CBP Mediates NF-?B-Dependent Histone Acetylation and Estrogen
Receptor Recruitment to an Estrogen Response Element in
the BIRC3 Promoter
Madhumita Pradhan, Sarah C. Baumgarten, Leslie A. Bembinster, and Jonna Frasor
Department of Physiology and Biophysics, University of Illinois at Chicago, Chicago, Illinois, USA
treated with endocrine therapies, such as tamoxifen or aromatase
inhibitors. However, not all ER-positive tumors respond to these
therapies. Through gene expression profiling studies, ER-positive
tumors have been delineated into two intrinsic subtypes, luminal
A and luminal B (48, 49). Women with the luminal A subtype of
whereas the outcome is poor for women with the luminal B sub-
type of tumors, nearly as poor as that seen in the case of ER-
negative tumors. Our lab recently identified a gene signature syn-
ergistically upregulated by cross talk between ER and NF-?B that
(16). This signature is enriched for cell survival genes, including
the cellular inhibitor of apoptosis gene, cIAP2, which is also
known as BIRC3. We have previously shown that BIRC3 is up-
regulated by estradiol (E2) and the proinflammatory cytokine tu-
not ER-negative cell lines. Using chemical inhibitors and a small
interfering RNA (siRNA) approach, our lab has further demon-
?-induced cell death (51). Understanding the mechanism by
which BIRC3 is upregulated by cross talk between ER and NF-?B
is therefore of clinical relevance.
The ER subtypes, ER? and ER?, are ligand-dependent tran-
of ER target genes in response to estradiol (E2). In breast cancer
cells, thousands of ER target genes and binding sites have been
the classical mechanism of ER action, binding to DNA occurs at
palindromic estrogen response elements (EREs). However, many
ER binding sites do not contain recognizable EREs (5, 35, 38);
hence, in addition to direct binding to DNA sequences, ER teth-
he estrogen receptor (ER) is expressed in approximately 75%
of breast cancers, and women with such tumors are generally
ering to other transcription factors appears to play a significant
role in mediating estrogen action (21, 27, 33, 39, 47). The NF-?B
pathway is activated by a variety of extracellular stimuli, such as
proinflammatory cytokines and growth factors. In the canonical
plex leads to downstream phosphorylation and subsequent pro-
NF-?B family members p65 and p50, which then translocate to
the nucleus, where they bind to their response elements (NF-?B-
act with many other transcription factors to regulate target gene
ER and NF-?B family members have been shown to influence
delineate the multiple mechanisms by which ER can repress
NF-?B action to exert an anti-inflammatory effect (7, 13, 28, 41).
Similarly, there are several pieces of evidence indicating that
NF-?B can repress ER expression and transcriptional activity (2,
10, 14, 23, 36). However, there are relatively few examples of ER
and NF-?B working together to increase transcription (1, 30, 45,
53). Recently, we found that these factors can work cooperatively
on a number of genes, including PTGES, which codes for prosta-
glandin E synthase, and ABCG2, which codes for a drug efflux
Received 1 July 2011 Returned for modification 25 July 2011
Accepted 21 October 2011
Published ahead of print 14 November 2011
Address correspondence to J. Frasor, firstname.lastname@example.org.
Supplemental material for this article may be found at http://mcb.asm.org/.
Copyright © 2012, American Society for Microbiology. All Rights Reserved.
0270-7306/12/$12.00Molecular and Cellular Biologyp. 569–575mcb.asm.org
tional EREs. For the PTGES gene, cross talk occurs at the ERE
itself, whereas for the ABCG2 gene, a weak NF-?B-RE located
specific mechanisms of positive cross talk can occur between ER
arrangement of the regulatory elements in the gene.
However, the mechanism by which ER and NF-?B work to-
gether to increase expression of BIRC3 to promote cell survival is
not known. In the present study, we show that ER can potentiate
TNF-?-dependent BIRC3 expression by binding to an ERE di-
rectly upstream of two NF-?B-REs. The ability of ER to access its
CBP-mediated changes in histone acetylation around the ERE.
tween two potent transcription factors that are known to be im-
portant regulators of breast cancer growth and progression. An
can alter where ER binds in the genome and that ER can, in turn,
enhance the effect of inflammatory factors on the regulation of
their target genes.
MATERIALS AND METHODS
Materials. 17?-Estradiol (E2) and 4-hydroxytamoxifen (TAM) were ob-
tained from Sigma. Cytokines were obtained from R&D Systems. ICI
182,780 (ICI) was purchased from Tocris. The small molecule theophyl-
David Shapiro (University of Illinois at Urbana-Champaign [UIUC]).
Adenoviral vectors for green fluorescent protein (GFP) and I?B?-DN
Chicago [UIC]) and Ruxana Sadikot (UIC), respectively, and used as
previously described (17, 43). Antibodies for chromatin immunoprecipi-
tation (ChIP) assays, ER? (sc-543), p65 (sc-372), and CBP (sc-369) were
obtained from Santa Cruz Biotechnology; anti-histone H3 (ab1791-100)
was obtained from Abcam, and anti-histone H4 (17-10047), anti-
bogen (UIUC). These cells express ER? but not ER? (8). Cells were cul-
tured in minimal essential medium (MEM) containing 5% calf serum
with antibiotics. Prior to E2 treatment, cells were cultured in phenol-red
qPCR) using primers specific for BIRC3 and for 36B4, which served as an
promoter (from ?527 to ?55 and ?247 to ?55) subcloned into the
(Yonsei University, South Korea). Mutations to the ERE and NF-?B-REs
(Stratagene). Wild-type and mutated sequences are given in Table S1 in
the supplemental material. MCF-7 cells were transiently transfected with
reporter constructs along with the Renilla luciferase construct pGL4.70
(Promega), using Lipofectamine 2000 (Invitrogen) in antibiotic-free
Opti-MEM (Invitrogen). Dual-luciferase assays (Promega) were carried
out after 4 h of treatment with E2 and/or TNF-?.
ChIP. ChIP assays were carried out as previously described (43). For
initial ChIP assays, chromatin was sonicated three times for 10 s to gen-
primers tiled along the BIRC3 promoter, sonication was increased to 10 s
30 times to generate fragments ?200 bp in size. Inputs were serially di-
luted to generate standard curves and DNA enrichment was calculated as
percent input for each sample. The percents input for individual experi-
ments were converted to fold change relative to an untreated control.
Average fold changes from a minimum of 3 independent experiments
were then plotted. Primer sequences for ChIP qPCR are listed in Table S1
in the supplemental material.
siRNA transfections. siRNA targeting p65, ER, or CBP or a nonspe-
cific control (siNeg) was purchased from Ambion and transfected using
ments for RNA measurements or ChIP assays were carried out 48 h after
Statistical analysis. qPCR and reporter data were analyzed by two-
Significance for all statistical tests was set at P ? 0.05. The data shown are
the mean ? standard error of the mean (SEM) from at least three inde-
In MCF-7 breast cancer cells, BIRC3 expression is upregulated by
ther enhanced by the combination of TNF-? and E2 (Fig. 1A). E2
in combination with interleukin-1? (IL-1?), but not IL-6, can
also enhance BIRC3 expression over that seen with the respective
cytokine alone (Fig. 1B). In the absence of cytokines, E2 has no
effect on BIRC3 expression across a wide range of doses (Fig. 1C).
dose-dependent increase in BIRC3 expression is observed. The
TAM (Fig. 1D) and ICI 182,780 (16), indicating that ER is re-
FIG 1 Estradiol (E2) cannot induce expression of BIRC3 but potentiates regulation by proinflammatory cytokines. MCF-7 cells were treated for 2 h with
increasing doses of TNF-? in the absence or presence of 10 nM E2 (A), 10 ng/ml IL-1? or IL-6 in the absence or presence of 10 nM E2 (B), increasing doses of
BIRC3 mRNA was measured by qPCR, and fold change was calculated using the ??CTmethod with 36B4 as an internal control. The data represent the means
? SEM for three independent replicates. ?, P ? 0.05 compared to treatment with either E2 or cytokine alone. nd, not detectable; ns, not significant.
Pradhan et al.
mcb.asm.orgMolecular and Cellular Biology
quired for E2 activity. These data demonstrate that although E2
cannot regulate BIRC3 expression on its own, it is capable of po-
tentiating cytokine action on this gene.
Previous studies have shown that cytokines regulate BIRC3
expression in an NF-?B-dependent manner through the first
(NF1) and third (NF3) of three NF-?B-REs located in the pro-
moter of the gene (24). We find that inhibition of the NF-?B
(Fig. 2B) not only prevents TNF-? from regulating expression of
BIRC3, but also completely blocks E2 from potentiating TNF-?
to mediate their effects on the BIRC3 gene. We confirmed that
members, p65 and p50, in MCF-7 cells (see Fig. S1 in the supple-
to bind to the promoter region containing NF1 and NF3 in re-
sponse to cytokines (24). p65 recruitment to this region was as-
sessed in breast cancer cells by ChIP assays, which demonstrated
that the combination of E2 and TNF-? had little effect on p65
therefore unlikely that E2 potentiates TNF-? action by influenc-
ing p65 recruitment to the promoter, which is in contrast to what
we have previously shown for the ABCG2 gene (43).
Further analysis of the BIRC3 promoter sequence revealed a
putative ERE located 156 bp upstream of NF1 (Fig. 3A). Deletion
from potentiating TNF-? action but had little effect on TNF-?
alone (Fig. 3B). This finding suggests that although the ERE does
not appear to be functional in response to E2 alone, it possesses a
latent functionality that becomes apparent in response to both E2
and TNF-?—or, in other words, TNF-? allows this ERE to func-
tion. Although the sequence of the ERE is near consensus
(GGGCA TAT TGACC), we mutated it to a full consensus site
(GGTCA CCG TGACC) according to MatInspector (44) because
FIG 2 The NF-?B pathway is required for enhanced BIRC3 expression by the combination of E2 and TNF-?. (A) BIRC3 mRNA expression was examined in
MCF-7 cells following 24 h of exposure to adenoviral vectors for GFP (control) or a dominant-negative form of I?B? (I?B?-DN) and an additional 2 h of
treatment with E2, TNF-?, or both. (B) MCF-7 cells were transiently transfected with a BIRC3 promoter reporter construct spanning bp ?527 to ?55 of the
promoter, in which the NF-?B response elements (NF1 and NF3) were intact (?527) or mutated (mNF1/3). Dual-luciferase assays were carried out following
to untreated controls from four independent experiments. ?, P ? 0.05 compared to treatment with TNF-? alone. nd, not detectable.
FIG 3 The ability of estrogen to enhance BIRC3 expression requires an intact estrogen response element (ERE) and ER binding to DNA. (A) Schematic
(C) Luciferase activity of the ?527 promoter fragment, in which the ERE was intact (wild type [WT-ERE]) or mutated to a consensus ERE (Cons-ERE), was
or presence of an ER-DNA binding inhibitor, TPBM (20 ?M). ?, P ? 0.05 compared to treatment with TNF-? alone. ns, not significant.
NF-?B and Estrogen Receptor Cross Talk at BIRC3
January 2012 Volume 32 Number 2mcb.asm.org 571
the single-nucleotide difference is at an essential position in the
sequence. A BIRC3 promoter construct containing the consensus
ERE also failed to respond to E2, indicating that the lack of func-
tionality of the ERE is not because of a weak binding sequence
(Fig. 3C). In addition, TPBM, a small molecule that prevents ER
by E2 plus TNF-? over TNF-? alone, suggesting that ER binding
to the BIRC3 ERE is essential for enhanced regulation of the gene
To examine recruitment of ER to the BIRC3 promoter, ChIP
assays were carried out. In agreement with the lack of an E2 effect
on BIRC3 expression, E2 had no effect on ER recruitment to the
the supplemental material). However, the combination of E2 and
TNF-? led to a rapid and robust recruitment of ER, which was
sustained over 60 min and completely prevented by blockade of
the NF-?B pathway with I?B?-DN (Fig. 4A and B). These results
indicate that E2 alone cannot bring about ER recruitment to the
BIRC3 gene and that activation of NF-?B is necessary to allow ER
to be recruited by the combination of E2 and TNF-?. This mech-
anism appears to be gene specific since TNF-? can actually en-
hance (17, 43), repress, or have no effect on (see Fig. S3 in the
supplemental material) ER recruitment to other ER target genes.
TNF-? influenced ER recruitment through chromatin remodel-
ing around the ERE of the BIRC3 gene. Formaldehyde-assisted
isolation of regulatory elements (FAIRE), micrococcal nuclease
failed to detect any changes in chromatin architecture at the ERE,
but chromatin remodeling was observed at the NF-?B-REs (see
TNF-? induced a robust enrichment of acetylated histones 3 and
the supplemental material). A similar increase in acetylation
scription start site, was observed in response to E2 plus TNF-?
(see Fig. S6). The effect of TNF-? on acetylation around the ERE
was blocked by siRNA targeting p65 (Fig. 5B and C), indicating
that TNF-? acting through NF-?B leads to an increase in histone
acetylation at this site. This finding suggests that TNF-?-
stimulated histone acetylation may be a potential mechanism by
which NF-?B influences ER recruitment to the BIRC3 promoter.
involved in histone acetylation around the ERE and subsequent
ER recruitment, siRNAs for a number of coactivators and HATs
known to interact with ER and p65 were used (see Fig. S7 in the
supplemental material). As expected, knockdown of ER and p65
reduced regulation of BIRC3. Of the HATs, we found that CBP
significantly reduced BIRC3 expression in response to E2 plus
down, significantly reduced ER recruitment to the BIRC3 pro-
moter in the presence of E2 plus TNF-? (Fig. 6B). Furthermore,
and/or TNF-? treatment (Fig. 6D). Taken together, these data
be due to its ability to acetylate histones around the ERE and
promote ER access to the gene, thereby leading to potentiation of
BIRC3 gene expression.
FIG 4 ER recruitment to the BIRC3 promoter requires E2, TNF-? and the
NF-?B pathway. (A) ChIP assays for ER were carried out following treatment
of MCF-7 cells with E2, TNF-?, or both for up to 60 min, and recruitment to
the BIRC3 promoter was examined. (B) ER recruitment to BIRC3 was carried
out by ChIP assay following 24 h of exposure to I?B?-DN and 45 min of
or TNF-? alone.
cells. PCR was carried out using small amplicons that tile along the BIRC3 promoter. ?, P ? 0.05 compared to an untreated control. (B) Acetylation of H3 and
H4 at the ERE was assessed following transfection with siNeg (control) or sip65 and treatment with or without TNF-? for 15 min. Fold change was calculated
siNeg control. (C) p65 protein levels were examined by Western blotting in MCF-7 cells transfected with siNeg or sip65 for 48 h.
Pradhan et al.
mcb.asm.orgMolecular and Cellular Biology
In this study, we find that BIRC3, an important antiapoptotic
cannot regulate the gene on its own. This occurs through a novel
mechanism of cross talk between ER and NF-?B not previously
observed. We find that E2 potentiates TNF-? action through a
near-consensus ERE that is located upstream of two functional
in the presence of E2 alone, but NF-?B activation leads to a high
tically with NF-?B to potently and rapidly increase transcription
of the BIRC3 gene.
At first we expected that the lack of functionality of the ERE
could be the result of its sequence. It has been demonstrated that
gene (18, 32). Furthermore, we have shown that cooperativity
between ER and NF-?B occurs between a nonconsensus NF-
by E2 and TNF-? (43). However, the nature of the BIRC3 ERE is
fect consensus sequence does not significantly increase ER activ-
ity, suggesting another factor may be preventing ER from inter-
that far more high-confidence ER binding sites have been identi-
fied computationally than are actually detected by genome-wide
ChIP-on-chip studies for ER recruitment with E2 alone (6, 52).
We also considered that the endogenous chromatin structure
may be preventing ER from interacting with its binding site. The
ing DNA packaging has been proposed as a common mechanism
to confer a higher degree of regulation to gene expression in re-
sponse to various stimuli (11). Furthermore, previous studies
have demonstrated that other transcription factors, such as
FOXA1, are necessary to facilitate ER recruitment to specific re-
gions of chromatin (26). NF-?B has previously been shown to
enhance DNA accessibility around the NF-?B-RE to which it
binds. For example, at the granulocyte-macrophage colony-
stimulating factor gene (GM-CSF) promoter, NF-?B causes in-
crease in chromatin accessibility via remodeling through recruit-
ment of the ATPase component of the SWI/SNF chromatin
remodeling complex, BRG1 (4, 22). More recently, constitutive
activation of NF-?B along with AP1 has been associated with in-
cancer cells (40). However, in the case of the BIRC3 gene, NF-?B
had little to no effect on the chromatin structure around the ERE,
suggesting that NF-?B may be influencing ER recruitment
ment of acetylated histones around the ERE in the presence of
with HATs may increase accessibility of the ERE to ER. Previous
studies have shown that histone acetylation can contribute to
DNA accessibility to transcription factors by loosening histone-
DNA contacts (3).
In our study, a number of HATs were identified that are re-
which are known to interact independently with ER or NF-?B.
CBP was of particular interest to us since it was required not only
for enhanced BIRC3 expression, but also for ER recruitment as
well as histone acetylation around the ERE. Previous studies have
shown that ER recruitment to the CRH promoter is accompanied
by an increase in H3 acetylation, as well as CBP recruitment (34).
present constitutively at the BIRC3 promoter. This has also been
FIG 6 Role of CBP in BIRC3 expression, ER recruitment, and histone acety-
(B) ER recruitment to the BIRC3 promoter was assessed by ChIP assays fol-
lowing transfection with siNeg, siER, or siCBP and treatment with E2 plus
TNF-? for 15 min. Data are represented as a percentage of ER recruitment
ined following transfection with siNeg or CBP. ?, P ? 0.05, ??, P ? 0.01, and
were carried out after 15 min of treatment with E2, TNF-?, or both.
NF-?B and Estrogen Receptor Cross Talk at BIRC3
January 2012 Volume 32 Number 2mcb.asm.org 573