©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
DNA Binding by the Heterodimeric Ah Receptor
RELATIONSHIP TO DIOXIN-INDUCED CYP1A1 TRANSCRIPTION IN VIVO(*)

(Received for publication, December 19, 1995)

Liqun Dong Qiang Ma James P. Whitlock Jr.

From the Department of Molecular Pharmacology, Stanford University School of Medicine, Stanford, California 94305-5332

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin induces the microsomal enzyme cytochrome P4501A1 by increasing the transcription rate of the CYP1A1 gene. Induction requires two basic helix-loop-helix proteins, the ligand-binding aromatic hydrocarbon receptor (AhR) and its heterodimerization partner, the AhR nuclear translocator (Arnt). The AhR/Arnt heterodimer induces transcription by binding to dioxin-responsive elements (DREs) within an enhancer upstream of the CYP1A1 gene. The basic regions of AhR and Arnt are crucial for DRE binding. We have mutated these regions in order to analyze the relationship between DRE binding (determined in vitro using an electrophoretic mobility shift assay) and induction of CYP1A1 transcription (determined in vivo by genetic complementation of AhR-defective and Arnt-defective mouse hepatoma cells, using an RNase protection assay to measure mRNA accumulation). Our findings reveal the amino acids in the basic regions of AhR/Arnt that are important for both DRE binding and induction of transcription. This information provides biological background for the interpretation of structural (e.g. crystallographic) studies of the interactions between AhR/Arnt and the DRE. Our findings also indicate that the in vitro behavior of the mutants does not consistently predict their functional activity in vivo. Thus, genetic complementation constitutes an important and stringent test for analyzing the effects of mutations on AhR/Arnt function.


INTRODUCTION

Induction of the microsomal enzyme cytochrome P4501A1 by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, dioxin) (^1)constitutes an interesting response for analyzing the mechanism by which a small, hydrophobic compound can activate the transcription of a specific mammalian gene (1) . Cytochrome P4501A1 oxygenates certain lipophilic xenobiotics, such as the environmental carcinogen benzo(a)pyrene. This metabolic processing usually results in detoxification and elimination of the xenobiotic from the cell; however, under some conditions, the enzyme generates chemically-reactive, toxic and/or mutagenic metabolites. Thus, cytochrome P4501A1 plays a key role in certain types of xenobiotic-induced cancer(2, 3) .

TCDD is the most potent known inducer of cytochrome P4501A1. Enzyme induction reflects an increased rate of transcription of the corresponding CYP1A1 gene(1) . TCDD is also a widespread and persistent environmental contaminant, which elicits numerous adaptive and adverse effects in experimental animals, presumably by altering gene expression(4, 5, 6, 7, 8) . TCDD's risk to human health is a matter of current debate. Studies of the mechanism of TCDD action may contribute to a better understanding of this public health issue in the future.

The aromatic hydrocarbon receptor (AhR) is an intracellular protein that binds TCDD and mediates its biological effects(9, 10, 11) . The AhR undergoes ligand-induced heterodimerization with a second intracellular protein, the AhR nuclear transporter (Arnt); heterodimerization generates a DNA-binding transcription factor, designated here as AhR/Arnt, that binds to dioxin-responsive elements (DREs) within an enhancer upstream of the CYP1A1 gene.

The core nucleotide sequence of the DRE (5`-TNGCGTG-3`/3`-ANCGCAC-5`), is asymmetric, and each DRE binds one AhR/Arnt heterodimer(1, 11) . The binding of AhR/Arnt to the DREs leads to changes in the chromatin structure of the CYP1A1 enhancer/promoter region and to the induction of transcription(12, 13, 14, 15) . Studies of their cDNAs reveal that both AhR and Arnt contain basic helix-loop-helix (bHLH) motifs, as well as domains that exhibit homologies to the Drosophila regulatory proteins Per and Sim(9, 10, 11) . The presence of the latter domains, which are designated as PAS (for Per, AhR/Arnt, and Sim), distinguishes AhR and Arnt from other bHLH transcription factors and may confer novel regulatory properties upon the proteins. Thus, analyses of AhR/Arnt function can provide new insights into the control of mammalian transcription.

The basic and HLH domains of AhR and Arnt play important roles in DNA binding and heterodimerization, respectively(9, 10, 11) . Arnt's basic region resembles those of class B bHLH proteins, which bind the palindromic sequence (5`-CACGTG-3`/3`-GTGCAC-5`). In contrast, AhR's basic region is atypical; it bears relatively little resemblance to the basic regions of other bHLH proteins. Here, we have analyzed the basic regions of AhR and Arnt in order to better understand the relationship between AhR/Arnt's ability to bind the DRE in vitro and to induce CYP1A1 transcription in vivo. In these studies, we have utilized a mouse hepatoma cell system, in order to exploit the availability of AhR-defective and Arnt-defective cells, which permit genetic analyses of AhR/Arnt(1, 11) . Thus, we have been able to use genetic complementation as a stringent test of AhR/Arnt function in vivo. Our findings provide new insights into the regulation of CYP1A1 transcription and the mechanism of dioxin action.


EXPERIMENTAL PROCEDURES

Materials

Taq polymerase and reverse transcription-PCR kit were from Perkin-Elmer (Branchburg, NJ). Restriction endonucleases, T4 DNA ligase, and polynucleotide kinase were from Life Technologies, Inc. (Grand Island, NY), Promega (Madison, WI), and New England Biolabs (Beverly, MA). pRc/CMV was from Invitrogen (San Diego, CA). TNT-coupled reticulocyte lysate and alkaline phosphatase-conjugated goat anti-rabbit antibody were from Promega. [-P]ATP, [alpha-P]UTP, [S]dATP, and [S]methionine were from Amersham. Total RNA isolation kit and DNA purification kit were from QIAGEN (Chatsworth, CA). In vitro transcription kit was from Ambion (Austin, TX). DNA sequencing kit (Sequenase version 2.0) was from U. S. Biochemical Corp. (Cleveland, OH).

Plasmid Construction

We used a PCR-based method to generate wild-type AhR and Arnt cDNA plasmids. An AhR clone from C57BL/6 mouse liver was used as template(16) . The forward primer for AhR PCR was GTCACCGCGGAAGCTTCCGCCACCATGGCCAGCAGCGGCGCCAACATC, which contains nucleotides 1-21 of mouse AhR (underlined), a Kozak consensus sequence (bold), and SacII and HindIII restriction sites. The reverse primer was GTCATCTAGACTCGAGACTCTGCACCTTGCTTAG, which contains nucleotides 2415-2398 (underlined) and a XhoI restriction site. The PCR product was digested with SacII and XhoI and was subcloned into the pBabe-Neo vector(17) . A fragment containing full-length AhR and a Kozak sequence was excised from pBabe using HindIII and ApaI and was inserted into pRc/CMV (Invitrogen).

An Arnt cDNA from Hepa 1c1c7 mouse hepatoma cells was used as template for Arnt PCR(18) . The forward primer was CTGATCTAGAAAGCTTATGGCGGCGACTACAGC, which contains nucleotides 1-17 of mouse Arnt (underlined) and a HindIII restriction site. The reverse primer was GTCATCTAGATTCGGAAAAGGGGGGAAAC, which contains nucleotides 2328-2310 of mouse Arnt (underlined) and a XbaI site. The PCR product was cloned into the pRc/CMV vector at HindIII and XbaI sites.

Site-directed Mutagenesis

We used a PCR-based site-directed mutagenesis approach(19) . Each amino acid of AhR or Arnt within its basic region was mutated to alanine. The primers for mutation were 17-18 nucleotides in length with the mutated nucleotide(s) in the center. A SacII and EcoRI DNA fragment containing an AhR mutation was used to replace the corresponding wild-type fragment in pBabe-AhR, and then the full-length AhR mutant was transferred into the pRc/CMV vector. The Arnt mutants were directly cloned into the pRc/CMV vector. AhR, Arnt, and their mutants in pRc/CMV were used for in vitro transcription/translation. For retroviral infection, AhR, Arnt, and their mutants were subjected to PCR with primers containing restriction sites for cloning into the MFG vector(15) . Each mutation was confirmed by DNA sequencing.

In Vitro Transcription/Translation

AhR, Arnt, and their mutant cDNAs in pRc/CMV were transcribed and translated in vitro from the T7 promoter using TNT-coupled rabbit reticulocyte lysate (Promega), according to the manufacturer's instructions. In a 10-µl reaction mixture, 0.4 µg of plasmid DNA were used. The reactions were incubated at 30 °C for 90 min. Protein expression was verified in parallel experiments, in which [S]methionine was incorporated into the in vitro translated proteins. The expressed proteins were analyzed by SDS-polyacrylamide gel electrophoriesis and fluorography.

Electrophoretic Mobility Shift Assay (EMSA)

The EMSA was carried out as described previously(20) . In vitro transcribed/translated AhR (1 µl) and Arnt (1 µl) (an approximately equimolar ratio) were incubated with Me(2)SO or 20 nM TCDD in Me(2)SO for 2 h at room temperature in HEDG buffer (25 mM Hepes, 1.5 mM EDTA, 5 mM dithiothreitol, 10% glycerol). Poly(dI-dC) was added to the mixture and incubated for 15 min at room temperature. The radiolabeled probe was then added and incubated for 15 min at room temperature, followed by nondenaturing gel electrophoresis and autoradiography.

Co-immunoprecipitation and Immunoblot Analyses

In vitro transcribed/translated AhR (5 µl) and Arnt (5 µl) (an approximately equimolar ratio) were incubated with Me(2)SO or 20 nM TCDD in Me(2)SO for 2 h at room temperature in HEDG buffer. Antiserum (10 µl) against AhR was added and incubated for 1 h at 4 °C followed by incubation with protein A-Sepharose. Precipitated proteins were separated by electrophoresis on a 7.5% SDS-polyacrylamide electrophoresis gel. For detection of Arnt, the gel was dried and analyzed by fluorography. For detection of AhR, proteins were transferred from the gel to nitrocellulose. Blots were blocked with 5% non-fat dry milk in Tris-buffered saline (20 mM Tris-Cl (pH 7.4), 0.5 M NaCl) and were probed with affinity-purified anti-AhR polyclonal antibody, followed by alkaline phosphatase-conjugated goat anti-rabbit antibody (Promega).

Cell Culture

Wild-type (Hepa 1c1c7), AhR-defective (TAOc1BP^rc1), and Arnt-defective (BP^rC1) cells were maintained in minimal essential medium containing 10% fetal bovine serum, as described previously(21) . Bosc 23 cells were maintained in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum(22) .

Gene Transfer by Retroviral Infection

The ecotropic packaging cell line Bosc 23 was seeded at 2 times 10^6 cells in 60-mm dishes the day before transfection. cDNA in the MFG vector (3 µg) was transfected into Bosc 23 cells using a modified calcium-phosphate method(22) . Conditioned medium containing virus was collected about 48 h after transfection. Aliquots (1 ml) were transferred to 60-mm dishes containing 1 times 10^5 wild-type cells or 2 times 10^5 AhR-defective or Arnt-defective cells, as described(22) . Infection efficiencies were determined in parallel cultures by introducing LacZ cDNA into cells and staining for 5-bromo-4-chloro-3-indoyl beta-D-galactoside. Infection efficiencies were 85% for wild-type cells and 95% for AhR-defective and Arnt-defective cells.

RNase Protection Assay

Total RNA was isolated from uninduced and TCDD-induced (1 nM, 16 h) cells by extraction with 4 M guanidine isothiocyanate using a QIAGEN total RNA isolation kit. A CYP1A1 cDNA fragment was synthesized by reverse transcription-PCR. The primers were 5`-GGGTCCTAGAGAACACTCTTCACTT-3` (nucleotides 41-65) and 5`-CTTCTCAAATGTCCTGTAGTGCTCT-3` (nucleotides 3294-3318)(23) . The PCR fragment was digested with ApaI and PstI to generate a 96-base pair cDNA fragment which was used as a template for riboprobe synthesis. This fragment was subcloned into pBluescript and sequenced. The riboprobe was synthesized in vitro in the presence of [alpha-P]UTP using an Ambion transcription kit.

Total RNA (5-10 µg) was hybridized with the CYP1A1-specific riboprobe at 50 °C for 16 h. tRNA was used as a negative control. Single-stranded RNA was digested with RNase A and RNase T1. Protected fragments were separated on a 6% polyacrylamide/urea gel and detected by autoradiography. The size of protected RNA was estimated by comparison with a DNA sequence reaction in parallel lanes.


RESULTS

Mutations in Arnt That Affect the Binding of AhR/Arnt to DNA in Vitro

Deletion analyses reveal that the basic region of Arnt is important for the binding of AhR/Arnt to the DRE in vitro(24) . As shown in Table 1, Arnt's basic region resembles the basic regions of class B bHLH proteins(25) ; the similarity includes several conserved amino acids. To determine the residues that contribute to DRE binding, we performed an alanine-scanning mutational analysis of Arnt's basic region, which spans amino acids 75 to 87; we assessed the DRE-binding capability of the AhR/Arnt mutants using in vitro transcription/translation and an EMSA. Neither Arnt nor AhR binds by itself to the DRE; the formation of a protein-DNA complex in this system requires heterodimerization between AhR and Arnt, a process which is inducible by TCDD(18, 24) . Our findings reveal that mutations at each of the six amino acids (Arg-76, His-79, Glu-83, Arg-84, Arg-86, and Arg-87) that are conserved between Arnt and other class B bHLH proteins markedly reduces or eliminates the ability of the AhR/Arnt complex to bind the DRE in vitro (Fig. 1). In addition, mutation at the non-conserved amino acid Ile-82 substantially reduces DNA binding. Mutations at other positions in Arnt's basic region have no detectable effect on the binding of AhR/Arnt to the DRE in vitro (Fig. 1). These observations tend to implicate the six conserved amino acids and Ile-82 in DRE recognition and binding by AhR/Arnt. However, these in vitro findings do not always predict the functional consequences of the Arnt mutations in vivo, as we show later.




Figure 1: Effect of mutations in Arnt's basic region on the ability of AhR/Arnt to bind to a DRE in vitro. Panel A, amino acid sequence of Arnt's basic region (amino acids 75-87). The boxed residues are conserved between Arnt and the basic regions of class B bHLH proteins. ``+'' indicates a positively-charged amino acid. Each amino acid was individually mutated to alanine and was designated as indicated in the column on the left. Panel B, DRE binding by AhR/Arnt heterodimers containing mutant Arnt proteins. The wild-type and mutant Arnt proteins described in panel A were incubated with wild-type AhR in the absence or presence of TCDD (20 nM, 2 h), as indicated, and the resulting AhR/Arnt heterodimers were analyzed by EMSA, using a radiolabeled DRE as probe. The arrow indicates the position of the TCDD-inducible AhR/Arnt-DRE complex.



Mutations in AhR That Affect the Binding of AhR/Arnt to DNA in Vitro

In contrast to the situation for Arnt, the sequence of AhR's basic region, which spans amino acids 27-39, is atypical; in fact, AhR's basic region exhibits only one amino acid that is conserved among class B bHLH proteins (Table 1). Deletion of this region abrogates the binding of AhR/Arnt to the DRE in vitro(26) . In addition, another AhR domain, spanning amino acids 12-16, contains five consecutive basic residues; because of its composition, this domain appears potentially capable of contributing to DNA binding. We performed an alanine-scanning mutational analysis of these two regions of AhR to determine which amino acids contribute to DRE binding in vitro. Our findings (Fig. 2) reveal that mutation of the conserved amino acid (Arg-39) in AhR's basic region abolishes the ability of AhR/Arnt to bind the DRE in vitro. In addition, mutation of several non-conserved amino acids (Asn-33, Ser-35, Lys-36, and His-38) reduces DRE binding. Mutations at other amino acids in the basic region have no detectable effect. Analysis of the five consecutive basic residues spanning positions 12-16 reveals that mutation at Arg-14 eliminates AhR/Arnt's DRE-binding capability in vitro. Mutations at the other four positions have no effect. These findings imply that Arg-14, His-38, and Arg-39 (and, to a lesser extent, Asn-33, Ser-35, and Lys-36) contribute to DRE binding by AhR/Arnt in vitro. The effects of these mutations on function in vivo are described below.


Figure 2: Effect of mutations in AhR on the ability of AhR/Arnt to bind to a DRE in vitro. Panel A, amino acid sequences of a domain that contains five consecutive basic residues (amino acids 12-16) and of AhR's basic region (amino acids 27-39). The boxed residue is conserved between AhR and the basic regions of class B bHLH proteins. ``+'' indicates a positively-charged amino acid. Each amino acid was individually mutated to alanine and was designated as indicated in the column on the left. Panels B and C, DRE binding by AhR/Arnt heterodimers containing mutant AhR proteins. The wild-type and mutant AhR proteins described in panel A were incubated with wild-type Arnt in the absence or presence of TCDD (20 nM, 2 h), as indicated, and the resulting AhR/Arnt heterodimers were analyzed by EMSA, using a radiolabeled DRE as probe. The arrow indicates the position of the TCDD-inducible AhR/Arnt-DRE complex. Panel B, analysis of amino acids 27-39. Panel C, analysis of amino acids 12-16.



Heterodimerization Capability of Arnt and AhR Mutants

In order to bind the DRE, Arnt and AhR must heterodimerize(24) . We used a co-immunoprecipitation assay to verify that heterodimerization capability is intact in the Arnt and AhR mutants that exhibit defects in DRE binding. The proteins analyzed in the co-immunoprecipitation studies were the same as those analyzed for DRE binding, except that Arnt and its mutants were labeled with [S]methionine during in vitro transcription/translation. Our findings (Fig. 3) reveal that heterodimerization is TCDD-dependent, as expected, and that each AhR and Arnt mutant heterodimerizes appropriately with its cognate partner. Thus, defective heterodimerization cannot account for diminished or absent DRE binding in the electrophoretic mobility shift assay.


Figure 3: Heterodimerization analyses. Arnt and AhR mutants that exhibited defective DRE binding in EMSA studies ( Fig. 1and Fig. 2) were analyzed for heterodimerization capability using a co-immunoprecipitation assay, as described under ``Experimental Procedures.'' Panel A, Arnt mutants. Panel B, AhR mutants from the region spanning amino acids 27-39. Panel C, AhR mutants from the region spanning amino acids 12-16. I.P. Ab, immunoprecipitating antibody.



Effect of Arnt Mutations on AhR/Arnt Function in Vivo

To assess the functional consequences of Arnt mutations, we used a retroviral-mediated gene transfer technique to reconstitute Arnt-defective cells with wild-type or mutant Arnt proteins; we then assayed the response of AhR/Arnt's native target, the chromosomal CYP1A1 gene, using an RNase protection assay to measure TCDD-inducible cytochrome P4501A1 mRNA. This complementation analysis constitutes a stringent test of AhR/Arnt function; it avoids potential artifacts associated with the use of a plasmid-based reporter gene, whose chromatin structure may not reflect that of a chromosomal gene. The reconstituted cell strains expressed the mutant proteins at similar levels, as measured by immunoblotting (data not shown); therefore, defective expression of the mutant proteins cannot account for the lack of CYP1A1 gene expression in the reconstituted cells.

Our findings (Fig. 4) reveal that Arnt mutations at Arg-86 and Arg-87 abolish the response of the CYP1A1 gene to TCDD, and mutations at His-79 and Glu-83 markedly attenuate responsiveness. In contrast, mutations at Arg-76, Ile-82, and Arg-84, which decrease DRE binding in vitro, have no effect on function in vivo. Together, the results in Fig. 1and Fig. 4imply that amino acids His-79, Glu-83, Arg-86, and Arg-87 of Arnt (each of which is conserved among class B bHLH proteins) are major determinants of both DRE recognition and CYP1A1 transcription by AhR/Arnt. Our findings also indicate the importance of analyzing the functional effects of Arnt mutations, because studies of DRE binding in vitro do not consistently predict AhR/Arnt function in vivo.


Figure 4: Effect of Arnt mutations on AhR/Arnt function in vivo. Arnt mutants that exhibited defective DRE binding in vitro (Fig. 1) were introduced into Arnt-defective cells by retroviral infection, and the accumulation of CYP1A1 mRNA in response to TCDD (1 nM, 16 h) was measured using an RNase protection assay.



Effect of AhR Mutations on AhR/Arnt Function in Vivo

We used an analogous complementation assay to measure the functional effects of AhR mutations in vivo. Again, immunoblotting experiments revealed that AhR-defective cells reconstituted with wild-type or mutant AhR expressed similar levels of AhR protein (data not shown). Therefore, deficits in CYP1A1 transcription observed in the reconstituted cells do not reflect inadequate expression of AhR. Our findings (Fig. 5) reveal that an AhR mutation at Arg-39 (the conserved residue in the basic region) markedly diminishes the ability of AhR/Arnt to induce CYP1A1 transcription in response to TCDD. Similarly, mutation of His-38 produces a substantial decrease in AhR/Arnt function. In contrast, the mutation at Arg-14, which abolishes DRE binding in vitro, has relatively little effect on in vivo function. Mutations at Asn-33 and Ser-35 produce modest decreases in both DRE binding and function, whereas mutation at Lys-36 has no effect on function, even though it diminishes DRE binding in vitro. Taken together, our findings imply that amino acids His-38 and Arg-39 of AhR are of primary importance for both DRE binding and CYP1A1 transcription by AhR/Arnt. As with Arnt, the lack of correspondence between the in vitro binding data and the in vivo functional data emphasizes the importance of the complementation test in analyzing the effects of AhR mutations.


Figure 5: Effect of AhR mutations on AhR/Arnt function in vivo. AhR mutants that exhibited defective DRE binding in vitro (Fig. 2) were introduced into AhR-defective cells by retroviral infection, and the accumulation of CYP1A1 mRNA in response to TCDD (1 nM, 16 h) was measured using an RNase protection assay. Panel A, mutants from the region spanning amino acids 27-39. Panel B, mutants from the region spanning amino acids 12-16.



Dominant Negative Effect of Arnt and AhR Mutants

The generation of non-functional AhR/Arnt heterodimers has the potential to interfere with the normal response to TCDD. To examine this possibility, we introduced Arnt and its mutants R86A and R87A into wild-type cells by retroviral infection and measured the response of the CYP1A1 gene to TCDD. Immunoblotting studies revealed about a doubling of Arnt protein in the infected cells (data not shown). The functional data (Fig. 6) indicate that increased expression of wild-type Arnt in wild-type cells produces about a 2-fold increase in the extent of CYP1A1 induction by TCDD; this result is consistent with our previous observations(18) . In contrast, expression of the R86A and R87A mutants in wild-type cells produces about a 50% reduction in the induction response. Thus, these mutants exert a dominant negative effect and interfere with the ability of wild-type cells to respond to TCDD. We envision that the mutant Arnt protein produces this effect by heterodimerizing with wild-type AhR, thereby generating a non-functional AhR/Arnt complex.


Figure 6: Dominant negative effect of Arnt and AhR mutants. Arnt and AhR mutants that exhibited defective function in vivo ( Fig. 4and Fig. 5) were introduced into wild-type mouse hepatoma cells by retroviral infection, and the accumulation of CYP1A1 mRNA in response to TCDD (1 nM, 16 h) was measured using an RNase protection assay. Panel A, Arnt mutants. Panel B, AhR mutant.



We performed analogous studies with the R39A AhR mutant. Expression of the mutant AhR in wild-type cells reduces the extent of CYP1A1 induction by TCDD by about 50% (Fig. 6). Thus, this mutant also interferes with the ability of wild-type cells to respond to TCDD. We suspect that the mutant AhR protein produces a dominant negative effect by forming a non-functional heterodimer with wild-type Arnt.


DISCUSSION

Mouse hepatoma cells constitute a powerful experimental system for analyzing the mechanism by which AhR/Arnt induces transcription, due to the availability of AhR-defective and Arnt-defective cells; with such cells, induction can be studied using both genetic and biochemical approaches(1, 11) . We have exploited the recessive nature of these mutant cells and have used cDNA to complement their defects. The high efficiency of the retroviral gene transfer method makes it feasible to employ a stringent assay for AhR/Arnt function, namely, transcriptional induction of the CYP1A1 gene in its native chromosomal setting. Our findings reveal the amino acids in the basic regions of AhR and Arnt that are important for both DRE binding and the induction of transcription. Such functional information will be crucial for interpreting structural data that may be generated in future crystallographic analyses of the AhR/Arnt-DRE complex.

Some Arnt mutants (R76A, I82A, and R84A) and AhR mutants (R14A and K36A) exhibit poor DRE binding in vitro, yet function normally in vivo. This discrepancy could reflect (a) the existence of other proteins that influence the AhR/Arnt-DRE interaction in vivo; (b) differences in the nucleotides that flank each of the eight DREs in the CYP1A1 enhancer; and/or (c) differences in the configuration of the DRE in naked DNA versus chromatin. In any event, our findings emphasize the importance of using both in vitro and in vivo assays to analyze the properties of AhR/Arnt.

Arnt's basic region contains six amino acids that are conserved in common with other class B bHLH DNA-binding proteins. Our results show that mutating each of these residues to alanine decreases the ability of AhR/Arnt to bind the DRE in vitro. However, only mutations at His-79, Gln-83, Arg-86, and Arg-87 diminish AhR/Arnt function in vivo. If Arnt's basic region assumes an alpha-helical configuration when it lies in the major DNA groove, then these four amino acids should be on the same face of the helix, in position to interact with the DRE sequence. Several kinds of evidence predict that these four amino acids interact with the 5`-GTG-3`/3`-CAC-5` half of the DRE. First, the amino acid sequence of Arnt's basic region is similar to that of proteins that bind the palindromic E-box sequence 5`-CACGTG-3`/3`-GTGCAC-5`, which contains two 5`-GTG-3`/3`-CAC-5` half-sites(25) . Second, protein-DNA cross-linking studies using a bromodeoxyuridine-substituted DRE imply that Arnt binds to 5`-GTG-3`/3`-CAC-5`(27) . Third, binding site selection studies, using various combinations of Arnt, AhR, and Sim, reveal that AhR/Arnt and Sim/Arnt heterodimers bind asymmetric sequences that contain 5`-GTG-3`/3`-CAC-5` half-sites, whereas Arnt/Arnt homodimers bind the E-box sequence containing two 5`-GTG-3`/3`-CAC-5` half-sites(28) . Thus, we envision that Arnt interacts with the 5`-GTG-3`/3`-CAC-5` half-site of the DRE in much the same way as Max interacts with the 5`-GTG-3`/3`-CAC-5` half-site of the E-box, as determined crystallographically(29) .

Protein-DNA cross-linking studies and binding site selection analyses imply that the AhR component of AhR/Arnt interacts with the 5`-TNGC-3`/3`-ANCG-5` half-site of the DRE(27, 28) . Our mutational analyses indicate that AhR's conserved amino acid Arg-39 is critical for both DRE binding and induction of transcription. The non-conserved residue His-38 also makes important contributions to both binding and function. We envision that both Arg-39 and His-38 face the major DNA groove and contact the DRE at or near the GC base pairs of the 5`-TNGC-3`/3`-ANCG-5` half-site. The non-conserved amino acids Asn-33 and Ser-35 also influence DRE binding and CYP1A1 transcription, but to a lesser extent than Arg-39 and His-38. We note that AhR contains a proline at position 34; therefore, its basic region may be unable to assume an alpha-helical configuration when interacting with the DRE. This structural feature may contribute to the distinctive DNA recognition properties of AhR/Arnt.

Our mutational analyses lead us to envision that the basic regions of AhR/Arnt contact the DRE in the vicinity of the central 5`-CG-3`/3`-GC-5` base pairs. We have shown earlier that cytosine methylation at these CpG dinucleotides prevents the binding of AhR/Arnt to the DRE and blocks the induction of CYP1A1 transcription by TCDD(30) . These previous findings constitute additional evidence that the central GC base pairs are important for the AhR/Arnt-DRE interaction. Presumably, cytosine methylation sterically hinders the binding of AhR/Arnt to the DRE.

We observe that mutations in AhR and Arnt that abolish DRE binding (but leave heterodimerization capability intact) interfere with wild-type AhR/Arnt function. The simplest explanation for this dominant inhibitory effect is that the mutant AhR or Arnt protein heterodimerizes with its cognate wild-type partner, thereby making it unavailable to participate in the response to TCDD; however, this hypothesis remains to be tested. In principle, the availability of such AhR and Arnt mutants enhances the feasibility of using gene transfer techniques to generate novel cell strains that are functionally deficient in AhR/Arnt. Such strains may be useful for implicating AhR/Arnt in responses to TCDD in systems where AhR-defective cells and Arnt-defective cells are not available.


FOOTNOTES

*
This work was supported by Grant R01 ES 03719 from National Institute of Environmental Health Sciences and Grant R35 CA 53887 from the National Cancer Institute (to J. P. W.), and by National Research Service Award ES 05679 (to Q. M.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

(^1)
The abbreviations used are: TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin; AhR, aromatic hydrocarbon receptor; Arnt, aromatic hydrocarbon receptor nuclear translocator; DRE, dioxin-responsive element; bHLH, basic helix-loop-helix; PCR, polymerase chain reaction; EMSA, electrophoretic mobility shift assay; CMV, cytomegalovirus.


ACKNOWLEDGEMENTS

We thank Margaret Tuggle for secretarial assistance and I. Kent Reed for comments on the manuscript.


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