(Received for publication, December 19, 1995)
From the
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.
Induction of the microsomal enzyme cytochrome P4501A1 by
2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, dioxin) ()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.
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.
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.
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.
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.
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.
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.
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.
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.
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
-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 -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.