ligand-binding domain located within the PAS region and, surprisingly, the bHLH domain. Whereas ligand-binding activity correlated with association with hsp90, bHLH-hsp90 interaction appeared to be important for DNA-binding activity but not for dimerization of the receptor. Several distinct roles for hsp90 in modulating dioxin receptor function are therefore likely: correct folding of the ligand-binding domain, interference with Arnt heterodimerization, and folding of a DNA-binding conformation of the bHLH domain. Thus, the dioxin receptor system provides a complex and interesting model of the regulation of transcription factors by hsp90.The intracellular dioxin receptor (also termed the aryl hydrocarbon receptor) is a ubiquitous basic helix-loop-helix (bHLH) factor (4, 10) that mediates signal transduction by the toxic environmental contaminant dioxin (2,3,7,8-tetrachlorodibenzo-p-dioxin) (for recent reviews, see references 33, 42, and 47). In the absence of ligand the receptor exists in an inducible cytoplasmic form. Dioxin induces nuclear translocation of the receptor (reference 34 and references therein) and regulates dimerization with the bHLH partner factor Arnt, enabling both proteins to specifically recognize cognate response elements (xenobiotic response elements [XREs]) within regulated genes (9,24,38,46). Individually, neither the receptor nor Arnt shows any detectable affinity for this target sequence (23, 46). Moreover, they do not appear to bind the twofold symmetric CACGTG or CAGCTG E box motifs (23, 46) that are recognized by the great majority of bHLH and bHLH-leucine zipper (bHLH-Zip) proteins, including lymphoid transcription factors, the oncoproteins Myc and Max, and factors involved in vertebrate myogenesis and Drosophila neurogenesis (for recent r...
The dioxin receptor is a ligand-regulated transcription factor that mediates signal transduction by dioxin and related environmental pollutants. The receptor belongs to the basic helix-loop-helix (bHLH)-Per-ArntSim (PAS) family of factors, which, in addition to the bHLH motif, contain a PAS region of homology. Upon activation, the dioxin receptor dimerizes with the bHLH-PAS factor Arnt, enabling the receptor to recognize xenobiotic response elements in the vicinity of target genes. We have studied the role of the PAS domain in dimerization and DNA binding specificity of the dioxin receptor and Arnt by monitoring the abilities of the individual bHLH domains and different bHLH-PAS fragments to dimerize and bind DNA in vitro and recognize target genes in vivo. The minimal bHLH domain of the dioxin receptor formed homodimeric complexes, heterodimerized with full-length Arnt, and together with Arnt was sufficient for recognition of target DNA in vitro and in vivo. In a similar fashion, only the bHLH domain of Arnt was necessary for DNA binding specificity in the presence of the dioxin receptor bHLH domain. Moreover, the bHLH domain of the dioxin receptor displayed a broad dimerization potential, as manifested by complex formation with, e.g., the unrelated bHLHZip transcription factor USF. In contrast, a construct spanning the dioxin receptor bHLH domain and an Nterminal portion of the PAS domain failed to form homodimers and was capable of dimerizing only with Arnt. Thus, the PAS domain is essential to confer dimerization specificity of the dioxin receptor.Members of the large family of basic helix-loop-helix (bHLH) transcription factors are often involved in regulation of cell type differentiation and proliferation and are characterized by the requirement of formation of homo-or heterodimeric complexes with bHLH partner factors for DNA binding activity (for reviews, see references 13, 28, and 51). In the case of a number of these factors, dimerization specificity has been shown to be determined by residues within the HLH domain itself, whereas structural studies have demonstrated that the basic region binds DNA (18; reviewed in reference 32). In addition, regions outside the bHLH motif, most notably the leucine zipper (Zip) domain in the bHLH-Zip subclass of transcription factors, have been shown to influence dimerization specificity and DNA binding activity of bHLH proteins. Zip domains form amphipathic coiled-coil structures (15,38) and have the ability to interact with one another or various other dimerization interfaces in a highly specific manner. In the case of the transcription factor TFE3, which binds the symmetrical CACGTG E-box motif recognized by a distinct subclass of bHLH transcription factors (29), mutation of the Zip domain yields a protein that binds the E box with reduced affinity. Moreover, mutation of conserved amino acids in the HLH domain completely inhibits specific DNA binding activity (43).The c-myc oncoprotein binds DNA poorly by itself and lacks the ability to form homodimers. The bHLH-Zip ...
Arnt is a nuclear basic helix-loop-helix (bHLH) transcription factor that, contiguous with the bHLH motif, contains a region of homology (PAS) with the Drosophila factors Per and Sim. Arnt dimerizes in a ligand-dependent manner with the bHLH dioxin receptor, a process that enables the dioxin-(2,3,7,8-tetrachlorodibenzo-p-dioxin)-activated Arnt-dioxin receptor complex to recognize dioxin response elements of target promoters. In the absence of dioxin, Arnt does not bind to this target sequence motif. The constitutive function of Arnt is presently not understood. Here we demonstrate that Arnt constitutively bound the E box motif CACGTG that is also recognized by a number of distinct bHLH factors, including USF and Max. Importantly, amino acids that have been identified to be critical for E box recognition by Max and USF are conserved in Arnt. Consistent with these observations, full-length Arnt, but not an Arnt deletion mutant lacking its potent C-terminal transactivation domain, constitutively activated CACGTG E box-driven reporter genes in vivo. These results indicate a role of Arnt in regulation of a network of target genes that is distinct from that regulated by the Arnt-dioxin receptor complex in dioxin-stimulated cells.
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