Biomedical subjects

M L Whitelaw

Publications and source records attributed to M L Whitelaw.

At least 19 recordsLinked to original sources

PubMed2003

The hypoxia-inducible factors: key transcriptional regulators of hypoxic responses.

Oxygen depravation in mammals leads to the transcriptional induction of a host of target genes to metabolically adapt to this deficiency, including erythropoietin and vascular endothelial growth factor. This response is primarily mediated by the hypoxia-inducible factors (HIFs) which are members of the basic-helix-loop-helix/Per-ARNT-Sim (bHLH/PAS) transcription factor family. The HIFs are primarily regulated via a two-step mechanism of HIF post-translational modification, increasing both protein stability and transactivation capacity. This review aims to summarise our current understanding of these processes, and discuss the important role of the HIFs in the pathophysiology of many human diseases.

Animals↗

PubMed2002

Effect of ARA9 on dioxin receptor mediated transcription.

The dioxin (Aryl hydrocarbon) receptor (DR) is a unique bHLH transcription factor which is activated by binding of planar aromatic hydrocarbons typified by dioxin (TCDD). The active receptor is key to metabolism of aryl hydrocarbon xenobiotics by being a potent inducer of CYP1A1 gene activity. Chlorinated dioxins are inert to metabolism and initiate multifarious toxicities, including potent tumour promotion. These ill-effects are mediated by the activated DR and we are studying the mechanisms by which the ligand binding domain of the DR controls activity of the protein. The DR ligand binding domain resides within a PAS (Per/Arnt/Sim homology) region which is contiguous with the bHLH. The latent bHLH/PAS dioxin receptor (DR) is found in the cytoplasm of most mammalian cell types in a complex with heat shock protein 90, a novel immunophilin like protein termed ARA9/XAP2/AIP, and the co-chaperone p23. Here we use antisense ARA9 constructs to reveal that in the absence of ARA9, the DR is unable to form a transcriptionally active complex. Co-expression of antisense ARA9 with a form of the DR which is constitutively targeted to the nucleus leads to dramatically decreased levels of the nuclear DR protein, implying that ARA9 may function beyond its currently proposed role in cytoplasmic retention of the latent DR.

Animals↗

PubMed2001

Definition of a dioxin receptor mutant that is a constitutive activator of transcription: delineation of overlapping repression and ligand binding functions within the PAS domain.

The intracellular dioxin (aryl hydrocarbon) receptor is a ligand-activated transcription factor that mediates the adaptive and toxic responses to environmental pollutants such as 2,3,7,8-tetrachlorodibenzo-p-dioxin and structurally related congeners. Whereas the ligand-free receptor is characterized by its association with the molecular chaperone hsp90, exposure to ligand initiates a multistep activation process involving nuclear translocation, dissociation from the hsp90 complex, and dimerization with its partner protein Arnt. In this study, we have characterized a dioxin receptor deletion mutant lacking the minimal ligand-binding domain of the receptor. This mutant did not bind ligand and localized constitutively to the nucleus. However, this protein was functionally inert since it failed to dimerize with Arnt and to bind DNA. In contrast, a dioxin receptor deletion mutant lacking the minimal PAS B motif but maintaining the N-terminal half of the ligand-binding domain showed constitutive dimerization with Arnt, bound DNA, and activated transcription in a ligand-independent manner. Interestingly, this mutant showed a more potent functional activity than the dioxin-activated wild-type receptor in several different cell lines. In conclusion, the constitutively active dioxin receptor may provide an important mechanistic tool to investigate receptor-mediated regulatory pathways in closer detail.

Animals↗

PubMed2000

A redox mechanism controls differential DNA binding activities of hypoxia-inducible factor (HIF) 1alpha and the HIF-like factor.

Hypoxia-inducible factor 1alpha (HIF-1alpha) and the HIF-like factor (HLF) are two highly related basic Helix-Loop-Helix/Per-Arnt-Sim (bHLH/PAS) homology transcription factors that undergo dramatically increased function at low oxygen levels. Despite strong similarities in their activation mechanisms (e.g. they both undergo rapid hypoxia-induced protein stabilization, bind identical target DNA sequences, and induce synthetic reporter genes to similar degrees), they are both essential for embryo survival via distinct functions during vascularization (HIF-1alpha) or catecholamine production (HLF). It is currently unknown how such specificity of action is achieved. We report here that DNA binding by HLF, but not by HIF-1alpha, is dependent upon reducing redox conditions. In vitro DNA binding and mammalian two-hybrid assays showed that a unique cysteine in the DNA-binding basic region of HLF is a target for the reducing activity of redox factor Ref-1. Although the N-terminal DNA-binding domain of HIF-1alpha can function in the absence of Ref-1, we found that the C-terminal region containing the transactivation domain requires Ref-1 for full activity. Our data reveal that the hypoxia-inducible factors are subject to complex redox control mechanisms that can target discrete regions of the proteins and are the first to establish a discriminating control mechanism for differential regulation of HIF-1alpha and HLF activity.

Amino Acid Sequence↗

PubMed1999

Degradation of the basic helix-loop-helix/Per-ARNT-Sim homology domain dioxin receptor via the ubiquitin/proteasome pathway.

The basic helix-loop-helix/Per-ARNT-Sim homology domain dioxin receptor (DR) translocates to the nucleus upon binding of aromatic hydrocarbon ligands typified by dioxin, whereupon it partners the Ah receptor nuclear translocator and initiates transcription. Concurrently, ligand binding down-regulates receptor levels via an unknown mechanism. In this study we show that receptor levels are dependent upon cellular compartmentalization, with entry into the nucleus leading to the rapid destruction of the DR. Ligand-induced DR translocation was bypassed by adding a heterologous nuclear localization signal to the DR, creating a constitutively nuclear form of the dioxin receptor (DRNLS). The DRNLS protein was shown to be unstable with a half-life of </=1 h whether partnering ARNT or HSP90. Thus, the structural changes induced by ligand binding have no inherent effect on DR stability but are critical in transporting the receptor prior to degradation. The proteolytic pathway that degrades the nuclear receptor is suggested to involve ubiquitination as it was inhibited by the proteasome inhibitor MG132 or co-expression of DRNLS with the ubiquitin mutant UbK48R. Incubation of cells expressing DRNLS with the phosphatase inhibitor calyculin resulted in the rapid phosphorylation and ubiquitination of DRNLS, suggesting that a nuclear kinase is required to trigger receptor proteolysis. Overall, this study demonstrates a novel mechanism of proteolysis whereby the simple relocation of a transcription factor from cytoplasm to nucleus initiates its rapid destruction.

Animals↗

PubMed1999

Multiple roles of ligand in transforming the dioxin receptor to an active basic helix-loop-helix/PAS transcription factor complex with the nuclear protein Arnt.

The dioxin receptor is a ligand-activated transcription factor belonging to an emerging class of basic helix-loop-helix/PAS proteins which show interaction with the molecular chaperone hsp90 in their latent states and require heterodimerization with a general cofactor, Arnt, to form active DNA binding complexes. Upon binding of polycyclic aromatic hydrocarbons typified by dioxin, the dioxin receptor translocates from the cytoplasm to the nucleus to allow interaction with Arnt. Here we have bypassed the nuclear translocation step by creating a cell line which expresses a constitutively nuclear dioxin receptor, which we find remains in a latent form, demonstrating that ligand has functional roles beyond initiating nuclear import of the receptor. Treatment of the nuclear receptor with dioxin induces dimerization with Arnt to form an active transcription factor complex, while in stark contrast, treatment with the hsp90 ligand geldanamycin results in rapid degradation of the receptor. Inhibition of degradation by a proteasome inhibitor allowed geldanamycin to transform the nuclear dioxin receptor to a heterodimer with Arnt (DR-Arnt). Our results indicate that unchaperoned dioxin receptor is extremely labile and is consistent with a concerted nuclear mechanism for receptor activation whereby hsp90 is released from the ligand-bound dioxin receptor concomitant with Arnt dimerization. Strikingly, artificial transformation of the receptor by geldanamycin provided a DR-Arnt complex capable of binding DNA but incapable of stimulating transcription. Limited proteolysis of DR-Arnt heterodimers indicated different conformations for dioxin versus geldanamycin-transformed receptors. Our studies of intracellular dioxin receptor transformation indicate that ligand plays multiple mechanistic roles during receptor activation, being important for nuclear translocation, transformation to an Arnt heterodimer, and maintenance of a structural integrity key for transcriptional activation.

Adrenal Glands↗

PubMed1998

Transcriptional activation of cytochrome P450 genes by different classes of chemical inducers.

1. We review here the molecular mechanisms underlying the xenobiotic induction of genes encoding cytochrome P450 (CYP) enzymes in the liver and other tissues. We will focus on four major families of CYP genes. 2. Members of the CYP1 gene family are induced by polycyclic aromatic hydrocarbons and this process is mediated by the basic helix-loop-helix proteins: the Ah receptor and its heterodimeric partner Arnt. Considerable progress has been made in elucidating the molecular details of this induction process. 3. CYP4 genes are activated by peroxisomal proliferators, a group of structurally diverse chemicals that also induce peroxisome proliferation. The transcriptional response is dependent on the peroxisome proliferator-activated receptor and its partner RXR, both members of the nuclear receptor superfamily; their role in the induction process has been well characterized at the molecular level. 4. In contrast, the mechanism of gene induction of CYP2 genes by phenobarbital and other structurally diverse inducers is not well understood and a specific phenobarbital-responsive receptor has not been identified. 5. Induction of the CYP3 gene family by the glucocorticoid dexamethasone appears to involve the glucocorticoid receptor, but this receptor is not apparently required for induction by metapyrone and a complete molecular understanding of the induction processes is lacking at present.

Animals↗

PubMed1998

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

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.

Animals↗

PubMed1995

The basic helix-loop-helix/PAS factor Sim is associated with hsp90. Implications for regulation by interaction with partner factors.

Sim is a Drosophila developmental basic helix-loop-helix (bHLH) transcription factor containing a Per-Arnt-Sim (PAS) region of homology. Here we demonstrate that Sim, in analogy to the structurally related bHLH/PAS dioxin receptor, was stably associated with the molecular chaperone hsp90. In the case of the dioxin receptor, release of hsp90 and derepression of receptor function appear to be regulated by ligand binding and dimerization with Arnt, a non-hsp90-associated bHLH/PAS factor. Dimerization with Arnt very efficiently disrupted Sim-hsp90 interaction, a process that required both the bHLH and PAS dimerization motifs of Arnt. Moreover, hsp90 was also released upon dimerization of Sim with the Drosophila PAS factor Per, whereas the hsp90-associated dioxin receptor failed to interact with Sim. These results indicate that hsp90 may play a role in conditional regulation of Sim function, and that Per and possibly bHLH/PAS partner factors may activate Sim by inducing release of hsp90 during the dimerization process.

Amino Acid Sequence↗

PubMed1995

Definition of a minimal domain of the dioxin receptor that is associated with Hsp90 and maintains wild type ligand binding affinity and specificity.

The dioxin receptor is a cytoplasmic basic helix-loop-helix/Per-Arnt-Sim homology (bHLH/PAS) protein known to bind planar polycyclic ligands including polycyclic aromatic hydrocarbons, benzoflavones, heterocyclic amines, and halogenated aromatic hydrocarbons, e.g. dioxins. Ligand-induced activation of the dioxin receptor initiates a process whereby the receptor is transformed into a nuclear transcription factor complex with a specific bHLH/PAS partner protein, Arnt. In analogy to the glucocorticoid receptor, the latent dioxin receptor is found associated with the molecular chaperone hsp90. We have defined and isolated a minimal ligand binding domain of the dioxin receptor from the central PAS region, comprising of amino acids 230 to 421, and found this domain to interact with hsp90 in vitro. Expression of the minimal ligand binding domain in wheat germ lysates or bacteria, systems which harbor hsp90 homologs unable to interact with the glucocorticoid or dioxin receptors, resulted in non-ligand binding forms of this minimal 230 to 421 fragment. Importantly, affinity of the minimal ligand binding domain for dioxin was similar to the affinity inherent in the full-length dioxin receptor, and a profile of ligand structures which specifically bound the minimal ligand binding domain was found to be conserved between this domain and the native receptor. These experiments show that the minimal ligand binding domain maintains the quantitative and qualitative aspects of ligand binding exhibited by the full-length receptor, implying that the central ligand binding pocket may exist to accommodate all classes of specific dioxin receptor ligands, and that this pocket is critically dependent upon hsp90 for its ligand binding conformation.

Animals↗

PubMed1995

Protein-protein interaction via PAS domains: role of the PAS domain in positive and negative regulation of the bHLH/PAS dioxin receptor-Arnt transcription factor complex.

Gene regulation by dioxins is mediated by the dioxin receptor-Arnt heterodimer, a ligand generated complex of two basic helix-loop-helix (bHLH)/Per-Arnt-Sim (PAS) transcription factors. By using dioxin receptor chimeras where the dimerization and DNA binding bHLH motif has been replaced by a heterologous DNA binding domain, we have detected an ability of Arnt to interact with the dioxin receptor via the PAS domain in a mammalian 'hybrid interaction' system. By coimmunoprecipitation assays, we have confirmed the ability of PAS domains of the dioxin receptor and Arnt to mediate independent heterodimerization in vitro. Selectivity for PAS dimerization was noted in our hybrid interaction system, as dioxin receptor or Arnt PAS-mediated homodimers were not detected. Surprisingly, however, the PAS domain of Per could dimerize with both the dioxin receptor and Arnt subunits in vitro, and disrupt the ability of these subunits to form a DNA binding heterodimer. Moreover, ectopic expression of Per blocked dioxin signalling in mammalian cells. The PAS domains of the dioxin receptor and Arnt are therefore novel dimerizing regions critical in formation of a functional dioxin receptor-Arnt complex, while the PerPAS domain is a potential negative regulator of bHLH/PAS factor function.

Animals↗

PubMed1995

Constitutive function of the basic helix-loop-helix/PAS factor Arnt. Regulation of target promoters via the E box motif.

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.

Amino Acid Sequence↗

PubMed1995

Heat shock protein hsp90 regulates dioxin receptor function in vivo.

The dioxin (aryl hydrocarbon) receptor is a ligand-dependent basic helix-loop-helix (bHLH) factor that binds to xenobiotic response elements of target promoters upon heterodimerization with the bHLH partner factor Arnt. Here we have replaced the bHLH motif of the dioxin receptor with a heterologous DNA-binding domain to create fusion proteins that mediate ligand-dependent transcriptional enhancement in yeast (Saccharomyces cerevisiae). Previously, our experiments indicated that the ligand-free dioxin receptor is stably associated with the 90-kDa heat shock protein, hsp90. To investigate the role of hsp90 in dioxin signaling we have studied receptor function in a yeast strain where hsp90 expression can be down-regulated to about 5% relative to wild-type levels. At low levels of hsp90, ligand-dependent activation of the chimeric dioxin receptor construct was almost completely inhibited, whereas the activity of a similar chimeric construct containing the structurally related Arnt factor was not affected. Moreover, a chimeric dioxin receptor construct lacking the central ligand- and hsp90-binding region of the receptor showed constitutive transcriptional activity in yeast that was not impaired upon down-regulation of hsp90 expression levels. Thus, these data suggest that hsp90 is a critical determinant of conditional regulation of dioxin receptor function in vivo via the ligand-binding domain.

Animals↗

PubMed1995

Distinct roles of the molecular chaperone hsp90 in modulating dioxin receptor function via the basic helix-loop-helix and PAS domains.

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.

Animals↗

PubMed1994

A tyrosine kinase-dependent pathway regulates ligand-dependent activation of the dioxin receptor in human keratinocytes.

Signal transduction by dioxin is mediated by the intracellular basic helix-loop-helix dioxin receptor which, in its ligand-activated state, binds to target DNA as a heteromeric complex with the partner factor Arnt. In contrast, the repressed form of the receptor is a complex with hsp90 which appears to maintain the receptor in an inducible conformation. In human keratinocytes dioxin receptor activation has previously been shown to depend on phosphorylation processes. To further dissect mechanisms regulating dioxin receptor function the importance of tyrosine phosphorylation was investigated by the use of specific tyrosine kinase inhibitors. Here we report that the inhibitor genistein inhibited dioxin-dependent induction of expression of the target gene cytochrome P-450IA1. This effect was rapid and reversible and did not lead to altered levels of dioxin receptor protein. Analyses of dioxin receptor or Arnt fusion proteins that function independently of one another showed that the target for genistein action was the dioxin receptor, and, more specifically, a region of the receptor harboring its ligand-binding domain. In addition, function of an unrelated transactivator, the glucocorticoid receptor, was inhibited by genistein while a truncated form lacking the ligand-binding domain was not. A common denominator between the ligand-binding domains of both receptors is their ability to interact with hsp90. Importantly, co-immunoprecipitation experiments showed that genistein inhibited ligand-induced release of hsp90 from the glucocorticoid receptor. Thus, the interaction of these transactivators with hsp90 may be regulated by a tyrosine kinase-dependent pathway.

Animals↗

PubMed1994

Agonistic and antagonistic effects of alpha-naphthoflavone on dioxin receptor function. Role of the basic region helix-loop-helix dioxin receptor partner factor Arnt.

The dioxin receptor is a ligand-dependent transcription factor that binds to target DNA sequences (xenobiotic responsive elements, XREs) following ligand-dependent dimerization with its partner factor, Arnt (aryl hydrocarbon receptor nuclear translocator). Both factors contain an N-terminal basic region helix-loop-helix motif mediating dimerization and subsequent DNA binding. In this study we investigate the possible role of Arnt in agonistic and antagonistic effects of the dioxin receptor ligand alpha-naphthoflavone (ANF). Using specific antisera for the ligand binding dioxin receptor and Arnt, respectively, we show that exposure of the dioxin receptor to ANF in vitro induced recruitment of Arnt, thus stimulating binding of the heteromeric complex to XRE. In transient transfection assays, ANF at high concentrations stimulated expression of an XRE-driven reporter gene. This agonistic effect of ANF is, therefore, most likely attributable to ANF stimulation of dioxin receptor-Arnt heterodimerization and subsequent binding of the complex to XRE. Using a minimal XRE-driven reporter gene construct, we could further confirm earlier studies showing that ANF antagonizes the effect of a dioxin receptor agonist, 2,3,7,8-tetrachlorodibenzo-p-dioxin. Next we employed chimeric receptor constructs containing amino acids 1-500 of the human glucocorticoid receptor fused to dioxin receptor fragments lacking the very N-terminal basic region helix-loop-helix dimerization and DNA binding motif. These chimeric receptor constructs show dioxin responsiveness upon transient transfection into mutant Arnt-deficient hepatoma cells and are, thus, functionally uncoupled from Arnt. Importantly, dioxin-dependent activation of the chimeric receptors was inhibited in the presence of ANF, demonstrating that dimerization of dioxin receptor with Arnt was not necessary for manifestation of the antagonistic effect of ANF. Rather, dioxin receptor sequences, which confer dioxin regulation upon a heterologous DNA binding and transactivating domain, also mediated the antagonistic effects of ANF.

Aryl Hydrocarbon Receptor Nuclear Translocator↗

PubMed1994

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

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.

Animals↗

PubMed1994

Identification of transactivation and repression functions of the dioxin receptor and its basic helix-loop-helix/PAS partner factor Arnt: inducible versus constitutive modes of regulation.

Gene regulation by dioxins is mediated via the dioxin receptor, a ligand-dependent basic helix-loop-helix (bHLH)/PAS transcription factor. The latent dioxin receptor responds to dioxin signalling by forming an activated heterodimeric complex with a specific bHLH partner, Arnt, an essential process for target DNA recognition. We have analyzed the transactivating potential within this heterodimeric complex by dissecting it into individual subunits, replacing the dimerization and DNA-binding bHLH motifs with heterologous zinc finger DNA-binding domains. The uncoupled Arnt chimera, maintaining 84% of Arnt residues, forms a potent and constitutive transcription factor. Chimeric proteins show that the dioxin receptor also harbors a strong transactivation domain in the C terminus, although this activity was silenced by inclusion of 82 amino acids from the central ligand-binding portion of the dioxin receptor. This central repression region conferred binding of the molecular chaperone hsp90 upon otherwise constitutive chimeras in vitro, indicating that hsp90 has the ability to mediate a cis-repressive function on distant transactivation domains. Importantly, when the ligand-binding domain of the dioxin receptor remained intact, the ability of this hsp90-binding activity to confer repression became conditional rather than irreversible. Our data are consistent with a model in which crucial activities of the dioxin receptor, such as dimerization with Arnt and transactivation, are conditionally repressed by the central ligand- and-hsp90-binding region of the receptor. In contrast, the Arnt protein appears to be free from any repressive activity. Moreover, within the context of the dioxin response element (xenobiotic response element), the C terminus of Arnt conferred a potent, dominating transactivation function onto the native bHLH heterodimeric complex. Finally, the relative transactivation potencies of the individual dioxin receptor and Arnt chimeras varied with cell type and promoter architecture, indicating that the mechanisms for transcriptional activation may differ between these two subunits and that in the native complex the transactivation pathway may be dependent upon cell-specific and promoter contexts.

Animals↗