THE JOURNAL OF BIOLOGICAL CHEMISTRY
© 2005 by The American Society for Biochemistry and Molecular Biology, Inc.
Vol. 280, No. 22, Issue of June 3, pp. 21607–21611, 2005
Printed in U.S.A.
ER␣-AHR-ARNT Protein-Protein Interactions Mediate Estradioldependent Transrepression of Dioxin-inducible Gene Transcription*
Received for publication, March 3, 2005, and in revised form, April 13, 2005
Published, JBC Papers in Press, April 18, 2005, DOI 10.1074/jbc.C500090200
Timothy V. Beischlag and Gary H. Perdew‡
From the Center for Molecular Toxicology and Carcinogenesis and Department of Veterinary Sciences, The Pennsylvania
State University, University Park, Pennsylvania 16802
The aryl hydrocarbon receptor (AHR) and the aryl
hydrocarbon receptor nuclear translocator (ARNT)
form a heterodimeric transcription factor upon binding
a wide variety of environmental pollutants, including
2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). AHR target
gene activation can be repressed by estrogen and estrogen-like compounds. In this study, we demonstrate that
a significant component of TCDD-inducible Cyp1a1
transcription is the result of recruitment of estrogen
receptor (ER)-␣ by AHR/ARNT as a transcriptional corepressor. Both AHR and ARNT were capable of interacting directly with ER␣, as ascertained by glutathione
S-transferase pull-down. 17␤-estradiol repressed TCDDactivated Cyp1a1 and Cyp1b1 gene transcription in
MCF-7 cells in the presence of cycloheximide, as determined by reverse transcription/real-time PCR. Furthermore, chromatin immunoprecipitation (ChIP) assays
have shown that ER␣ is present at the Cyp1a1 enhancer
only after co-treatment with E2 and TCDD, in MCF-7
cells. Sequential two-step ChIP assays were performed
which demonstrate that AHR and ER␣ are present together at the same time on the Cyp1a1 enhancer during
transrepression. Taken together these data support a
role for ER-mediated transrepression of AHR-dependent gene regulation.
AHR1 and ER are both ligand activated transcription factors
that transduce extracellular signals through DNA-binding-dependent and -independent mechanisms (1– 4). AHR and the
aryl hydrocarbon receptor nuclear translocator (ARNT) form a
heterodimeric transcription factor, the aryl hydrocarbon receptor complex (AHRC), that binds a wide variety of environmen* This work was supported by NIEHS, National Institutes of Health
Grant ES04869. The costs of publication of this article were defrayed in
part by the payment of page charges. This article must therefore be
hereby marked “advertisement” in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
‡ To whom correspondence should be addressed: Center for Molecular
Toxicology and Carcinogenesis, Life Sciences Bldg., Rm. 309A, Dept. of
Veterinary Sciences, The Pennsylvania State University, University
Park, PA 16802. Tel.: 814-865-0400; Fax: 814-863-1696; E-mail:
ghp2@psu.edu.
1
The abbreviations used are: AHR, aryl hydrocarbon receptor;
AHRC, aryl hydrocarbon receptor complex (AHR/ARNT); ARNT, aryl
hydrocarbon receptor nuclear translocator; CBP, CREB-binding protein
(where CREB indicates cAMP-responsive element-binding protein);
p/CIP, p300/CBP-interacting protein; ChIP, chromatin immunoprecipitation; DBD, DNA-binding domain; DRE, dioxin response element; ER,
estrogen receptor; GRIP, glucocorticoid receptor-interacting protein;
HSP90, heat-shock protein 90; NR, nuclear receptor; TCDD, 2,3,7,8tetrachlorodibenzo-p-dioxin; TR, thyroid hormone receptor; ERE, estrogen response element; GR, glucocorticoid receptor; GST, glutathione
S-transferase; E2, estradiol; P/S/T, proline-serine-threonine-rich; PAH,
polycyclic aromatic hydrocarbon; HAH, halogenated aromatic
hydrocarbon.
This paper is available on line at http://www.jbc.org
tal pollutants including polycyclic aromatic hydrocarbons
(PAHs), and halogenated aromatic hydrocarbons (HAHs) (5),
such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (dioxin, TCDD).
The binding of these compounds and subsequent activation of
target genes are part of an organism’s adaptive response to
environmental contaminants (6). Furthermore, studies in AHR
knock-out mice have revealed an important role for AHR in
development and physiological homeostasis (7–11).
Unliganded AHR exists in the cytoplasm as part of a multimeric complex containing two molecules of HSP90, the
HSP90 co-chaperone p23, and a 36-kDa protein termed hepatitis B virus X-associated protein 2 (XAP2) (12–16). Upon ligand binding, AHR translocates to the nucleus where it associates with ARNT to form a functional transcription factor
complex, the AHRC. As an activated complex, the AHRC is
capable of recruiting several classes of co-activators such as
SRC-1 (steroid receptor co-activator-1), NCoA2 (nuclear coactivator-2)/GRIP1/TIF2, and p/CIP (17, 18), RIP140 (receptorinteracting protein 140) (19), BRG-1 and components of the
mediator complex (20, 21), and CBP and TRIP230 (thyroid
hormone receptor/retinoblastoma protein-interacting protein)
(22–25). These proteins are incorporated into multimeric complexes, which interact with and modulate the activity of the
core transcriptional machinery, as well as modifying local chromatin structure (26). However, the identity and mechanisms
whereby ancillary proteins are recruited by the AHRC to its
cognate response element are still, largely, unknown. Elucidation of the molecular mechanisms underlying activated transcription by the AHRC is central to our understanding of development, physiological homeostasis, and the complex
pathologies responsible for a wide spectrum of human diseases
including chemical carcinogenesis and solid tumor growth.
Like AHR, the estrogen receptor (ER) is a ligand activated
transcription factor. ER belongs to the superfamily of nuclear
hormone receptors (NR) (27), which upon ligand binding forms
a functional homodimer and binds its cognate response elements, the estrogen response element (ERE). As with most
NRs, ER is thought to manifest its main biological function by
transducing the transcriptional information contained in its
response elements. This has been questioned over the past
decade by several independent findings. Investigators in
Gunther Schutz’s laboratory made the startling observation
that DNA-binding/dimerization deficient glucocorticoid receptor (GR) mutant mice were viable (28), while null mutations are
lethal (29). These observations demonstrated unequivocally
that the DNA binding capability of a transcription factor was
not necessarily essential for survival and that a prototypic
transcription factor had vital non-DNA binding properties (28).
Furthermore, AP-1 regulation of interstitial collagenase was
repressed by GR by a direct protein-protein interaction between GR and AP-1 (30 –33). GR-mediated transrepression of
NF-␬B signaling is also well documented (4, 34, 35). Subse-
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ER␣ Repression of AHR Signaling
quently, other nuclear hormone receptors, including the thyroid hormone receptor (TR), and ER, were shown to be able to
repress AP-1 and NF-␬B activity via direct protein-protein
interactions (4, 34, 35). Furthermore, the effects of endocrine
disruptors and compounds that mimic the activities of endogenous estrogens affect not only ERE-regulated genes but also
genes transrepressed by ER and the relative importance of
each phenomenon has yet to be established.
The mechanisms by which PAH/HAH repress ER signaling
are well understood and are attributable to direct proteinprotein interactions with liganded AHR and ER at the regulatory regions of ER target genes and non-transcriptional or
downstream events. However, the mechanisms by which ER
down-regulates aromatic hydrocarbon signaling remain unclear. Furthermore, the general mechanism in which NRs
tether to other transcription factors and modulate their activities reveals another level of specificity of NR function. In this
report, we provide evidence that ER␣ represses TCDD-inducible Cyp1a1 and Cyp1b1 transcription through direct proteinprotein interactions with the AHRC in the regulatory regions of
these genes.
EXPERIMENTAL PROCEDURES
Cell Lines and Reagents—Polyclonal anti-ER␣ and CYP1A1 antibodies were purchased from Santa Cruz Biotechnology, Inc. Rabbit antiAHR polyclonal antibody (SA-210) was purchased from Biomol. Affinity-purified anti-acetyl histone H4 (Lys12) was purchased form Upstate
Biotechnology, Inc. Rabbit anti-ARNT polyclonal antibody was the gift
of Dr. Oliver Hankinson. HEK-293 and MCF-7 cells were maintained in
serum free ␣-minimal essential medium lacking phenol red with high
glucose for at least 48 h prior to any treatment or experimental manipulation to ensure that unnecessary activation/down-regulation of ER or
other signal transduction pathways would not confound any experimental parameters.
GST Pull-down Assays and Transient Transfections and Reporter
Gene Assays—GST pull-down assays were performed as described previously (18). Six-well plates containing 293 cells were transfected with
100 ng of the GAL4 upstream activating sequence reporter pG5E4T,
CMV-␤-Gal (100 ng), and either 200 ng of pGAL-AHR419 – 805 or pGALARNT, with or without 200 ng of pG5-ER␣. A total of 600 ng of DNA
was used for each transfection using empty expression vector when
necessary. Each transfection was achieved using 15 ␮l of Superfect
transfection reagent (Qiagen). Medium was changed 3 h after transfection, and cells were treated with 100 nM E2. Cells were harvested 16 –20
h after transfection and assayed as described previously (17).
Reverse Transcription and Real-time PCR—Prior to treatment with
ligand MCF-7 cells were exposed to cycloheximide (10 ␮g/ml) for 1 h to
halt protein translation. Subsequently, cells where treated either with
vehicle (Me2SO), 2 nM TCDD, 100 nM E2, or a combination of TCDD and
E2 for 8 h. Cells were harvested in TRIzol, and total RNA was isolated
and subjected to reverse transcription using a high capacity cDNA
archive kit (Applied Biosystems). cDNAs were amplified by real-time
PCR using a DyNAmo HS SYBR Green kit (Finnzymes) according to
manufacturer’s protocols. Oligonucleotide pairs used to amplify human
cDNA sequences were: for Cyp1a1, 5⬘-TCTTCCTTCGTCCCCTTTAC-3⬘
and 5⬘-TGGTTGATCTGCCACTGGTT-3⬘ (forward and reverse, respectively); for Cyp1b1, 5⬘-CATGCGCTTCTCCAGCTTTGT-3⬘ and 5⬘-GGCCACTTCACTGGGTCATGA-3⬘; for pS2, 5⬘-AGTGGCCCCCCGTGAAAG-3⬘ and 5⬘-TCTGGAGGGACGTCGATGGT-3⬘; and for GAPDH, 5⬘-TGCACCACCAACTGCTTAGC-3⬘ and 5⬘-GGCATGGACTGTGGTCATCAG-3⬘.
Western Blot Analysis—Whole cell extracts of MCF-7 cells were used,
and Western blotting was performed as described previously (17), with
minor modifications. After incubation with primary antibodies, blots
were incubated with a biotin labeled goat anti-rabbit IgG. Blots were
washed and incubated with 125I-labeled strepavidin (Amersham Biosciences) and exposed to film overnight. Protein levels were quantified
using a Cyclone phosphor imaging system and OptiQuant software.
Single and Sequential Two-step ChIP Assays—Precipitations for
ChIPs and re-ChIPs from fixed MCF-7 cell lysates were performed in
quadruplicate. One sample from each set was chosen for analysis by
single step chromatin immunoprecipitations, and these reactions were
performed as described previously (23). For re-ChIP experiments, complexes from the primary ChIP were eluted in 50 ␮l of 10 mM dithiothre-
itol for 30 min at 37 °C, pooled, and precipitated with the indicated
antibody.
RESULTS AND DISCUSSION
Prenatal, perinatal, as well as long term exposures to estrogens, xenoestrogens, and endocrine disruptors such as TCDD,
put individuals at an increased risk of testicular, breast, and
prostate cancers and developmental defects of the urogenital
tract (36, 37). Many testicular cancer etiologies implicate the
activation of the aryl hydrocarbon receptor (37–39) or, conversely, the disruption of the estrogen-signaling pathways by
these compounds and other endocrines disruptors such as diethylstilbestrol. Subsequent studies continue to re-iterate
these findings (40). As a result, cross-talk between AHR and
NR pathways have been implicated but studies investigating
the molecular mechanisms underlying AHR-NR cross-talk are
scarce. Perhaps one of the most important and intriguing problems concerning the mechanism of NR function is how they
moderate gene transcription in a DNA-binding-independent
fashion.
AHR and ARNT Interactions with ER␣—Experiments employing GST-AHR/ARNT fusions were performed with radiolabeled in vitro translated 35S-labeled ER␣. Consistent with the
observations of other investigators (1, 2) we were able to demonstrate an interaction between ER␣ and AHR or ARNT. Furthermore, we were able to demonstrate that the ER␣ interaction domain within AHR resides within the P/S/T region of the
transactivation domain of AHR (Fig. 1A). GST fusion chimeras
of Sp1 and VP16 failed to precipitate in vitro translated ER␣.
To test the hypothesis, that ER directly repressed AHRC via
the P/S/T domain of AHR, we co-transfected HEK-293 cells
with a CYP1A1 promoter-driven luciferase plasmid, pGUDLUC, expression vectors encoding ER␣, and wild-type human
AHR, or a deletion mutant encoding human AHR deleted for its
P/S/T domain. Cells were treated with vehicle or E2 or E2 in
combination with TCDD overnight. Both wild-type AHR and
AHR⌬P/S/T were equally capable of activating transcription
from pCYP-GudLuc in a TCDD-dependent fashion (Fig. 1B).
However, co-transfection of ER␣ in the presence of 100 nM
E2 significantly diminished this activation in each instance
(Fig. 1B).
To determine whether ER␣ can repress either AHR, or
ARNT transactivation function, HEK-293 cells were co-transfected with expression cDNA plasmids encoding either GAL4DNA-binding domain AHR carboxyl terminus or full-length
ARNT and ER␣ the GAL4-driven luciferase vector, pG5E4T
(Fig. 1C). Activated ER␣ enhanced the GAL4-DBD-AHR chimera harboring the P/S/T putative ER␣ interaction domain
transactivation function significantly (Fig. 1D). However, transcriptional activity driven by the GAL4-ARNT chimera was
significantly repressed. This suggests that ER␣ transduces its
repressor function via its interaction with ARNT.
GST pull-down and in vivo co-immunoprecipitation studies
have demonstrated a direct interaction between AHR and ER
(2, 41) as well as ARNT and ER (1). Furthermore, dominant
negative studies have shown that the AHR transactivation
domain and the N-terminal region of ER can reciprocally repress transcription from ERE- and DRE-driven luciferase constructs, respectively (42). TCDD exposure causes a robust increase in mRNA levels and of DRE-driven reporter gene
activity, both of which are significantly decreased by co-treatment with E2 in Hepa-1 and MCF-7 cells (43). Likewise, in the
human endometrial carcinoma cell line ECC-1, E2 again
blocked DRE-driven luciferase activity as well as 7-ethoxycoumarin hydroxylation (44). This effect was reversed by the addition of the selective ER modulator 4-hydroxy-tamoxifen
(TAM). Despite a wealth of evidence suggesting that ER acti-
ER␣ Repression of AHR Signaling
FIG. 1. ER␣ interactions with AHR and ARNT. A, in vitro translated ER␣ interacts with GST-ARNT and GST-AHR-P/S/T fusions. B,
effect of ER␣ on wild-type AHR- and AHR⌬P/S/T-mediated transcription. HEK-293 cells were maintained in serum- and Phenol Red-free
media for at least 48 h prior to any experimental manipulation. C, ER␣
represses ARNT transactivation function and enhances AHR transactivation. A schematic of AHR/ARNT-GAL4-DBD mutants used in mammalian interaction studies is presented above. GAL4 DNA-binding domain (GAL4-DBD), the basic helix-loop-helix (bHLH), PAS A and B (A
and B), acidic (Ac), Q-rich (Q), and proline-serine-threonine-rich (P/
S/T) regions are shown. D, HEK-293 cells were co-transfected with
expression cDNA plasmids encoding either GAL-AHR419 – 805 or GALARNT and ER␣ and the GAL4-driven luciferase vector, pG5E4T (100 ng
each). After transfection cells were treated with 100 nM E2 to activate
ER␣. Cells were grown for an additional 18 –20 h and harvested, and
luciferase activity was determined. Luciferase activity was normalized
to that of ␤-galactosidase to control for transfection efficiency.
vation represses AHR target genes, little is known regarding
the molecular mechanisms involved. Furthermore, most experimental efforts to characterize the biochemical pathways involved in ER-AHR cross-talk have focused on the TCDD-inducible repression of ER target genes.
ER␣ Represses TCDD-inducible Transcription—We made
21609
FIG. 2. 17␤-Estradiol represses TCDD-inducible endogenous Cyp1a1
(A) and Cyp1b1 (B) transcription in MCF-7 cells. Cells were incubated
in the presence or absence of 10 ␮g/ml cylcoheximide (CHX) for 1 h prior
to treatment with either E2 (100 nM) or TCDD (2 nM) or both for 7 h.
Total RNA was reverse-transcribed, and Cyp1a1, Cyp1b1, and GAPDH
cDNA were amplified by real-time PCR. A paired t test was performed
on the indicated parameters: *, p ⬍ 0.001; **, p ⬍ 0/01. C, MCF-7 cells
were treated with Me2SO (lane C), 100 nM 17␤-estradiol (lane E2), 2 nM
TCDD (lane T), or E2 and TCDD (lane E⫹T) together for 18 h. Whole
cell extracts were subjected to SDS-PAGE, and relative amounts of
CYP1A1 and ARNT protein were assessed by Western blot analysis. D,
CYP1A1 protein band intensity levels were normalized to that of ARNT
and HSP86 with essentially identical results.
21610
ER␣ Repression of AHR Signaling
FIG. 3. ER␣ binds the Cyp1a1 enhancer only in response to a
combination of E2 and TCDD. A, sequential two-step ChIP of AHR
and ER␣ over the Cyp1a1 enhancer demonstrates that ER␣ and AHR
exist together on the Cyp1a1 enhancer. B, ChIP analysis of ER␣, AHR,
ARNT, and acetylated histone H4 (AcH4) over the Cyp1a1 enhancer
and pS2 promoter region. Cells were incubated with either vehicle (lane
C), 100 nM E2 (lane E), 1 nM TCDD (lane T), or E2 ⫹ TCDD (lane E⫹T).
the observation that co-transfection of ER␣ with a Cyp1a1
promoter driven luciferase vector in Hepa1 cells treated with
TCDD with or without E2 leads to E2-mediated repression of
TCDD-dependent luciferase activity (42). We have confirmed
and extended these observations in HEK-293 cells and in an in
vitro system. However, these systems cannot distinguish between direct transcriptional events at the target and secondary
transcriptional events such as the transcriptional activation/
repression of other regulatory genes. Therefore, to assess the
direct transcriptional effect of ER activation on AHRC-dependent gene transcription, we employed reverse transcription/realtime PCR of the AHRC target genes Cyp1a1 and Cyp1b1 in the
presence and absence of the protein synthesis inhibitor cycloheximide. TCDD but not 100 nM E2 caused a significant increase in Cyp1a1 gene transcription in MCF-7 cells (Fig. 2A).
However, E2 (like TCDD, alone) caused a significant increase
in Cyp1b1 gene transcription, consistent with reports that the
5⬘-regulatory region of the Cyp1b1 gene harbors an ERE (45)
(Fig. 2B). The addition of 100 nM estradiol significantly reduced
TCDD-induced Cyp1a1 gene transcription in the presence or
absence of cycloheximide (Fig. 2, A and B) and significantly
reduced Cyp1b1 transcription in the presence of cycloheximide.
The observation that maximal Cyp1a1 gene induction was repressed by ⬃50% in the absence or presence of cycloheximide
suggests that both direct transcriptional repression of Cyp1a1
and not secondary E2 mediated downstream events are responsible for the repressive effects of estrogens on AHRC signaling.
Furthermore, the E2-mediated decrease in Cyp1a1 mRNA production preceded a concomitant decrease in CYP1A1 protein
levels (Fig. 2, C and D) in MCF-7 cells. The observed decrease
in corrected CYP1A1 protein levels (⬃42%) is consistent with
the observed decrease in mRNA levels demonstrating that the
observed repression of TCDD-inducible transcription by E2 can
have an equally profound physiological outcome.
ER␣ Associates with the Cyp1a1 Promoter in an AHRC-dependent Fashion—We employed the ChIP assay to ascertain
the status of ER␣ at the Cyp1a1 enhancer in the presence and
absence of E2 and TCDD. Initially, consistent with other investigators’ observations, we observed the presence of AHR and
ARNT over the human Cyp1a1 enhancer in a TCDD-dependent
fashion (1, 2, 23). ER␣ was greatly enriched at the Cyp1a1
enhancer only after treatment with a combination of 1 nM
TCDD and 100 nM E2 (Fig. 3B). As a control, we monitored the
presence of AHR, ARNT and ER␣ over the pS2 promoter under
similar conditions (Fig. 3B) with results similar to those observed by other investigators (2). Furthermore, precipitation of
Cyp1a1 enhancer chromatin was enriched by antibodies directed against the acetylated form (Lys12) of histone H4 in cells
treated with TCDD, but this was seemingly reduced by the
addition of E2 (Fig. 3B), suggesting that ER␣ mediates its
repressive effects through a histone de-acetylase dependent
mechanism. Chromatin encompassing the pS2 promoter was
efficiently precipitated by this antibody under all conditions
tested reflecting the high level of pS2 expression observed in
MCF-7 cells (data not shown). Affinity-purified antibody to the
hemaglutinnin protein tag (control antibody) failed to precipitate either the Cyp1a1 enhancer or the pS2 promoter, indicating that anti-AHR, -ARNT, and -ER␣ were precipitating their
respective antigens in a specific fashion. Taken together, these
data indicate that liganded ER␣ associates with the Cyp1a1
enhancer only after TCDD activated transcription has been
initiated.
To determine unequivocally if ER␣ and AHR occupy the
same portion of chromatin at the same time we performed
sequential precipitations with antibodies for AHR and ER␣ in
MCF-7 cells. Chromatin samples from cells treated with either
vehicle, E2, TCDD, or E2 ⫹ TCDD were precipitated with either anti-AHR or anti-ER␣. After binding to agarose beads,
samples were eluted and those precipitated with an anti-AHR
were incubated with anti-ER␣ and those initially precipitated
with an anti-ER␣ were incubated with anti-AHR. Again, complexes were bound to the appropriate secondary antibody-conjugated agarose resin. Both AHR and ER␣ could be precipitated from ER␣ and AHR affinity purified samples,
respectively, on the Cyp1a1 enhancer (Fig. 3A). Thus, we have
established that ER␣ is present at the Cyp1a1 enhancer only in
the presence of both estradiol and TCDD. Furthermore, we
have demonstrated by two-step ChIP that AHR and ER␣ are
present at the Cyp1a1 enhancer at the same time. Taken together, these data strongly suggest that ER␣ directly interacts
with the AHRC multiprotein complex. This is in direct contrast
to the observations of another group that failed to record ER␣
on the Cyp1a1 enhancer in response to co-treatment with
3-methylcholanthrene, a PAH, and E2 (2). We cannot resolve
these differences except to note that in the study noted above
the AHRC was activated with the PAH, 3-methylcholanthrene,
and that ligand-specific differences may exist.
The nature of repression by tethering of ligand-activated
NRs is not well understood. The classical model of NR activation by ligand suggests that ligand facilitates an exchange of
NCoR/SMRT/Sin3/HDAC co-repressor complexes for co-activator complexes with ATP-dependent chromatin remodeling and
histone acetyltransferase activities (46, 47). It has been suggested that this happens only in the context of a receptor’s own
cognate positive response element. Studies regarding the repression of thyrotropin ␤ gene by TR suggest that this switch
does not occur in the context of a negative thyroid hormone
receptor element (48). Other studies have suggested that GRmediated repression of the IL-8 gene occurs through a direct
protein-protein interaction with the NF-␬B heterodimeric tran-
ER␣ Repression of AHR Signaling
scription factor and is independent of GRE binding (49). One
model put forth is that GR recruits GRIP1 in this context, but
a steric change in the complex due to tethering unmasks the
repressor function of GRIP1 (50). This, in turn, may play a role
in the GR-mediated inhibition of phosphorylation of the carboxyl-terminal catalytic domain of RNA polymerase II (51).
Whether ER␣ utilizes GRIP or recruits other ancillary factors
remains unclear; however, our data would suggest that ultimately, histone acetylation and chromatin condensation occur.
The mechanism by which PAHs mediate adverse biological
effects is understood in broad terms. PAHs induce expression of
Cyp1a1 and Cyp1b1 via the AHRC. These cytochrome P450
xenobiotic-metabolizing enzymes generate electrophilic derivatives that form DNA adducts thereby activating proto-oncogenes and inactivating tumor suppressor genes. However, P450
induction is also responsible for the ultimate metabolism and
clearance of many toxic substances. Furthermore, repression of
PAH- and HAH-inducible gene transcription by activated ER␣
could, in susceptible tissues, lead to a blunted or inappropriate
response to these carcinogens, such as the unmasking of other
drug metabolizing activities leading to an increased or untoward production of electrophilic metabolites. Conversely,
breast cancer treatments that include ER antagonists, such as
tamoxifen, might increase or exacerbate AHR activity in tumor
cells. Furthermore, AHR is important for development. Exposure to estrogens in an untoward fashion (i.e. pseudo- and
phyto-estrogens) or altered ER status in affected tissues (i.e.
certain small cell lung tumors, breast cancer) would ultimately
impact AHR function and disrupt physiological homeostasis.
We have demonstrated that ER␣ can repress AHRC target
gene induction through direct protein-protein interactions with
the AHRC. Endogenous or exogenous ligand availability for
both AHR and ER as well as target tissue and receptor availability will likely determine the degree of ER␣ transrepression.
Furthermore, the identification of ancillary factors recruited
during transrepression of AHRC-mediated gene induction
highlights an intriguing avenue for future research.
Acknowledgment—We thank Dr. Oliver Hankinson for the rabbit
anti-ARNT antibody.
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