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,
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
Received for publication, January 29, 2001, and in revised form, March 8, 2001
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ABSTRACT |
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Regulation of nuclear receptor gene expression
involves dynamic and coordinated interactions with histone acetyl
transferase (HAT) and deacetylase complexes. The estrogen
receptor (ER Nuclear receptors coordinate diverse physiological roles in
metabolism and development through ligand-dependent and
-independent mechanisms (1). Nuclear receptors form multiprotein
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). Conformational changes induced upon nuclear receptor ligand binding release corepressors, with subsequent transient association of coactivator proteins (2-4). Estrogen binds the estrogen
receptor (ER The two activation domains of 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 provides a scaffold,
interacting with numerous transcription factors 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.
Transcriptional activation in chromatin-containing systems has
correlated transcriptional activity with acetylation of specific
lysines in the NH2 termini of histones (13, 14). Histone
acetylation 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 induction 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 Reporter Genes, Expression Vectors, and Luciferase
Assays--
The ERE luciferase reporter gene ERE2TK81
pA3LUC (28), the Flag-tagged P/CAF mutants (29), the ER
Cell culture, DNA transfection, and luciferase assays were performed as
previously described (30, 34). Cells were incubated in media containing
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
Protein Expression and Western Blots--
The antibodies used in
Western blot analysis were anti-M2 Flag (Sigma), anti-guanine
nucleotide dissociation inhibitor (35), anti-acetyl lysine (16), and
GST (B-14) and ER
In vitro [35S]methionine-labeled proteins were
prepared by coupled transcription-translation with a Promega
TNT®-coupled reticulocyte lysate kit (Promega), using 1.0 µg of plasmid DNA in a total of 50 µl. 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 Immunoprecipitation Histone Acetyltransferase
Assays--
Immunoprecipitation histone acetyl transferase
(IP-HAT) assays were performed using p300 as described previously
(16, 37). For immunoprecipitation the protein concentration was
adjusted to 1 µg/µl in 500 µl. The relevant antibodies from Santa
Cruz Biotechnology (p300, N15) were added (2 µg/500 µg of extract) and incubated at 4 °C for 2 h. A standard HAT assay was
performed containing 5 µg 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
[14C]acetyl-CoA reaction was electrophoresed on a
SDS-polyacrylamide gel and viewed following autoradiography of the gel.
[14C]acetyl incorporation into the substrates was also
determined by liquid scintillation counting or filter assays.
In Vitro Protein-Protein Interactions and Mapping the
ER
In vitro acetylation assays were performed as
described previously 1(7). Synthetic peptide corresponding to the ER The ER The ER
P/CAF has been reported to associate with ER Identification of the ER
Peptides were synthesized to encompass the two lysine-containing motifs
identified within the region of the ER
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 unmodified ER1 peptide and the
higher molecular weight component corresponding to the acetylated ER1
product (Fig. 4A). Following
in vitro acetylation of the ER1 peptide, Edman degradation
assays were performed. As only monoacetylated lysine-containing
peptides were detected in the samples by MALDI-TOF mass spectrometry,
the product analyzed by Edman degradation was a heterogeneous
population of polypeptides, each acetylated at a single site (Fig.
4A). These studies demonstrated that lysines 302 and 303 of
the ER The ER MAPK-induced ER
Assessment was made of the AF-1 function mediated by MAPK signaling.
Growth factors induce ligand-independent activity of the ER The ER
We next assessed the role of the hinge domain lysine residues in
p300-dependent regulation of ER
In the current studies, the selective histone deacetylase inhibitor TSA
induced ER The regulation of estradiol signaling by direct ER The mechanisms governing substrate specificity of HATs are not well
understood at this time (49). P/CAF did not acetylate ER In the current studies, mutation of the ER In the current studies, ER Our findings that p300 efficiently acetylated ER These studies raise several important new types of question regarding
the direct acetylation of the ER) contains two transactivation domains regulating
ligand-independent and -dependent gene transcription (AF-1
and AF-2 (activation functions 1 and 2)). ER
-regulated gene
expression 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
induction by MAPK signaling, suggesting that direct ER
acetylation
normally suppresses ligand sensitivity. These ER
lysine residues
also regulated transcriptional 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 metabolism and development.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
),1 thereby
regulating important functions in development and reproduction and in
human diseases including breast cancer, cardiovascular disease,
osteoporosis, and Alzheimer's disease. The ER
contains domains
conserved with other members of the "classical" receptor subclass
(termed A
F) and two activation domains, AF (activation function)-1
and AF-2.
contribute synergistically 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 contributes
to ER
induction through several separable subdomains including the
histone acetyl transferase (HAT) and the bromodomain (4, 8, 10), which
make separate contacts to distinct domains of the ER
.
transcriptional regulatory proteins, which include
p300, CBP, P/CAF, SRC1, and ACTR (26, 27). Redundancy of the HAT
function among cointegrators 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 -independent 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 phylogenetically related
nuclear receptors.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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 sequence-specific primers. Both
the wild type ER
and ER
mutants were cloned into
pCI-neo (Promega, Madison, WI). The integrity of all
constructs was confirmed by sequence analysis.
-galactosidase reporters or Renilla luciferase as
an internal control exactly as described previously (20).
(H-184) antibodies from Santa Cruz
Biotechnology (Santa Cruz, CA).
was obtained from Affinity
Bioreagents, Inc. (Golden, CO).
Acetylation Sites--
The interactions between in
vitro expressed proteins was performed as described previously
(38). The in vitro translated protein (15 µl of ER
), 1 µg of rabbit anti-ER
polyclonal antibody (H184, Santa Cruz
Biotechnology), and 5 µg of purified Flag-tagged baculovirus-expressed P/CAF were incubated in 300 µl of binding buffer.
(ER1, residues 293-310, NH2-PSPLMIKRSKKNSLALSL-OH, and
ER2, residues 353-370, NH2-ELVHMINWAKRVPGFVDL-OH) were
synthesized by Bio·Synthesis (Lewisville, TX) and purified to 95%
purity by HPLC. The peptides were acetylated in vitro by
incubation with 5 mM acetyl-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 products 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
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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
baculovirus-expressed P/CAF did not acetylate ER
, although it was
capable of acetylating histone H3 and itself (Fig. 1B) as
shown previously (39).
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Fig. 1.
p300 acetylates the ER
C-terminal to the zinc finger DNA binding 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 [14C]acetyl-Co-A
([14C]Ac-p300). The arrow
indicates the autoradiogram of the acetylated ER
fusion 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 baculovirus-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
autoacetylated and acetylated core histones H3 and H4 but did not
acetylate 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 acetylate 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
[14C]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 selectively acetylated by p300 in vitro.
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Fig. 2.
ER is an efficient
substrate for p300 acetylation. A
and B, HAT assays were performed using a constant amount of
enzyme and equimolar amounts of either ER
or histone H3 substrate.
B, the acetylated bands were excised 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 antibody.
Western blotting was used to detect P/CAF, and ER
was visualized by
autoradiography. Interactive domains identified 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.
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 failed binding. These findings are
consistent with the observation that p300 and P/CAF have
distinguishable substrate specificities (21).
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 recombinant p300
as a source of HAT activity and the previously described filter assay
(16). As shown in Fig. 3, B
and C, the ER
from residues 282-337 was sufficient to
function as a substrate for acetylation by p300.
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Fig. 3.
Mapping p300-mediated acetylation sites of
the ER . A, schematic
representation of the ER
(indicating 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 14C-labeled ER
proteins (lower
panel). C, p300-mediated in vitro IP-HAT
assays were performed using equal amounts of GST-ER
fusion protein.
The products corresponding to the expected molecular weight were
excised and HAT activity quantitated by liquid scintillation counting.
D, ER
peptide corresponding to either ER-(293-310)
(ER1) or ER-(353-370) (ER2) were used as
in vitro substrates with 14C-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 homologous to the acetylation
motif of the murine GATA-1 and human p53 proteins. The ER-(293-310)
peptide was selectively acetylated by p300.
acetylated 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 acetylate ER2 (Fig. 3D). Recombinant P/CAF
failed to acetylate either ER polypeptides.
were preferentially acetylated by p300 with an additional
acetylation site at lysine 299 (Fig. 4B).
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Fig. 4.
A conserved acetylation motif in the
ER . A parallel reaction to that used in
Fig. 3D using 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
X corresponds 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.
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 luciferase
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 (ERE2TKpA3LUC) 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 normalization 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 abundance 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.
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Fig. 5.
Histone deacetylase regulation of
ER is dependent upon the ER
acetylation site. A and 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 nucleotide 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.
Functions Independently of the ER
Acetylation Site--
To investigate further the in vivo
consequence of the ER
acetylation site, point mutation substitutions
were introduced into the wild type ER
at the lysine residues
acetylated in vitro. It was reasoned that the acetylation 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
residue (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 substitution 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.
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Fig. 6.
The ER acetylation
site mutants convey enhanced ligand sensitivity in cultured cells
with altering MAPK responsiveness. A,
regulation of wild type or mutant ER
activity by activating 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 separate experiments. The S.E. was
<3% for the data points. The ER
mutants were increased
significantly in ligand-induced activity at each ligand concentration
compared 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 differences 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 blotting 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 immunoreactive band detected with
the anti-acetyl lysine antibody is of identical molecular weight to 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-dependent 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
acetylation 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.
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 luciferase 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 ERE2TK81LUC reporter is not
induced by 10
10 M E2 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-dependent 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
enhanced ligand sensitivity. As each of the
ER
mutants exhibited 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.
function. The modest
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 observed 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 to the ER
hydrophobic
fold was not determined2
(46).
activity, indicating that histone
acetylase-dependent regulation of ER
activity can occur in
the absence of ligand in cultured cells (Fig. 5, A and
B). The previous findings that p300 can bind ER
in a
ligand-independent manner (3, 4, 6, 8), together with the current
findings that p300 acetylates ER
in the absence of ligand, raised
the possibility that ER
may be acetylated in living cells in the
absence 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
acetylated 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. 6D shows that the ER
antibody specifically
recognized ER
protein within the anti-acetylated lysine
immunoprecipitate (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 immunoreactive 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
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
acetylation
reveals an unexpected and novel role for histone acetyltransferase in
hormone signaling. Nuclear receptors have been shown to form multiprotein complexes with coregulatory proteins that possess either
histone acetylase or histone deacetylase 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 demonstrated. Here we have shown that ER
is
acetylated in vivo and is a substrate for selective
acetylation by p300 in vitro. Although cointegrators
recruited to ER
share a redundant capacity 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 divergent HAT domains (36, 48). Mutagenesis
demonstrated a critical 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
sensitivity suggests these residues may be involved in
ligand-dependent 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.
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 TAFII250 it is known that the
bromodomain modules form selective interactions with multiple
acetylated histone H4 peptides (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 surprising 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 subsequent acetylase
activity. Alternatively, the acetylation motif of the substrate may be
critical. The ER
acetylation motif resembles 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 differentially 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 vitro
acetylation site enhanced ligand sensitivity. The 2-fold induction of the synthetic estrogen-responsive enhancer reporter gene
ERE2TK81pA3LUC at 10
8
M 17
-estradiol with the wild type ER
was identical to
the induction observed by other investigators in HeLa cells using a
similar luciferase reporter gene (41). Although the magnitude of
induction of synthetic estrogen-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 hydrophobic 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 hormone 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).
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 regulation of ER
involves
both direct phosphorylation and regulation of coactivators themselves.
Our finding that the ER
acetylation mutation does not affect
MAPK signaling distinguishes regulation of ER
activity from the
mechanisms governing ER
regulation by the p160 coactivator ACTR/AIB1. ACTR is phosphorylated and activated by MAPK, contributing to the Ser118-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
attenuation (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 understanding of ER
mitogenic signaling.
in vitro
and that acetylated ER
is present in MCF7 cells are consistent 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 repressed 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 suggest that p300 conveys important
ligand-dependent and -independent 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 unknown2 (46). Future studies will
discern whether the increased ligand sensitivity 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.
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 analysis 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 repression 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) together 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 enhancement 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 premalignant 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 homeostasis (1), these studies may have possible implications in
understanding regulation and function of many nuclear receptors.
View larger version (40K):
[in a new window]
Fig. 7.
Phylogenetic conservation of the
acetylation motif. The phylogenetic tree connecting nuclear
receptor genes in vertebrates, arthropods, and nematodes is shown
(adapted from Ref. 58). Nuclear receptors containing the acetylation
motif are in yellow, and nuclear receptors lacking the motif
in the 4A and 2B subgroups are in pink.
![]() |
FOOTNOTES |
---|
* 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 Institutes of Health Grant 5-P30-CA13330-26. The proteomic analysis performed 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. The 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.
Published, JBC Papers in Press, March 9, 2001, DOI 10.1074/jbc.M100800200
2 G. Greene, personal communication.
![]() |
ABBREVIATIONS |
---|
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-response element-binding protein)-binding
protein;
IP, immunoprecipitation;
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.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. |
Glass, C. K.,
and Rosenfeld, M. G.
(2000)
Genes Dev.
14,
121-141 |
2. |
McKenna, J.,
Lanz, R. B.,
and O'Malley, B. W.
(1999)
Endocr. Rev.
20,
321-344 |
3. | Shang, Y., Hu, X., DiRenzo, J., Lazar, M., and Brown, M. (2000) Cell 103, 843-852[Medline] [Order article via Infotrieve] |
4. | Chen, H., Lin, R. J., Xie, W., Wilpitz, D., and Evans, R. M. (1999) Cell 98, 675-686[Medline] [Order article via Infotrieve] |
5. | Kato, S., Endoh, H., Masuhiro, Y., Kitamoto, T., Uchiyama, S., Sasaki, H., Masushige, S., Gotoh, Y., Nishida, E., Kawashima, H., Metzger, D., and Chambon, P. (1995) Science 270, 1491-1494[Abstract] |
6. |
Kobayashi, Y.,
Kitamoto, T.,
Masuhiro, Y.,
Watanabe, M.,
Kase, T.,
Metzger, D.,
Yanagisawa, J.,
and Kato, S.
(2000)
J. Biol. Chem.
275,
15645-15651 |
7. |
Endoh, H.,
Maruyama, K.,
Masuhiro, Y.,
Kobayashi, Y.,
Goto, M.,
Tai, H.,
Yanagisawa, J.,
Metzger, D.,
Hashimoto, S.,
and Kato, S.
(1999)
Mol. Cell. Biol.
19,
5363-5372 |
8. |
Kraus, W. L.,
and Kadonaga, J. T.
(1998)
Genes Dev.
12,
331-342 |
9. |
Hanstein, B.,
Eckner, R.,
DiRenzo, J.,
Halachmi, S.,
Liu, H.,
Searcy, B.,
Kurokawa, R.,
and Brown, M.
(1996)
Proc. Natl. Acad. Sci. U. S. A.
93,
11540-11545 |
10. |
Kraus, W. L.,
Manning, E. T.,
and Kadonaga, J. T.
(1999)
Mol. Cell. Biol.
19,
8123-8135 |
11. | Nakajima, T., Uchida, C., Anderson, S. F., Lee, C.-G., Hurwitz, J., Parvin, J. D., and Montminy, M. (1997) Cell 90, 1107-1112[Medline] [Order article via Infotrieve] |
12. | Giordano, A., and Avantaggiati, M. L. (1999) J. Cell. Physiol. 181, 218-230[CrossRef][Medline] [Order article via Infotrieve] |
13. |
Wang, L.,
Liu, L.,
and Berger, S. L.
(1998)
Genes Dev.
12,
640-653 |
14. |
Kuo, M.-H.,
Zhou, J.,
Jambeck, P.,
Churchill, M. E. A.,
and Allis, C. D.
(1998)
Genes Dev.
12,
627-639 |
15. |
Struhl, K.
(1998)
Genes Dev.
12,
599-606 |
16. | Gu, W., and Roeder, R. G. (1997) Cell 90, 595-606[Medline] [Order article via Infotrieve] |
17. | Boyes, J., Byfield, P., Nakatani, Y., and Ogryzko, V. (1998) Nature 396, 594-598[CrossRef][Medline] [Order article via Infotrieve] |
18. | Munshi, N., Merika, M., Yie, J., Senger, K., Chen, G., and Thanos, D. (1998) Mol. Cell 2, 457-467[Medline] [Order article via Infotrieve] |
19. |
Zhang, W.,
and Bieker, J. J.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
9855-9860 |
20. |
Martinez-Balbas, M. A.,
Bauer, U.-M.,
Nielson, S., J.,
Brehm, A.,
and Kouzarides, T.
(2000)
EMBO J.
19,
662-671 |
21. |
Liu, L.,
Scolnick, D. M.,
Trievel, R. C.,
Zhang, H. B.,
Marmorstein, R.,
Halazonetis, T. D.,
and Berger, S. L.
(1999)
Mol. Cell. Biol.
19,
1202-1209 |
22. |
Hung, H. L.,
Lau, J.,
Kim, A. Y.,
Weiss, M. J.,
and Blobel, G. A.
(1999)
Mol. Cell. Biol.
19,
3496-3505 |
23. | Berger, S. L. (1999) Curr. Opin. Cell Biol. 11, 336-341[CrossRef][Medline] [Order article via Infotrieve] |
24. | Walter, L., and Bienz, M. (1998) Nature 395, 521-525[CrossRef][Medline] [Order article via Infotrieve] |
25. | Kouzarides, T. (1999) Curr. Opin. Genet. Dev. 9, 40-48[CrossRef][Medline] [Order article via Infotrieve] |
26. | Kadonaga, J. T. (1998) Cell 92, 307-313[Medline] [Order article via Infotrieve] |
27. |
Martinez-Balbas, M. A.,
Bannister, A. J.,
Martin, K.,
Haus-Seuffert, P.,
Meisterernst, M.,
and Kouzarides, T.
(1998)
EMBO J.
17,
2886-2893 |
28. |
Schlegel, A.,
Wang, C.,
Katzenellenbogen, B.,
Pestell, R. G.,
and Lisanti, M. P.
(1999)
J. Biol. Chem.
274,
33551-33556 |
29. | Hamamori, Y., Sartorelli, V., Ogryzko, V., Puri, P. L., Wu, H. Y., Wang, J. Y., Nakatani, Y., and Kedes, L. (1999) Cell 96, 405-413[Medline] [Order article via Infotrieve] |
30. |
Fan, S.,
Wang, J.-A.,
Yuan, R.,
Ma, Y.,
Meng, Q.,
Erdos, M. R.,
Pestell, R. G.,
Yuan, F.,
Auborn, K. J.,
Goldberg, I. D.,
and Rosen, E. M.
(1999)
Science
284,
1354-1356 |
31. |
Albanese, C.,
D'Amico, M.,
Reutens, A. T.,
Fu, M.,
Watanabe, G.,
Lee, R. J.,
Kitsis, R. N.,
Henglein, B.,
Avantaggiati, M.,
Somasundaram, K.,
Thimmapaya, B.,
and Pestell, R. G.
(1999)
J. Biol. Chem.
274,
34186-34195 |
32. | Mansour, S. J., Matten, W. T., Hermann, A. S., Candia, J. M., Rong, S., Fukasawa, K., Vande Woude, G. F., and Ahn, N. G. (1994) Science 265, 966-969[Medline] [Order article via Infotrieve] |
33. |
Watanabe, G.,
Howe, A.,
Lee, R. J.,
Albanese, C.,
Shu, I.-W.,
Karnezis, A. N.,
Zon, L.,
Kyriakis, J.,
Rundell, K.,
and Pestell, R. G.
(1996)
Proc. Natl. Acad. Sci. U. S. A.
93,
12861-12866 |
34. |
Watanabe, G.,
Albanese, C.,
Lee, R. J.,
Reutens, A.,
Vairo, G.,
Henglein, B.,
and Pestell, R. G.
(1998)
Mol. Cell. Biol.
18,
3212-3222 |
35. |
Lee, R. J.,
Albanese, C.,
Fu, M.,
D'Amico, M.,
Lin, B.,
Watanabe, G.,
Haines, G. K. I.,
Siegel, P. M.,
Hung, M. C.,
Yarden, Y.,
Horowitz, J. M.,
Muller, W. J.,
and Pestell, R. G.
(2000)
Mol. Cell. Biol.
20,
672-683 |
36. | Ogryzko, V. V., Schiltz, R. L., Russanova, V., Howard, B. H., and Nakatani, Y. (1996) Cell 87, 953-959[Medline] [Order article via Infotrieve] |
37. | Bannister, A. J., and Kouzarides, T. (1996) Nature 384, 641-643[CrossRef][Medline] [Order article via Infotrieve] |
38. | Neuman, E., Ladha, M. H., Lin, N., Upton, T. M., Miller, S. J., DiRenzo, J., Pestell, R. G., Hinds, P. W., Dowdy, S. F., Brown, M., and Ewen, M. E. (1997) Mol. Cell. Biol. 17, 5338-5347[Abstract] |
39. |
Schiltz, R. L.,
Mizzen, C. A.,
Vassilev, A.,
Cook, R. G.,
Allis, C. D.,
and Nakatani, Y.
(1999)
J. Biol. Chem.
274,
1189-1192 |
40. |
McMahon, C.,
Suthiphongchai, T.,
DiRenzo, J.,
and Ewen, M. E.
(1999)
Proc. Natl. Acad. Sci. U. S. A.
96,
5382-5387 |
41. | Henttu, P. M., Kalkhoven, E., and Parker, M. G. (1997) Mol. Cell. Biol. 17, 1832-1839[Abstract] |
42. |
Font de Mora, J.,
and Brown, M.
(2000)
Mol. Cell. Biol.
20,
5041-5047 |
43. | Chen, H., Lin, R. J., Schiltz, R. L., Chakravarti, D., Nash, A., Nagy, L., Privalsky, M. L., Nakatani, Y., and Evans, R. M. (1997) Cell 90, 569-580[Medline] [Order article via Infotrieve] |
44. |
Webb, P.,
Nguyen, P.,
Shinsako, J.,
Anderson, C.,
Feng, W.,
Nguyen, M. P.,
Chen, D.,
Huang, S. M.,
Subramanian, S.,
McKinerney, E.,
Katzenellenbogen, B. S.,
Stallcup, M. R.,
and Kushner, P. I.
(1998)
Mol. Endocrinol.
12,
1605-1618 |
45. | Tremblay, A., Tremblay, G. B., Labrie, F., and Giguere, V. (1999) Mol. Cell. 3, 513-519[Medline] [Order article via Infotrieve] |
46. | Shiau, A. K., Barstad, D., Loria, P. M., Cheng, L., Kushner, P. J., Agard, D. A., and Greene, G. L. (1998) Cell 95, 927-937[Medline] [Order article via Infotrieve] |
47. |
Lavinsky, R. M.,
Jepsen, K.,
Heinzel, T.,
Torchia, J.,
Mullen, T. M.,
Schiff, R.,
Del-Rio, A. L.,
Ricote, M.,
Ngo, S.,
Gemsch, J.,
Hilsenbeck, S. G.,
Osborne, C. K.,
Glass, C. K.,
Rosenfeld, M. G.,
and Rose, D. W.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
2920-2925 |
48. | Ogryzko, V. V., Kotani, T., Zhang, X., Schlitz, R. L., Howard, T., Yang, X. J., Howard, B. H., Qin, J., and Nakatani, Y. (1998) Cell 94, 35-44[Medline] [Order article via Infotrieve] |
49. |
Kouzarides, T.
(2000)
EMBO J.
19,
1176-1179 |
50. |
Jackson, R. H.,
Ladurner, A. G.,
King, D. S.,
and Tjian, R.
(2000)
Science
288,
1422-1425 |
51. | Sartorelli, V., Puri, P. L., Hamamori, Y., Ogryzko, V., Chung, G., Nakatani, Y., Wang, J. Y. J., and Kedes, L. (1999) Mol. Cell. 4, 725-734[Medline] [Order article via Infotrieve] |
52. | Berry, M., Metzger, D., and Chambon, P. (1990) EMBO J. 2811-8 |
53. |
Espinos, E.,
Le Van Thai, A.,
Pomies, C.,
and Weber, M. J.
(1999)
Mol. Cell. Biol.
19,
3474-3484 |
54. | Jepsen, K., Hermanson, O., Onami, T. M. G., A. S., Lunyak, V., Kumar, V., Liu, F., Seto, E., Hedrick, S. M., Mandel, G., Glass, C. K., Rose, D. W., and Rosenfeld, M. G. (2000) Cell 102, 753-763[Medline] [Order article via Infotrieve] |
55. | Alland, L., Muhle, R., Hou, H. J., Potes, J., Chin, L., Schreiber-Agus, N., and DePinho, R. A. (1997) Nature 387, 49-55[CrossRef][Medline] [Order article via Infotrieve] |
56. | Taunton, J., Hassig, C. A., and Schreiber, S. L. (1996) Science 272, 408-411[Abstract] |
57. |
Fuqua, S. A.,
Wiltschke, C.,
Zhang, Q. X.,
Borg, A.,
Castles, C. G.,
Friedrichs, W. E.,
Hopp, T.,
Hilsenbeck, S.,
Mohsin, S.,
O'Connell, P.,
and Allred, D. C.
(2000)
Cancer Res.
60,
4026-4029 |
58. | Auwerx, J., Baulieu, E., Beato, M., Becker-Andre, M., Burbach, P. H., Camerino, G., Chambon, P., Cooney, A., Dejean, A., Dreyer, C., Evans, R. M., Gannon, F., Giguere, V., Gronemeyer, H., Gustafsson, J. A., Laudet, V., Lazar, M. A., Mangelsdorf, D. J., Milbrandt, J., Milgrom, E., et al. (1999) Cell 161-163 |