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Direct Acetylation of the Estrogen Receptor
␣
Hinge Region by p300
Regulates Transactivation and Hormone Sensitivity*
Received for publication, January 29, 2001, and in revised form, March 8, 2001
Published, JBC Papers in Press, March 9, 2001, DOI 10.1074/jbc.M100800200
Chenguang Wang‡, Maofu Fu‡, Ruth H. Angeletti‡, Linda Siconolfi-Baez‡, Anne T. Reutens‡,
Chris Albanese‡, Michael P. Lisanti§, Benita S. Katzenellenbogen¶, Shigeaki Kato储,
Torsten Hopp**, Suzanne A. W. Fuqua**, Gabriela N. Lopez‡‡, Peter J. Kushner‡‡,
and Richard G. Pestellत
From the ‡Department of Developmental and Molecular Biology, Albert Einstein Cancer Center, Albert Einstein College of
Medicine, Bronx, New York 10461, and §Department of Pharmacology, Albert Einstein College of Medicine, Bronx, New
York 10461, the ¶Departments of Molecular and Integrative Physiology and Cell and Structural Biology, University of
Illinois, and the College of Medicine, Urbana, Illinois, 61801-3704, 储The University of Tokyo, Bunkyo-ku, Tokyo, 113-0032
and CREST, Japan Science and Technology, Kawaguchi, Saitama 332-0012, Japan, the **Breast Center, Baylor College
of Medicine, Houston, Texas 77030, and the ‡‡Metabolic Research Unit, University of California School of Medicine,
San Francisco, California 94143-0540
Regulation of nuclear receptor gene expression in-
volves dynamic and coordinated interactions with his-
tone acetyl transferase (HAT) and deacetylase com-
plexes. The estrogen receptor (ER
␣
) contains two
transactivation domains regulating ligand-independent
and -dependent gene transcription (AF-1 and AF-2 (ac-
tivation functions 1 and 2)). ER
␣
-regulated gene expres-
sion involves interactions with cointegrators (e.g. p300/
CBP, P/CAF) that have the capacity to modify core
histone acetyl groups. Here we show that the ER
␣
is
acetylated in vivo. p300, but not P/CAF, selectively and
directly acetylated the ER
␣
at lysine residues within the
ER
␣
hinge/ligand binding domain. Substitution of these
residues with charged or polar residues dramatically
enhanced ER
␣
hormone sensitivity without affecting in-
duction by MAPK signaling, suggesting that direct ER
␣
acetylation normally suppresses ligand sensitivity.
These ER
␣
lysine residues also regulated transcrip-
tional activation by histone deacetylase inhibitors and
p300. The conservation of the ER
␣
acetylation motif in a
phylogenetic subset of nuclear receptors suggests that
direct acetylation of nuclear receptors may contribute
to additional signaling pathways involved in metabo-
lism and development.
Nuclear receptors coordinate diverse physiological roles in
metabolism and development through ligand-dependent and
-independent mechanisms (1). Nuclear receptors form multi-
protein complexes with coactivator and corepressor proteins to
orchestrate dynamic transcriptional events in response to
ligand. In the absence of ligand, nuclear receptors repress
transcription through a dominant association with corepressor
complexes with histone deacetylase activity (2). Conforma-
tional changes induced upon nuclear receptor ligand binding
release corepressors, with subsequent transient association of
coactivator proteins (2–4). Estrogen binds the estrogen recep-
tor (ER
␣
),
1
thereby regulating important functions in develop-
ment and reproduction and in human diseases including breast
cancer, cardiovascular disease, osteoporosis, and Alzheimer’s
disease. The ER
␣
contains domains conserved with other mem-
bers of the “classical” receptor subclass (termed A—F) and two
activation domains, AF (activation function)-1 and AF-2.
The two activation domains of ER
␣
contribute synergisti-
cally to transcription of target genes. The AF-1 function is both
constitutive and induced by mitogen-activated protein kinases
(MAPKs) induced by growth factors or oncoproteins (5). p300
(6) and a p300/CBP-binding protein, p68 RNA helicase A (7),
also induce AF-1 activity. Thus, p300 binds AF-1 in the absence
of ligand (6, 8) inducing ER
␣
activity 2–3-fold in either reporter
or in vitro transcription assays (6, 8). p300/CBP binding to ER
␣
is also detectable in MCF7 cells in the absence of ligand (4). The
ligand-dependent transactivation function (AF-2) domain of
ER
␣
consists of a conserved carboxyl-terminal helix. The AF-2
domain contributes to ligand-induced activity through further
recruitment of coactivator proteins including the p160 family,
(SRC-1, TIF2/GRIP1, AIB1/ACTR), the cointegrators (CBP,
p300), and p300/CBP-associated factor (P/CAF) (2, 8, 9). The
role of p300 as an ER
␣
cointegrator is complex; p300 contrib-
utes to ER
␣
induction through several separable subdomains
including the histone acetyl transferase (HAT) and the bromo-
domain (4, 8, 10), which make separate contacts to distinct
domains of the ER
␣
.
The enhancement of transcriptional activity by p300/CBP
involves several different functions. The cointegrators provide
a bridging function, which associates transcription factors with
the basal transcription apparatus (11). Second, p300/CBP pro-
* This work was supported by National Institutes of Health Grants
RO1CA70897 and RO1CA75503 (to R. G. P.), NIHCA18119 and
CA60514 (to B. S. K.), R01-CA-80250 (to M. P. L.), and R01-
CA72038-01 (to S. A. W. F.) and by Cancer Center Core National Insti-
tutes of Health Grant 5-P30-CA13330-26. The proteomic analysis per-
formed by the Laboratory for Macromolecular Analysis and Proteomics
at the Albert Einstein College of Medicine was supported by the Albert
Einstein Comprehensive Cancer Center (CA13330) and the Diabetes
Research and Training Center (DK20541). 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: Albert Einstein
Cancer Center, Chanin 302, 1300 Morris Park Ave., Bronx, NY 10461.
Tel.: 718-430-8662; Fax: 718-430-8674; E-mail: pestell@aecom.yu.edu.
1
The abbreviations used are: Er
␣
, estrogen receptor
␣
; AF, activation
function; MAPK, mitogen-activated protein kinase; MEK, MAPK/ERK
(extracellular signal-related kinase) kinase; CBP, CREB (cAMP-re-
sponse element-binding protein)-binding protein; IP, immunoprecipi-
tation; HAT, histone acetyl transferase; HPLC, high pressure liquid
chromatography; GST, glutathione S-transferase; TSA, trichostatin A;
MALDI-TOF, matrix-assisted laser desorption/ionization time-of-flight;
E2, estradiol; P/CAF, p300/CBP-associated factor; EKLF, erythroid
Kruppel-like factor; ERE, estrogen response element.
THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 276, No. 21, Issue of May 25, pp. 18375–18383, 2001
© 2001 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.
This paper is available on line at http://www.jbc.org 18375
by guest on November 24, 2015http://www.jbc.org/Downloaded from
vides a scaffold, interacting with numerous transcription fac-
tors through dedicated domains to assemble high molecular
weight “enhanceosomes” (reviewed in Ref. 12). Third, the HAT
activity of p300/CBP, which may be either intrinsic or mediated
through the recruitment of associated proteins such as P/CAF,
contributes to the transcriptional coactivator function. Tran-
scriptional activation in chromatin-containing systems has cor-
related transcriptional activity with acetylation of specific
lysines in the NH
2
termini of histones (13, 14). Histone acety-
lation is thought to facilitate binding of transcription factors to
specific target DNA sequences by destabilizing nucleosomes
bound to the promoter region of a target gene (15). In addition,
p300/CBP and P/CAF directly acetylate non-histone proteins
including a subset of transcription factors and coactivators
(p53, EKLF, HMG1(Y), GATA-1, E2F-1, and ACTR (16–20).
Transcription factor acetylation by cointegrators has divergent
effects. p300/CBP-dependent acetylation enhanced the activity
of the tumor suppressor p53 (21), the Kruppel-like factor
(EKLF) (19), and the erythroid cell differentiation factor,
GATA-1 (22) (reviewed in Ref. 23). In contrast, CBP repressed
the transcriptional activity of T cell factor (24), and direct
acetylation of the coactivator ACTR by p300 contributed to an
inhibition of hormone-induced nuclear receptor signaling (4).
Together these studies are consistent with a model in which
cointegrator proteins, through their acetylation function, are
engaged in a dynamic interplay to coordinate both the induc-
tion and repression of gene expression.
Although transcription factors can serve as substrates for
HATs, no direct role for such molecules in hormone signaling
had been identified (25). Intrinsic HAT activity for histone
lysines is shared redundantly by ER
␣
transcriptional regula-
tory proteins, which include p300, CBP, P/CAF, SRC1, and
ACTR (26, 27). Redundancy of the HAT function among cointe-
grators raises the fundamental question of whether alternate
substrates to histones may be involved in hormonal signaling.
In the current studies we show that the ER
␣
is acetylated in
vivo and is directly and selectively acetylated by p300, but not
by P/CAF, within the ER
␣
hinge region at conserved lysines in
vitro. Substitution mutation established an important role for
these acetylated residues in both ligand-dependent and -inde-
pendent functions, suggesting local conformational changes
may regulate interactions between the two activation domains
of the ER
␣
. Conservation of the ER
␣
motif acetylated in vitro
between a subset of nuclear receptors raises the possibility that
direct acetylation may regulate diverse functions of phyloge-
netically related nuclear receptors.
MATERIALS AND METHODS
Reporter Genes, Expression Vectors, and Luciferase Assays—The
ERE luciferase reporter gene ERE
2
TK81 pA
3
LUC (28), the Flag-tagged
P/CAF mutants (29), the ER
␣
fusion proteins (30), pcDNA3-HA-p300
(31), the constitutively active MEK1 plasmids, pCMV-⌬N3, pCMV-R⌬F
(⌬N3-S218E-S222D), and the catalytically inactive mutant MEK1
(K97M) (32, 33) were described previously. The ER
␣
mutants were
derived by polymerase chain reaction-directed amplification using se-
quence-specific primers. Both the wild type ER
␣
and ER
␣
mutants were
cloned into pCI-neo (Promega, Madison, WI). The integrity of all con-
structs was confirmed by sequence analysis.
Cell culture, DNA transfection, and luciferase assays were performed
as previously described (30, 34). Cells were incubated in media contain-
ing 10% charcoal-stripped fetal bovine serum prior to experimentation
using estradiol and transfected by calcium phosphate precipitation or
Superfect transfection reagent (Qiagen, Valencia, CA). The medium
was changed after 5 h and luciferase activity determined after 24 h.
Luciferase activity was normalized for transfection using

-galactosid-
ase reporters or Renilla luciferase as an internal control exactly as
described previously (20).
Protein Expression and Western Blots—The antibodies used in West-
ern blot analysis were anti-M2 Flag (Sigma), anti-guanine nucleotide
dissociation inhibitor (35), anti-acetyl lysine (16), and GST (B-14) and
ER
␣
(H-184) antibodies from Santa Cruz Biotechnology (Santa Cruz,
CA).
In vitro [
35
S]methionine-labeled proteins were prepared by coupled
transcription-translation with a Promega TNT
®
-coupled reticulocyte
lysate kit (Promega), using 1.0
g of plasmid DNA in a total of 50
l.
Flag-tagged P/CAF proteins were expressed in Sf9 cells by infecting
with recombinant baculovirus and purified using an anti-Flag antibody
(Sigma, M2) (36). Full-length recombinant baculovirus ER
␣
was ob-
tained from Affinity Bioreagents, Inc. (Golden, CO).
Immunoprecipitation Histone Acetyltransferase Assays—Immu-
noprecipitation histone acetyl transferase (IP-HAT) assays were
performed using p300 as described previously (16, 37). For immunopre-
cipitation the protein concentration was adjusted to 1
g/
lin500
l.
The relevant antibodies from Santa Cruz Biotechnology (p300, N15)
were added (2
g/500
g of extract) and incubated at 4 °C for 2 h. A
standard HAT assay was performed containing 5
g of substrate and
enzyme, either 200 ng of purified histone acetyl transferase (purified
baculovirus p300 or P/CAF) or immunoprecipitated p300 from cultured
cells (16, 37). The mixture was incubated at 30 °C for 1 h. 90 pmol of
[
14
C]acetyl-CoA reaction was electrophoresed on a SDS-polyacrylamide
gel and viewed following autoradiography of the gel. [
14
C]acetyl incor-
poration into the substrates was also determined by liquid scintillation
counting or filter assays.
In Vitro Protein-Protein Interactions and Mapping the ER
␣
Acetyla-
tion Sites—The interactions between in vitro expressed proteins was
performed as described previously (38). The in vitro translated protein
(15
lofER
␣
), 1
g of rabbit anti-ER
␣
polyclonal antibody (H184, Santa
Cruz Biotechnology), and 5
g of purified Flag-tagged baculovirus-
expressed P/CAF were incubated in 300
l of binding buffer.
In vitro acetylation assays were performed as described previously
1(7). Synthetic peptide corresponding to the ER
␣
(ER1, residues
293–310, NH
2
-PSPLMIKRSKKNSLALSL-OH, and ER2, residues 353–
370, NH
2
-ELVHMINWAKRVPGFVDL-OH) were synthesized by
Bio䡠Synthesis (Lewisville, TX) and purified to 95% purity by HPLC. The
peptides were acetylated in vitro by incubation with 5 mMacetyl-CoA
and baculovirus-purified Flag-p300 or P/CAF at 30 °C for 2 h. After
incubation, acetylated peptides were separated from contaminating
p300 by passage through a micron filter (Amicon Inc., Beverly, MA) and
further purified by analytical reversed phase HPLC. The reaction prod-
ucts were analyzed with a PE-Biosystems DE-STR MALDI-TOF mass
spectrometer. Further analysis by Edman degradation was performed
on a PE-Biosystems Procise sequencer. Phenylthiohydantoin-acetyl-
lysine was measured by absorbance at 259 nm.
RESULTS
The ER
␣
Is Acetylated by p300 in Vitro and in Vivo—The
p300/CBP coactivator proteins have been shown to regulate
several promoters in a manner dependent upon their histone
acetylase activity (25), and p300 can both bind and stimulate
the activity of the ER
␣
(4, 8, 10). In addition, p300/CBP and
P/CAF have been shown to acetylate non-core histone-related
transcription factors directly through a conserved motif. We
assessed whether p300 could acetylate recombinant ER
␣
in
vitro. Recombinant p300 acetylated recombinant ER
␣
but did
not acetylate GST (Fig. 1A). In contrast, recombinant baculo-
virus-expressed P/CAF did not acetylate ER
␣
, although it was
capable of acetylating histone H3 and itself (Fig. 1B) as shown
previously (39).
The ER
␣
Is an Efficient and Selective Substrate for p300
Acetylation in Vitro—Two fundamental types of questions
raised by these studies are, first, the relative efficiency of ER
␣
acetylation and, second, whether the failure of P/CAF to acet-
ylate the ER
␣
is due to failed binding or substrate selectivity.
To assess the relative efficiency with which p300 acetylates the
ER
␣
, a direct comparison was made between equimolar
amounts of ER
␣
and histone H3. The products acetylated by
increasing amounts of p300 were electrophoresed on a SDS-
polyacrylamide gel and the incorporation of [
14
C]acetyl-CoA
assessed (Fig. 2A). The efficiency of incorporation on an
equimolar basis was ⬃3-fold greater for histone H3 (16 kDa)
than ER
␣
(66 kDa) (Fig. 2B), suggesting ER
␣
is acetylated with
substantial efficiency. Thus the ER
␣
is efficiently and selec-
tively acetylated by p300 in vitro.
Conserved ER
␣
Acetylation Motif in Nuclear Receptors18376
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P/CAF has been reported to associate with ER
␣
in vitro (40).
We examined whether the recombinant P/CAF used in the HAT
assays bound to the ER
␣
. As shown in Fig. 2C, recombinant
P/CAF bound with high affinity to ER
␣
, and binding required
the HAT domain. Thus, although P/CAF acetylates histone H3
and H4, the failure of P/CAF to acetylate ER
␣
is not due to
FIG.1. p300 acetylates the ER
␣
C-
terminal to the zinc finger DNA bind-
ing domain. A, IP-HAT assays were
performed as described previously (16,
37). Equal amounts of either the GST-
ER
␣
fusion protein or GST protein were
incubated with p300 and [
14
C]acetyl-Co-A
([
14
C]Ac-p300). The arrow indicates the
autoradiogram of the acetylated ER
␣
fu-
sion protein and autoacetylated p300. The
autoradiogram of the electrophoresed
products demonstrates equal amounts of
autoacetylated p300 in both lanes and the
presence of acetylated ER
␣
.B, the bacu-
lovirus-expressed full-length ER
␣
protein
or core histones were used as substrates
in HAT assays using either full-length
p300 or P/CAF. p300 acetylated the ER
␣
and autoacetylated. P/CAF autoacety-
lated and acetylated core histones H3 and
H4 but did not acetylate the ER
␣
.
FIG.2.ER
␣
is an efficient substrate
for p300 acetylation. Aand B, HAT as-
says were performed using a constant
amount of enzyme and equimolar
amounts of either ER
␣
or histone H3 sub-
strate. B, the acetylated bands were ex-
cised and counted. C, affinity-purified
Flag-P/CAF proteins were incubated with
equal amounts of full-length in vitro
translated ER
␣
. Protein complexes were
immunoprecipitated by anti-Flag anti-
body. Western blotting was used to detect
P/CAF, and ER
␣
was visualized by auto-
radiography. Interactive domains identi-
fied by pull-down were scored as ⫹or ⫺.
Western blotting of the P/CAF mutant
proteins using the anti-Flag antibody
(upper panel) confirmed that equal
amounts of wild type and mutant P/CAF
proteins were incubated in the pull-down
experiment.
Conserved ER
␣
Acetylation Motif in Nuclear Receptors 18377
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failed binding. These findings are consistent with the observa-
tion that p300 and P/CAF have distinguishable substrate spec-
ificities (21).
Identification of the ER
␣
Acetylation Sites—To identify the
residues required for ER
␣
acetylation in vitro, recombinant
GST-ER
␣
fusion fragments were expressed, their integrity was
confirmed by Western blotting using a GST antibody, and equal
amounts of proteins were assayed in HAT assays using recom-
binant p300 as a source of HAT activity and the previously
described filter assay (16). As shown in Fig. 3, Band C, the ER
␣
from residues 282–337 was sufficient to function as a substrate
for acetylation by p300.
Peptides were synthesized to encompass the two lysine-con-
taining motifs identified within the region of the ER
␣
acety-
lated in vitro (Fig. 3D). We identified residues resembling an
acetylation motif found in the p53 and GATA-1 transcription
factors, which were conserved between species (Fig. 3D). An
additional lysine, residue 362, was identified that had been
implicated previously in ligand-regulated ER
␣
function (41).
Polypeptides were synthesized therefore to include residues
encoding the consensus acetylation motif ER1-(293–310) (ER1)
and a second polypeptide including lysine 366 (ER2-(353–370))
(ER2). HAT assays were performed using recombinant p300 or
P/CAF. p300 acetylated the ER1 polypeptide but did not acet-
ylate ER2 (Fig. 3D). Recombinant P/CAF failed to acetylate
either ER polypeptides.
Mass analysis of the acetylated ER1 peptide confirmed the
presence of two major ions differing by 42 mass units, with the
smaller molecular weight product corresponding to the unmod-
ified ER1 peptide and the higher molecular weight component
corresponding to the acetylated ER1 product (Fig. 4A). Follow-
ing in vitro acetylation of the ER1 peptide, Edman degradation
assays were performed. As only monoacetylated lysine-contain-
ing peptides were detected in the samples by MALDI-TOF
mass spectrometry, the product analyzed by Edman degrada-
tion was a heterogeneous population of polypeptides, each
acetylated at a single site (Fig. 4A). These studies demon-
strated that lysines 302 and 303 of the ER
␣
were preferentially
acetylated by p300 with an additional acetylation site at lysine
299 (Fig. 4B).
The ER
␣
Acetylated Residues Regulate Basal Activation of
the ER
␣
by TSA—To examine the role of histone acetylases in
the regulation of ER
␣
activity, an estrogen-responsive lucifer-
ase reporter gene was assessed in ER
␣
-deficient cells (MDA
MB231). Inhibitors of histone deacetylase(s) trichostatin A
(TSA) and sodium butyrate were added to transfected cells for
24 h. TSA induced the ERE-LUC reporter (ERE
2
TKpA
3
LUC)
4–6-fold (Fig. 5A). Similarly, sodium butyrate (1 mM) induced
ER reporter activity 2-fold (Fig. 5B). To examine the functional
consequence of lysines 302 and 303 in ER
␣
function, point
mutation of the ER
␣
acetylation sites was performed. The
ER-responsive reporter was assessed in ER
␣
-deficient cells
(MDA MB231 and HeLa). Activity was assessed through nor-
malization to the internal standard

⫺galactosidase reporter.
The 2-fold induction of wild type ER
␣
by sodium butyrate was
abolished by the ER
(K302A/K303A)
mutant (Fig. 5C). The abun-
dance of the ER
␣
K302A/K303A
mutant was similar to ER
␣
wild
type in cultured cells (Fig. 5D). HeLa cells were transfected
with either wild type ER
␣
or mutants of the acetylation site
and assessed for ERE activity. The wild type ER
␣
was induced
3-fold by the addition of TSA in a dose-dependent manner (Fig.
5E). Both the alanine and threonine substitutions failed to
respond to TSA (Fig. 5E). Together these findings suggest that
direct ER
␣
acetylation contributes to induction by histone
deacetylase inhibitors.
MAPK-induced ER
␣
Functions Independently of the ER
␣
Acetylation Site—To investigate further the in vivo conse-
quence of the ER
␣
acetylation site, point mutation substitu-
tions were introduced into the wild type ER
␣
at the lysine
residues acetylated in vitro. It was reasoned that the acetyla-
FIG.3. Mapping p300-mediated
acetylation sites of the ER
␣
.A, sche-
matic representation of the ER
␣
(indicat-
ing the A–F domains, DNA binding
domain (DBD), the ligand binding domain
(LBD), and the conserved RXKK motif)
and the GST-ER
␣
fusion proteins. B, the
Coomassie-stained gel corresponding to
the GST-ER
␣
fusion proteins (upper
panel) and the
14
C-labeled ER
␣
proteins
(lower panel). C, p300-mediated in vitro
IP-HAT assays were performed using
equal amounts of GST-ER
␣
fusion pro-
tein. The products corresponding to the
expected molecular weight were excised
and HAT activity quantitated by liquid
scintillation counting. D,ER
␣
peptide cor-
responding to either ER-(293–310) (ER1)
or ER-(353–370) (ER2) were used as in
vitro substrates with
14
C-labeled acetyl-
Co-A and either p300 or P/CAF. The motif
identified in the human ER
␣
is shown as
conserved between species and is homol-
ogous to the acetylation motif of the mu-
rine GATA-1 and human p53 proteins.
The ER-(293–310) peptide was selectively
acetylated by p300.
Conserved ER
␣
Acetylation Motif in Nuclear Receptors18378
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tion of a lysine, a positively charged, hydrophobic residue, is
thought to both reduce its charge and increase its polarity. If
acetylation augments activity through increasing the polarity
or reducing the charge, a mutation of the two ER
␣
lysines to
polar residues, ER
(K302Q/K303Q)
, may function as an activating
mutant. The introduction of a large positively charged amino
acid with a significant side chain (ER
(K302R/K303R
) might be
anticipated to mimic acetylation if increasing polarity is of
greater importance. Substitution of lysine to alanine,
(ER
(K302A/A303A)
) or another small hydrophobic threonine resi-
due (ER
(K302T/K303T)
) was anticipated to result in a loss of
function. If the post-translational modification of acetylation
itself were important in regulating ER
␣
activity, the substitu-
tion of the lysine residues with any of these other residues
would be expected to have a similar effect. The results of these
studies are shown in Fig. 6. The mutant ER
␣
proteins were
expressed equally in transfected cells (data not shown). HeLa
cells were transfected with either wild type ER
␣
or mutants of
the acetylation site and assessed for their ability to regulate
the activity of a synthetic ERE in the absence of ligand.
Assessment was made of the AF-1 function mediated by
MAPK signaling. Growth factors induce ligand-independent
activity of the ER
␣
through activation of MAPK (5) and the
p160 coactivator AIB1 (also named RAC3, ACTR, or SRC3)
(42). p160 proteins bind p300 (43) and contact both the AF-1
and AF-2 of the ER
␣
(44, 45). To determine whether the lysine
substitutions within the ER
␣
hinge regulated MAPK-depend-
ent ER
␣
activity, constitutively activated MEK1 (⌬N3, ⌬N3-
S218E-S222D) were coexpressed with the ER
␣
mutants (Fig.
6A). The wild type ER
␣
was induced 3.5-fold by activated
MEK1 but was not significantly induced by the catalytically
defective MEK1 (K97 M). The basal activity of the ER
␣
(K302A/
K303A)
mutant was reduced 2.5-fold; however, the magnitude of
induction by activated MEK1 was not significantly changed for
any of the mutants (Fig. 6A). The finding that the ER
␣
acety-
lation mutants are not altered in their responsiveness to
MAPK activation suggests the mechanisms governing ligand-
induced ER
␣
activity through the ER
␣
acetylation site are
distinct from those governed by ACTR.
The ER
␣
Acetylation Site Governs Ligand Sensitivity—In
previous studies of ER
␣
activity in HeLa cells using a similar
reporter assay, estradiol (10
⫺8
M) induced ERE-dependent lu-
ciferase activity 2-fold (41). In our studies the wild type ER
␣
gave a similar 2-fold induction upon the addition of estradiol
(10
⫺8
M) (Fig. 6B). This ERE
2
TK81LUC reporter is not induced
by 10
⫺10
ME2 in HeLa cells with the wild type ER
␣
; however,
both the glutamine and arginine substitutions were induced by
2–3-fold, suggesting the positive charge of these residues may
contribute to ligand sensitivity (Fig. 6B). The hinge domain
mutants were compared with the wild type ER
␣
for ligand-de-
pendent transactivation using increasing concentrations of E2.
Enhanced E2-dependent activity was observed for each of the
ER
␣
mutations of the hinge region lysine residues. Thus,
uncharged, polar, or hydrophobic substitutions of the ER
␣
en-
hanced ligand sensitivity. As each of the ER
␣
mutants exhib-
ited similar levels of expression to wild type ER
␣
, and the wild
type ER
␣
functioned in the same manner as the ER
␣
wild type
in other studies in this cell type (41), these findings suggest
that the wild type lysine residues within the ER
␣
hinge region
may play a role in normally repressing ligand-dependent ER
␣
activity.
We next assessed the role of the hinge domain lysine resi-
dues in p300-dependent regulation of ER
␣
function. The mod-
est induction of wild type ER
␣
activity by p300 in the absence
of ligand (Fig. 6C) is consistent with studies by others. Binding
of p300 to the ER
␣
in the absence of ligand and a 2–3-fold
induction of ER
␣
activity in the absence of ligand were ob-
served both in reporter assays (6) and in in vitro transcription
assays (8). Conformational changes induced by the addition of
estradiol recruits p160 coactivators to a hydrophobic fold in the
ER
␣
with the p300 cointegrator (9). Because mutation of the
lysine residues of the ER
␣
enhanced ligand sensitivity, we
hypothesized that substitutions of these lysines may also
enhance p300-dependent transactivation of the ER
␣
in the
presence of E2. In keeping with this model each of the ER
␣
acetylation mutants demonstrated enhanced activation by
p300 in the presence of hormone (Fig. 6C). These findings raise
the possibility that this region of the ER
␣
may serve to dock
repressor proteins or that direct acetylation of the ER
␣
may
play a role in ligand-dependent transcriptional attenuation, as
was recently described for the direct acetylation of ACTR by
p300 (4). Crystal structural analyses showed the LXXLL motif
of the coactivator GRIP1 forms the core of a short amphipathic
␣
helix that contacts helices 3, 5/6, 11, and 12 of the ER
␣
;
however, the exact proximity of the ER
␣
(K302A/K303A)
residues
FIG.4.A conserved acetylation motif in the ER
␣
.A parallel reaction to that used in Fig. 3Dusing unlabeled acetyl Co-A was analyzed by
MALDI-TOF mass spectrometry (A) and sequenced by Edman degradation (B). In A, the resulting ER-(293–310) peptide mass spectrum is shown
with mass/charge expressed in atomic mass units (amu). The major peak labeled Xcorresponds to the expected mass of the unmodified ER
␣
peptide. The major peak labeled Y, larger by 42 atomic mass units, represents singly acetylated peptide. The minor peaks are methionine oxidation
products present in the starting material. In B, the bars represent the amount of phenylthiohydantoin-acetyl-lysine present in the corresponding
positions. The major acetylated products correspond to residues 302 and 303.
Conserved ER
␣
Acetylation Motif in Nuclear Receptors 18379
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to the ER
␣
hydrophobic fold was not determined
2
(46).
In the current studies, the selective histone deacetylase in-
hibitor TSA induced ER
␣
activity, indicating that histone
acetylase-dependent regulation of ER
␣
activity can occur in the
absence of ligand in cultured cells (Fig. 5, Aand B). The
previous findings that p300 can bind ER
␣
in a ligand-indepen-
dent manner (3, 4, 6, 8), together with the current findings that
p300 acetylates ER
␣
in the absence of ligand, raised the pos-
sibility that ER
␣
may be acetylated in living cells in the ab-
sence of ligand. Alternatively, the addition of ligand may be
required for the acetylation of ER
␣
in cultured cells. This would
seem unlikely, however, as mutations of the ER
␣
acetylation
site, which could not be acetylated in vitro, conveyed enhanced
ligand sensitivity in cultured cells. To determine whether ER
␣
is acetylated in vivo, a polyclonal antibody raised against acety-
lated lysines (16) was used to immunoprecipitate acetylated
proteins from MCF7 cells. The IP product was subjected to
SDS-polyacrylamide gel electrophoresis and probed with an
ER
␣
antibody. Fig. 6Dshows that the ER
␣
antibody specifically
recognized ER
␣
protein within the anti-acetylated lysine im-
munoprecipitate (upper panel). Because the coactivator ACTR
is acetylated by itself (4), the co-immunoprecipitation of the
ER
␣
may potentially be due to cross-reactivity with ACTR.
Therefore, a reciprocal immunoprecipitation was performed in
which we used the ER
␣
antibody to IP ER
␣
from MCF7 cells,
and Western blotting was performed with the anti-acetyl lysine
antibody (Fig. 6D, lower panel). The acetyl lysine immunore-
active band corresponding to the molecular weight of the ER
␣
was observed in the ER
␣
IP but not with the control IgG IP.
Together these studies indicated that the ER
␣
is acetylated in
cultured cells consistent with previous findings that p300 binds
and regulates ER
␣
in the absence of ligand in vivo (4, 6, 8).
DISCUSSION
The regulation of estradiol signaling by direct ER
␣
acetyla-
tion reveals an unexpected and novel role for histone acetyl-
transferase in hormone signaling. Nuclear receptors have been
shown to form multiprotein complexes with coregulatory pro-
teins that possess either histone acetylase or histone deacety-
lase activity (4, 47). The evidence that the ER
␣
is a direct
substrate for HAT activity and may thereby regulate hormone-
dependent transactivation function remained to be demon-
strated. Here we have shown that ER
␣
is acetylated in vivo and
is a substrate for selective acetylation by p300 in vitro. Al-
though cointegrators recruited to ER
␣
share a redundant ca-
pacity to acetylate histones, herein the ER
␣
was selectively
acetylated by p300. The select enzymatic activities of p300 and
PCAF toward ER
␣
are consistent with their structurally diver-
gent HAT domains (36, 48). Mutagenesis demonstrated a crit-
ical role for the ER
␣
acetylation site in regulation by histone
deacetylase inhibitors. The finding that mutations with the
ER
␣
hinge domain lysine residues enhanced hormone sensitiv-
ity suggests these residues may be involved in ligand-depend-
ent transcriptional repression or transcriptional attenuation.
The finding that the lysine residues within the ER
␣
that
are substrates for the HAT activity of p300 may function in
transcriptional repression suggests that cointegrator proteins
acetylate several distinct substrates with distinct effects to
coordinate genomic responses.
The mechanisms governing substrate specificity of HATs are
not well understood at this time (49). P/CAF did not acetylate
ER
␣
but was capable of efficiently acetylating histone H3 and
binding ER
␣
. These findings suggest that p300 and P/CAF,
although both capable of binding ER
␣
, convey select enzymatic
activities, consistent with the lack of sequence similarity
within their HAT domains (36, 48). From previous studies of
TAF
II
250 it is known that the bromodomain modules form
selective interactions with multiple acetylated histone H4 pep-
tides (50). To understand the mechanisms responsible for the
failure of P/CAF to acetylate ER
␣
, we performed an analysis of
P/CAF domain mutants to identify the sites of interaction with
the ER
␣
lysine motif peptide. These studies revealed the sur-
prising result that the P/CAF bromodomain was dispensable
and that the HAT domain was required for binding to ER
␣
.It
is possible that the interaction surfaces may determine subse-
quent acetylase activity. Alternatively, the acetylation motif of
the substrate may be critical. The ER
␣
acetylation motif re-
2
G. Greene, personal communication.
FIG.5. Histone deacetylase regulation of ER
␣
is dependent
upon the ER
␣
acetylation site. Aand B, the ERE-LUC reporter was
co-transfected with expression vectors for the wild type (wt)ER
␣
; cells
were treated with either trichostatin A (TSA)(A) or sodium butyrate
(NaB)(B), and luciferase activity was assessed. In B, cells were also
treated with estradiol (10
⫺7
M) or vehicle for 24 h. C, the point mutant
of the ER
␣
,ER
␣
K302A/K303A
, was assessed for TSA responsiveness and
expression in cultured cells. Mutation of the acetylation site abrogates
induction by TSA but does not affect expression in cultured cells. -Fold
induction of ERE-LUC reporter activity by sodium butyrate is shown in
the presence or absence of E2. The data are the mean ⫾S.E. for at least
nine separate transfections. D, Western blotting for ER
␣
was performed
on ER-deficient 293T cells transfected with the expression plasmids
encoding the wild type ER
␣
and ER
(K302A/K303A)
. Western blotting is
shown using the ER
␣
antibody (upper panel) and the guanine nucleo-
tide dissociation inhibitor (GDI) antibody as a loading control (lower
panel). E, the expression plasmids encoding the wild type ER
␣
and
point mutants of the ER
␣
acetylation site were transfected into HeLa
cells with the ERE-LUC reporter and treated with TSA for 24 h at the
indicated concentrations. Luciferase activity was normalized to the
internal control of Rous sarcoma virus-

-galactosidase. A comparison
was made with equal amounts of empty expression vector cassette. The
-fold induction is shown for wild type ER
␣
and the acetylation point
mutants. The ER
(K302A/K303A)
and ER
(K302T/K303T)
were not induced by
TSA.
Conserved ER
␣
Acetylation Motif in Nuclear Receptors18380
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sembles the GATA-1 and p53 acetylation sites. GATA-1, EKLF,
and ACTR are selectively acetylated by p300/CBP (4, 19, 22).
By contrast, P/CAF preferentially acetylates E2F-1 and MyoD
in vitro (20, 51). p53 contains two acetylation sites differen-
tially acetylated by either p300 (16) or P/CAF (21). Although
the determinants of the histone acetylase substrate preference
are poorly understood, this substrate specificity may form the
biochemical basis for functional synergy and promoter
selectivity.
In the current studies, mutation of the ER
␣
in vitro acetyla-
tion site enhanced ligand sensitivity. The 2-fold induction of
the synthetic estrogen-responsive enhancer reporter gene
ERE
2
TK81pA
3
LUC at 10
⫺8
M17

-estradiol with the wild type
ER
␣
was identical to the induction observed by other investi-
gators in HeLa cells using a similar luciferase reporter gene
(41). Although the magnitude of induction of synthetic estro-
gen-responsive reporters can be enhanced by increasing the
number of ERE enhancer sites, changing the type of minimal
promoter, or altering the cell type (52), the high sensitivity of
the assays allowed clear discrimination of basal compared with
induced activity in the current studies. The expression of the
acetylation site ER
␣
mutants was identical in cultured cells,
allowing a clear comparison of their functional activities. When
comparing between the double point mutants, there was a
tendency for the mutant with substitution of threonine (a hy-
drophobic polar residue) to have higher induction by E2 than
other substitutions (3-fold versus 2-fold). Nonetheless, each
mutation of the lysines within the acetylation motif enhanced
hormone sensitivity compared with wild type ER
␣
(p⬍0.05),
suggesting that the acetylation modification itself govern hor-
mone sensitivity. These findings are consistent with recent
observations in which mutation of an acetylation motif within
the coactivator ACTR resulted in transcriptional attenuation of
ER
␣
signaling (4).
In the current studies, ER
␣
acetylation site mutations that
enhanced ligand sensitivity did not affect ER
␣
activation by the
MAPK signaling pathway, suggesting direct acetylation of the
ER
␣
affects a specific subset of ER
␣
activities. MAPK regula-
tion of ER
␣
involves both direct phosphorylation and regula-
tion of coactivators themselves. Our finding that the ER
␣
acetylation mutation does not affect MAPK signaling distin-
guishes regulation of ER
␣
activity from the mechanisms gov-
erning ER
␣
regulation by the p160 coactivator ACTR/AIB1.
ACTR is phosphorylated and activated by MAPK, contributing
FIG.6.The ER
␣
acetylation site mu-
tants convey enhanced ligand sensi-
tivity in cultured cells with altering
MAPK responsiveness. A, regulation of
wild type or mutant ER
␣
activity by acti-
vating MEK1 mutants ⌬N3-S218E-
S222D and ⌬N3 is shown compared with
either vector or the catalytically inactive
mutant MEK1 (K97M). Results are
shown on the left as the mean ⫾S.E. for
the luciferase activity. B, E2-induced
transactivation of the ERE-LUC reporter
was determined for the wild type and ER
␣
mutants; the mean -fold induction is
shown at each of the E2 concentrations
used. The data are the mean of six sepa-
rate experiments. The S.E. was ⬍3% for
the data points. The ER
␣
mutants were
increased significantly in ligand-induced
activity at each ligand concentration com-
pared with wild type (wt)ER
␣
(p⬍0.05).
C, the effect of p300 on wild type and ER
␣
mutant activity was determined in the
presence and absence of ligand. Data are
the mean ⫾S.E. with significant differ-
ences shown (*, p⬍0.05) compared with
wild type ER
␣
.D, upper panel, MCF7
cells were subjected to IP with polyclonal
anti-acetylated lysine antibody (New
England Biolabs, Beverly, MA), and the
IP product was subjected to Western blot-
ting with the ER
␣
antibody. Lower panel,
MCF7 cells were immunoprecipitated
with an anti-ER
␣
antibody or control IgG
and the electrophoresed product was
subjected to Western blotting with an
anti-acetyl-lysine antibody (16). The im-
munoreactive band detected with the an-
ti-acetyl lysine antibody is of identical
molecular weight to the ER
␣
.
Conserved ER
␣
Acetylation Motif in Nuclear Receptors 18381
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to the Ser
118
-independent, MAPK-dependent activation of ER
␣
(42). ACTR/AIB1 contacts AF-2 and enhances the ER
␣
AF-1
function while recruiting p300 (42). p300 also acetylates ACTR/
AIB1, contributing to ER
␣
ligand-mediated transcriptional at-
tenuation (4). Our observations that ER
␣
acetylation by p300
did not affect MAPK signaling in cultured cells is consistent
with findings that the p300 HAT subdomain is distinct from
the p160 recruitment domain (10). Although post-translational
modification by acetylation and phosphorylation may, under
some circumstances, be integrated processes (1, 53), it is likely
that a subset of specific acetylation events may be regulated
independently of MAPK signaling. The identification of specific
components of the cross-talk between hormone sensitivity and
acetylation will contribute substantially to an improved under-
standing of ER
␣
mitogenic signaling.
Our findings that p300 efficiently acetylated ER
␣
in vitro
and that acetylated ER
␣
is present in MCF7 cells are consist-
ent with a number of recent studies supporting a model in
which the net acetylation of specific transcription factors
within the cell and at sites of local transcriptionally active
promoters are both under dynamic regulation and are re-
pressed coordinate with acetylation events (25, 49). ER
␣
was
found at the estrogen-responsive pS2 promoter in MCF7 cells
together with the coactivators p300, CBP, and ACTR (4). Upon
the addition of estradiol, p300 was recruited quite transiently
to the pS2 promoter prior to dissociation from the site (4).
Ligand-independent binding of p300 to the ER
␣
(6) and a 2-fold
induction of ER
␣
activity in the absence of ligand, using in vitro
transcription assays (8) or in reporter assays (6), together sug-
gest that p300 conveys important ligand-dependent and -inde-
pendent functions. Estradiol treatment of MCF7 for 24 h cells
does not change the abundance of p300, histone deacetylase-1,
or ER
␣
(4), and the induction of histone H4 acetylation at
target promoters in response to ligand is quite transient (4).
Conformational changes induced by the addition of estradiol
are known to recruit p160 coactivators to a hydrophobic fold in
the ER
␣
with the p300 cointegrator (9). As noted above, the
LXXLL motif of the coactivator GRIP1 forms the core of a short
amphipathic
␣
helix that contacts helices 3, 5/6, 11, and 12 of
the ER
␣
; however, the exact proximity of the ER
␣
(K302A/K303A)
residues to the ER
␣
hydrophobic fold remain unknown
2
(46).
Future studies will discern whether the increased ligand sen-
sitivity of these ER
␣
acetylation mutants is due to enhanced
recruitment of coactivators within the local promoter context or
to loss of binding to transcriptional repressors.
These studies raise several important new types of question
regarding the direct acetylation of the ER
␣
affects interactions
with other coactivators and corepressors, DNA binding within
native chromatin at estrogen-responsive promoters of target
genes, the function of the ER
␣
in in vitro transcription assays,
and the effect of these mutations on selectivity of estrogen
signaling pathways. In the current studies, mutational analy-
sis of the ER
␣
acetylation site demonstrated dissociable effects
of histone deacetylase inhibitors (TSA) and the addition of
ligand on ER
␣
activity. The induction of ER
␣
activity by the
histone deacetylase inhibitors TSA and sodium butyrate was
abolished upon substitution of the acetylated lysine residues
with small hydrophobic residues, either alanine or threonine,
suggests that basal ER
␣
activity is under constitutive repres-
sion by histone deacetylase-containing complexes and that the
lysine residues may contribute to a surface recruiting such
complexes. In the absence of ligand, nuclear receptors have
been shown to exist in multiprotein complexes containing N-
CoR (nuclear receptor corepressor) or related proteins (54) to-
gether with histone deacetylases and homologues of the yeast
corepressor Sin3, which repress gene transcription (47, 55, 56).
As estrogen is mitogenic in mammary epithelial cells, the en-
hancement of ligand-dependent transactivation induced by
mutation of these ER
␣
target lysines may be predicted to confer
a growth advantage. The same mutant that we demonstrated
as conveying enhanced ligand sensitivity for transactivation
(ER
␣
(K303R)
) was recently shown to occur in 34% of premalig-
nant human breast lesions, suggesting that these acetylated
residues play an important role in ER
␣
function and biology
(57). The ER
␣
acetylation motif is conserved between species
and between phylogenetically related nuclear receptors (58)
(Fig. 7). Mutations of the conserved lysine motif have been
identified in the ER
␣
in breast cancer as has the androgen
receptor in prostate cancer. Because nuclear receptors that
contain the candidate acetylation motif contribute to diverse
roles in the regulation of growth, development, and homeosta-
sis (1), these studies may have possible implications in under-
standing regulation and function of many nuclear receptors.
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Conserved ER
␣
Acetylation Motif in Nuclear Receptors 18383
by guest on November 24, 2015http://www.jbc.org/Downloaded from
Richard G. Pestell
Gabriela N. Lopez, Peter J. Kushner and
Torsten Hopp, Suzanne A. W. Fuqua,
Benita S. Katzenellenbogen, Shigeaki Kato,
Reutens, Chris Albanese, Michael P. Lisanti,
Angeletti, Linda Siconolfi-Baez, Anne T.
Chenguang Wang, Maofu Fu, Ruth H.
Transactivation and Hormone Sensitivity
Hinge Region by p300 Regulatesα
Direct Acetylation of the Estrogen Receptor
AND DEGRADATION:
POST-TRANSLATION MODIFICATION
PROTEIN SYNTHESIS
doi: 10.1074/jbc.M100800200 originally published online March 9, 2001
2001, 276:18375-18383.J. Biol. Chem.
10.1074/jbc.M100800200Access the most updated version of this article at doi:
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