Direct Acetylation of the Estrogen Receptor alpha  Hinge Region by p300 Regulates Transactivation and Hormone Sensitivity*

Chenguang WangDagger , Maofu FuDagger , Ruth H. AngelettiDagger , Linda Siconolfi-BaezDagger , Anne T. ReutensDagger , Chris AlbaneseDagger , Michael P. Lisanti§, Benita S. Katzenellenbogen, Shigeaki Kato||, Torsten Hopp**, Suzanne A. W. Fuqua**, Gabriela N. LopezDagger Dagger , Peter J. KushnerDagger Dagger , and Richard G. PestellDagger §§

From the Dagger  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 Dagger Dagger  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


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
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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 (ERalpha ) contains two transactivation domains regulating ligand-independent and -dependent gene transcription (AF-1 and AF-2 (activation functions 1 and 2)). ERalpha -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 ERalpha is acetylated in vivo. p300, but not P/CAF, selectively and directly acetylated the ERalpha at lysine residues within the ERalpha hinge/ligand binding domain. Substitution of these residues with charged or polar residues dramatically enhanced ERalpha hormone sensitivity without affecting induction by MAPK signaling, suggesting that direct ERalpha acetylation normally suppresses ligand sensitivity. These ERalpha lysine residues also regulated transcriptional activation by histone deacetylase inhibitors and p300. The conservation of the ERalpha 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
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 (ERalpha ),1 thereby regulating important functions in development and reproduction and in human diseases including breast cancer, cardiovascular disease, osteoporosis, and Alzheimer's disease. The ERalpha 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.

The two activation domains of ERalpha 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 ERalpha activity 2-3-fold in either reporter or in vitro transcription assays (6, 8). p300/CBP binding to ERalpha is also detectable in MCF7 cells in the absence of ligand (4). The ligand-dependent transactivation function (AF-2) domain of ERalpha 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 ERalpha cointegrator is complex; p300 contributes to ERalpha 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 ERalpha .

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 ERalpha 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 ERalpha is acetylated in vivo and is directly and selectively acetylated by p300, but not by P/CAF, within the ERalpha 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 ERalpha . Conservation of the ERalpha 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
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MATERIALS AND METHODS
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Reporter Genes, Expression Vectors, and Luciferase Assays-- The ERE luciferase reporter gene ERE2TK81 pA3LUC (28), the Flag-tagged P/CAF mutants (29), the ERalpha fusion proteins (30), pcDNA3-HA-p300 (31), the constitutively active MEK1 plasmids, pCMV-Delta N3, pCMV-RDelta F (Delta N3-S218E-S222D), and the catalytically inactive mutant MEK1 (K97M) (32, 33) were described previously. The ERalpha mutants were derived by polymerase chain reaction-directed amplification using sequence-specific primers. Both the wild type ERalpha and ERalpha mutants were cloned into pCI-neo (Promega, Madison, WI). The integrity of all constructs was confirmed by sequence analysis.

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 beta -galactosidase reporters or Renilla luciferase as an internal control exactly as described previously (20).

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 ERalpha (H-184) antibodies from Santa Cruz Biotechnology (Santa Cruz, CA).

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 ERalpha was obtained from Affinity Bioreagents, Inc. (Golden, CO).

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 ERalpha Acetylation Sites-- The interactions between in vitro expressed proteins was performed as described previously (38). The in vitro translated protein (15 µl of ERalpha ), 1 µg of rabbit anti-ERalpha 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.

In vitro acetylation assays were performed as described previously 1(7). Synthetic peptide corresponding to the ERalpha (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
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The ERalpha 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 ERalpha (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 ERalpha in vitro. Recombinant p300 acetylated recombinant ERalpha but did not acetylate GST (Fig. 1A). In contrast, recombinant baculovirus-expressed P/CAF did not acetylate ERalpha , 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 ERalpha 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-ERalpha 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 ERalpha 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 ERalpha . B, the baculovirus-expressed full-length ERalpha protein or core histones were used as substrates in HAT assays using either full-length p300 or P/CAF. p300 acetylated the ERalpha and autoacetylated. P/CAF autoacetylated and acetylated core histones H3 and H4 but did not acetylate the ERalpha .

The ERalpha 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 ERalpha acetylation and, second, whether the failure of P/CAF to acetylate the ERalpha is due to failed binding or substrate selectivity. To assess the relative efficiency with which p300 acetylates the ERalpha , a direct comparison was made between equimolar amounts of ERalpha 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 ERalpha (66 kDa) (Fig. 2B), suggesting ERalpha is acetylated with substantial efficiency. Thus the ERalpha is efficiently and selectively acetylated by p300 in vitro.


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Fig. 2.   ERalpha 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 ERalpha 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 ERalpha . Protein complexes were immunoprecipitated by anti-Flag antibody. Western blotting was used to detect P/CAF, and ERalpha 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.

P/CAF has been reported to associate with ERalpha in vitro (40). We examined whether the recombinant P/CAF used in the HAT assays bound to the ERalpha . As shown in Fig. 2C, recombinant P/CAF bound with high affinity to ERalpha , and binding required the HAT domain. Thus, although P/CAF acetylates histone H3 and H4, the failure of P/CAF to acetylate ERalpha is not due to failed binding. These findings are consistent with the observation that p300 and P/CAF have distinguishable substrate specificities (21).

Identification of the ERalpha Acetylation Sites-- To identify the residues required for ERalpha acetylation in vitro, recombinant GST-ERalpha 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 ERalpha 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 ERalpha . A, schematic representation of the ERalpha (indicating the A-F domains, DNA binding domain (DBD), the ligand binding domain (LBD), and the conserved RXKK motif) and the GST-ERalpha fusion proteins. B, the Coomassie-stained gel corresponding to the GST-ERalpha fusion proteins (upper panel) and the 14C-labeled ERalpha proteins (lower panel). C, p300-mediated in vitro IP-HAT assays were performed using equal amounts of GST-ERalpha fusion protein. The products corresponding to the expected molecular weight were excised and HAT activity quantitated by liquid scintillation counting. D, ERalpha 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 ERalpha 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.

Peptides were synthesized to encompass the two lysine-containing motifs identified within the region of the ERalpha 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 ERalpha 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.

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 ERalpha 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 ERalpha . 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 ERalpha 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.

The ERalpha Acetylated Residues Regulate Basal Activation of the ERalpha by TSA-- To examine the role of histone acetylases in the regulation of ERalpha activity, an estrogen-responsive luciferase reporter gene was assessed in ERalpha -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 ERalpha function, point mutation of the ERalpha acetylation sites was performed. The ER-responsive reporter was assessed in ERalpha -deficient cells (MDA MB231 and HeLa). Activity was assessed through normalization to the internal standard beta -galactosidase reporter. The 2-fold induction of wild type ERalpha by sodium butyrate was abolished by the ER(K302A/K303A) mutant (Fig. 5C). The abundance of the ERalpha K302A/K303A mutant was similar to ERalpha wild type in cultured cells (Fig. 5D). HeLa cells were transfected with either wild type ERalpha or mutants of the acetylation site and assessed for ERE activity. The wild type ERalpha 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 ERalpha acetylation contributes to induction by histone deacetylase inhibitors.


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Fig. 5.   Histone deacetylase regulation of ERalpha is dependent upon the ERalpha acetylation site. A and B, the ERE-LUC reporter was co-transfected with expression vectors for the wild type (wt) ERalpha ; 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 ERalpha , ERalpha 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 ERalpha was performed on ER-deficient 293T cells transfected with the expression plasmids encoding the wild type ERalpha and ER(K302A/K303A). Western blotting is shown using the ERalpha 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 ERalpha and point mutants of the ERalpha 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-beta -galactosidase. A comparison was made with equal amounts of empty expression vector cassette. The -fold induction is shown for wild type ERalpha and the acetylation point mutants. The ER(K302A/K303A) and ER(K302T/K303T) were not induced by TSA.

MAPK-induced ERalpha Functions Independently of the ERalpha Acetylation Site-- To investigate further the in vivo consequence of the ERalpha acetylation site, point mutation substitutions were introduced into the wild type ERalpha 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 ERalpha 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 ERalpha 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 ERalpha proteins were expressed equally in transfected cells (data not shown). HeLa cells were transfected with either wild type ERalpha 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 ERalpha acetylation site mutants convey enhanced ligand sensitivity in cultured cells with altering MAPK responsiveness. A, regulation of wild type or mutant ERalpha activity by activating MEK1 mutants Delta N3-S218E-S222D and Delta 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 ERalpha 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 ERalpha mutants were increased significantly in ligand-induced activity at each ligand concentration compared with wild type (wt) ERalpha (p < 0.05). C, the effect of p300 on wild type and ERalpha 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 ERalpha . 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 ERalpha antibody. Lower panel, MCF7 cells were immunoprecipitated with an anti-ERalpha 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 ERalpha .

Assessment was made of the AF-1 function mediated by MAPK signaling. Growth factors induce ligand-independent activity of the ERalpha 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 ERalpha (44, 45). To determine whether the lysine substitutions within the ERalpha hinge regulated MAPK-dependent ERalpha activity, constitutively activated MEK1 (Delta N3, Delta N3-S218E-S222D) were coexpressed with the ERalpha mutants (Fig. 6A). The wild type ERalpha 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 ERalpha (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 ERalpha acetylation mutants are not altered in their responsiveness to MAPK activation suggests the mechanisms governing ligand-induced ERalpha activity through the ERalpha acetylation site are distinct from those governed by ACTR.

The ERalpha Acetylation Site Governs Ligand Sensitivity-- In previous studies of ERalpha 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 ERalpha 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 ERalpha ; 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 ERalpha for ligand-dependent transactivation using increasing concentrations of E2. Enhanced E2-dependent activity was observed for each of the ERalpha mutations of the hinge region lysine residues. Thus, uncharged, polar, or hydrophobic substitutions of the ERalpha enhanced ligand sensitivity. As each of the ERalpha mutants exhibited similar levels of expression to wild type ERalpha , and the wild type ERalpha functioned in the same manner as the ERalpha wild type in other studies in this cell type (41), these findings suggest that the wild type lysine residues within the ERalpha hinge region may play a role in normally repressing ligand-dependent ERalpha activity.

We next assessed the role of the hinge domain lysine residues in p300-dependent regulation of ERalpha function. The modest induction of wild type ERalpha activity by p300 in the absence of ligand (Fig. 6C) is consistent with studies by others. Binding of p300 to the ERalpha in the absence of ligand and a 2-3-fold induction of ERalpha 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 ERalpha with the p300 cointegrator (9). Because mutation of the lysine residues of the ERalpha enhanced ligand sensitivity, we hypothesized that substitutions of these lysines may also enhance p300-dependent transactivation of the ERalpha in the presence of E2. In keeping with this model each of the ERalpha acetylation mutants demonstrated enhanced activation by p300 in the presence of hormone (Fig. 6C). These findings raise the possibility that this region of the ERalpha may serve to dock repressor proteins or that direct acetylation of the ERalpha 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 alpha  helix that contacts helices 3, 5/6, 11, and 12 of the ERalpha ; however, the exact proximity of the ERalpha (K302A/K303A) residues to the ERalpha hydrophobic fold was not determined2 (46).

In the current studies, the selective histone deacetylase inhibitor TSA induced ERalpha activity, indicating that histone acetylase-dependent regulation of ERalpha activity can occur in the absence of ligand in cultured cells (Fig. 5, A and B). The previous findings that p300 can bind ERalpha in a ligand-independent manner (3, 4, 6, 8), together with the current findings that p300 acetylates ERalpha in the absence of ligand, raised the possibility that ERalpha may be acetylated in living cells in the absence of ligand. Alternatively, the addition of ligand may be required for the acetylation of ERalpha in cultured cells. This would seem unlikely, however, as mutations of the ERalpha acetylation site, which could not be acetylated in vitro, conveyed enhanced ligand sensitivity in cultured cells. To determine whether ERalpha 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 ERalpha antibody. Fig. 6D shows that the ERalpha antibody specifically recognized ERalpha protein within the anti-acetylated lysine immunoprecipitate (upper panel). Because the coactivator ACTR is acetylated by itself (4), the co-immunoprecipitation of the ERalpha may potentially be due to cross-reactivity with ACTR. Therefore, a reciprocal immunoprecipitation was performed in which we used the ERalpha antibody to IP ERalpha 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 ERalpha was observed in the ERalpha IP but not with the control IgG IP. Together these studies indicated that the ERalpha is acetylated in cultured cells consistent with previous findings that p300 binds and regulates ERalpha in the absence of ligand in vivo (4, 6, 8).

    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The regulation of estradiol signaling by direct ERalpha 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 ERalpha is a direct substrate for HAT activity and may thereby regulate hormone-dependent transactivation function remained to be demonstrated. Here we have shown that ERalpha is acetylated in vivo and is a substrate for selective acetylation by p300 in vitro. Although cointegrators recruited to ERalpha share a redundant capacity to acetylate histones, herein the ERalpha was selectively acetylated by p300. The select enzymatic activities of p300 and PCAF toward ERalpha are consistent with their structurally divergent HAT domains (36, 48). Mutagenesis demonstrated a critical role for the ERalpha acetylation site in regulation by histone deacetylase inhibitors. The finding that mutations with the ERalpha 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 ERalpha 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 ERalpha but was capable of efficiently acetylating histone H3 and binding ERalpha . These findings suggest that p300 and P/CAF, although both capable of binding ERalpha , 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 ERalpha , we performed an analysis of P/CAF domain mutants to identify the sites of interaction with the ERalpha 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 ERalpha . It is possible that the interaction surfaces may determine subsequent acetylase activity. Alternatively, the acetylation motif of the substrate may be critical. The ERalpha 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 the current studies, mutation of the ERalpha in vitro acetylation site enhanced ligand sensitivity. The 2-fold induction of the synthetic estrogen-responsive enhancer reporter gene ERE2TK81pA3LUC at 10-8 M 17beta -estradiol with the wild type ERalpha 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 ERalpha 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 ERalpha (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 ERalpha signaling (4).

In the current studies, ERalpha acetylation site mutations that enhanced ligand sensitivity did not affect ERalpha activation by the MAPK signaling pathway, suggesting direct acetylation of the ERalpha affects a specific subset of ERalpha activities. MAPK regulation of ERalpha involves both direct phosphorylation and regulation of coactivators themselves. Our finding that the ERalpha acetylation mutation does not affect MAPK signaling distinguishes regulation of ERalpha activity from the mechanisms governing ERalpha regulation by the p160 coactivator ACTR/AIB1. ACTR is phosphorylated and activated by MAPK, contributing to the Ser118-independent, MAPK-dependent activation of ERalpha (42). ACTR/AIB1 contacts AF-2 and enhances the ERalpha AF-1 function while recruiting p300 (42). p300 also acetylates ACTR/AIB1, contributing to ERalpha ligand-mediated transcriptional attenuation (4). Our observations that ERalpha 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 ERalpha mitogenic signaling.

Our findings that p300 efficiently acetylated ERalpha in vitro and that acetylated ERalpha 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). ERalpha 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 ERalpha (6) and a 2-fold induction of ERalpha 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 ERalpha (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 ERalpha with the p300 cointegrator (9). As noted above, the LXXLL motif of the coactivator GRIP1 forms the core of a short amphipathic alpha  helix that contacts helices 3, 5/6, 11, and 12 of the ERalpha ; however, the exact proximity of the ERalpha (K302A/K303A) residues to the ERalpha hydrophobic fold remain unknown2 (46). Future studies will discern whether the increased ligand sensitivity of these ERalpha 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 ERalpha affects interactions with other coactivators and corepressors, DNA binding within native chromatin at estrogen-responsive promoters of target genes, the function of the ERalpha 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 ERalpha acetylation site demonstrated dissociable effects of histone deacetylase inhibitors (TSA) and the addition of ligand on ERalpha activity. The induction of ERalpha 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 ERalpha 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 ERalpha target lysines may be predicted to confer a growth advantage. The same mutant that we demonstrated as conveying enhanced ligand sensitivity for transactivation (ERalpha (K303R)) was recently shown to occur in 34% of premalignant human breast lesions, suggesting that these acetylated residues play an important role in ERalpha function and biology (57). The ERalpha 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 ERalpha 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.


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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: Eralpha , estrogen receptor alpha ; 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
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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