Role of the PAS domain in regulation of dimerization and DNA binding specificity of the dioxin receptor

Abstract

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-Arnt-Sim (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 bHLH-Zip transcription factor USF. In contrast, a construct spanning the dioxin receptor bHLH domain and an N-terminal 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.

FIG. 1

Bacterial expression and purification of dioxin receptor-GST fusion proteins. (A) Schematic representation of the dioxin receptor fragments that were expressed as GST fusion proteins. The wild-type mouse dioxin receptor (mDR) is presented for comparison. (B) Bacterial fusion proteins were affinity purified on glutathione-Sepharose, and 0.5 μg of protein was analyzed by SDS–10% PAGE and silver staining. Arrows indicate the various purified bHLH-PAS fragments.

FIG. 2

Bacterially expressed dioxin receptor bHLH– and bHLH-PAS–GST fusion proteins interact with Arnt. (A) Full-length Arnt was in vitro translated in the presence of [³⁵S]methionine and incubated with 0.1 μg of bacterially expressed receptor proteins, as indicated, or the GST protein as a negative control. The mixtures were precipitated by adsorption to glutathione-Sepharose and centrifugation. Eluted material was separated by SDS-PAGE and visualized by fluorography. (B) bHLH-PAS fragments of the dioxin receptor form non-DNA-binding or DNA-binding complexes with Arnt. The indicated bacterially expressed dioxin receptor fragments (0.1 μg) were incubated in the absence or presence of bacterially expressed Arnt, and XRE binding activity was assayed by gel mobility shift analysis. (C) XRE binding activity of the DR-1-82–Arnt complex was analyzed in the absence or presence of polyclonal antibodies against Arnt (α-Arnt) (lane 4), the GST domain of the recombinant protein (α-GST) (lane 5), or preimmune serum (P.I.S.) (lane 6). (D) Certain dioxin receptor bHLH-PAS fragments function as dominant negative regulators of XRE binding activity. A cytosolic extract from untreated Hepa 1c1c7 cells was treated with 10 nM dioxin (+) or with dimethyl sulfoxide vehicle alone (−) for 2 h at 30°C and incubated with 0.1 μg of purified dioxin receptor fragments for 30 min at 25°C. XRE binding activity was assayed by gel mobility shift analysis. The asterisk indicates nonspecific DNA binding activity occasionally observed with some DR-1-82 preparations. Specific dioxin receptor-Arnt DNA complexes are indicated by arrows.

FIG. 3

The bHLH domain of the dioxin receptor is sufficient for XRE recognition. COS-7 cells were cotransfected with 0.5 μg of the XRE-driven pTXIXI luciferase reporter gene, 0.2 μg of pCMV-Arnt, and increasing amounts (10 to 100 ng) of pCMX-VP16 expression vectors containing either the DR-1-82 or the DR-1-287 fragment fused to the VP16 transactivation domain and were assayed for luciferase activity. Results of a representative experiment are shown.

FIG. 4

The bHLH domain of Arnt is sufficient to reconstitute XRE binding activity of the bHLH-PAS dioxin receptor. (A) Schematic representation of the different Arnt fragments expressed in E. coli. (B) Bacterially expressed dioxin receptor fragment DR-1-82 and Arnt-1-140 or Arnt-1-407 were coincubated for 30 min at 25°C. XRE binding activity was analyzed by gel mobility shift assay as described in the legend to Fig. 2B.

FIG. 5

Homodimerization and XRE binding activities of the bHLH domain of the dioxin receptor. (A) In vitro-translated full-length dioxin receptor was labeled with [³⁵S]methionine incubated with 0.1 μg of the indicated dioxin receptor fusion proteins, precipitated with glutathione-Sepharose, and analyzed by SDS-PAGE and fluorography. (B) Homodimerization activity of the minimal DR-1-82 receptor fragment was monitored in vivo by using a mammalian two-hybrid assay. COS-7 cells were cotransfected with 0.2 μg of pCMX DR-1-82–Gal4 containing the dioxin receptor bHLH domain fused to the Gal4 DNA binding domain and up to 100 ng of a DR-1-82–VP16 fusion construct together with 0.5 μg of a Gal4 luciferase reporter construct. The cells were assayed for luciferase activity, and results of a representative experiment are shown.

FIG. 5

Homodimerization and XRE binding activities of the bHLH domain of the dioxin receptor. (A) In vitro-translated full-length dioxin receptor was labeled with [³⁵S]methionine incubated with 0.1 μg of the indicated dioxin receptor fusion proteins, precipitated with glutathione-Sepharose, and analyzed by SDS-PAGE and fluorography. (B) Homodimerization activity of the minimal DR-1-82 receptor fragment was monitored in vivo by using a mammalian two-hybrid assay. COS-7 cells were cotransfected with 0.2 μg of pCMX DR-1-82–Gal4 containing the dioxin receptor bHLH domain fused to the Gal4 DNA binding domain and up to 100 ng of a DR-1-82–VP16 fusion construct together with 0.5 μg of a Gal4 luciferase reporter construct. The cells were assayed for luciferase activity, and results of a representative experiment are shown.

FIG. 6

Constitutive dimerization of dioxin receptor bHLH and bHLH–PAS-A proteins with a dioxin receptor bHLH swap mutant. (A) Structural organizations of the dioxin receptor (DR), Arnt, and a bHLH swap mutant (DRbHLH/Arnt) in which the bHLH domain of Arnt has been replaced by that of the dioxin receptor. (B) DRbHLH/Arnt was expressed and labeled with [³⁵S]methionine by in vitro translation in reticulocyte lysates and incubated with either DR-1-82, DR-1-287, or the purified GST domain, as indicated, prior to precipitation with glutathione-Sepharose. Precipitated material was analyzed by SDS-PAGE and fluorography.

FIG. 7

The PAS domain restricts a broad dimerization activity of the minimal bHLH domain of the dioxin receptor. (A) USF dimerizes with the dioxin receptor bHLH domain. Wild-type USF was in vitro translated in the presence of [³⁵S]methionine and incubated with the bacterially expressed dioxin receptor proteins DR-1-82, DR-1-165, DR 1-188, and DR 1-287. GST precipitation experiments were performed and analyzed as described in the legend to Fig. 2A. (B and C) Alteration in DNA specificity upon formation of USF–DR-1-82 heterodimeric complexes. Purified, bacterially expressed His₁₀-tagged USF was incubated with DR-1-82 in the absence (B) or presence (C) of Arnt and analyzed for DNA binding activity in gel mobility shift experiments using either a ³²P-labeled E-box probe from the adenovirus major late promoter (B) or an XRE probe (C) as described in the legend to Fig. 2B.