From the
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.
The environmental contaminant
2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, dioxin)
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
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.
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.
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
The forward primers for the
NH
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.
The transfected cells were
treated with 1 nM TCDD in dimethyl sulfoxide for 16 h prior to
harvest. Control cells were treated with Me
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.
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
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
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
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.
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.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
(
)
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).
-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.
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.
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.
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.
-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`.
-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).
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).
SO 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.
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.
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.
-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.
-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).
-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).
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.