©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Transcriptional Activation by the Mouse Ah Receptor
INTERPLAY BETWEEN MULTIPLE STIMULATORY AND INHIBITORY FUNCTIONS (*)

Qiang Ma, Liqun Dong, and James P. Whitlock, Jr.

From the (1) Department of Molecular Pharmacology, Stanford University School of Medicine, Stanford, California 94305-5332

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The aromatic hydrocarbon receptor (AhR) is a ligand-dependent transcription factor that mediates cellular responses to the environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). We cloned AhR cDNA from C57BL/6 mouse liver and verified by transfection that it encodes a functional protein. Analyses of deletion mutants indicate that the carboxyl half of AhR contains several types of transactivation domain, which function independently of domains that mediate TCDD recognition, DNA binding, and heterodimerization with the Ah receptor nuclear translocator (Arnt) protein. The transactivation domains function independently of each other, display different levels of activity, and act synergistically when linked. In addition, AhR contains an 82-amino acid domain that inhibits transactivation. The inhibitory domain displays specificity, in that it blocks the transactivating functions of AhR and Arnt, but not that of the herpes simplex protein VP16. The inhibitory activity depends upon the cell type in which AhR is expressed, implying that a cell-specific protein mediates the effect.


INTRODUCTION

The environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, dioxin)() serves as the prototype for a group of halogenated aromatic hydrocarbons that pose a potential hazard to human health. In animals, TCDD elicits a variety of biochemical, metabolic, reproductive, and neoplastic effects (Poland and Knutson, 1982; Safe, 1986). The risk that dioxin poses to humans remains uncertain and controversial (Bailar, 1991; Johnson, 1992; Peterson et al., 1993).

An intracellular protein, designated as the aromatic hydrocarbon receptor (AhR), mediates the biological effects of TCDD (Poland and Knutson, 1982; Okey et al., 1993; Swanson and Bradfield, 1993). Cloning of its cDNA reveals that AhR is a basic helix-loop-helix (bHLH) type of ligand-dependent transcription factor (Burbach et al., 1992: Ema et al., 1992). AhR functions in partnership with a second bHLH protein, designated as the Ah receptor nuclear translocator (Hoffman et al., 1991). Neither AhR nor Arnt binds to DNA by itself (Reyes et al., 1992; Whitelaw et al., 1993a; Matsushita et al., 1993). The liganded AhR heterodimerizes with Arnt to form a species that activates transcription by recognizing a specific DNA sequence within a transcriptional enhancer in the vicinity of the target gene (Whitlock, 1993).

Transcription factors are often modular in nature and contain distinct domains that confer specific functions upon the proteins (Tjian and Maniatis, 1994). In the case of AhR, the NH-terminal half is responsible for ligand binding, heterodimerization, and DNA binding (Dolwick et al., 1993b; Whitelaw et al., 1993b; Poland et al., 1994; Ema et al., 1994), whereas the COOH-terminal half has been implicated in transactivation (Jain et al., 1994; Whitelaw et al., 1994). Here, we have analyzed the modular organization of AhR in greater detail, with a focus on its transactivation function. We find that AhR's transactivation capability reflects an interplay between multiple stimulatory and inhibitory components. This functional complexity may constitute a mechanism by which AhR is able to mediate a broad spectrum of biological effects.


EXPERIMENTAL PROCEDURES

Materials

Vent polymerase was from New England Biolabs (Beverly, MA). The plasmids pRc/CMV and pCH110 were from Invitrogen Corp. (San Diego, CA) and Pharmacia LKB Biotechnology Inc. (Piscataway, NJ), respectively. Other molecular biological reagents were from Life Technologies, Inc. (Grand Island, NY), Promega (Madison, WI), Stratagene (La Jolla, CA), and Clontech (Palo Alto, CA). [C]Chloramphenicol (57 mCi/mmol) was purchased from Amersham Corp. TCDD was from the National Cancer Institute Chemical Carcinogen Reference Standard Repository.

Cell Culture

Wild type Hepa1c1c7, Ah receptor-defective (AhR-D) cells, and Ah receptor nuclear translocator-defective (Arnt-D) cells were grown as monolayers in -minimal essential medium containing 10% fetal bovine serum, 5% CO, at 37 °C, as described previously (Miller et al., 1983). HeLa, COS-1, and HepG2 cells were cultured under identical conditions.

Cloning of Ah Receptor cDNA

For PCR cloning of Ah receptor cDNA, two primers were designed, based upon AhR cDNA cloned from Hepa-1 cells (Ema et al., 1992): A: TGTCGTCTAGAGATGAGCAGCGG-CGCCAACATCACC; B: CTGCCAAGCTTTCAACTCTGCACCTTGCTT-AGGAATGC. The underlined sequence in primer A is identical to the 5`-end of AhR mRNA beginning at its start codon. The underlined sequence of primer B anneals to the 3`-end of the AhR coding sequence. Primers A and B contain an XbaI and a HindIII restriction site, respectively, at their 5`-ends to facilitate subcloning of the PCR products. Total RNA was prepared from C57BL/6 mouse liver and was used as a template for first strand cDNA synthesis using reverse transcriptase (Sambrook et al., 1989). PCR was used to amplify the single-stranded AhR cDNA. The PCR reaction mixture contained 50 µl of the single-stranded cDNA product, 4 units of Vent polymerase, 1 µM of each primer, 400 µM each of dATP, dCTP, dGTP, and dTTP, 100 µg of bovine serum albumin, 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl, 0.001% (w/v) gelatin in a final volume of 100 µl. The mixture was amplified for 25 cycles under the following conditions: denature, 94 °C, 1 min; primer anneal, 55 °C, 1 min; primer extension, 72 °C, 2 min. To increase the yield of the cDNA, 0.5 µl of the PCR product was amplified a second time under the same conditions. For sequencing, the product of the second PCR was digested with XbaI and HindIII and subcloned into a pBluescript plasmid to generate the plasmid designated pAhR/Bluescript.

Construction of the Expression Plasmid for Mouse AhR cDNA

The pRc/CMV expression vector, in which a cDNA insert can be expressed under the control of the CMV promoter, was used for expression of the AhR cDNA both in mammalian cells and in vitro.

To subclone the full-length AhR cDNA from pAhR/Bluescript into pRc/CMV, a PCR reaction was conducted with a forward primer F1 and a reverse primer R1. Primer F1 contains the following elements: 1) a HindIII site to facilitate subcloning; 2) the Kozak sequence (italicized) (Kozak, 1987) to enhance expression of the AhR in vitro; 3) the ATG start codon (boldface); and 4) nucleotides 4-15 of the AhR coding sequence (underlined): 5`-GTCGAAGCTTCCGCCACC ATGGCCAGCA-GCGGCGCC-3`.

Primer R1 contains: 1) an ApaI and an XbaI site to facilitate subcloning and subsequent mutational studies; 2) a stop codon (boldface); and 3) nucleotides 2395-2415 of the AhR noncoding strand (underlined): 5`-CTGCCGGGCCCTCATCTAGAACTCTGCACCTTGCTTAG-GAA-3`. The PCR reaction conditions were as described above, except that 1 ng of pAhR/Bluescript was used as the DNA template. The PCR product was cloned into the pRc/CMV vector at the HindIII and ApaI restriction sites to generate the plasmid designated pAhR/CMV.

Generation of AhR Deletion Mutants

We used PCR to generate COOH-terminal deletions of AhR cDNA. The forward primer used in each case was the same as the primer F1 described above for PCR amplification of the full-length AhR cDNA.

To generate the mutant designated ``C1,'' the reverse primer R2 was 5`-TGCCGGGCCCTCAGCAAGCCGAGTTCAGCA-3`, which contains an ApaI site, a stop codon, and nucleotides 1763-1779 of AhR noncoding strand (underlined).

To generate the mutant designated ``C2,'' the reverse primer R3 was 5`-TGCCGGGCCCTCATCTAGATAGGGGATCCATTATGG-3`, which contains an ApaI site, a stop codon, an XbaI site, and nucleotides 1247-1263 of AhR noncoding strand (underlined).

The pAhR/Bluescript (1 ng) was used as the template for PCR, and the PCR conditions were as described above. The mutant cDNAs were digested with HindIII and ApaI and were ligated into pRc/CMV at the corresponding restriction sites.

Construction of Gal4:AhR and Gal4:Arnt Fusion cDNAs

To construct Gal4:AhR fusion cDNAs, we first generated the 5`-end of the Gal4 cDNA by PCR and designated it as G4(1-147). The primers used were as follows: forward primer GF, which contains a HindIII site, the Kozak sequence (italicized), and nucleotides 1-15 of Gal4 coding strand (underlined): 5`-TGTCGAAGCTTCCACCATGAAGCTACTGTCT-3`; reverse primer GR, which contains an XbaI site and nucleotides 422-441 of the Gal4 noncoding strand (underlined): 5`-CTGCCTCTAGACGATA-CAGTCAACTGTCTTT-3`. The plasmid pGal4:VP16, which contains a fusion cDNA of Gal4(1-147) and VP16(413-490), was used as the template. The PCR product was inserted into pRc/CMV at the HindIII and XbaI sites to generate the plasmid designated pG4/CMV.

The cDNA sequence encoding the carboxyl terminus(413-490) of VP16 was obtained by PCR using two primers: forward primer VF, which contains a XbaI site, a start codon, and nucleotides 1237-1255 of the VP16 coding sequence (underlined): 5`-CTGCCTCTAGAATGAC-CGCCCCCATTACCGACG-3`; reverse primer VR, which contains an ApaI site, stop codon, and nucleotides (underlined) downstream of the VP16 coding sequence in the template cDNA (pGal4:VP16): 5`-CTGCCGGGCCCTCAATCAATCAGGAATTC-3`. The PCR fragment was inserted into pRc/CMV at the XbaI and ApaI sites and was designated as pVP16/CMV(413-490).

The Gal4(1-147):VP16(413-490) fusion cDNA was generated by subcloning the VP16 fragment into pG4/CMV at the XbaI and ApaI sites and was designated as G4:VP16.

To generate fusion cDNAs of Gal4 and AhR mutants, PCR was used to obtain cDNA fragments of the AhR with pAhR/Bluescript as the template. To generate NH-terminal deletions, the same reverse primer R1B was used in each case, which contain an ApaI site and nucleotides 2399-2418 of the AhR noncoding strand (underlined): 5`-TGCCGGGCCCTCAACTCTGCACCTTGCTTA-3`.

The forward primers for the NH-terminal deletions were as follows: N2: primer F2, 5`-TGTCGTCTAGAATGAGCTTCTTTGATGTTGCATT-3`, which contains an XbaI site and nucleotides 238-257 of AhR coding sequence (underlined). N3: primer F3, 5`-CTGCCTCTAGACAGCTTC-CTCCAGAGAACGC-3`, which contains an XbaI site and nucleotides 601-620 of AhR coding sequence (underlined). N4: primer F4, 5`-CTGCCTCTAGAAGTGGCATGACAGTTTTCCG-3`, which contains an XbaI site and nucleotides 1017-1036 of AhR coding sequence (underlined). N5: primer F5, 5`-GTCGTCTAGACCAATACGCACCAAACGC-3`, which contains an XbaI site and nucleotides 1264-1281 of AhR coding sequence (underlined). N6: primer F6, 5`-CTGCCTCTAGAAGCTTTGCGGCCGCAGGAAG-3`, which contains an XbaI site and nucleotides 1468-1487 of AhR coding sequence (underlined). N7: primer F7, 5`-GTCGTCTAGATTGGACTTCCCTGGAAGG-3`, which contains an XbaI site and nucleotides 2155-2172 of AhR coding sequence (underlined). N8: primer F8, 5`-TGTCGTCTAGAAACTCGGCT-TGCCAGCAGCA-3`, which contains an XbaI site and nucleotides 1768-1787 of the AhR coding sequence (underlined).

To obtain internal fragments of the AhR cDNA, PCR was used with the following primer sets: NC1: forward primer F6 and reverse primer R2 as described above. NC2: forward primer F8 and reverse primer R4, 5`-TGCCGGGCCCTCACTGCACACTCTTGGAAT-3`, containing an ApaI site, a stop codon, and nucleotides 2138-2154 of the AhR noncoding strand (underlined). NC3: forward primer F6 and reverse primer R4. NC4: forward primer F4 and reverse primer R3. NC5: forward primer F5 and reverse primer R4. NC6: forward primer F4 and reverse primer R4. NC7: forward primer F2 and reverse primer R5: 5`-TGCCTCTAGATTCTCCAGTCTTAATCATGC-3`, which contains an XbaI site and nucleotides 998-1017 of the AhR noncoding strand (underlined).

The AhR cDNA fragments generated by PCR were inserted into pG4/CMV at XbaI and ApaI sites immediately downstream of the Gal4 DNA-binding domain to generate the corresponding Gal4:AhR fusion cDNAs.

To obtain the Ga14:Arnt fusion cDNA, an Arnt cDNA fragment encoding residues 128 to 776 was generated by PCR with pArnt/BK (Li et al., 1994) as the template. The forward primer was 5`-GTCGTCTAGACGGGGAACTGGCAACACATC-3`, which contains an XbaI site and nucleotides encoding residues 128-134 of mouse Arnt. The reverse primer was 5`-TGCCGGGCCCCTATTCTGAAAAGGGGGGAAAC-3`, which contains an ApaI site, a stop codon, and nucleotides encoding residues 771-776 of mouse Arnt. The PCR fragment was subcloned into pG4/CMV at the XbaI and ApaI sites to generate G4:Arnt.

To construct triple fusion cDNAs, the AhR fragment NC4 was digested with XbaI and ligated into pG4:N6, pG4:N8, pG4:Arnt, and pG4:VP16 at the XbaI sites. The resulting cDNAs were designated as G4:NC4:N6, G4:NC4:N8, G4:NC4:Arnt, and G4:NC4:VP16, respectively. The fusion cDNA G4:NC7:N5 was constructed by subcloning the AhR fragment NC7 into pG4:N5 at the XbaI site.

Each plasmid construct was analyzed by restriction endonuclease digestion and/or DNA sequencing to verify that the insert was in the proper orientation and in the correct translation frame. Each cDNA construct also was used as a template to direct protein synthesis in an in vitro transcription/translation system, as described below. These studies verified that the full-length AhR, its deletion mutants, and the Gal4:AhR/Arnt fusion proteins had the expected molecular weights and were expressed at similar levels.

Transfection and Chloramphenicol Acetyltransferase Assay

Transfection was carried out using Polybrene as described previously (Kawai and Nishizawa, 1984; Fisher et al., 1990). The AhR cDNA or its mutant derivatives were cotransfected with a reporter plasmid which expresses chloramphenicol acetyltransferase (CAT). The pMcat5.9 reporter plasmid contains an AhR-dependent enhancer inserted upstream of the mouse mammary tumor virus promoter and the bacterial CAT cDNA (Jones et al., 1986). For Gal4:AhR fusion derivatives, the G5E4T construct, which contains 5 copies of the Gal4 binding site upstream of the E4 promoter, was used as the reporter plasmid (Carey et al., 1990).

The transfected cells were treated with 1 nM TCDD in dimethyl sulfoxide for 16 h prior to harvest. Control cells were treated with MeSO only. Cell lysates were prepared in Reporter lysis buffer (Promega). CAT activity was measured using a differential extraction/liquid scintillation assay (Promega). -Galactosidase activity was measured using a colorimetric assay (Promega) according to the manufacturer's instructions. CAT activity was normalized to -galactosidase activity and/or to protein concentration to correct for differences in transfection efficiency and/or protein concentration. Cellular protein concentration was determined by the method of Bradford(1976).

In Vitro Transcription/Translation

The full-length AhR and mutant cDNAs in pRc/CMV were transcribed from the T7 promoter using the TNT coupled reticulocyte lysate system (Promega). Full-length mouse Arnt cDNA in pArnt/BK (Li et al., 1994) was expressed from a T3 promoter in pBK-CMV. Briefly, 0.4 µg of plasmid was used as the DNA template, and in vitro transcription and translation were performed at 30 °C for 90 min as suggested by the manufacturer. Expression of the cDNAs was verified by including [S]methionine in the incubation and analyzing the proteins by SDS-polyacrylamide gel electrophoresis and fluorography.

Electrophoretic Mobility Shift Assay (EMSA)

The EMSA was carried out as described previously, using the in vitro expressed proteins and, as a positive control, nuclear extract from Hepa1c1c7 cells. The wild type DNA probe contains the DNA recognition sequence for the AhR/Arnt heteromer designated DRE D (Lusska et al., 1993). The mutant DNA probe (Mt) was as described previously and contains two substitutions in the core recognition sequence for AhR/Arnt (Elferink and Whitlock, 1994). Preparation of nuclear extract from hepatoma cells was as described previously (Lusska et al., 1992). The in vitro expressed proteins or nuclear extract was incubated with poly(dI-dC) for 15 min at room temperature, followed by the addition of a P-labeled probe. The DNA-protein complexes were resolved by electrophoresis on a nonreducing 6% polyacrylamide gel.


RESULTS

Cloning of Ah Receptor cDNA

We used PCR amplification and primers based upon a published sequence to isolate a 2.5-kilobase cDNA from C57BL/6 mouse liver. Its nucleotide sequence agrees with that reported for the mouse b-1 allele (Poland et al., 1994), except at residues 604 (arginine instead of cysteine) and 626 (proline instead of leucine). Transfection experiments in mouse hepatoma cells reveal that the cDNA (a) restores TCDD-responsiveness to AhR-defective (AhR-D) cells and (b) encodes a protein that recognizes the expected DNA sequence in a dioxin-responsive enhancer (data not shown). Therefore, by both structural and functional criteria, the cDNA encodes a bona fide AhR. Functional Importance of the Receptor's COOH-terminal Half-Eukaryotic transcription factors often have a modular organization and contain domains that independently specify DNA binding and transcriptional activation (Ptashne, 1988). Previous studies reveal that the NH-terminal half of AhR contains domains responsible for ligand binding, heterodimerization with Arnt, and DNA recognition (Dolwick et al., 1993b; Whitelaw et al., 1993b; Poland et al., 1994; Ema et al., 1994). However, the function of the receptor's COOH-terminal half is less well understood. Here, we tested the hypothesis that the COOH-terminal half exhibits a transactivation function.

First, we analyzed the ability of AhR deletion mutants to activate the transcription of a reporter gene after transient transfection into AhR-D cells. Our findings reveal that removal of 212 amino acids (which includes the glutamine-rich region) from the receptor's COOH terminus is associated with a 50% decrease in its ability to activate the CAT gene (C1, Fig. 1). This finding is consistent with previous observations by others (Whitelaw et al., 1994). Because this mutant (C1) retained substantial activity, we analyzed additional mutants, which contained larger COOH-terminal deletions. Our findings reveal that removal of 384 amino acids from the COOH terminus results in virtually complete loss of AhR function (C2, Fig. 1). This finding is new. Our observations imply that the COOH-terminal half of AhR contains at least two functional domains.


Figure 1: Deletion analysis of the Ah receptor. Panel A, schematic diagrams of expression and reporter plasmids. Panel B, functional analyses. AhR-defective cells were cotransfected with the indicated expression plasmid (10 µg) and the pMcat5.0 reporter plasmid (5 µg); CAT activity was measured from uninduced (DMSO) and induced (TCDD; 1 nM, 16 h) cells and corrected for protein concentration. The data represent means and standard deviations from four experiments. The empty vector, pRc/CMV, was used as a negative control to measure background activity, which represents low levels of AhR in AhR-D cells. PAS-A and -B, domains that exhibit homology with Per, Arnt, and Sim; Q, glutamine-rich domain.



We analyzed the ability of the full-length and truncated receptors to respond to TCDD, to heterodimerize with Arnt, and to bind DNA, using in vitro transcribed/translated proteins and an EMSA. Control experiments reveal that the formation of a TCDD-inducible protein-DNA complex is AhR-dependent, Arnt-dependent, and DNA sequence-specific; thus, the assay accurately reflects the biological behavior of the AhR/Arnt system (data not shown). Analyses of full-length and mutant receptor cDNAs using this method reveal that the truncated AhR proteins (C1 and C2) participate in TCDD-inducible, Arnt-dependent protein-DNA interactions (Fig. 2). These findings indicate that the TCDD recognition, heterodimerization, and DNA-binding functions of AhR remain intact in the mutant proteins. Therefore, their failure to activate gene expression (Fig. 1) must reflect the loss of some other function(s). We note that, in contrast to our findings, others have reported that truncated AhR proteins comparable to those described here exhibit diminished activity in mobility shift studies (Dolwick et al., 1993b). The reason for this disagreement is unknown.


Figure 2: Analyses of full-length and truncated AhR by EMSA. Full-length (FL) and mutant AhR (C1, C2) and Arnt were expressed in vitro using the TNT coupled reticulocyte lysate system. Equimolar amounts of the in vitro translated proteins were incubated with TCDD (20 nM, 2 h) and were analyzed by EMSA, using P-labeled wild type DRE as the DNA recognition sequence.



Identification of Multiple Domains That Participate in Transactivation

The preceding observations suggested that the COOH-terminal half of AhR might exhibit a transactivation function. To test this idea, we analyzed AhR segments as Gal4:AhR fusion proteins in a standard transactivation assay. In these studies, we used AhR segments from which the bHLH domain had been deleted; this deletion prevents AhR from heterodimerizing with Arnt, which itself exhibits transactivation capability (Jain et al., 1994; Li et al., 1994; Whitelaw et al., 1994). Therefore, under the experimental conditions used here, transactivation is due to AhR, and not to Arnt.

Our findings (Fig. 3) reveal that relatively large AhR segments exhibit weak transactivation capability (see G4:N2 and G4:N3, Fig. 3B). Others have made similar observations (Whitelaw et al., 1994; Jain et al., 1994). However, when the NH-terminal half of AhR is removed, the COOH-terminal half displays a transactivation function comparable in magnitude to that of the prototype VP16 (see G4:N5 and G4:N6, Fig. 3B). These observations are consistent with our transfection experiments (Fig. 1), in that the loss of transactivation capability could account for the loss of AhR function associated with the COOH-terminal deletions. Our findings also indicate that the COOH-terminal half of AhR transactivates constitutively and does not require TCDD. These results are consistent with the findings of others, which suggest that the ligand-binding region of AhR is located in its NH-terminal half (Dolwick et al., 1993b; Whitelaw et al., 1993b; Poland et al., 1994; Ema et al., 1994).


Figure 3: Transactivation function of AhR. Panel A, schematic diagram of expression and pG5E4T reporter plasmids. Panel B, analyses of NH-terminal deletions of AhR. Wild type hepatoma cells were cotransfected with the indicated expression plasmid (10 µg) and the pG5E4T reporter plasmid (5 µg). CAT specific activity was measured in extracts from uninduced and TCDD-induced (1 nM, 16 h) cells and corrected for protein concentration. The data represent means and standard deviations from four to eight experiments. Panel C, transactivation function and synergistic action of AhR modules. Wild type cells were cotransfected and analyzed as described in Panel B. The data represent means and standard deviations from four experiments. PAS-A and -B, domains that exhibit homology with Per, Arnt, and Sim; Q, glutamine-rich domain.



More detailed analyses indicate that the COOH-terminal half of AhR contains distinct subdomains that exhibit independent transactivation capability. For example, the subdomain that spans amino acids 490 through 593 functions as a strong transactivator (see G4:NC1, Fig. 3C); this subdomain is rich in glutamic acid and aspartic acid residues, a characteristic that is typical of acidic transactivators (Hahn, 1993). In contrast, subdomains spanning amino acids 590 to 718 (G4:NC2, Fig. 3C) and amino acids 719 to 805 (G4:N7, Fig. 3C) exhibit weaker transactivating capabilities. The former region is rich in glutamine (28%) and proline (18%), and the latter region is rich in glutamine (13%), proline (10%), and serine (15%). Thus, these subdomains differ in both composition and sequence from the acidic transactivating region; such structural differences presumably account for the quantitative variation in function. The subdomains function synergistically (compare G4:NC1, G4:NC2, and G4:NC3; also compare G4:NC2, G4:N7, and G4:N8, Fig. 3C). The synergy that occurs when two activation domains are linked suggests that multiple segments of AhR simultaneously establish protein-protein interactions with other components of the transcriptional machinery.

Identification of a Domain That Inhibits the Transactivation Function of AhR

Progressive NH-terminal deletions of AhR reveal that removal of the 82-amino acid segment spanning residues 340 through 421 is associated with a marked increase in the transactivation capability of the receptor's COOH-terminal half (compare G4:N4 with G4:N5, Fig. 3B). These findings imply the existence of an inhibitory region, which blocks the transactivation function of AhR. To study in greater detail the functional properties of the putative inhibitory domain, we analyzed hybrid proteins, in which the 82-amino acid segment was linked to other AhR fragments that display transactivation capability. Our findings (Fig. 4A) reveal that the hybrid proteins have lost >90% of their transactivation capability. These data indicate that the 82-amino acid segment inhibits transactivation. To assess the specificity of inhibition, we asked whether the 82-amino acid segment can also inhibit the transactivation capabilities of Arnt and VP16. Our findings (Fig. 4B) indicate that the inhibitory segment blocks transactivation by Arnt but has no effect on transactivation by VP16. These results indicate that the inhibitory domain of AhR can block transactivation by a heterologous protein. However, the domain also exhibits some specificity, because it does not block the function of VP16.


Figure 4: Inhibition of transactivation by a domain of AhR. Wild type hepatoma cells were cotransfected with the indicated expression plasmid (10 µg) and the pG54E4T reporter plasmid (5 µg). CAT specific activity was measured in extracts and corrected for protein concentration. The data represent means and standard deviations from four experiments. Panel A, inhibition of AhR transactivation modules. Panel B, inhibition of Arnt and VP16 transactivation modules. Panel C, effect of an internal deletion on transactivation by AhR. PAS-A and -B, domains that exhibit homology with Per, Arnt, and Sim; Q, glutamine-rich domain.



In order to determine whether AhR contains additional domains that block transactivation, we analyzed a mutant AhR, from which the 82-amino acid inhibitory domain had been deleted. Our findings (Fig. 4C) reveal that the transactivation capability of the mutant is greater than that of the wild type AhR, as expected. However, the mutant's activity is still substantially less than that of the COOH-terminal half of AhR. These results confirm that the 82-amino acid segment inhibits transactivation; they also imply the existence of an additional inhibitory domain(s), located within the NH-terminal half of AhR. Taken together, our observations reveal that transactivation by AhR reflects an interplay between multiple stimulatory and inhibitory components and that the inhibitory effects dominate in the intact protein.

Influence of Cellular Context on Inhibitory Activity

Two general mechanisms might account for the inhibitory effect of the 82-amino acid segment. One possibility is that the segment interacts with an autoregulatory site, thereby maintaining the neighboring transactivation domains in an inactive configuration. A second possibility is that the segment binds an extrinsic factor, and the resulting protein-protein interaction inhibits transactivation. To distinguish between these hypotheses, we measured the inhibitory effect in different cell types using G4:N4 and G4:N5, which differ only in the presence of the inhibitory domain. Our findings (Fig. 5) reveal that, in each of the four cell types tested, the transactivation capability of G4:N4 (which contains the inhibitory domain) is lower than that of G4:N5 (which lacks the inhibitory domain). However, the inhibitory effect is strong in mouse hepatoma (Hepa 1c1c7) cells, intermediate in HeLa and COS-1 cells, and weak in human hepatoma (HepG2) cells. These findings indicate that the capacity to inhibit transactivation varies according to the cellular context in which the inhibitory domain is expressed. These observations argue in favor of the second hypothesis described above and suggest the existence of a cell-specific factor that interacts with the inhibitory domain and blocks transactivation.


Figure 5: Cell-type specificity of transactivation inhibition. Hepa1c1c7, HeLa, HepG2, and COS-1 cells were cotransfected with the indicated plasmid and pG5E4T. CAT activity was analyzed as described in the legend for Fig. 3. The data represent means and standard deviations from four experiments.




DISCUSSION

As a prelude to analyzing the biological function of AhR, we cloned its cDNA from mouse liver using a PCR-based approach. The deduced amino acid sequence of the protein is compatible with the primary structure of AhR reported previously (Ema et al., 1992; Burbach et al., 1992; Chang et al., 1993; Poland et al., 1994). The cDNA encodes a functional AhR as measured by transfection.

Transcription factors are often modular in organization and contain DNA-binding domains that function independently of transactivation domains (Ptashne, 1988). The findings in this report indicate that AhR, like other transcription factors, contains domains that function independently. The NH-terminal half of the AhR determines its TCDD recognition, heterodimerization, and DNA binding functions (Dolwick et al., 1993b; Whitelaw et al., 1993b; Poland et al., 1994; Ema et al., 1994). Our results indicate that these functions can be dissociated experimentally from transactivation, which is specified by the COOH-terminal half of AhR. Our results agree with recent observations that the COOH-terminal half of AhR has a transactivation function as well as an inhibitory function (Jain et al., 1994; Whitelaw et al., 1994). We extend these findings by demonstrating that transactivation by AhR is unusually complicated, in that it involves multiple stimulatory and inhibitory components and that one inhibitory component is cell-type specific. The complex nature of AhR's transactivation function may be related to its ability to mediate a diverse set of biological responses. For example, its multiplicity of transactivation domains may enable AhR to interact with different classes of transcription factors and, thus, to enhance transcription from a variety of promoters. The cellular specificity of the inhibitory function may constrain the transactivation to particular tissues. Together, the interplay between stimulatory and inhibitory functions may contribute to the diversity of effects that is characteristic of TCDD (Poland and Knutson, 1982; Safe, 1986).

The structural determinants that confer transactivation capability upon a protein are not well understood; acidic, glutamine-rich, and proline-rich domains exhibit such function (Hahn, 1993). The AhR region between amino acids 541 and 581 contains 30% glutamic acid and aspartic acid residues and transactivates strongly. These properties resemble those of the prototypical acidic transactivator VP16 (Sadowski and Ptashne, 1988). At two different locations within the 541-581 region, acidic and hydrophobic amino acids flank a phenylalanine residue, a motif that has been implicated in the transactivation capability of VP16 and NF-KB p65 (Regier et al., 1993; Blair et al., 1994). Analyses of the 541-581 region using the Chou-Fasman (Chou and Fasman, 1978) or Garnier et al. (1978) algorithms imply that it has the potential to form an -helix. The ability to adopt an -helical structure under hydrophobic conditions might facilitate protein-protein interactions and contribute to acidic activator function (Schmitz et al., 1994).

Analyses in other systems suggest at least two general mechanisms by which transactivation might occur. One possibility is that transactivation domains contact a component(s) of the general transcriptional machinery, either directly or indirectly, thereby stabilizing an active transcriptional complex at the promoter (Tjian and Maniatis, 1994). For example, VP16 may interact with the TATA-binding protein and/or the general transcription factor TFIIB (Ingles et al., 1991; Roberts et al., 1993). By analogy, the AhR's acidic transactivation domain may also contact these proteins in mediating the response to TCDD. Similarly, by analogy with findings for Sp1, the AhR's glutamine/proline-rich transactivating domains may interact with TATA-binding protein (Emili et al., 1994) and/or an accessory transcription factor (Hoey et al., 1993). A second possibility is that transactivators counteract the inhibitory effects of histone proteins and chromatin structure, thereby allowing general transcription factors access to the promoter (Wolffe, 1994). We have shown previously that the activation of CYP1A1 transcription by AhR/Arnt is associated with disruption of nucleosomes and increased accessibility of promoter DNA (Morgan and Whitlock, 1992; Wu and Whitlock, 1992). Thus, an activation domain(s) of AhR might contact a histone(s), thereby altering chromatin structure. These potential mechanisms are not mutually exclusive; given that both AhR and Arnt have independent transactivation functions and that multiple DNA-bound AhR/Arnt heteromers participate in CYP1A1 gene expression, both mechanisms could operate in vivo.

The mechanism by which the inhibitory domain exerts its effect remains to be elucidated. Our observations imply that a cell-specific factor(s) plays an important role in the inhibitory mechanism. Although the unliganded AhR can interact with hsp90 (Perdew, 1988; Denis et al., 1988; Chen and Perdew, 1994; Carver et al., 1994), it is unlikely that hsp90 is responsible for inhibiting transactivation, because (a) hsp90 is a not a cell-specific factor; it is present in many cell types at relatively high concentration; (b) in AhR fragments that do contain an intact ligand/hsp90 binding region, TCDD (which releases hsp90 from AhR) does not relieve the inhibition (G4:N2 and G4:N3, Fig. 3B; Whitelaw et al., 1994). Therefore, we infer that a cell-specific factor other than hsp90 mediates the inhibitory effect. Such a factor could act by binding to the inhibitory domain, thereby blocking transactivation. One potential candidate is a putative cycloheximide-sensitive inhibitory protein that modulates AhR function and ``superinduces'' gene transcription (Lusska et al., 1992; Reick et al., 1994). Another possibility is that the cell-specific factor enzymatically modifies the inhibitory domain, thereby converting AhR to an inactive form. AhR has several potential phosphorylation sites within the 82-amino acid inhibitory segment (Dolwick et al., 1993a). Whether modification at these sites influences transactivation remains to be determined.


FOOTNOTES

*
This research was supported by Research Grant ES03719 from the National Institute of Environmental Health Sciences. 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.

The abbreviations used are: TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin; CAT, chloramphenicol acetyltransferase; EMSA, electrophoretic mobility shift assay; AhR, aromatic hydrocarbon receptor; Arnt, Ah receptor nuclear translocator; bHLH, basic helix-loop-helix; hsp, heat shock protein; CMV, cytomegalovirus; PCR, polymerase chain reaction.


ACKNOWLEDGEMENTS

We thank Dr. G. R. Crabtree for providing the G4:VP16 plasmid. We thank M. L. Tuggle for secretarial assistance and I. D. Clair for comments on the manuscript.


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