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Two benzo[a]pyrene‐resistant mutant clones (c1 and c37) of the mouse hepatoma Hepa‐1 wild‐type (wt) cell line were examined for their lack of P(1)450 [aryl hydrocarbon (benzo[a]pyrene) hydroxylase (AHH)] activity. From lambda gt11 cDNA libraries, the nearly full‐length P(1)450 cDNAs of wt, c1 and c37 were isolated and sequenced. The c1 cDNA was found to have a single mutation leading to premature termination of the protein after Asn‐414; a rapidly migrating band corresponding to this truncated protein was found on Western immunoblots. The c37 cDNA was found to have two point mutations, leading to Leu‐118––Arg‐118 and Arg‐245––Pro‐245, but otherwise to encode the normal (524‐residue) protein; the mature protein was confirmed by Western blot analysis. P(1)450 cDNA from wt, c1 and c37 and chimeric cDNAs between wt and c37 were inserted into the expression vector pAAH5 and expressed in Saccharomyces cerevisiae strain 50.L4. The Leu‐118––Arg‐118 mutation alone was found to have negligible effect on AHH activity, while the Arg‐245––Pro‐245 mutation alone leads to a 2‐ to 3‐fold decrease in enzyme activity. The two mutations together totally abrogate AHH activity. The biologic mutant c37 provides the first evidence for the importance of Arg‐245, and the complementary function of Leu‐118, in normal P(1)450 enzymic function. This alteration in a single amino acid from arginine to proline might block electron flow directly, or change secondary structure of the protein, such that normal monooxygenation of benzo[a]pyrene cannot occur.

Revertants were selected from aryl hydrocarbon hydroxylase (AHH)-deficient recessive mutants belonging to three complementation groups and from a dominant mutant of the Hepa-1 cell line. The recessive mutants had low spontaneous reversion frequencies (less than 4 X 10(-7] that were increased by mutagenesis. The majority of these revertants also had reacquired only partial AHH activity. Revertants of group A mutants were identical to the wild type with respect to both in vivo and in vitro enzyme stability and the Km for the substrate, benzo [alpha]pyrene, and therefore failed to provide evidence that gene A is the AHH structural gene. Group B and group C mutants are defective in the functioning of the Ah receptor required for AHH induction. Revertants of these groups were normal with respect to in vivo temperature sensitivity for AHH induction and for the 50% effective dose for the inducer, 2,3,7,8-tetrachlorodibenzo-p-dioxin, and thus provided no evidence that the B and C genes code for components of the receptor. Two rare group C revertants possessed AHH activity in the absence of induction. The phenotype of one of these was shown to be recessive to the wild type. Spontaneous revertants of the dominant mutant occurred at a frequency 300-fold greater than those of the recessive mutants, and this frequency was not increased by mutagenesis. These revertants all displayed complete restoration of AHH activity to wild type levels. These observations and the results from cell hybridization studies suggest that the dominant revertants arose by a high frequency event leading to functional elimination of the dominant mutation.

Rare benzo[a]pyrene-resistant clones were previously isolated from the mouse hepatoma cell line, Hepa-1 (Hepa1c1c7), and shown to be deficient in induction of CYP1A1 mRNA by ligands for the aryl hydrocarbon receptor (AHR). Clones belonging to complementation group B were shown to have reduced levels of ligand binding to AHR. It is shown here that all 15 independently derived B clones analyzed had much reduced levels of AHR mRNA, but in each case, the mRNA was normal in size. Infection of B clones with a retroviral expression vector for AHR restores CYP1A1 inducibility (although viral AHR expression is progressively silenced and CYP1A1 expression progressively diminishes as the cells are maintained in culture). Treatment of the B clones with the histone deacetylase inhibitors sodium butyrate or trichostatin A restores AHR expression and also restores CYP1A1 inducibility to nearly 100% of the cells in the treated cultures. Fusion of a representative B clone with a rat hepatoma cell line restores expression to the mouse AHR gene encoded by the B clone's genome. These results demonstrate that the loss of CYP1A1 inducibility in B clones is probably totally ascribable to their reduced levels of AHR and that the clones are most probably not mutated in the AHR gene but are deficient in its expression. The evidence suggests that the reduction in expression of mRNA encoded by the endogenous AHR gene in the B clones is not due to an epigenetic alteration in chromatin structure but that the clones are probably defective either in a transcription factor for the AHR gene or in a protein required for generating an open chromatin configuration over the gene.

The 2,3,7,8-tetrachlorodibenzo-p-dioxin-transformed aryl hydrocarbon receptor (AHR) complex binds to xenobiotic-responsive element (XRE) sequences in the 5' flanking region of the CYP1A1 gene, resulting in initiation of transcription. Both components of the transformed AHR complex [the ligand-binding AHR monomer and the AHR nuclear translocator (ARNT)] directly contact the XRE. These proteins belong to a novel subclass of basic helix-loop-helix transcription factors. The binding sites of AHR and ARNT on the asymmetric XRE were determined using nuclear extracts of 2,3,7,8-tetrachlorodibenzo-p-dioxin-treated Hepa-1c1c7 cells and a panel of double-stranded oligonucleotides containing XRE1 of the CYP1A1 gene (5'-TTGCGTGAGAA-3'), in which all combinations of three, two, or one of the thymines indicated were substituted by the photoreactive thymine analog 5-bromodeoxyuracil. Covalent cross-linking analysis and immunoprecipitation with antibodies specific for AHR or ARNT demonstrated that ARNT directly contacts the 3'-most thymine position, that AHR directly contacts the second thymine position, and that neither protein contacts the 5'-most thymine position. The thymine position contacted by ARNT lies within a three-nucleotide sequence (5'-GTG-3') identical to a half-site of an E-box element (5'-CACGTG-3') that is recognized by a number of other basic helix-loop-helix transcription factors. AHR binds to a portion of the XRE that does not resemble an E-box. Additional experiments demonstrated that neither protein loops over to contact residues located beyond the other's binding site.

Hypoxia-inducible factor-1 (HIF-1), a DNA-binding complex implicated in the regulation of gene expression by oxygen, has been shown to consist of a heterodimer of two basic helix-loop-helix Per-AHR-ARNT-Sim (PAS) proteins, HIF-1alpha, and HIF-1beta. One partner, HIF-1beta, had been recognized previously as the aryl hydrocarbon receptor nuclear translocator (ARNT), an essential component of the xenobiotic response. In the present work, ARNT-deficient mutant cells, originally derived from the mouse hepatoma line Hepa1c1c7, have been used to analyze the role of ARNT/HIF-1beta in oxygen-regulated gene expression. Two stimuli were examined: hypoxia itself and desferrioxamine, an iron-chelating agent that also activates HIF-1. Induction of the DNA binding and transcriptional activity of HIF-1 was absent in the mutant cells, indicating an essential role for ARNT/HIF-1beta. Analysis of deleted ARNT/HIF-1beta genes indicated that the basic, helix-loop-helix, and PAS domains, but not the amino or carboxyl termini, were necessary for function in the response to hypoxia. Comparison of gene expression in wild type and mutant cells demonstrated the critical importance of ARNT/HIF-1beta in the hypoxic induction of a wide variety of genes. Nevertheless, for some genes a reduced response to hypoxia and desferrioxamine persisted in these mutant cells, clearly distinguishing ARNT/HIF-1beta-dependent and ARNT/HIF-1beta-independent mechanisms of gene activation by both these stimuli.

The aryl hydrocarbon receptor (AHR) and the aryl hydrocarbon receptor nuclear translocator (ARNT) belong to a novel subclass of basic helix-loop-helix transcription factors. The AHR.ARNT heterodimer binds to the xenobiotic responsive element (XRE). Substitution of each of four amino acids in the basic region of ARNT with alanine severely diminishes or abolishes XRE binding, intimating that these amino acids contact DNA bases. Three of these amino acids are conserved among basic helix-loop-helix proteins, and the corresponding amino acids of Max and USF are known to contact DNA bases. Alanine scanning mutagenesis of the basic domain of AHR and substitution with conservative amino acids at particular positions in this domain and in a more amino-proximal AHR segment previously shown to be required for XRE binding (Fukunaga, B. N., and Hankinson, O. (1996) J. Biol. Chem. 271, 3743-3749) demonstrate that the most carboxyl-proximal amino acid position of the basic domain and a position within the amino-proximal segment are intolerant to amino acid substitution with regard to XRE binding, suggesting that these two amino acids make base contacts. Amino acid positions in these AHR regions and in the ARNT basic region less adversely affected by substitution are also identified. The amino acids at these positions may contact the phosphodiester backbone. The apparent bipartite nature of the DNA binding region of AHR and the identity of those of its amino acids that apparently make DNA contacts impute a novel protein-DNA binding behavior for AHR.

We have isolated a new benzo(a)pyrene-resistant clone, c35, of the mouse hepatoma line, Hepa-1. Cytochrome P1-450 mRNA and P1-450-dependent aryl hydrocarbon hydroxylase (AHH) activity are no longer inducible by 2,3,7,8-tetrachlorodibenzo-p-dioxin in c35. The phenotype of c35 is unstable in long-term culture. A subclone, c35-1, having partially restored AHH inducibility, was studied in detail. The concentration of dioxin required to give half-maximal induction of AHH activity was 16-fold greater in c35-1 than in Hepa-1. Scatchard analysis showed that c35-1 contains reduced levels of the Ah (dioxin) receptor, which mediates induction of P1-450, but that the affinity of the receptor for dioxin is unaltered. In vivo assays confirmed that c35-1 possesses reduced levels of receptor but showed that it is even more severely affected in nuclear translocation of the receptor. Somatic cell hybridization experiments demonstrated that c35 is recessive and belongs to a new, third complementation group of mutants defective in Ah receptor activity. We propose that c35 is mutated either in the ligand-binding Ah receptor polypeptide or in another polypeptide required for receptor function and that in c35-1 partial reversion has occurred to generate a polypeptide which is still impaired in its role in promoting nuclear translocation and/or DNA binding.

Introduction of a retroviral expression vector for the aryl hydrocarbon receptor (AHR) restores CYP1A1 inducibility to a mutant derivative of the Hepa-1 cell line that is defective in induction of CYP1A1 by ligands for the receptor. An AHR protein with normal ligand binding activity is expressed in the mutant but ligand treatment of mutant cell extract fails to induce binding of the AHR. ARNT (aryl hydrocarbon receptor nuclear translocator) dimer to the xenobiotic responsive element (XRE). AHR cDNAs derived from the mutant encode a protein that is unimpaired in ligand-dependent dimerization with ARNT, but the AHR.ARNT dimer so formed is severely impaired in XRE binding activity. The mutant cDNAs contain a C to G mutation at base 648, causing a cysteine to tryptophan alteration at amino acid 216, located between the PER-ARNT-SIM homology region (PAS) A and PAS B repeats. Introduction of the same mutation in the wild-type AHR sequence by site-directed mutagenesis similarity impaired XRE binding activity. Substitution with the conservative amino acid, serine, had no effect on XRE binding. The tryptophan mutation, but not the wild-type allele, was detectable in genomic DNA of the mutant. The implication that an amino acid within the PAS region may be involved in DNA binding indicates that the DNA binding behavior of AHR may be more anomalous than previously suspected.

Functional domains of the mouse aryl hydrocarbon receptor (Ahr) were investigated by deletion analysis. Ligand binding was localized to a region encompassing the PAS B repeat. The ligand-mediated dissociation of Ahr from the 90-kDa heat shock protein (HSP90) does not require the aryl hydrocarbon receptor nuclear translocator (Arnt), but it is slightly enhanced by this protein. One HSP90 molecule appears to bind within the PAS region. The other molecule of HSP90 appears to require interaction at two sites: one over the basic helix-loop-helix region, and the other located within the PAS region. Each mutant was analyzed for dimerization with full-length mouse Arnt and subsequent binding of the dimer to the xenobiotic responsive element (XRE). In order to minimize any artificial steric hindrances to dimerization and XRE binding, each Ahr mutant was also tested with an equivalently deleted Arnt mutant. The basic region of Ahr is required for XRE binding but not for dimerization. Both the first and second helices of the basic helix-loop-helix motif and the PAS region are required for dimerization. These last results are analogous to those previously obtained for Arnt (Reisz-Porszasz, S., Probst, M.R., Fukunaga, B. N., and Hankinson, O. (1994) Mol. Cell. Biol. 14, 6075-6086) compatible with the notion that equivalent regions of Ahr and Arnt associate with each other. Deletion of the carboxyl-terminal half of Ahr does not affect dimerization or XRE binding but, in contrast to an equivalent deletion of Arnt, eliminates biological activity as assessed by an in vivo transcriptional activation assay, suggesting that this region of Ahr plays a more prominent role in transcriptional activation of the cyp1a1 gene than the corresponding region of Arnt.

The cytochrome P1-450-dependent activity, aryl hydrocarbon hydroxylase, and cytochrome P1-450 messenger RNA levels were studied in wild-type Hepa1c1c7 cells and in aryl hydrocarbon hydroxylase-deficient mutants derived from this line. Tetrachlorodibenzo-p-dioxin (TCDD) induced both parameters approximately 50-fold in Hepa1c1c7 cells. Mutants in genes B and C that are affected in the functioning of the Ah receptor required for hydroxylase induction and dominant mutants had nondetectable or much reduced P1-450 mRNA levels and hydroxylase activities after TCDD treatment. Hybrids between wild-type cells and the dominant mutants were also deficient in these parameters. The dominant mutants therefore appear to express a trans-acting repressor of cytochrome P1-450 transcription. Mutants in gene A were heterogenous. Some lacked the mRNA completely; others were inducible for it; while still others (subgroup IV) had high levels even when they were grown without TCDD. These results suggest strongly that gene A is the structural gene for cytochrome P1-450. When subgroup IV mutants were cultured together with wild-type cells, they failed to express P1-450 mRNA in the absence of TCDD treatment. These cells probably accumulate an inducer that can be metabolized by aryl hydrocarbon hydroxylase. The inducer did not appear to be a component of the medium and therefore may be an endogenous ligand of the Ah receptor.

Hepa-1c1c7, a mouse hepatoma cell line, was used to study the effect of cytochrome P1-450 inducers on the binding of 125I-epidermal growth factor (EGF), 125I-insulin, or [20-3H]phorbol 12,13-dibutyrate each to its specific cell-surface receptor. After a 24-h exposure to the cultured cells, several polycyclic hydrocarbon P1-450 inducers decrease the binding of EGF to EGF receptors much more than phenobarbital does. There appears to be a selectivity in the inhibitory effects: whereas EGF binding to EGF receptors is blocked, the binding of either phorbol ester or insulin each to its specific cell-surface receptors remains unaffected. The rank order of binding affinities of these chemicals to the cytosolic Ah receptor (2,3,7,8-tetrachlorodibenzo-p-dioxin much greater than benzo[a]pyrene greater than benzo[a]anthracene greater than 6-aminochrysene much greater than phenobarbital) is not correlated with their effects on EGF binding capacity. The effect of polycyclic hydrocarbons on EGF binding takes 24 h at 37 degrees C to be maximal, whereas phorbol 12-myristate 13-acetate, a potent tumor-promoting compound, inhibits EGF binding in less than 30 min. Removal of benzo[a]anthracene from the growth medium after 24 h results in a gradual recovery in EGF binding, indicating that the effect is reversible. Benzo[a]pyrene and benzo[a]anthracene are relatively ineffective at decreasing EGF binding to the EGF receptors in Hepa-1 mutant clones c2 and c4, which lack a normally functioning Ah receptor and inducible aryl hydrocarbon hydroxylase activity (P1-450). The very toxic metabolite (+)-7 beta,8 alpha-dihydroxy-9 alpha, 10 alpha-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene, when added directly to the growth medium of c4 cells, however, is effective at decreasing EGF binding. These data suggest that electrophilic metabolites of polycyclic aromatic compounds, formed by P1-450 induced during the exposure of Hepa-1 cells to these chemicals, are important in decreasing EGF binding to the EGF cell-surface receptor. Occupancy of the Ah receptor per se does not affect EGF binding.

The mouse hepatoma line Hepa-1 has high and inducible aryl hydrocarbon (benzo[a]pyrene) hydroxylase[benzo[a]pyrene, reduced-flavoprotein:oxygen oxidoreductase (3-hydroxylating), EC 1.14.14.2] activity. Fourteen subclones of a clonal derivative of Hepa-1 were isolated and shown not to display heterogeneity in aryl hydrocarbon hydroxylase activity beyond what could be ascribed to experimental error. Hepa-1 was found to be very sensitive to benzo[a]pyrene (BaP) toxicity and a single-step selection procedure for isolating clones resistant to BaP at 4 microgram/ml was designed. Those BaP-resistant variants tested had much reduced aryl hydrocarbon hydroxylase activities under both inducing and noninducing conditions and they retained their resistance to BaP and low aryl hydrocarbon hydroxylase activities over considerable periods of time of culture in the absence of BaP. The spontaneous rate of origin of the BaP-resistant clones was estimated, by Luria-Delbrück fluctuation analysis, to be 2 X 10(-7) events per cell per generation. It is concluded that the properties of the variants are consistent with the notion that they are mutational in origin and that this system is a promising one for carrying out a somatic cell genetic analysis of aryl hydrocarbon hydroxylase.

Drosophila single-minded, which acts as a positive master gene regulator in central nervous system midline formation in Drosophila, its two mouse homologs SIM1 and SIM2, and the mammalian aryl hydrocarbon receptor (AHR) and aryl hydrocarbon receptor nuclear translocator (ARNT) proteins are members of the basic-helix-loop-helix.PAS family of transcription factors. In the yeast two-hybrid system, we demonstrate strong constitutive interaction of ARNT with SIM1 and SIM2 and fully ligand-dependent interaction of ARNT with AHR. Both the helix-loop-helix and the PAS regions of SIM1 and of ARNT are required for efficient heterodimerization. SIM1 and SIM2 do not form homodimers, and they do not interact with AHR. We also failed to detect homodimerization of ARNT. The interaction of ARNT with SIM1 was confirmed with in vitro synthesized proteins. Like AHR, in vitro synthesized SIM1 associates with the 90-kDa heat shock protein. SIM1 inhibits binding of the AHR.ARNT dimer to the xenobiotic response element in vitro. Introduction of SIM1 into hepatoma cells inhibits transcriptional transactivation by the endogenous AHR.ARNT dimer. The mouse SIM1. ARNT dimer binds only weakly to a proposed DNA target for the Drosophila SIM.ARNT dimer. In adult mice mRNA for SIM1 was expressed in lung, skeletal muscle, and kidney, whereas the mRNA for SIM2 was found in the latter two. ARNT is also expressed in these organs. Thus mouse SIM1 and SIM2 are novel heterodimerization partners for ARNT in vitro, and they may function both as positive and negative transcriptional regulators in vivo, during embryogenesis and in the adult organism.

The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor that binds DNA in the form of a heterodimer with the AHR nuclear translocator protein (ARNT). Both proteins possess basic helix-loop-helix motifs. ARNT binds to the side of the xenobiotic responsive element (XRE) that resembles an E-box (the sequence recognized by the majority of other basic helix-loop-helix proteins), whereas AHR binds to the side of the XRE that does not conform to the E-box sequence. The basic region of ARNT closely resembles those of other E-box-binding proteins, whereas the "nominal basic region" of AHR (amino acids 27 39), although required for XRE binding, deviates from this consensus. By extensive mutational analysis it is shown here that an additional block of amino acids of AHR (from tyrosine 9 to lysine 20) that contains a highly basic segment is required for XRE binding and transcriptional activation. Deletion of the first nine amino acids negates XRE binding. Substitution of either tyrosine 9 or arginine 14 with alanine eliminates XRE binding, whereas alanine substitutions at certain other sites within the block reduce but do not eliminate binding. The reported absence of the first nine amino acids in the purified protein may therefore be artifactual. These results suggest that the amino acids of AHR involved in binding to the XRE constitute a novel DNA-binding domain, comprising amino acids located within and amino-terminal to the nominal basic region.

An aryl hydrocarbon hydroxylase (AHH)-deficient gene A- mutant of the mouse line Hepa-1 was treated with calcium phosphate precipitates of DNA from Hepa-1, the rat line H4IIEC3, or an A- -human hybrid in which the A- mutation is complemented by the corresponding human gene. AHH+ transfectants were isolated by selection with benzo[ghi]perylene plus near UV. In addition, a gene A- mutant which also carries a mutation for hypoxanthine phosphoribosyltransferase deficiency was treated with the above genomic DNAs together with pSV2-gpt DNA, and cotransfectants were isolated after treatment with both benzo[ghi]pereylene and HAT. All transfectants and cotransfectants were inducible for AHH by 2,3,7,8-tetrachlorodibenzo-p-dioxin. Both transfectants and cotransfectants were unstable during culture, rapidly losing AHH activity. Rat DNA-derived transfectants were probed in Southern blots with a cDNA probe to mouse cytochrome P1-450 that cross-hybridizes to the corresponding rat gene. All rat DNA-derived transfectants contained the rat P1-450 gene. In half of the transfectants, the rat gene was amplified four- to sevenfold. In one transfectant, the rat gene was truncated at the 3' end. The proportion of rat DNA in different transfectants, as determined by hybridization to a rat repetitive sequence, ranged from less than 1% to 5%. AHH activity and the rat P1-450 gene segregated together in subclones of one of the transfectants. These results demonstrate that the A gene is either the structural gene for cytochrome P1-450, or another very closely linked gene. Previous results (O. Hankinson et al., J. Biol. Chem. 260:1790-1795, 1985) favor the former alternative.

Immunoprecipitation experiments performed on cytosolic extracts of the mouse hepatoma cell line Hepa-1c1c7 (Hepa-1) confirm that the 9-S, unliganded, cytosolic aryl hydrocarbon (Ah) receptor complex contains the 90-kDa heat shock protein and the Ah receptor protein but reveal that it does not contain the Ah receptor nuclear translocator (ARNT) protein. These experiments confirm that the 6-S liganded form of the receptor identified in nuclear extracts of cells treated with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) contains the Ah receptor protein and ARNT but not the 90-kDa heat shock protein. The 6-S liganded Ah receptor complex activates transcription of the CYP1A1 gene via its binding to upstream xenobiotic-responsive elements (XREs). Treatment of cytosolic extracts of Hepa-1 cells with TCDD in vitro transforms the Ah receptor complex to the XRE-binding state. No such transformation occurs in a C- mutant deficient in ARNT activity. When in vitro synthesized ARNT was added concomitantly with TCDD to C- cytosolic extracts, it associated with the Ah receptor and restored Ah receptor-dependent XRE-binding activity to the extracts. Covalent cross-linking experiments in nuclear extracts of Hepa-1 and human LS180 cells treated with TCDD in vivo demonstrate that both ARNT and the Ah receptor bind directly to the XRE core sequence.