MOLECULAR AND CELLULAR BIOLOGY, Apr. 2009, p. 1749–1759
Copyright © 2009, American Society for Microbiology. All Rights Reserved.
Vol. 29, No. 7
Estrogen Receptor Alpha Represses Transcription of Early Target
Genes via p300 and CtBP1?
Fabio Stossi,1Zeynep Madak-Erdogan,2and Benita S. Katzenellenbogen1,2*
Department of Molecular and Integrative Physiology1and Department of Cell and Developmental Biology,2
University of Illinois at Urbana-Champaign, Urbana, Illinois 61801
Received 19 September 2008/Returned for modification 29 October 2008/Accepted 20 January 2009
The regulation of gene expression by nuclear receptors controls the phenotypic properties and diverse
biologies of target cells. In breast cancer cells, estrogen receptor alpha (ER?) is a master regulator of
transcriptional stimulation and repression, yet the mechanisms by which agonist-bound ER? elicits repression
are poorly understood. We analyzed early estrogen-repressed genes and found that ER? is recruited to ER?
binding sites of these genes, albeit more transiently and less efficiently than for estrogen-stimulated genes. Of
multiple cofactors studied, only p300 was recruited to ER? binding sites of repressed genes, and its knockdown
prevented estrogen-mediated gene repression. Because p300 is involved in transcription initiation, we tested
whether ER? might be trying to stimulate transcription at repressed genes, with ultimately failure and a shift
to a repressive program. We found that estrogen increases transcription in a rapid but transient manner at
early estrogen-repressed genes but that this is followed by recruitment of the corepressor CtBP1, a p300-
interacting partner that plays an essential role in the repressive process. Thus, at early estrogen-repressed
genes, ER? initiates transient stimulation of transcription but fails to maintain the transcriptional process
observed at estrogen-stimulated genes; rather, it uses p300 to recruit CtBP1-containing complexes, eliciting
chromatin modifications that lead to transcriptional repression.
The regulation of gene expression by nuclear receptors is
critical in controlling the phenotypic properties and diverse
biologies of their target cells (4). Estrogen receptor alpha
(ER?) and ER?, which are members of the nuclear receptor
superfamily of ligand-activated transcription factors, play cru-
cial roles in mammary gland development and also in breast
cancer etiology, progression, and treatment (2, 21, 23). From
genome-wide transcriptome studies of breast cancer cells, it is
clear that hormone-bound ER? is a pivotal regulator that can
both stimulate and repress gene transcription and influence,
over time, a vast number of target gene mRNAs and proteins,
thus creating a well-integrated hormonal response that affects
numerous cell processes (6, 14, 42). Global gene expression
profiling by microarray analysis has revealed that estradiol
(E2), acting through ER?, upregulates the expression of genes
encoding positive proliferation regulators, including multiple
growth factors, growth factor receptors, and proteins involved
in cell cycle progression, and downregulates transcriptional
repressors and antiproliferative and proapoptotic genes, these
together contributing to the stimulation of proliferation and
suppression of apoptosis by estradiol in breast cancer cells (14,
Most of the studies thus far have elucidated the mechanisms
by which ER? stimulates gene transcription, often focusing on
the TFF1/pS2 gene as a model of ER? action (18). At the
TFF1/pS2 enhancer, ER? works in a highly dynamic fashion as
a nucleation factor for a cohort of coregulators (e.g., p160
family members and others) and enzymatic complexes (e.g.,
histone acetyltransferases [HATs] and SWI/SNF) that relax
chromatin structure, allowing the basal transcriptional machin-
ery to increase the transcriptional output (31, 38).
In contrast to all that is known about the molecular mech-
anisms by which the estrogen-occupied ER? stimulates gene
expression, the mechanisms that this receptor utilizes to re-
press gene transcription are only starting to be elucidated. In
our laboratory and elsewhere, a direct role of ER? and of
corepressor/histone deacetylase (HDAC)-containing com-
plexes has been demonstrated in the regulation of some estro-
gen (E2)-repressed target genes (32, 40). Carroll et al. (5) have
highlighted a role for the coregulator NRIP1/RIP140 in the
regulation of late E2-repressed target genes, while other mech-
anisms proposed to be involved in ER?-mediated transcrip-
tional repression include physiological squelching of coactiva-
tor proteins and involvement of components of the basal
transcriptional machinery (7, 19).
We sought to investigate the mechanisms involved in ER?-
mediated transcriptional repression of endogenous genes
downregulated at early times, in contrast to most previous
studies of nuclear receptor-mediated gene expression that have
used artificial gene constructs or addressed repression events
at late time points of E2 treatment (?8 to 24 h). Herein, we
have characterized a group of E2 target genes whose down-
regulation by ER? is rapid and direct. These estradiol-re-
pressed genes include the cyclin G2 (CCNG2) gene, which
encodes a negative regulator of the cell cycle; SMAD6, impor-
tant in modulating transforming growth factor ? signaling
pathways; and monocyte-to-macrophage differentiation-asso-
ciated protein (MMD), highly expressed in the placenta and
some cancer cell lines and believed to play critical roles in
extracellular matrix remodeling and wound healing.
Our studies reveal that ER? is transiently recruited to the
* Corresponding author. Mailing address: University of Illinois, De-
partment of Molecular and Integrative Physiology, 524 Burrill Hall,
407 South Goodwin Avenue, Urbana, IL 61801-3704. Phone: (217)
333-9769. Fax: (217) 244-9906. E-mail: email@example.com.
?Published ahead of print on 2 February 2009.
ER binding sites of these estrogen-repressed genes and that at
these repressed genes there is also a transient increase in
transcriptional rate. Further, p300 is observed to be recruited
upon E2 treatment both at stimulated and repressed gene
binding sites, and notably, p300 knockdown blocked both E2-
mediated gene stimulation and repression. The repressive ac-
tion of p300 at estrogen-repressed genes appears to be exerted
through its partnering with the repressive CtBP1 complex, and
through a variety of studies we confirm the central role of
CtBP1 in specifically mediating transcriptional repression by
this nuclear receptor.
Our study thus provides evidence that ER? is directly uti-
lizing p300 and CtBP1 in transcriptional repression, and we
propose that this is due to a failure in sustaining a stable
transcriptionally active complex at these sites that causes p300
to partner with repressive rather than activating coregulator
MATERIALS AND METHODS
Cell culture, treatments, RNA extraction, and real-time qPCR. MCF-7 cells
were cultured in minimal essential medium (MEM) (Sigma Chemical Co., St.
Louis, MO) supplemented with 5% heat-inactivated calf serum (HyClone, Lo-
gan, UT) and 1% antibiotics. Before experiments, the cells were maintained in
phenol red-free MEM containing 5% charcoal-stripped calf serum (CD-CS) for
a minimum of 4 days with the medium changed every other day. Cells were
treated with E2 or vehicle control (0.1% ethanol) alone or in combination with
other ligands at the concentrations and for various times. Total RNA was har-
vested and isolated using Trizol reagent (Invitrogen, Carlsbad, CA), following
the manufacturer’s instructions. Quantitative real-time PCR (qPCR) was per-
formed as previously described (39). All PCR primer sequences are available
ChIP and sequential ChIP (reChIP) assays. Chromatin immunoprecipitation
assays (ChIPs) were performed with minor modifications as described in refer-
ence 31. The antibodies used were purchased from Santa Cruz Biotechnology
(ER? [HC-20], NRIP1 [H-300], RNA polymerase II [N-20], CBP [A-22], p300
[N-15], SRC-3 [H-270], CtBP1 [C-1], and rabbit, goat, and mouse immunoglob-
ulin G [IgG]), Millipore-Upstate (histone H3 [no. 07-690]), and Abcam
(H3K14ac [ab52946] and H3K9ac [ab10812]). The DNA isolated was subjected
to qPCR. Data were normalized to the results for 36B4 used as an internal
control (measuring total input DNA in every sample), and a recruitment index
was calculated (difference between specific antibody signals over Ig signal). For
histone mark experiments, data were also normalized to total histone H3 con-
tent. For ?-amanitin (Sigma) experiments, MCF-7 cells were kept for 4 days in
MEM containing 2% CD-CS and then treated with 5 ?M ?-amanitin in serum-
free medium for 2 h. After treatment, the cells were washed four times and
treated with 1 nM E2 for 1 h in 5% CD-CS medium.
ChIP/reChIP experiments were done following the same ChIP protocol. After
the first pull-down, immunoprecipitated material was recovered with 10 mM
dithiothreitol in IP buffer at 37°C for 30 min, diluted, and submitted to a second
round of immunoprecipitation.
RNA interference and Western immunoblotting. MCF-7 cells were transfected
with small interfering RNAs (siRNA) for ER? (3); p300, CBP, CtBP1, or CtBP2
SMARTpool; or GL3 luciferase control (Dharmacon), following the manufac-
turer’s instructions. After 72 h, cells were treated with 1 nM E2 for 4 h. Total
RNA was prepared from cells and analyzed as described in the previous section.
Western immunoblotting was performed on MCF-7 whole-cell extracts following
standard protocols. The antibodies used were the same as for ChIP assays, and
antibody for CtBP2 was from BD Biosciences.
Nuclear run-on assay. Nuclear run-on assays were carried out as described
previously (30, 33). Briefly, MCF-7 cells were treated with 10 nM E2 or vehicle
for different times, washed with PBS, harvested, and lysed (lysis buffer consisted
of 0.5% NP-40, 10 mM KCl, 10 mM MgCl2, 10 mM HEPES [pH 7.9], 0.5 mM
?-mercaptoethanol) on ice for 5 min. After centrifugation, nuclei were washed
with lysis buffer without NP-40 and resuspended in 100 ?l of storage buffer (50
mM Tris-HCl, 5 mM MgCl2, 0.5 mM ?-mercaptoethanol, 40% glycerol) before
being frozen at ?80°C. A 100-?l amount of transcription buffer (10 mM Tris-
HCl [pH 8.00], 0.3 M MgCl2, 5 mM dithiothreitol, 40 U RNase inhibitor [Roche],
1? biotin labeling mix [Roche]) was added to the nuclei, and the reaction
mixture was incubated at 30°C for 45 min. RNA was isolated by using Trizol
reagent (Invitrogen). A 50-?l amount of streptavidin-conjugated magnetic beads
(Invitrogen) resuspended in binding buffer (10 mM Tris-HCl [pH 7.5], 1 mM
EDTA, 100 mM NaCl) was added to each sample and incubated at room
temperature for 2 h. Beads were washed twice in 500 ?l of 2? SSC (1? SSC is
0.15 M NaCl plus 0.015 M sodium citrate), 15% formamide for 15 min and once
in 500 ?l of 2? SSC for 5 min and then dissolved in 12 ?l of diethyl pyrocar-
bonate water. RNA was reverse transcribed, and qPCR reactions were carried
out as described above.
Characterization of early estrogen-repressed target genes.
To explore the mechanisms involved in E2-mediated transcrip-
tional repression, we identified a subset of early E2-repressed
target genes in MCF-7 breast cancer cells based on previous
cDNA microarray studies performed in our laboratory (14,
15). Using qPCR, we examined the effect of E2 on this group
of genes and found that their expression was repressed rapidly
(by 2 h) and remained repressed in a sustained manner fol-
lowing E2 treatment (Fig. 1A). To determine whether ER?
was necessary for the transcriptional repression of these genes,
we used both the ER? antagonist ICI182,780 and ER? knock-
down with siRNA (Fig. 1B and C). The results of both ap-
proaches demonstrated that ER? was required for gene re-
pression, because the E2-elicited repression was completely
abrogated by ICI182,780 cotreatment along with the E2 or by
To examine whether there was a direct, primary transcrip-
tional mechanism of ER? regulation of these target genes, we
treated MCF-7 cells with the protein translation inhibitor cy-
cloheximide or the transcriptional inhibitor actinomycin D
(Fig. 1D and E). We found that repression of the expression of
these genes did not require protein synthesis, whereas actino-
mycin D greatly reduced basal gene mRNA and eliminated the
E2-induced downregulation. The results of these experiments
therefore imply that these genes are primary ER? targets.
Further, there was no effect of E2 on the stability of these
mRNAs, as assessed by actinomycin D treatment (Fig. 1E) and
time course studies (data not shown).
We next examined the status of RNA polymerase II at these
genes, in particular whether after E2 treatment RNA polymer-
ase II would be pausing or be displaced from the promoters of
these E2-repressed genes, because both mechanisms have been
shown to be possible at ER?-regulated genes (25, 40). We
performed ChIP time course experiments using a specific an-
tibody directed against RNA polymerase II. As shown in Fig.
1F, RNA polymerase II was displaced from the transcriptional
start sites of all E2-repressed genes within 15 min of E2 treat-
ment, indicating that dismissal, rather than stalling or pausing,
of RNA polymerase II occurred rapidly at these repressed
target genes. In contrast, recruitment of RNA polymerase II
continued to increase over time at the E2-stimulated gene
TFF1 (Fig. 1F).
Characterization of ER? binding sites for early E2-re-
pressed genes. Genome-wide studies using ChIP-on-chip
(combined use of ChIP and microarrays) and ChIP–paired-
end-tag (ChIP-PET) technologies have identified putative
ER? binding sites in MCF-7 breast cancer cells (5, 28). We
used this available information to analyze ER? binding sites
associated with ER?-repressed genes. The early E2-repressed
1750STOSSI ET AL.MOL. CELL. BIOL.
target genes that we characterized in the experiments whose
results are shown in Fig. 1 (CCNG2, MMD, and SMAD6) were
chosen because each has a single ER? binding site in the
proximity of the gene (Fig. 2A). As a positive control for our
experiments, we used the well-characterized enhancer of the
E2-stimulated gene TFF1/pS2. To examine the presence of
ER? at these putative binding sites after E2 treatment, an ER?
time course ChIP assay was performed (Fig. 2B). As expected,
at the TFF1/pS2 enhancer region, ER? recruitment was
greatly increased, starting after 5 min of E2 treatment and
increasing over the 45 min studied. Interestingly, the time
pattern of ER? recruitment at the binding sites of E2-re-
pressed genes was different. ER? was recruited after 5 min of
E2 treatment, as observed for the E2-stimulated TFF1/pS2
gene, but then ER? occupancy decreased rapidly or remained
constant and only mildly elevated over the time analyzed (Fig.
2B). Thus, the magnitudes and time courses of ER? recruit-
ment to the binding sites of repressed and stimulated genes are
Characterization of the recruitment of coregulators at the
ER? binding sites of early E2-repressed genes. The role of
specific ER? coregulatory factors in E2-mediated gene repres-
sion is still an open question. Therefore, we performed ChIP
assays on MCF-7 cells treated with 1 nM E2 for 5, 15, and 45
FIG. 1. Characterization of early estradiol (E2)-repressed genes in MCF-7 breast cancer cells. (A) Time course of E2 regulation. MCF-7 cells
were treated with 1 nM E2, and RNA was isolated at various time points. cDNA was measured by qPCR using primers for the genes indicated
and internal control 36B4. (B) ICI182,780 antagonist experiments. MCF-7 cells were treated with 1 ?M ICI182,780 (ICI) with or without 1 nM
E2. (C) ER? siRNA experiments. MCF-7 cells were transfected with siRNA for ER? (siER?) or for GL3 control (siGL3) (luciferase) for 48 h
prior to treatment with 1 nM E2. (D) Protein translation inhibitor experiments. MCF-7 cells were treated with cycloheximide (10 ?g/ml) (CHX)
with or without 1 nM E2. (E) Transcriptional inhibitor experiments. MCF-7 cells were treated with actinomycin D (5 ?g/ml) (ActD) with or without
1 nM E2. In the experiments whose results are shown in panels B to E, the 1 nM E2 treatment was for 4 h, and total RNA was extracted and
quantified by qPCR using 36B4 as the internal control. (F) RNA polymerase II ChIP was performed as described in Materials and Methods after
15, 25, or 45 min of 1 nM E2 treatment. Data are represented as the recruitment index (RNA polymerase II signal/IgG signal ratio). Data shown
in all panels are averages ? standard errors of the means of the results of three or more independent experiments. In all panels, an asterisk
indicates a P value of ?0.05 for results of E2 treatment versus results for vehicle control. In panel D, a dagger indicates a P value of ?0.05 for
results for cycloheximide with E2 versus results for cycloheximide alone. Veh, vehicle.
VOL. 29, 2009 ESTROGEN RECEPTOR GENE REPRESSION AND p300/CtBP11751
min using specific antibodies directed against HATs (CBP and
p300), the p160 coactivator SRC-3, and the mixed coactivator/
corepressor NRIP1 (Fig. 2C, D, E, and F) to examine their
patterns of recruitment over early times of hormone treatment.
Since one of the main features of E2-mediated gene repres-
sion appears to be histone deacetylation (25, 40) and because
CBP has been found to be squelched away at the GnRH
receptor gene (7), we tested whether physiological squelching
of HATs was also occurring at early E2-repressed genes. As
shown in Fig. 2C, p300 recruitment increased at the binding
sites of all E2-repressed genes with a time profile that was
similar and at levels that were comparable (CCNG2) or some-
what less (MMD and SMAD6) than that for the E2-stimulated
TFF1/pS2 gene. In the case of CBP (Fig. 2D), its occupancy
increased at TFF1/pS2 and, more slightly, at the CCNG2 site,
while being low at the SMAD6 and MMD binding sites.
Hence, although we cannot exclude that squelching of CBP or
p300 might occur from other regions of DNA, we find that this
does not occur from the ER? binding sites or from regions
close to the transcriptional start site (Fig. 2D and data not
shown). Regarding p160/SRC coactivators (Fig. 2E), ChIP as-
says revealed SRC-3, the most abundant SRC in MCF-7 cells,
to be either absent or present at very low levels at the ER?
binding sites of the examined genes prior to E2 treatment.
Upon E2 treatment, SRC-3 was strongly recruited to the
TFF1/pS2 enhancer while remaining absent at the binding
sites of E2-repressed genes. An intriguing exception was the
transient increase in occupancy of SRC-3 at the MMD bind-
FIG. 2. Characterization of occupancy across time of ER? and selected coregulators at ER? binding sites associated with E2-repressed genes.
(A) UCSC Genome Browser schematics of selected ER? binding sites according to Carroll et al. (5). TSS, transcription site. (B to F) ChIP assays
were performed at various times of 1 nM E2 treatment, using specific antibodies against ER? (B), p300 (C), CBP (D), SRC-3 (E), NRIP1 (F),
or IgG negative control antibody. Data are averages ? standard errors of the means of the results of four or more independent experiments and
are represented as the recruitment index (specific antibody signal/IgG signal ratio).
1752 STOSSI ET AL.MOL. CELL. BIOL.
ing site at the 15-min time point, possibly indicating the
formation of a transient ER?-p160 complex at this E2-
repressed gene (Fig. 2E).
Since NRIP1 is itself upregulated by the E2-ER? complex
and has been proposed to be an important factor mediating
ER? repression of late/secondary genes (5), we examined
whether this coregulator might also be employed by ER? in
the repression of early primary target genes. NRIP1 occupancy
increased strongly at the ER? binding site of the TFF1/pS2
gene after E2 treatment, with a profile similar to that observed
for SRC-3 (Fig. 2F), whereas at ER? binding sites of E2-
repressed genes, NRIP1 was present at generally low levels in
control vehicle-treated cells (Fig. 2F, zero time), and its occu-
pancy did not change or increased mildly over time after E2
treatment, especially at the MMD binding site, suggesting that
NRIP1 plays only a limited role in early gene repression (Fig.
2F). Likewise, we observed no recruitment of the corepressors
NCoR or SMRT to the ER? binding sites of the three E2-
repressed genes and no recruitment to the E2-stimulated TFF1
gene (data not shown).
In summary, the results of our ChIP time course experi-
ments reveal that p300 was the only coregulator recruited at
the ER? binding sites of E2-repressed, as well as E2-stimu-
lated, genes, whereas other factors, like SRC-3, CBP, and
NRIP1 may have more gene-specific roles.
p300 knockdown blocks E2-mediated gene repression. To
directly address the roles of p300 and CBP in E2-mediated
gene repression, we used RNA interference knockdown (Fig.
3) that selectively reduced p300 or CBP without affecting ER?
(Fig. 3A and B). As seen in Fig. 3C, knockdown of p300
resulted in a nearly complete loss of E2-mediated gene repres-
sion, indicating a crucial role for p300 in the repression pro-
cess. We also observed that the E2-stimulated increase in
TFF1/pS2 mRNA was also greatly reduced by p300 knockdown
(Fig. 3C), confirming the already known role of p300 in E2-
mediated stimulation of gene expression. Since p300 and CBP
are homologous proteins and their pattern of occupancy at the
binding sites of repressed genes was different, we wanted to
verify whether their character was retained after CBP-specific
knockdown. As shown in Fig. 3C, CBP knockdown blocked the
stimulation of TFF1/pS2 and also the repression of CCNG2 by
E2, which was the only E2-repressed gene where CBP recruit-
ment at the ER? binding site was detected. In contrast, CBP
knockdown had little impact on the repression of MMD or
SMAD6 gene expression by E2. These experiments highlight
the central role for an ER?-p300-containing complex in the
repression of transcription, while CBP appears to have a more
ER? transiently activates gene transcription at E2-re-
pressed gene sites. Because we found p300 to be involved in
the repression of gene activity by the E2-ER? complex and
p300 is known to be an essential factor for ER?-mediated gene
activation, especially having a role in the establishment of the
transcription initiation complex (1, 27), we explored the pos-
sibility that ER? might initially stimulate gene transcription
also at early E2-repressed genes while ultimately failing to
FIG. 3. p300 knockdown prevents E2-induced gene repression and E2-induced gene stimulation. (A) MCF-7 cells were transfected with siRNA
against p300, CBP, or control GL3 luciferase for 72 h prior to 0.1% control ethanol vehicle or 1 nM E2 treatment for 4 h. p300 and CBP levels
were assessed by immunoblotting. WB, Western blotting. (B and C) RNA was extracted after GL3, p300, or CBP siRNA treatment of cells followed
by control vehicle or E2 treatment for 4 h, and the levels of E2 target genes were evaluated by qPCR. Data are means ? standard errors of the
means of the results of three independent experiments and are expressed relative to results for GL3 siRNA vehicle-treated cells, set at 1. Veh,
vehicle;*, P ? 0.05 for results of E2 treatment versus results for vehicle control.
VOL. 29, 2009 ESTROGEN RECEPTOR GENE REPRESSION AND p300/CtBP1 1753
continue the process, with subsequent inhibition of transcrip-
tion. To test this possibility, we took two different approaches.
In the first, we performed nuclear run-on assays to determine
whether there was a transient increase in newly synthesized
mRNA at E2-repressed genes. As shown in Fig. 4A, we ob-
served that E2 did transiently increase the production of newly
made mRNA at the MMD and also, to a lesser extent, at the
CCNG2 and SMAD6 genes. This stimulation was more rapid
than that seen for the E2-stimulated gene TFF1/pS2, for which
nuclear run-on-assays showed a continued increase over time
in TFF1 mRNA production (Fig. 4A).
In the second approach, we examined the effect of E2 treat-
ment on RNA polymerase II levels at the regulated genes in
MCF-7 cells by performing ChIP using an RNA polymerase
II-specific antibody. These studies were done either before or
after treatment with the RNA polymerase II inhibitor ?-aman-
itin, which is known to block ongoing transcription (31, 38) and
to clear the coding sequences from transcribing RNA polymer-
ase II. In cells not treated with ?-amanitin, E2 treatment
greatly increased RNA polymerase II levels at the TFF1/pS2
gene and lowered RNA polymerase II levels at the repressed
genes, consistent with repression of the latter genes (Fig. 4B).
In contrast, when MCF-7 cells were first treated with ?-aman-
itin for 2 h, followed by extensive washes, E2 treatment actually
increased RNA polymerase II levels in the coding sequence of
all three of the early E2-repressed genes, although to a lesser
extent (3 times or 14 times or 3 times) (Fig. 4B) than at the
TFF1/pS2 gene (26 times) (Fig. 4B). Because ER? is able to
stimulate gene transcription after clearance of the transcrip-
tional machinery even at the E2-repressed genes, it suggests
that certain “transcriptional barriers” may be playing an im-
portant role in deciding the direction of gene regulation by
ER? after an initial phase of stimulation.
CtBP1 is recruited together with p300 to the binding sites of
E2-repressed genes and is essential for E2-mediated gene re-
pression. To address how p300 might be working in opposite
ways at E2-repressed versus E2-stimulated genes, we analyzed
the recruitment of the CtBP1 corepressor complex, since it has
been shown to directly interact with p300 and to block its HAT
activity by binding to its bromodomain (24). As shown in Fig.
5A, CtBP1 was recruited to the binding sites of the three
E2-repressed genes, whereas CtBP1 was dismissed from the
TFF1/pS2 enhancer. This confirms that its presence in E2-
stimulated genes before hormone treatment might be impor-
tant for maintaining low basal activity and its hormone-regu-
lated clearance might be important for transcriptional
activation (35). Consistent with this pattern, at genes repressed
by E2, CtBP1 levels were low prior to hormone treatment
when basal gene activity is high, and the hormone-regulated
increased recruitment of CtBP1 (Fig. 5A) coincides with tran-
As seen in Fig. 5B, by performing ChIP/reChIP experiments
we observed CtBP1 to be in the same complex with p300
uniquely at E2-repressed target genes, whereas they were not
present together at the TFF1/pS2 ER? binding site (Fig. 5B).
To test the functional role of CtBP1, we used siRNA that
effectively reduced CtBP1 levels in MCF-7 cells (Fig. 5C).
CtBP1 knockdown completely blocked E2-mediated gene re-
pression, whereas it had little impact on TFF1/pS2 gene stim-
ulation, thus implicating CtBP1 as a key factor in E2-mediated
gene repression (Fig. 5D).
Because of the effect of CtBP1 depletion in preventing gene
repression by estrogen, we also examined the effect of the
closely related protein CtBP2. Of interest, depletion of CtBP2
had no effect on gene repression; repression remained as ro-
bust as that observed in control siRNA-treated cells (Fig. 5D),
suggesting that gene repression uniquely requires CtBP1. Sur-
prisingly, however, CtBP2 knockdown greatly reduced estro-
gen stimulation of the TFF1 gene, suggesting that CtBP2 is
likely to be a factor involved in restricting transcriptional ac-
CtBP1 recruitment requires the presence of p300 and elicits
lysine 9 of histone 3 (H3K9) and H3K14 deacetylation. To
determine whether p300 recruitment is a pioneer event for
CtBP1 action, we reduced cellular p300 levels by using specific
siRNA and then examined CtBP1 recruitment. As shown in
Fig. 6A, CtBP1 recruitment to the E2-repressed gene CCNG2
was increased by E2 treatment in control (GL3) siRNA-ex-
posed cells and was greatly impaired after p300 knockdown,
indicating p300 to be essential for CtBP1-mediated transcrip-
tional repression at E2-inhibited target genes and suggesting
that p300 might act as a bridge between ER? and CtBP1.
FIG. 4. ER? transiently increases gene transcription at E2-repressed genes. (A) MCF-7 cells were treated with 10 nM E2, and nascent mRNA
was labeled with biotin-UTP, isolated, and quantified via qPCR using 36B4 as the internal control. Data shown are means ? standard errors of
the means of the results of three independent experiments. (B) RNA polymerase II ChIP was performed after 2 h of treatment with 5 ?M
?-amanitin followed by 1 h of 1 nM E2 treatment (?-ama?E2) or vehicle (?-ama). Change for ?-ama?E2 versus ?-ama is indicated above the
red bars. Data are means ? standard errors of the means of the results of three independent experiments. Veh, vehicle.
1754 STOSSI ET AL.MOL. CELL. BIOL.
Similar observations were made for the E2-repressed genes
MMD and SMAD6 (data not shown). In contrast, at the TFF1
gene, CtBP1 recruitment was similar after p300 or control
siRNA knockdowns. On the other hand, p300 recruitment was
not impaired after knockdown of CtBP1 at either E2-repressed
or E2-stimulated genes (Fig. 6B), indicating that CtBP1 is
probably recruited after p300 interaction with the ER?-con-
taining complex. Hence, the presence of CtBP1 requires p300,
but the reverse is not the case.
Since CtBP1-containing complexes have been shown to pos-
sess multiple histone-modifying activities (8) and transcrip-
tional repression has been correlated with histone deacetyla-
tion, we assessed changes in specific histone marks after E2
treatment at the repressed and stimulated genes.
H3K14ac is a major site for p300-mediated histone acetyla-
tion and has been correlated with transcriptional activation
(10). As shown in Fig. 6C, H3K14 was deacetylated at two out
of three ER? binding sites near E2-repressed genes, indicating
that p300 does not act as a HAT at these sites and that, after
recruitment, its activity may be blocked by CtBP1 binding. The
opposite was observed at the TFF1/pS2 enhancer, where
H3K14 acetylation was increased after E2 treatment of cells
The second acetylation event that we checked was at the
H3K9 mark (H3K9ac) (Fig. 6C). At the ER? binding sites of
the three E2-repressed genes, H3K9 was deacetylated, con-
firming that one of the important features of gene repression is
general histone deacetylation, while at the TFF1 binding site
we observed strong acetylation (Fig. 6C).
To demonstrate a direct link between the histone deacety-
lation events and CtBP1-containing complexes, we performed
ChIP assays after CtBP1 knockdown and probed for H3K9ac,
which showed the strongest deacetylation at estrogen-re-
pressed gene sites (Fig. 6C). As shown in Fig. 6D, after knock-
down of CtBP1, the deacetylation of H3K9 observed with E2 in
control (GL3) siRNA-treated cells at the CCNG2, MMD, and
SMAD6 genes was nearly completely prevented, while acety-
lation at the TFF1/pS2 enhancer was not affected. Our findings
shown in Fig. 6 suggest that the actions of CtBP1 are unique
for E2-mediated gene repression and that the histone deacety-
lation observed is likely due to HDAC activities in the CtBP1
In this study, we have examined the mechanisms involved in
E2-ER? mediated transcriptional repression of early primary
target genes, and we describe a new mechanism for ER-medi-
ated transcriptional repression that involves the recruitment of
a p300-dependent CtBP1 corepressor complex following ER?
failure to activate gene transcription. We demonstrate that
ER? can be recruited directly, albeit transiently and less
strongly, to DNA elements close to E2-repressed genes where
ER? recruits p300 and is able to transiently increase the tran-
FIG. 5. CtBP1, in a complex with p300, is required for E2-mediated gene repression. (A) ChIP assay using CtBP1 antibody was performed on
MCF-7 cells after 45 min of control (0.1% ethanol) vehicle or 1 nM E2 treatment. (B) ChIP/reChIP was performed on MCF-7 cells after 45 min
of 1 nM E2 treatment, using p300 antibody for the first pull-down and CtBP1 antibody or IgG control for the second pull-down. (C and D) CtBP1,
CtBP2, or GL3 control siRNA was transfected into MCF-7 cells for 72 h prior to treatment with 1 nM E2 for 4 h. mRNA levels of target genes
were measured by qPCR, and Western immunoblotting (WB) was used to confirm CtBP protein knockdown. Data are means ? standard errors
of the means of the results of three independent experiments. In panels A, B, and D, an asterisk indicates a P value of ?0.05 for results of E2
treatment versus results for vehicle control. For panel C, an asterisk indicates a P value of ?0.05 versus results for GL3 siRNA. Veh, vehicle; si,
VOL. 29, 2009 ESTROGEN RECEPTOR GENE REPRESSION AND p300/CtBP11755
scriptional output; however, ER? and p300 are unable to be-
come a nucleation site for p160 coactivators and to sustain
positive transcriptional regulation. This leads to recruitment of
the corepressor CtBP1, via p300, with RNA polymerase II
eviction and histone deacetylation that result in transcriptional
repression. Of note, CtBP1 was a crucial factor for gene re-
pression, whereas it was irrelevant for gene stimulation, by
estrogen. Furthermore, the important effects of CtBP1 in E2-
ER?-mediated gene repression were unique to CtBP1 and
were not reproduced by the related CtBP2 protein.
Modulation of gene transcription by ER?: stimulation ver-
sus repression. It is now well accepted that the ER is a master
regulator of gene transcription, as demonstrated by numerous
studies using both cell culture models and whole-animal target
tissues (11, 14, 22). From these studies it is evident that, upon
E2 treatment, gene transcription is widely impacted, creating
highly complex regulatory networks whose ultimate goal is the
stimulation or suppression of specific biological processes. In
fact, in MCF-7 breast cancer cells, the expression of more
genes is repressed than stimulated by the E2-occupied ER?
(14, 15). Hence, it was of interest to understand how the ER,
being a strong transcriptional activator, can also behave as a
Thus far, several mechanisms have been hypothesized for
E2-mediated gene repression, including physiological squelch-
ing of cofactors (e.g., p160s and CBP), direct action of core-
pressors (NCoR, SMRT, and NRIP1) accompanied by histone
deacetylation, and participation of elements of the basal tran-
scriptional machinery (e.g., TAFII30). Most of these studies,
though, employed exogenous reporter systems or overexpres-
sion of selected factors and/or considered events mostly at late
time points of E2 treatment (8 to 24 h). What we addressed in
this study is the analysis of early, primary ER?-repressed target
genes and the mechanisms that occur at their ER? binding
sites in the endogenous cell chromatin setting.
From genome-wide studies of ER? binding sites, it appears
that ca. 60% of E2-repressed genes at early time points (1 to
4 h) possess at least one ER? binding site in their proximity,
indicating that direct effects of ER? are likely and that they
may account for at least a portion of the repressive events. In
this study, we characterized a group of primary E2-repressed
target genes and document that ER? is recruited to these
binding sites by E2 treatment but, interestingly, in a manner
that is different from recruitment to the enhancer of the TFF1/
pS2 gene, which is strongly E2 stimulated. At the ER? binding
sites that we studied, ER? occupancy increased comparatively
similarly to that at the TFF1/pS2 enhancer in the first 5 to 15
min of E2 treatment, after which ER? occupancy decreased or
remained constant at approximately 10 to 15% of the level of
TFF1/pS2 enhancer occupancy. This indicates that ER? may
interact less efficiently and more transiently with these sites
and also points to the fact that the number of binding sites
close to E2-repressed genes may have been underestimated by
these genome-wide techniques, because only the strongest in-
teractions would be detected and also because, based on our
study, a different result might be obtained by using earlier time
points (i.e., 15 min) of E2 treatment versus the typical 45-min
time point most commonly examined (5, 31). From our bioin-
formatic analysis of the composition of the ER? binding sites,
there appears not to be any preferential factor linked to E2-
FIG. 6. p300 is required for CtBP1 recruitment, and CtBP1 is important for changes in histone marks. (A and B) ChIP assays were performed
on MCF-7 cells after knockdown of p300 (A), CtBP1 (B), or GL3 control (A and B) with siRNA for 72 h. Antibodies for CtBP1 (A) or p300
(B) were used. (C) ChIP assays for histone marks (H3K14ac and H3K9ac) were performed after 45 min of 1 nM E2 or vehicle treatment, and data
were normalized to total histone H3 content. (D) Histone H3K9ac changes were evaluated after CtBP1 or control GL3 (luciferase) knockdown
as described for panel A. In panels A, B, and C, an asterisk indicates a P value of ?0.05 for results of E2 treatment versus results for vehicle control.
In panel D, an asterisk indicates a P value of ?0.05 versus results for siGL3 vehicle. Veh, vehicle.
1756STOSSI ET AL.MOL. CELL. BIOL.
repressed versus E2-stimulated genes (F. Stossi, unpublished
observation), suggesting that it may be difficult to isolate spe-
cific transcription factors that are associated only with E2-
mediated transcriptional repression.
p300 plays a central role in both gene activation and repres-
sion. Nuclear receptor coregulators encompass a large family
of proteins with multiple enzymatic activities that appear to be
essential in performing and fine-tuning the actions of the ER at
the chromatin level (29). Several studies (16, 31, 34, 38) have
highlighted a very dynamic picture of multiple coactivating and
corepressing complexes exchanging during the transcriptional
process, adding an important level of complexity and control in
the regulation and direction (up/down) of the transcriptional
Using time-course ChIP assays, we could establish that p300
was the only cofactor that appeared to be recruited at all the
sites analyzed, while other factors, like CBP, NRIP1, and
p160s, might play more gene-specific roles. Also of note, an
important finding in our study was the essential role of p300 in
gene repression as well as gene stimulation. p300 was found to
be recruited to the binding sites of both E2-stimulated and
E2-repressed genes, and p300 knockdown fully prevented E2-
mediated gene repression and also markedly reduced E2-me-
diated gene stimulation.
The role of p300 in ER?-mediated gene stimulation has
been extensively characterized where it plays a central role in
transcriptional initiation, but not reinitiation (27), and is nor-
mally seen before recruitment of SRCs to the TFF1 gene (31,
38). There is evidence that ER? and p300 interact directly (12,
26), although some have suggested that this may involve me-
diating proteins (e.g., SRC-3).
p300 is also known to elicit negative roles in transcription, as
recently shown in a completely purified in vitro transcription
system (36) where p300 acted as a negative cofactor whose
repressive activity was reversed by the addition of acetyl-coen-
zyme A. Moreover, p300 contains a strong repressive domain
(cell cycle regulatory domain 1, amino acids 1017 to 1029) that
functions independently from the HAT domain via sumoyla-
tion and HDAC6 recruitment (17).
Since a role for p300 in transcriptional initiation has been
extensively characterized, we hypothesized that the p300-ER?-
containing complex might be trying to stimulate gene tran-
scription also at E2-repressed targets but ultimately fails to
continue the process. To investigate this possibility, we first
performed nuclear run-on assays that clearly demonstrated
that, at early time points, ER? can transiently stimulate the
transcription of E2-repressed genes. Second, after we cleared
the coding sequences of the genes from transcribing RNA
polymerase II, E2 treatment resulted in the reloading of RNA
polymerase II at both E2-repressed and E2-stimulated genes,
indicating that ER? is capable of driving positive transcription
from binding sites close to E2-repressed genes. The results of
these two experiments also lead us to speculate that elements
in the basal transcription machinery and/or in the elongation
complexes might be important in choosing the direction of
regulation of transcription by ER? after this initial phase of
stimulation at both types of genes.
The corepressor CtBP1 is utilized by ER?, via p300, to
repress gene transcription. In addressing how ER? and p300
elicit transcriptional repression, we focused on the corepressor
CtBP1. Although CtBP1 had not previously been directly
linked to ER? action, it had been shown to interact directly
with p300 and inhibit the HAT activity of p300 via interaction
with its bromodomain, thus impeding p300’s recognition and
acetylation of histone tails (24, 37). Moreover, CtBP1-contain-
ing complexes have been characterized as containing numer-
ous enzymatic activities, including histone deacetylation via
multiple HDACs (i.e., HDAC1 and HDAC2). Although the
relationships between histone posttranslational modifications
and positive or negative gene activities are known to be very
complex (9, 20), it was striking that robust H3K14 and H3K9
deacetylation accompanied the gene repression by E2 and that
these were prevented by depletion of CtBP1.
We demonstrated that CtBP1, in complex with p300, is re-
cruited to E2-repressed genes and is essential for the repres-
sive process and histone tail deacetylation events, highlighting
a central role for this factor in ER?-mediated transcriptional
repression. In addition, p300 recruitment appeared to be a
prerequisite for CtBP1 recruitment, although there may also
be additional mechanisms, because CtBP1 has been shown to
interact with the corepressors NRIP1/RIP140 and LCoR,
which can directly interact with ER? (13, 41).
A model for E2-mediated gene repression of early target
genes. Based on our observations, we present a model for
E2-mediated transcriptional repression of early target genes
(Fig. 7). In this scenario, ER? would interact either directly,
indirectly, or cooperatively with DNA elements in a manner
that is comparable for stimulated and repressed target genes in
the first 5 to 15 min after E2 treatment. During this first phase,
p300 and, in a gene-specific fashion (i.e., MMD), other cofac-
tors (i.e., SRC-3) are being recruited by ER?, causing a spike
in transcriptional activation, possibly due to the RNA polymer-
ase II already cycling at these genes. After this first phase, ER?
occupancy starts to decrease or remains constant, and this is
followed by a loss of capacity for sustaining a steady increase in
transcription that causes RNA polymerase II loss, recruitment
of CtBP1-containing complexes, and histone deacetylation at
early repressed genes.
It is notable that even at E2-repressed genes, one sees some
molecular attributes of gene stimulation, albeit transiently: re-
cruitment of ER?, p300, and to some genes, CBP, and a tran-
sient increase in nuclear run-on and RNA polymerase II re-
cruitment on ?-amanitin-cleared genes. While it might seem
paradoxical to see such changes at genes that show a net
reduction of RNA levels, it is clear that elevated RNA pro-
duction from repressed genes is, at most, brief. Also, at the
E2-repressed genes, there is a net dismissal of RNA polymer-
ase II upon E2 treatment, RNA polymerase II recruitment by
E2 being evident only on the artificially lowered background
following ?-amanitin treatment.
The most significant and durable differences between the
E2-repressed genes and E2-stimulated genes we have studied
appear to be the relative instability of the recruitment of ER?
and the clear differential recruitment of certain coregulator
complexes, e.g., the corepressor CtBP1 to repressed genes
versus the coactivator SRC-3 to stimulated genes. It is the
consequences of the known differential chromatin-modifying
activities of these coregulators that appear to be responsible
VOL. 29, 2009ESTROGEN RECEPTOR GENE REPRESSION AND p300/CtBP11757
for the ultimate differential effects on the production of RNA
from the repressed versus the stimulated genes.
Our work raises two interesting questions. First, what is
responsible for the fact that the E2-ER?-p300 complex, which
forms at both stimulated and repressed genes, recruits CtBP1
only to E2-repressed genes? It is possible that differential post-
translational modifications of p300 or other coregulators by
specific enzymatic complexes will determine the choice of pro-
tein partners for p300. It is indeed known that a “posttransla-
tional code” exists for coactivators like SRC-3 (29), and p300 is
known to possess multiple sites of posttranslational modifica-
tion that influence its activity (17, 43, 44). Second, if E2 treat-
ment results in dismissal of the CtBP1 corepressor system from
E2-stimulated genes, by what mechanism is CtBP1 present in
the absence of hormone, ER?, and p300? Presumably, in the
absence of hormone, CtBP1 is held at stimulated genes
through other transcription factors via different coregulatory
proteins, like TBL1 (35). It would be interesting to investigate
this system, because such transcription factor-CtBP1 corepres-
sor complexes might be responsible for maintaining the low
basal activity expected of genes poised to be stimulated by
estrogens acting through ER?.
Our studies highlight a new mechanism utilized by the ER to
elicit transcriptional repression of target genes. This mecha-
nism includes a new role for p300 as a bridging factor between
ER? and coregulator complexes that appears to be crucial in
deciding the direction of transcription after ER? activation
and binding to DNA. Moreover, we demonstrate for the first
time the involvement of the corepressor CtBP1 in estrogen-
mediated gene repression. Thus, the cooperation between
CtBP1 and p300 appears to be central in discriminating nu-
clear receptor repression versus stimulation of genes at early
times after hormone exposure.
This work was supported by NIH grant CA18119 (B.S.K.) and a
grant from The Breast Cancer Research Foundation (B.S.K.). Z.M.-E.
received partial support from NIH T32 ES07326.
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