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INTRODUCTION |
The halogenated aromatic hydrocarbon
2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD,
dioxin)1 is a widespread
environmental contaminant that elicits both adverse and adaptive
responses in animals and humans (1, 2). An intracellular protein known
as the aromatic hydrocarbon receptor (AhR) mediates the biological
effects of TCDD (3-6). The most well characterized action of TCDD is
an AhR-dependent adaptive response that leads to increased
transcription of the CYP1A1 gene, which encodes the microsomal enzyme
cytochrome P4501A1 (7-9). To activate transcription, AhR dimerizes
with a second protein, the AhR nuclear translocator (Arnt) (10, 11)
thereby generating a transcription factor with specific DNA recognition
properties. AhR and Arnt are structurally related, prototypical members
of an interesting class of transcription factors that contain bHLH motifs juxtaposed to PAS domains (12, 13); the latter are regions of
homology between Drosophila Per, mammalian Arnt, and Drosophila Sim (14-16). bHLH/PAS proteins mediate
transcriptional responses to a variety of environmental and
developmental signals (17).
Like other transcriptional activators, AhR and Arnt contain domains
that function independently of each other. For example, their
N-terminal segments mediate heterodimerization and DNA recognition whereas their C-terminal segments contain transactivation domains (12,
13, 18-20). Using AhR-defective mouse hepatoma cells, we have shown
previously that the C-terminal segment of AhR is required for
TCDD-inducible occupancy of the CYP1A1 promoter and transactivation of the gene in situ (21, 22). In these
studies, we also found that an AhR chimera containing the N-terminal
segment of AhR (amino acids 1-494) linked to a 69-amino acid domain
from the C-terminal segment of AhR (amino acids 515-583) facilitates CYP1A1 promoter occupancy and restores its responsiveness to
TCDD in AhR-defective cells, whereas the N-terminal segment by itself does not (22). The domain spanning amino acids 515-583 is rich in
glutamate and aspartate residues (24%); by this criterion, it
resembles an acidic activation domain (AAD) (23, 24). Here, we have
analyzed the function of this acidic domain in more detail. In
particular, we have studied its transactivation function in a
chromosomal setting by reconstituting AhR-defective cells with chimeric
AhR mutants and measuring the response of the native CYP1A1
target gene to TCDD. Our findings reveal similarities, as well as
notable differences, between the function of the acidic domain of AhR
and that of previously characterized AADs.
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EXPERIMENTAL PROCEDURES |
Materials--
The pGalO vector was provided by
Dr. Chi V. Dang (Johns Hopkins University, Baltimore, MD) and
contains a Gal4 DNA-binding domain (amino acids 1 to 147) juxtaposed to
a multicloning site (25). The plasmid pFR-Luc, the QuikChange
site-directed mutagenesis kit, and Pfu DNA polymerase were
purchased from Stratagene (La Jolla, CA). The
pRL-cytomegalovirus vector and dual-luciferase reporter assay
system were purchased from Promega (Madison, WI). The retroviral vector
pMFG was derived from the Moloney murine leukemia virus (26).
Transcription of the inserted coding sequences is driven by the
retroviral long terminal repeat. The Phoenix-eco retroviral producer
cell line was provided by Dr. Garry Nolan (Stanford University,
Stanford, CA) (27). [
-32P]dCTP (3,000 Ci/mmol) and the
Renaissance chemiluminescence kit were purchased from PerkinElmer Life
Sciences. The RNeasy kit was from Qiagen (Valencia, CA).
Reagents for SDS-PAGE were from Bio-Rad. Hyperfilm MP was from
Amersham Pharmacia Biotech. Tissue culture reagents were from Life
Technologies, Inc. The AhR antibody was kindly provided by
Dr. Gary H. Perdew (Pennsylvania State University, University Park, PA).
Cell Culture--
Wild-type (Hepa1c1c7) and AhR-defective
(Taoc1BPrc1) mouse hepatoma cells were cultured as
described previously (28). Phoenix cells were cultured as described
previously (27).
Plasmid Construction--
Deletion mutants (
1-5) were
generated by cloning PCR-amplified fragments of AhR cDNA into the
multicloning site of plasmid pGALO at BamHI and
ClaI sites. The plasmid pGAhR515-583 (22) was
used as a template in PCR reactions, and amplifications were performed
using Pfu DNA polymerase according to the manufacturer's instructions. A linker sequence containing a BamHI site was
attached to forward primers, whereas reverse primers contained a
ClaI site. The following primers were used for
amplification: Primer A, 5'-CGCGGATCCTCTCTGGCGGCCCCTCAGAG-3'; Primer B,
5'-TCCATCGATTCAGGTCAGGATTTCGTCCGTTAT-3'; Primer C,
5'-TCCATCGATTCAGAACTCCTCGTTCTGCATGCA-3'; Primer D,
5'-CGCGGATCCAATACAGCATCATGAGGAACCTT-3'; Primer E,
5'-CGCGGATCCAATTCTTCAGAACTGACTCCACC-3'; and Primer F,
5'-TCCATCGATTCACAGGGAATCCTGCACGTAGGT-3'. Primers A, D, and E are
forward primers, and B, C, and F are reverse primers. Mutant
1
(containing AhR amino acids 515-553) was constructed using primer sets
A and C; mutant
2 (amino acids 553-583), primer sets E and F;
mutant
3 (amino acids 532-583), primer sets D and F; mutant
4
(amino acids 515-577), primer sets A and B; and mutant
5 (amino
acids 532-577), primer sets D and B. The junctions between Gal4 and
AhR sequences were confirmed to be in-frame by nucleotide sequencing.
Single or multiple point mutations were made in the acidic segment,
515-583, of AhR using the QuikChange site-directed mutagenesis kit
(Stratagene) according to the manufacturer's instructions. Plasmid
pGAhR515-583 was used as a template for the appropriate sense and antisense mutation primers. The specific amino acid changes
introduced by mutagenic primers are indicated in each figure. All
mutations were confirmed by nucleotide sequencing.
The pGAhR515-583 mutant constructs were used as templates
to PCR amplify sequences encoding AhR amino acids 515-583 for
insertion into pMFGAhR494 (22). Both the forward primer (5'-ACTACTGCAGCGGCCGCACTCTCTGGCGGCCCCTCAGAG-3') and reverse
primer (5'-ACTACTGCAGCGGCCGCTCACAGGGAATCCTGCACGTAGGT-3')
contained NotI sites (underlined), and the reverse primer
contained a stop codon (bold). The PCR products were digested with
NotI and subcloned into the internal NotI site
(amino acids 492 to 494) of AhR in plasmid pMFGAhR494.
Transient Transfections and Transactivation
Assays--
Wild-type mouse hepatoma cells were plated in 35-mm 6-well
tissue culture dishes and incubated overnight. Cells were cotransfected with 2 µg of pGAhR515-583 (or mutants), 1 µg of
pFR-Luc, a reporter plasmid that contains five Gal4-binding sites
upstream of the firefly luciferase gene (Stratagene), and 10 ng of
pRL-Luc (expression plasmid for Renilla luciferase, used to
control for transfection efficiency) (Promega) using a polybrene method
(29). 24 h after transfection, transactivation capabilities of
pGAhR515-583 or pGAhR515-583 mutants were
determined using the dual-luciferase reporter assay system (Promega)
according to the manufacturer's instructions. Light production was
measured using a Lumat LB 9507 luminometer, and firefly luciferase
activities were normalized to Renilla luciferase activities.
All experiments were repeated at least three times, and the data are
expressed as mean ± S.E.
Retroviral Expression of AhR--
5 µg of pMFGAhR,
pMFGAhR494, pMFGAhR494/515-583 (22), and the
pMFGAhR494/515-583 mutants were transfected into the
ecotropic packaging cell line, Phoenix, as described previously (27).
Infection of AhR-defective mouse hepatoma cells was carried out as
described previously (21).
Analysis of CYP1A1 Gene Expression--
Wild-type,
AhR-defective, and reconstituted mouse hepatoma cells were grown to
~80% confluence on 100-mm-diameter tissue culture dishes and treated
with 1 nM TCDD or 0.1% Me2SO for
18 h. Total RNA was isolated using RNeasy spin columns (Qiagen).
Total RNA (5 µg) was fractionated on 1.2% agarose, 2.2 M formaldehyde gels, transferred to Nytran by capillary
blotting in 20× SSC, and cross-linked to the membrane in a UV
Stratalinker 2400 (Stratagene). Blots were hybridized with
32P-labeled P4501A1 or actin cDNA overnight at 55 °C
using ExpressHyb hybridization solution (CLONTECH).
Blots were washed as described previously (30) and then
autoradiographed with Hyperfilm MP (Amersham Pharmacia Biotech).
Immunoblotting Analysis--
Whole cell extracts were prepared
from wild-type, AhR-defective, and reconstituted cells as described
previously (31). 40 µg of cellular proteins were dissolved in 2×
Laemmli sample buffer (Bio-Rad), fractionated on a 7.5% polyacrylamide
gel using SDS-PAGE running buffer (Bio-Rad), and transferred to a
polyvinylidene difluoride membrane. Blots were blocked in TBS-T (20 mM Tris (pH 7.6), 137 mM NaCl, 0.1% Tween 20)
containing 5% nonfat milk (blocking buffer) overnight at 4 °C.
Incubation with primary antibody (anti-AhR; 1:2000) was carried out for
1 h at room temperature. After several washes in blocking buffer,
blots were incubated with secondary antibody (anti-mouse-horseradish
peroxidase; 1:2000) for 1 h at room temperature. After washing in
TBS-T, blots were developed using the Renaissance chemiluminescence kit
(PerkinElmer Life Sciences) and visualized on Hyperfilm MP.
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RESULTS |
Acidic Amino Acids and Transactivation--
Previously, we have
shown that a 69-amino acid region (amino acids 515-583) of the
C-terminal segment of AhR can transactivate a dioxin-responsive target
gene when linked to the N-terminal segment of AhR (22). The region
containing amino acids 515-583 is rich in glutamate and
aspartate residues (24%); thus, it resembles an AAD. We have
analyzed the acidic domain in more detail to identify structural
features important for transactivation. The specific role(s) of acidic
amino acids in AAD function is uncertain (32-35). Therefore, we first
asked whether acidic residues contribute to transactivation. For these
studies, we constructed single or double mutants in which we
substituted alanine for aspartate or glutamate. We fused the mutants to
the DNA-binding domain of Gal4 and measured their ability to
transactivate a Gal4-dependent luciferase reporter gene in transient transfection experiments. Table
I shows the results of these studies.
Five mutants (E521A, E563A, D565A, D568A, and D581A) exhibit little
change in function. Four mutants (D525A, D541A, E551A/E552A, and D557A)
reveal modest reductions (20-30%) in transactivation activity. Four
other mutants (D530A, E543A/D544A, D570A, and D573A/E574A) show more
substantial (40-60%) losses of function. Our results imply that the
acidic side chains at these four sites are particularly important for
transactivation capability. However, individual acidic residues in
these four regions apparently do not contribute equally to function.
For example, the decreased activity of single mutant D530A resembles that of double mutant E543A/D544A. Therefore, on an individual basis,
Asp530 may be more important for transactivation
than either Glu543 or Asp544. In general, our
results indicate that acidic residues vary substantially in the
contribution that each makes to transactivation. This finding imposes
an important constraint on the possible mechanism by which the acidic
residues influence function.
Hydrophobic Amino Acids and Transactivation--
Transactivation
domains can interact with their target proteins via hydrophobic
contacts (35-37). Therefore, we asked whether hydrophobic amino acids
in the acidic segment of AhR contribute to transactivation capability.
We mutated hydrophobic residues to alanine and tested the resulting
mutants (schematically represented in Fig.
1 as
1-
7) for their ability to
transactivate the luciferase reporter gene. Our findings reveal that
four of the mutants (
3,
4,
6, and
7) show reduced activity
in transactivation assays. Of these four, mutant
4, exhibits the
largest decrease in function (25-30% of wild-type), whereas mutants
3,
6, and
7 exhibit somewhat smaller changes (45-75% of
wild-type). In contrast, mutants
1,
2, and
5 show no loss of
function (Fig. 1). These results imply that the hydrophobic residues
targeted in mutants
3,
4,
6, and
7 are important for
function and that hydrophobic amino acids vary considerably in the
degree to which they influence transactivation.

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Fig. 1.
Transactivation capabilities of AhR
mutants. The amino acid sequence of the acidic segment of AhR is
shown; boxes indicate hydrophobic residues mutated to
alanine (A). Transactivation capabilities were measured as
described under "Experimental Procedures" and are expressed as a
percentage of wild-type (wt) ± standard error
(n = 3). Luc, luciferase.
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To extend these studies further, we analyzed mutants
3,
4,
6,
and
7 using a more stringent assay. Reconstitution of AhR-defective cells allows us to measure the transactivation of a known AhR target
gene (CYP1A1) in its native chromosomal setting (21). For
these studies, we linked mutants
3,
4,
6, and
7 to the N-terminal segment of AhR (amino acids 1-494), introduced the resulting constructs into AhR-defective cells by retroviral infection, and assessed the ability of the reconstituted cells to respond to TCDD
(as measured by the induction of CYP1A1 mRNA). Fig.
2, A and C
schematically depicts the constructs we analyzed. Positive and negative
control experiments indicate that the acidic region of AhR, 515-583,
restores TCDD responsiveness to AhR-defective cells when linked to the
N-terminal segment of AhR (construct AhR494/AAD) whereas
the N-terminal segment of AhR alone (construct AhR494) does
not (Fig. 2B). Our analyses also reveal that each of the
four mutants is defective in its ability to activate CYP1A1 transcription (Fig. 2C). The mutations in
4 have the
greatest adverse impact on function, substantiating the results in Fig. 1 and revealing the importance of these hydrophobic residues for function in vivo. Immunoblotting studies confirm that the
wild-type and mutant proteins are expressed at similar levels in the
reconstituted cell lines (Fig. 2D). Therefore, our findings
do not represent artifacts because of unequal expression of the mutant
proteins.

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Fig. 2.
In Vivo analysis of AhR mutants.
A and C, schematic representation of AhR
constructs. B and C, TCDD-inducible P4501A1
mRNA accumulation in reconstituted cells. AhR-defective cells were
reconstituted by retroviral infection with AhR, AhR494,
AhR494/AAD, or AhR mutants
(AhR494/ 3,
AhR494/ 4,
AhR494/ 6, and
AhR494/ 7). Total RNA was isolated from
uninduced ( ) and TCDD-induced (1 nM, 18 h) (+)
wild-type (wt), AhR-defective, and reconstituted cells.
CYP1A1 and actin mRNAs were measured by Northern analysis.
D, immunoblot analysis of AhR in wild-type, AhR-defective,
and reconstituted cells. Cell extracts were fractionated by SDS-PAGE
(40 µg of protein per lane), transferred to a
polyvinylidene difluoride membrane, and probed with an antibody to the
N-terminal sequence of AhR. Molecular masses are indicated in
kilodaltons.
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Bipartite Organization of Acidic Segment--
Our findings
indicate that the hydrophobic residues and the acidic residues that are
important for transactivation map to two smaller regions of the acidic
segment of AhR (Fig. 3). For example, the
mutations in
3,
4, D530A, and E543A/D544A map to the region
spanning amino acids 530-545 (which we designate R1), and the
mutations in
6,
7, D570A, and D573A/E574A map to the region
spanning amino acids 564-579 (which we designate R2). These results
imply that regions R1 and R2 are crucial for transactivation. To assess
the relative contributions of R1 and R2 to transactivation in
vivo, we substituted alanine for all of the hydrophobic residues in R1 or R2 (the mutants are designated mR1 and mR2, respectively). We
linked each mutant to the N-terminal segment of AhR, introduced the
resulting constructs into AhR-defective cells by retroviral infection,
and measured the response of the CYP1A1 gene to TCDD. Our
findings (Fig. 4) reveal that mutation of
the hydrophobic residues in either R1 (construct
AhR494/mR1) or R2 (construct AhR494/mR2)
substantially (>80%) impairs the ability of the acidic segment of AhR
to transactivate the CYP1A1 gene in response to TCDD.
Furthermore, mutation of the hydrophobic residues in R1 and R2
simultaneously (construct AhR494/mR1/mR2) abolishes
function. These results imply that both R1 and R2 are crucial for
transactivation and R1 and R2 function synergistically, because
mutation of either one produces >50% loss of function. The mechanism
responsible for the synergy remains to be determined.

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Fig. 3.
Amino acid sequence of regions 1 and 2 in the
acidic segment of AhR. A, arrows indicate
hydrophobic residues, and asterisks indicate acidic residues
that are important for transactivation capability (see Table I and Fig.
1).
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Fig. 4.
In Vivo analysis of AhR mutants.
A, schematic representation of mutants. Constructs
containing mutations in hydrophobic residues in regions 1 and 2 are
designated mR1 and mR2, respectively. The construct designated
AhR494/mR1/mR2 contains mutations in hydrophobic residues
in both R1 and R2. B, TCDD-inducible CYP1A1 mRNA
accumulation in AhR-defective cells reconstituted with the indicated
constructs. Total RNA was isolated from uninduced ( ) and TCDD-induced
(1 nM, 18 h) (+) wild-type (wt),
AhR-defective, and reconstituted cells. CYP1A1 and actin mRNAs were
measured by Northern analysis.
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We further evaluated the importance of R1 and R2 by measuring the
ability of deletion mutants to transactivate a luciferase reporter gene
in response to TCDD. Our findings (Fig.
5) indicate that mutant
1 (which is
missing R2) and mutant
2 (which is missing R1) exhibit little
transactivation capability (15-25% of wild-type). These results are
consistent with those in Fig. 4 and imply that both R1 and R2 are
necessary for maximal transactivation. Mutants
3 and
4, which are
missing portions of R1 and R2, respectively, but which otherwise
contain most of the acidic segment, display substantial losses in
function. In addition, mutant
5, which spans amino acids 539-577
and contains partial deletions in both R1 and R2, exhibits virtually no
transactivation capability. Taken together, the findings in Fig. 5 are
internally consistent with those in Fig. 4 and imply that both R1 and
R2 must be intact for complete transactivation activity. Our results
indicate that the acidic segment of AhR is composed of two subdomains;
this bipartite organization suggests that the segment makes multiple
contacts with its target protein(s).

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Fig. 5.
Transactivation capability of AhR deletion
mutants. Transactivation capabilities of the indicated deletion
constructs were measured as described under "Experimental
Procedures." Values are expressed as a percentage of wild-type
(wt) ± standard error (n = 3).
Luc, luciferase.
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-Helices and Transactivation--
Computer analyses of
the primary sequence of AhR using the Chou and Fasman (38) and Garnier
et al. (39) algorithms predict that the amino acid sequences
in the vicinity of R1 and R2 form
-helices. Furthermore,
interspecies comparisons indicate that the predicted
-helical
regions are >90% conserved among mouse, rat, and human. Therefore, we
asked whether the ability to form an
-helix is required for
transactivation. We constructed mutants that contain a
helix-incompatible proline residue within each of the two predicted
-helices, thereby generating mutants F542P and F566P (Fig.
6). Assays for function reveal that both
mutants transactivate a luciferase reporter gene to about the same
extent as wild-type (89 and 96%, respectively). Furthermore, a double mutant containing proline substitutions at both positions displays ~75% of wild-type activity (Fig. 6). These observations imply that
the capacity for
-helix formation is not a major factor in the
transactivation capability of the acidic segment of AhR.

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Fig. 6.
Transactivation capabilities of AhR proline
substitution mutants. Phenylalanine (F) residues that
were mutated to proline (P) and putative -helical regions
1 and 2 ( H1 and H2)
are indicated. Transactivation capabilities of the constructs were
measured as described under "Experimental Procedures" and are
expressed as a percentage of wild-type ± standard error
(n = 3). Luc, luciferase.
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The results in Fig. 6 were unexpected, because studies on other AADs
have stressed the importance of
-helix formation for transactivation
(32, 36, 40, 41). Therefore, we confirmed our findings using the more
stringent reconstitution type of experiment. Our results again
demonstrate that reconstitution with the double mutant
AhR494/2XPro restores TCDD responsiveness to the
CYP1A1 gene in AhR-defective cells (Fig.
7). Thus, substitution of proline at
positions 542 and 566 does not impair TCDD-inducible transactivation of
a chromosomal gene in intact cells. Immunoblotting experiments (Fig.
7C) reveal similar levels of AhR protein in the
reconstituted cell lines; therefore, the activity of the double mutant
(AhR494/2XPro) is not because of its overexpression
relative to wild-type (AhR494/AAD). Our findings indicate
that the ability to form an
-helix is not required in order for the
acidic segment of AhR to transactivate a chromosomal target gene. These
observations reveal an interesting contrast between AhR and
transactivators that form
-helices as part of their function.

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Fig. 7.
In Vivo analysis of AhR proline substitution
mutants. A, schematic representation of AhR constructs.
B, TCDD-inducible CYP1A1 mRNA accumulation in
AhR-defective cells reconstituted with AhR, AhR494,
AhR494/AAD, or the mutant AhR494/2XPro, which
contains proline substitutions at positions 542 and 566 in the acidic
segment of AhR. Total RNA was isolated from uninduced ( ) and
TCDD-induced (1 nM, 18 h) (+) wild-type
(wt), AhR-defective, and reconstituted cells. CYP1A1 and
actin mRNAs were measured by Northern analysis. C,
immunoblot analysis of AhR in wild-type, AhR-defective, and
reconstituted cells. Cell extracts were fractionated by SDS-PAGE (40 µg of protein per lane), transferred to a polyvinylidene
difluoride membrane, and probed with an antibody to the N-terminal
sequence of AhR. Molecular masses are indicated in kilodaltons.
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DISCUSSION |
The mechanism by which AADs increase transcription in
vivo is poorly understood. Here, we have analyzed an AhR segment
that exhibits transactivation capability and resembles an AAD in its primary amino acid sequence. In addition to using standard reporter gene assays to measure transactivation, we have studied the ability of
AhR to transactivate a native chromosomal target gene. Previous studies
in other systems imply that transcriptional activators interact with
the general transcriptional machinery, either directly or via
multivalent coactivators (31, 35, 37, 42, 43). Structural analyses of
the prototypical activator VP16, as well as other AADs, suggest that
the protein-protein interactions involve a transition from random coil
to
-helix with hydrophobic residues along one face of the AAD helix
contacting the target protein (35, 37, 43). Our studies of the AAD of
AhR reveal notable differences between it and AADs in other systems.
Our experiments reveal that the acidic segment of AhR contains two
regions that contribute to transactivation. Both regions contain
important acidic and hydrophobic residues, and the two regions function
synergistically. From a mechanistic standpoint, synergy could reflect
either of two scenarios. First, the two regions may contact different
sites within a single target protein (such as a coactivator or general
transcription factor). For example, the interaction of the
transactivation domain of p53 with MDM2 displays this type of
synergy (44). Alternatively, the two regions may interact with separate
target proteins to produce synergy; for example, two consecutive
LXXLL motifs in SRC-1 contact distinct subunits of a
PPAR-
homodimer (45). Although its mechanism remains to be
elucidated, the synergy we observe for transactivation by AhR resembles
that in other systems where hydrophobic regions mediate interactions
with target proteins.
Our studies also implicate acidic side chains in transactivation by
AhR. We envision that electrostatic interactions complement hydrophobic
protein-protein interactions, because the acidic and hydrophobic
residues that strongly affect function are located adjacent to each
other. The finding that only certain acidic side chains are important
for function suggests that these residues confer local charge to
specific regions of the acidic segment of AhR. In this respect, AhR is
distinctly different from transactivators whose function depends more
on overall negative charge than on the positions of individual acidic
residues (33, 35, 46, 47).
Structural analyses in other systems (for example, p53-MDM2 (36),
VP16-TAF31 (35), and cAMP-responsive element-binding protein
(CREB)-CREB-binding protein (37, 48) suggest that transcriptional
activation domains are largely unstructured in solution but adopt an
-helical conformation upon interaction with their target proteins.
Mutational analyses of the AADs of Pho4 (49), peroxisome
proliferator-activated receptor
(32), and the ETS family member
ERM (41) support this concept, because their capacity to form an
-helix correlates with their transactivation potential. In contrast,
our studies of proline substitution mutants imply that the capacity for
-helix formation is not a major factor in transactivation by the
acidic domain of AhR even though the potential for
-helix formation
has been conserved across species. Furthermore, we note that although
they prevent
-helix formation, proline substitutions maintain
hydrophobicity, reinforcing our impression that hydrophobicity is more
important than
-helicity for transactivation by the acidic segment
of AhR. Thus, our studies reveal an interesting example of an AAD whose
action at a mammalian chromosomal target gene does not require that it
form an
-helix. This property distinguishes the AAD of AhR from
those described previously.
Another noteworthy difference between the acidic segment of AhR and
other transcriptional activation domains is the absence of an obvious
signature motif. For example, the acidic activators p53 (43) and VP16
(35) contain FXX
motifs (
, hydrophobic residues), which are essential for function. Furthermore, Uesugi et al. (43) have suggested that the FXX
motif is a general recognition sequence for the coactivator
hTAFII31. Similarly, the activation domain of CREB binds to
CREB-binding protein via a related motif, YXXIL (37). In
contrast, the AAD of AhR contains no comparable amino acid sequence.
Furthermore, there is no obvious similarity between the amino acid
sequences of R1 and R2 within the AAD of AhR. The absence of known
signature motifs in the AAD of AhR implies that its interactions with
target proteins are novel and differ from those described for other
AADs. A more complete understanding of the interactions between the
acidic segment of AhR and target proteins awaits structural studies.
Because AhR is a prototypical bHLH/PAS transcription factor, its
mechanism of transactivation might be representative of other members
of this class of regulatory proteins. If so, future studies of the AhR
system may generate insights into transactivation of genes involved in
responses to hypoxia, circadian rhythms, development, and other
pathways (17).
Finally, our studies indicate that different mutations in the acidic
segment of AhR produce quantitatively different effects on
transactivation capability. If analogous mutations were to occur in
human populations, we envision that they could account, in part, for
genetic polymorphisms among individuals in their responsiveness to
TCDD. Whether such polymorphisms would be associated with different
susceptibilities to dioxin-induced disease is an interesting issue for
future research.