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Molecular Endocrinology, doi:10.1210/me.2005-0178
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Molecular Endocrinology 19 (12): 2930-2942
Copyright © 2005 by The Endocrine Society

Recruitment of Histone Deacetylase 4 to the N-Terminal Region of Estrogen Receptor {alpha}

Hoyee Leong, John R. Sloan, Piers D. Nash and Geoffrey L. Greene

The Ben May Institute for Cancer Research, The University of Chicago, Chicago, Illinois 60637

Address all correspondence and requests for reprints to: Geoffrey L. Greene, The Ben May Institute for Cancer Research, The University of Chicago, Center for Integrative Sciences, Room W330, 929 East 57th Street, Chicago, Illinois 60637. E-mail: ggreene{at}uchicago.edu.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Transcriptional activation of estrogen receptor {alpha} (ER{alpha}) is regulated by the ligand-dependent activation function 2 and the constitutively active N-terminal activation function 1. To identify ER{alpha} N-terminal-specific coregulators, we screened a breast cDNA library by T7 phage display and isolated histone deacetylase 4 (HDAC4). HDAC4 interacts with the ER{alpha} N terminus both in vitro and in vivo. Presence of the ER{alpha} DNA binding domain and hinge region reduce HDAC4 recruitment whereas full-length ER{alpha} enhances recruitment. HDAC4 interaction is selective for the ER{alpha} and not ERß N terminus and occurs in the nucleus. We demonstrate in vivo that HDAC4 is recruited by the N terminus to the promoter of an endogenous estrogen responsive gene. HDAC4 suppresses transcriptional activation of ER{alpha} by estrogen and selective ER modulators (SERMs) such as tamoxifen in a cell type-dependent manner. Consistently, silencing of HDAC4 promotes the agonist effect of SERMs (tamoxifen and raloxifene) in a cell type-specific manner. These findings indicate a role for HDAC4 in regulating ER{alpha} activity as a novel N-terminal coregulator and uncover a mechanism by which certain cell types regulate SERM behavior.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
THE ESTROGEN RECEPTOR (ER) plays a critical role in the growth and development of reproductive tissues and breast cancer (1). The ER, expressed as {alpha}- and ß-subtypes, is a member of the superfamily of hormone-activated transcription factors that induces transcription of genes containing hormone responsive elements. The structure of the ER is separated into six functional domains, designated A–F, that cooperate to activate gene transcription (2). The DNA-binding (DBD) domain is located in the C domain whereas the transcriptional activation function (AF) domains are located in the N-terminal A/B region (AF-1) and the C-terminal E/F region (AF-2). The AF-1, located between amino acids 51 and 150 of the A/B domain, is autonomous and constitutively active whereas the AF-2 contains the ligand-binding domain (LBD) and mediates ligand-specific activation of the ER (3, 4, 5, 6). Both activation domains cooperate to activate transcription in a cell- and promoter type-dependent manner (2, 7). Several selective ER modulators (SERMs), such as tamoxifen and raloxifene, have been developed for the prevention and treatment of hormone-responsive breast cancers. SERMs compete with estrogens for binding to the ER and effectively block the mitogenic effects of estrogens in breast tissue. Some SERMs, such as tamoxifen, exert estrogenic effects on other tissues such as the bone and uterus. Others, like raloxifene, are estrogenic in the bone but do not promote uterine growth (8). The mechanisms by which tamoxifen and raloxifene exert diverse effects in these tissues are not fully understood (9).

Evidence suggests that nuclear receptor coregulators may determine the tissue specificity of SERMs (10). After activation, the ER recruits coactivators that stabilize the preinitiation transcriptional complex and facilitate the disruption of chromatin for transcription of target genes. Recruitment of these coactivators occurs through a signature motif called the NR box, which consists of the LxxLL sequence, in which L represents leucine and x represents any amino acid (11). Different classes of coactivators recognize distinct and overlapping regions in the AF-1 and AF-2 (12, 13). The majority of known coactivators identified are predominantly AF-2-interacting proteins, including steroid receptor coactivator-1 (SRC-1, NCoA-1), steroid receptor coactivator-2 (SRC-2, GRIP-1, TIF-2, NCoA-2), steroid receptor coactivator-3 (SRC-3, p/CIP, RAC3, ACTR, TRAM-1, AIB1), ER-associated protein-140 (ERAP140), receptor interacting protein-160 (RIP160), and cAMP response element binding protein (CREB)-binding protein (CBP) (14, 15). Coactivators recognize distinct ligand-bound receptors and exert varying effects on transcriptional activation (12, 16). The recruitment of such coactivators to the AF-2 is blocked by ER antagonists. Thus, the tissue-selective behavior of SERMs is likely dependent on their ability to regulate AF-1 activity (4, 17).

Relatively few coregulators that bind exclusively to the AF-1 have been reported. Examples include p68 RNA helicase and repressor of tamoxifen transcriptional activity (RTA) (18, 19). p68 RNA helicase interaction selectively potentiates ER{alpha} AF-1 and full-length ER{alpha} transcriptional activation induced by estrogen and tamoxifen (19). RTA interaction with the AF-1, however, inhibits ER transcriptional activation by tamoxifen with minimal effect on estrogen-induced activation (18). Altogether, these observations demonstrate a role of AF-1-specific coregulators in mediating both the agonist and antagonist effects of SERMs. Thus, an understanding of AF-1 regulation is required to determine the pharmacology of ER ligands. We report here the identification of histone deacetylase 4 (HDAC4) as a novel ER{alpha} N-terminal interacting coregulator. HDAC4 inhibits the transcriptional activation of ER{alpha} whereas silencing of HDAC4 enhances the agonist activity of SERMs in a cell type-dependent manner. These findings demonstrate that HDAC4 plays a complex role in transcriptional regulation of ER{alpha} and indicate the importance of HDAC4 in determining the tissue-specific effects of ER antagonists.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Isolation of HDAC4 as an ER{alpha} N-Terminal Interacting Protein
To identify proteins involved in AF-1 regulation, we screened a T7 phage library displaying human breast cDNA using the ER{alpha} A/B domain as bait. After three rounds of biopanning, 60 phage clones showing high affinity for the A/B domain were sequenced for analysis. We identified one of these clones as HDAC4, which has been previously identified in a corepressor complex that is recruited to steroid hormone receptors (20, 21). Others clones identified include proteins involved in cell signaling, angiogenesis, and transcriptional activation.

HDAC4 Interacts with the ER{alpha} N-Terminal Domain in Vitro and in Vivo
The interaction between HDAC4 and the ER{alpha} N terminus was confirmed by glutathione-S-transferase (GST) pull-down analysis. When GST-ER{alpha}-A/B was incubated with COS-7 cells transiently transfected with HDAC4, protein interaction was detected by Western analysis (Fig. 1AGo). HDAC4 overexpression was required because the pull-down method was not sensitive enough to demonstrate an interaction with endogenous HDAC4.



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  		Fig. 1. HDAC4 Interacts with the ER{alpha} A/B Domain

A, GST-Sepharose beads bound with GST alone or GST-ER{alpha}-A/B fusion protein were incubated in the absence or presence of whole cell extracts obtained from either nontransfected or HDAC4-transfected COS-7 cells. Bound protein was subjected to SDS-PAGE and detected by Western analysis using anti-HDAC4 antibody. B, Whole-cell extracts were obtained from COS-7 cells transfected with ER{alpha}-A/B or Flag-tagged ER{alpha} and immunoprecipitated (IP) with ER21 or anti-Flag antibody. Immunoprecipitates were analyzed by Western blotting using anti-HDAC4 and ER21 or anti-FLAG antibodies to confirm ER immunoprecipitation. Arrows indicate full-length (FL) and ER{alpha}-A/B proteins.

 
We performed coimmunoprecipitation studies to further assess the interaction of HDAC4 with the A/B domain. Whole-cell extracts from COS-7 cells transfected with ER{alpha}-A/B or Flag-tagged ER{alpha} were immunoprecipitated with ER21 or Flag antibody, respectively. Western analysis for HDAC4 demonstrated that endogenous HDAC4 associated with ER{alpha}-A/B and full-length ER{alpha} (Fig. 1BGo). After normalization for immunoprecipitated ER levels, treatment with estradiol (E2) did not further enhance the association of HDAC4 with full-length ER{alpha} (Fig. 1BGo). As controls, HDAC4 was not detected in the absence of ER{alpha}, and ER{alpha}-A/B and full-length ER{alpha} were both detected after immunoprecipitation. A reciprocal experiment using the anti-HDAC4 antibody for immunoprecipitation also demonstrated the physical interaction between the A/B domain and endogenous HDAC4 (data not shown).

Mammalian two-hybrid experiments were performed to study HDAC4 interaction in vivo and to determine the effect of ER{alpha} N-terminal structure. Two-hybrid assays were performed using COS-7 cells transfected with a chimeric HDAC4 protein fused to the GAL4 DBD and ER{alpha} mutants containing the A/B, ABC, ABCD domains, or full-length receptor fused to the VP16 activation domain (Fig. 2AGo). As indicated by Western analysis, VP16-ABC and full-length ER{alpha} are expressed poorly in COS-7 cells compared with VP16-A/B and ABCD (Fig. 2BGo). After normalization for both differences in protein expression and activity level of ER fragments in the presence of GAL4 DBD alone, two-hybrid experiments indicated that HDAC4 differentially interacts with the A/B domain, depending on its context (Fig. 2CGo). The presence of the C and D domains reduces the ability of HDAC4 to interact with the A/B domain. However, HDAC4 interaction is greatly enhanced when the A/B domain is found in context of the full-length receptor. These results indicate that HDAC4 recruitment is dependent on ER{alpha}-A/B structure and that interaction may additionally occur with the ER{alpha} C terminus.



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  		Fig. 2. The Context of the ER{alpha} N Terminus Influences Its Interaction with HDAC4

A, VP16-ER{alpha} fusion proteins containing the A/B domain in the presence or absence of the DBD (region C) and hinge region (region D) or full-length ER{alpha} (regions A–F) were used to perform mammalian two-hybrid experiments. B, COS-7 cells were transfected with VP16-ER{alpha} fusion proteins. Expression of fusion proteins was detected by Western analysis using H226 antibody. C, Interaction between HDAC4 and ER{alpha} was detected by transfecting COS-7 cells with VP16-ER{alpha} and Gal4-HDAC4 along with pG5-Luc and ß-galactosidase reporter plasmids. Activity was assayed as described in Materials and Methods. Results are normalized by VP16 fusion expression and activity in the presence of Gal4 alone and are presented as a fold induction over VP16 activity. VP16-FL, VP16-full-length ER{alpha}.

 
To determine the selectivity of HDAC4 interaction and further assess the role of the ER{alpha} C terminus, we performed additional two hybrid analyses in COS-7 cells using VP16-ER{alpha}-LBD (LBD containing the E and F domains) and VP16-ERß-A/B. After normalization for both VP16-ER expression (Fig. 3AGo) and VP16-ER activity displayed with GAL4 DBD alone, we observed that HDAC4 interaction with the A/B domain is highly selective for ER{alpha} and not ERß (Fig. 3BGo). Furthermore, we observed that HDAC4 does not interact strongly with the ER{alpha} C terminus in isolation. These observations suggest that HDAC4 interaction with the ER{alpha} N terminus is dominant, and that C terminus interaction is secondary and produces a stabilizing effect. Accordingly, we observed such stabilizing effect with full-length ER{alpha} (Fig. 2CGo). The results were confirmed by coimmunoprecipitation assays using COS-7 protein extracts (Fig. 3CGo). Whole-cell extracts from cells expressing ER{alpha}-LBD or full-length ERß were immunoprecipitated with H222 or CO1531 antibody, respectively. Western analysis demonstrated that HDAC4 was not associated with either the ER{alpha}-LBD or ERß immunoprecipitates. We also failed to detect an interaction in reciprocal experiments using anti-HDAC4 antibody for immunoprecipitation (data not shown). Finally, to investigate whether ligands influence the interaction of HDAC4 with full-length ER{alpha}, two-hybrid experiments were performed in the presence or absence of E2, ICI 182,780 (ICI), tamoxifen, raloxifene, diethylstilbestrol, and genestein (Fig. 3DGo). Except for the small but statistically significant promoting effect of E2, none of the ligands tested had an effect on HDAC4 interaction. Altogether, these experiments demonstrate that HDAC4 interaction is selective for the ER{alpha} N terminus and suggest that although interaction with the C terminus occurs, the event is subsequent and not dependent on ligands.



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  		Fig. 3. HDAC4 Binds Selectively to the ER{alpha} N Terminus

A, COS-7 cells were transfected with the indicated VP16-ER{alpha} and ERß fusion plasmids. Expression of fusion proteins was detected by Western analysis using an anti-VP16 antibody. B, COS-7 cells were transfected with VP16-ER{alpha}-A/B, VP16-ER{alpha}-LBD (containing the E and F domains), or VP16-ERß-A/B and Gal4-HDAC4 along with pG5-Luc and ß-galactosidase reporter plasmids. HDAC4 interaction with the ER fragments was determined by mammalian two-hybrid assay. Results were normalized by VP16-ER expression and activity in the presence of Gal4 alone and presented as a fold induction over VP16 activity. C, Whole-cell extracts were obtained from COS-7 cells transfected with ER{alpha}-LBD or ERß and immunoprecipitated (IP) with H222 or CO1531 antibody, respectively. Immunoprecipitates were analyzed by Western blotting using anti-HDAC4 and H222 or CO1531 antibodies to confirm ER{alpha}-LBD or ERß immunoprecipitation. D, COS-7 cells were transfected with VP16 (–ER) or VP16-ER{alpha} (+ER) and the pG5-Luc and ß-galactosidase reporter plasmids. ER{alpha} and HDAC4 interaction was determined by mammalian two-hybrid assay after 24-h exposure to vehicle (ethanol), E2 (10–8 M), diethylstilbestrol (DES, 10–7 M), genestein (GEN, 10–7 M), ICI (10–8 M), tamoxifen (OHT, 10–8 M), or raloxifene (RAL, 10–7 M). All values are reported as the fold induction over VP16 in the absence of ligand. Asterisks indicate a significant difference (P < 0.05) from vehicle control in the presence of ER using Student’s t test for data analysis.

 
We next confirmed the nuclear localization of HDAC4 and ER interaction by immunofluorescence staining. Various forms of ER{alpha}, including ER{alpha}-A/B and full-length ER{alpha}, were distributed throughout the nucleus (Fig. 4AGo). ER{alpha}-A/B showed weak staining in the cytoplasm and stronger but diffuse nuclear staining. To our knowledge, this is the first report demonstrating nuclear translocation of the AF-1 in the absence of the DBD. HDAC4 was localized in the nucleus and cytoplasm and not affected by the presence of wild-type or mutant forms of ER{alpha} (data not shown). Cell fractionation studies (Fig. 4BGo) similarly showed an overlapping but distinct localization of ER{alpha} and HDAC4 in the cell nucleus.



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  		Fig. 4. HDAC4 and ER{alpha} Associate in the Nucleus

COS-7 cells were transfected with full-length (FL) or mutant (A/B, ABC, ABCD) ER{alpha} expression plasmids. A, Cells were immunostained for ER{alpha} and HDAC4 as described in Materials and Methods. B, Cells were lysed in extraction buffer in the absence of detergent to obtain cytosolic fractions. Loosely bound nuclear proteins (nuclear loose) were released from the nuclear pellet by addition of 0.5% Triton-X100 whereas tightly bound nuclear proteins (nuclear matrix) were obtained from the resultant pellet. Equal volumes of cytosolic and loosely and tightly bound nuclear proteins were analyzed by Western blotting (IB) using H226 and anti-HDAC4 antibodies as described in Materials and Methods.

 
HDAC4 Displays Functional Activity in Complex with the A/B Domain
We determined whether HDAC activity is associated with the A/B domain as a functional confirmation of HDAC4 recruitment to the ER. Cellular extracts were obtained from COS-7 cells cotransfected with HDAC4 and ER{alpha}-A/B and immunoprecipitated with ER21 antibody. Significant HDAC activity was associated with the A/B domain immunoprecipitate only in the presence of transfected HDAC4 compared with the negative control (absence of ER{alpha}-A/B) (Fig. 5AGo). Although the possibility exists that other HDACs are present in the immunoprecipitate, the observed activity is likely attributed to HDAC4 because no increase in activity was seen in the absence of exogenous HDAC4. Comparable HDAC activity was recruited by immunoprecipitation with the anti-HDAC4 antibody and effectively blocked by the addition of the HDAC inhibitor, trichostatin A (TSA).



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  		Fig. 5. HDAC4 Displays Functional Activity on ER{alpha}

COS-7 cells were transfected with HDAC4 and ER{alpha}-A/B (A) or ER{alpha}-ABC, ER{alpha}-ABCD, or full-length ER{alpha} (B). HDAC4 protein was immunoprecipitated (IP) with ER21, anti-FLAG, or anti-HDAC4 antibody and tested for HDAC activity in the presence or absence of E2 (10–8 M) and TSA as described in Materials and Methods. Results are reported as arbitrary fluorescent units (AFU). Asterisks indicate a significant difference (P < 0.05) from samples not expressing any ER{alpha} fragments in the absence of TSA using Student’s t test for data analysis. C, ER{alpha}-A/B and full-length ER{alpha} immunoprecipitates (IP) were analyzed by Western blotting using anti-HDAC4 antibody to confirm HDAC4 immunoprecipitation.

 
Results also indicate that HDAC activity was recruited by the A/B domain in the presence of the C and D domains as well as in the context of the full-length receptor (Fig. 5BGo). We did not observe a reduction of activity in the presence of the C and D domains as anticipated from the binding assays, which indicate a reduced interaction of HDAC4 with the A/B domain (Fig. 2CGo). Such discrepancy may reflect a saturation of HDAC4 activity in the assay or may reflect the activity of other possible HDACs present in the immunoprecipitates. Although HDAC4 is immunoprecipitated much more efficiently by the HDAC4 antibody compared with the ER antibody (Fig. 5CGo), HDAC assays show comparable activity between the immunoprecipitates (Fig. 5AGo), suggesting that activity saturation is likely. Additionally, E2 reduced the association of HDAC4 activity with ER{alpha}, indicating that ER agonists inhibit HDAC activity.

Recruitment of HDAC4 Occurs on an ER-Responsive Promoter
Using chromatin immunoprecipitation (ChIP) analysis, we further examined whether the N terminus of ER{alpha} recruits HDAC4 to an endogenous ER-regulated gene promoter. XBP-1, a member of the CREB/activating transcription factor family of transcription factors, directly interacts with and enhances ER{alpha} transcriptional activation (26). Additionally, XBP-1 expression is regulated by an ER-responsive promoter (M. Brown, personal communication). In SKBR3 cells, HDAC4 was recruited by ER{alpha}-ABCD to the XBPI promoter under basal conditions and in the presence of ER ligands (Fig. 6Go). In comparison, only estrogen and SERMs such as tamoxifen induced the recruitment of full-length ER{alpha} and HDAC4 to XBP1, as previously observed by others on other ER-responsive promoters (20, 27, 28, 29, 30). These results confirm that the ER{alpha} N terminus, in isolation from the C terminus, can recruit HDAC4 to an endogenous ER-regulated gene promoter.



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  		Fig. 6. The ER{alpha} A/B Domain Recruits HDAC4 to the XBPI Gene Promoter

SKBR3 cells were transfected with ER{alpha}-ABCD or full-length ER{alpha}. Cells were treated with vehicle (V), estrogen (E, 10–7 M), tamoxifen (T, 10–6 M), or ICI (I, 10–7 M) for 45 min. ChIP assays were performed using antibodies against ER{alpha} (H226) and HDAC4.

 
Identification of HDAC4 Interaction Sites
To identify the residues required for HDAC4 interaction, 15 mer peptides were generated with a three-residue overlap to encompass the entire ER{alpha} sequence. The peptides were expressed, spotted onto a membrane, and incubated with HDAC4-transfected COS-7 cell extracts. We detected by Western blotting the interaction of HDAC4 with two peptide sequences, ER{alpha} residues 173–187 and 349–363, located in the A/B domain adjacent to the AF-1 and in the AF-2, respectively (Fig. 7AGo). HDAC4 showed a stronger interaction with the A/B domain peptide. As a negative control, no interaction was detected in the absence of COS-7 extract (data not shown). Alanine scanning mutagenesis of the A/B domain peptide revealed that residues T182 and Y184 are required for HDAC4 interaction (Fig. 7BGo). Additional mutagenesis studies indicated that no other amino acid can substitute for T182, suggesting that phosphorylation of T182 may be required for HDAC4 interaction (Fig. 7CGo). Furthermore, aromatic side chains at residue 184 are critical for HDAC4 interaction, as phenylalanine and tryptophan are able to substitute for Y184. Amino acid substituting of residues immediately surrounding T182 and Y184 also indicated that these residues are critical for HDAC4 interaction. For example, substitution of C185 with large amino acids, such as phenylalanine or tyrosine, results in the loss of interaction. Altogether, these results demonstrate that the A/B domain residues, T182 and Y184, are essential for HDAC4 interaction.



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  		Fig. 7. Mapping of HDAC4 Interaction Sites on ER{alpha}

Whole-cell extract from HDAC4-transfected COS-7 cells was incubated with ER{alpha} peptides spotted on membranes. Membranes were analyzed by Western blotting using anti-HDAC4 antibody. A, Peptides, 15 amino acids in length, encompassing the entire sequence of ER{alpha}, were used to determine the region of HDAC4 interaction. B, Alanine scanning mutagenesis was performed on the N terminus-interacting peptide (amino acids 173–187). C, Amino acid substitutions on interacting (amino acids 182, 184) and surrounding residues were performed.

 
Expression of HDAC4 Modulates ER{alpha} Transcriptional Activity
We predicted that association of HDAC4 with the ER should inhibit ER{alpha} transcriptional activity as HDAC4 is associated with the corepressors SMRT (silencing mediator of retinoid and thyroid hormone receptor) and NCoR (nuclear receptor corepressor) (21, 22, 23). Overexpression of HDAC4 in a variety of breast (MCF7, SKBR3, MDA-MB231), endometrial (Ishikawa), and cervical (HeLa) cancer cell lines resulted in a significant suppression of 3ERE-Luc reporter activity with unliganded ER{alpha} in a concentration-dependent manner (Fig. 8AGo). HeLa cells showed the least sensitivity to HDAC4, whereas MDA-MB231 showed the most sensitivity. Overexpression of HDAC4 resulted in a similar concentration-dependent reduction of ER{alpha}-ABCD transcriptional activity but had little or no effect on ER{alpha}-LBD activity (data not shown). These results demonstrate the significance of HDAC4 in regulating full-length ER{alpha} activity and suggest that the regulation occurs through specific inhibition of the N terminus.



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  		Fig. 8. HDAC4 Reversibly Inhibits Transcriptional Activation of ER{alpha}

A, COS-7 cells were transfected with increasing concentrations of HDAC4, full-length ER{alpha}, and 3ERE-Luc and ß-galactosidase (internal control) reporter plasmids. COS-7 (B and D) and MDA-MB231 (C and E) cells were transfected with full-length ER{alpha}, 60 ng HDAC4, and 3ERE-Luc and ß-galactosidase reporter plasmids. Cells were pretreated with vehicle (ethanol) or 25 ng/ml TSA for 2 h before treatment with vehicle, E2 (10–8 M), E2 after 2 h treatment with ICI (10–8 M) (B and C), or tamoxifen (OHT, 10–8 M) (D and E). Cells were assayed as described in Materials and Methods. Luciferase activity is reported as the fold induction over activity in the absence of transfected HDAC4 and any treatment. A significant difference (P < 0.05) exists between a and b using Student’s t test for data analysis.

 
Modulation of ER transcriptional activation by HDAC4 was additionally examined in the presence of ER ligands. Treatment of COS-7 cells with E2 elicited a strong transcriptional activation of full-length ER{alpha} that was suppressed by ICI (Fig. 8BGo). Combined treatment of cells with E2 and the HDAC inhibitor TSA resulted in a synergistic activation of the ER. The observed synergism was inhibited by ICI, indicating that the effects of TSA are dependent on the ER. Similar results were observed in MDA-MB231 cells (Fig. 8CGo). In COS-7 cells, tamoxifen produced a significant agonist effect that was further promoted by TSA, in agreement with previous reports (24, 25) (Fig. 8DGo). The activating effects of TSA on both E2 and tamoxifen were concentration dependent (data not shown). Coexpression of HDAC4 showed a small inhibitory effect on E2 activity (Fig. 8BGo) and significantly reduced the agonist effect of tamoxifen (Fig. 8DGo). The inhibitory action of HDAC4 was more significant in the presence of TSA for both E2 and tamoxifen. In comparison, HDAC4 showed a complete inhibitory effect on E2 activation that was reversed by TSA treatment in MDA-MB231 cells (Fig. 8CGo). HDAC4 had no effect on tamoxifen activity in the presence or absence of TSA as tamoxifen lacked an agonist effect in MDA-MB231 cells (Fig. 8EGo). Altogether, these results indicate that HDAC4 suppresses the agonist effect of tamoxifen and that the level of sensitivity to HDAC inhibition of E2 activity is dependent on cell type.

HDAC4 protein expression was evaluated to determine whether ER transcriptional responses to HDAC4 are dependent on endogenous protein levels. As demonstrated by Western analysis, relative HDAC4 expression is lowest in COS-7 and MDA-MB231 cells whereas expression is highest in HeLa, SKBR3, and Ishikawa cells (Fig. 9AGo). Accordingly, cells expressing the lowest levels of HDAC4 display the most ER transcriptional sensitivity to HDAC4 overexpression (Fig. 8AGo). Relative HDAC4 protein levels, however, do not account for the observed cell type-specific ER responses to ligands in the presence of exogenous HDAC4 (Fig. 8Go, B–E), suggesting that other cell-specific factors are involved.



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  		Fig. 9. Endogenous HDAC4 Protein Expression

A, Relative HDAC4 protein levels were detected in whole-cell extracts from COS-7, MDA-MB231, MCF7, Ishikawa, HeLa, and SKBR3 cells using anti-HDAC4 and ß-actin antibodies. HDAC4 expression levels were normalized by ß-actin and reported as an average of three independent experiments. B, COS-7 cells were transfected with 100 nM control or HDAC4 siRNA in the presence or absence of HDAC4 expression plasmid. Knockdown of HDAC4 expression from whole-cell extracts was confirmed by Western analysis using anti-HDAC4 antibody.

 
To determine the overall contribution of HDAC4 in regulating ER response, ER transcriptional activity was examined after HDAC4 silencing. Western analysis of cells transfected with HDAC4 small interfering RNA (siRNA) confirmed the complete knockdown of both endogenous and strongly overexpressed exogenous HDAC4 protein in COS-7 cells whereas nonspecific siRNA control had no effect (Fig. 9BGo). The knockdown of HDAC4 was also confirmed in HeLa, SKBR3, Ishikawa, MCF-7, and MDA-MB231 cells (data not shown). Transfection of cells with HDAC4 siRNA produced cell type-dependent effects on liganded ER{alpha} (Fig. 10Go, A–F). HDAC4 siRNA enhanced the activation of ER{alpha} in combination with agonists (E2 and genestein) in SKBR3 cells (Fig. 10FGo). Interestingly, genestein showed strong (Fig. 10Go, A and B), weak (Fig. 10Go, C and F), or no (Fig. 10Go, D and E) agonist activity in a cell type-dependent manner. Additionally, expression of HDAC4 siRNA converted raloxifene into a weak agonist in COS-7 and MDA-MB231 cells (Fig. 10Go, A and B). A similar weak promotion of tamoxifen agonist activity was observed in SKBR3 cells (Fig. 10FGo). These studies clearly demonstrate a cell type-specific role for HDAC4 in regulating liganded ER{alpha} response. Most significantly, these results suggest that expression of HDAC4 is required to suppress the agonist behavior of SERMs in certain cell types.



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  		Fig. 10. Silencing of HDAC4 Exerts Cell Type-Dependent Effects on Liganded ER{alpha}

Full-length ER{alpha} was transfected into COS-7 (A), MDA-MB231 (B), Ishikawa (C), HeLa (D), MCF7 (E), and SKBR3 (F) cells with 100 nM control or HDAC4 siRNA and 3ERE-Luc and ß-galactosidase reporter plasmids. Cells were treated with vehicle, E2 (10–8 M), tamoxifen (OHT, 10–8 M), ICI (10–8 M), raloxifene (Ral, 10–7 M), genestein (Gen, 10–7 M), or ICI (10–8 M). Luciferase activity was assayed as described in Materials and Methods and reported as the fold induction over activity in the absence of both HDAC4 siRNA and treatment. Significant differences (P < 0.05) exist between a and b, c and d, and e and f using Student’s t test for data analysis.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
In the present study, we identified HDAC4 as a novel ER{alpha} N-terminal coregulator by phage display screening of a human breast cDNA library. Our studies demonstrate that the structure of the N terminus influences HDAC4 recruitment. Presence of the DBD and hinge region reduces interaction whereas full-length ER{alpha} enhances interaction. Although the N and C termini coordinate to exert a stabilizing effect, results suggest that the interaction of HDAC4 with the C terminus is a secondary event as HDAC4 does not interact with or modulate activity of the ER{alpha}-LBD in isolation in vivo. HDAC4 is recruited by the N terminus to an estrogen responsive promoter and inhibits the agonist behavior of estradiol and SERMs in certain cell types. Our findings suggest that the specific inhibition of N-terminal activity by HDAC4 plays a role in antagonizing overall ER{alpha} activity. Altogether, these studies support the function of HDAC4 as an ER{alpha} AF-1 modulator that regulates tissue-specific SERM behavior.

HDACs play a key role in the regulation of gene expression. During transcriptional activation, chromatin structure is remodeled by posttranslational histone modifications, including methylation, phosphorylation, ubiquitylation, and acetylation (31). Chromatin remodeling is tightly regulated by the balance of histone acetyltransferases and HDACs. In general, hyperacetylation is associated with transcriptional activation whereas hypoacetylation is associated with transcriptional repression (32). The association of HDACs with ER{alpha} is well established. Several ER-associated cofactors including NCoR, SMRT, receptor-interacting protein 140, L-CoR, MTA1, and REA are able to recruit HDACs (21, 23, 33, 34, 35). HDAC1 and HDAC7 are recruited to an ER-regulated gene promoter activated by estrogen (20, 36). In the presence of SERMs, such as tamoxifen and raloxifene, HDAC1, HDAC2, HDAC3, and HDAC4 are recruited (20, 30). Others have found that a direct interaction exists between HDAC1 and the ER{alpha} DNA-binding and AF-2 domains (37). The present study is the first to demonstrate a direct interaction of an HDAC protein to the ER{alpha} A/B domain.

Previous studies demonstrate that ER{alpha} responsiveness to both estrogens and partial antiestrogens involves HDAC activity (24, 25, 37, 38, 39). We observed that overexpression of HDAC4 suppresses ER activity in the presence and absence of estrogen and tamoxifen. Similar effects are observed on estrogen-activated ER{alpha} by the overexpression of HDAC1 (37). Conversely, inhibition of HDAC activity by TSA promotes basal and synergistic activation of the ER{alpha} in combination with E2 (24, 25, 38). Furthermore, we and others show that TSA promotes the agonist activity of tamoxifen (24). Such an effect appears to be dependent on the ER{alpha} subtype as TSA promotes tamoxifen inhibition of ERß activity (25, 38). Using HDAC4 siRNA, we demonstrate a role for HDAC4 in regulating ER{alpha} responsiveness to SERMs. The selective inhibition of HDACs by siRNA has also revealed a role for HDACs in regulating cell growth and survival. Silencing of HDAC1 and HDAC3, but not HDAC7 or HDAC4, produces antiproliferative and apoptotic effects (40). These observations highlight the diverse functions of various HDACs and support the development of specific HDAC inhibitors for cancer treatment.

Transcriptional repression of ER{alpha} and other nuclear receptors occurs via the recruitment of corepressors such as NCoR, SMRT, and MTA1 (41). The majority of corepressors are recruited to the ER LBD/AF-2 and function by associating with multiple HDACs. For example, the NCoR/SMRT complex associates with HDACs 1, 2, 3, 4, and 5 (23, 29, 30, 41). Previous studies have demonstrated that SERMs, such as tamoxifen and raloxifene, induce the recruitment of the NCoR/SMRT complex to ER-responsive promoters (20, 29, 30). Although the function of multiple HDACs on the corepressor complex remains unclear, these HDACs may have specific molecular targets or regulate transcription by bridging the complex to other transcriptional factors (21, 23). For example, others have suggested that HDAC4 is enzymatically inactive and serves only to recruit active HDAC3-containing NCoR/SMRT complexes (21). However, we demonstrate here that ER{alpha}-A/B-associated HDAC4 is enzymatically active, similar to other reports that HDAC4 displays functional activity on the NCoR/SMRT complex (23).

In the present study, we show that HDAC4 is directly recruited to the ER{alpha} A/B domain on an ER-regulated gene promoter. HDAC4 interaction with the A/B domain is strengthened in the presence of the C-terminal AF-2 domain even though HDAC4 does not interact strongly with the AF-2 alone. These observations suggest that recruitment of HDAC4 to the A/B domain induces a conformational change in ER{alpha} that further stabilizes HDAC4 through an interaction with the AF-2 domain. Whereas direct interaction with the A/B domain occurs independently of ligand, we and others have observed that recruitment of HDAC4 to an ER-responsive promoter by full-length ER{alpha} occurs only in the presence of estrogen or tamoxifen, suggesting that differential recruitment of HDAC4 to the N and C termini may occur. Additional experiments are required to determine whether multiple HDAC4 proteins interact with ER{alpha}. We propose that N-terminal-bound HDAC4 associates with corepressor complexes that are recruited to the AF-2 in a ligand-dependent manner. A similar cooperative interaction of SRC-1 between the AF-1 and AF-2 has already been demonstrated to mediate synergism between the two transcriptional activation function domains (42). In the case of SERMs such as tamoxifen, association of the recruited NcoR-SMRT complex with N-terminal-bound HDAC4 may enhance transcriptional repression activity. Alternatively, tamoxifen may induce the indirect recruitment of HDAC4 already associated with the NcoR-SMRT complex. In such a model, the regulation of SERM behavior would likely involve a coordination of both N- and C-terminal HDAC4 activities. In either case, whether HDAC4 is selectively recruited to the N terminus or both the N and C termini, overall HDAC4 activity is likely influenced by additional factors that are associated with ER{alpha}.

Further studies are required to define the relative contribution of HDAC4 activity and that of recruited coregulators in determining ER{alpha} response to SERMs. For example, HDAC4 activity may have little influence in cells expressing high levels of the AF-1-specific RTA corepressor, which inhibits the agonist activity of tamoxifen in an HDAC-independent manner (18). The expression of such coregulators may explain the discrepancies we observed between the effects of HDAC4 overexpression and knockdown on liganded ER{alpha} activity in COS-7 and MDA-MB231 cells (Figs. 8DGo and 10AGo, 8CGo, and 10BGo). HDAC4 overexpression suppressed the agonist effects of E2 and tamoxifen whereas knockdown had no effect, suggesting that these cells may express coregulators that exert an overlapping function with HDAC4. Ultimately, the effect of HDAC4 on SERM response is dependent on the expression of cell type-specific transcriptional coregulators. The direct acetylation of ER{alpha} at residues within the hinge/LBD has also been found to regulate the transcriptional response to HDAC inhibitors and suppress ligand sensitivity (43). Thus, in addition to the expression of cell type-specific factors, the balance between recruited HDACs and histone acetyltransferases can influence the overall response of ER{alpha} to ligands.

In summary, our studies demonstrate a novel role for HDAC4 as an ER{alpha} N-terminal coregulator and a determinant in cell type-specific SERM response. Further studies are necessary to define the regulation of ER{alpha}-associated HDAC4 activity. A better understanding of the molecular mechanisms of HDAC4 will be necessary to fully comprehend AF-1 function and its role in determining SERM pharmacology.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Plasmids
The ER{alpha} (HEGO) and GAL4-HDAC4 plasmids were kindly provided by Dr. Pierre Chambon and Dr. Xiang-Jiao Yang. The 3ERE-Luc reporter, VP16-ER{alpha}-LBD, and pGEX-KG plasmids were gifts from Dr. Donald McDonnell and Dr. Elizabeth Taparowsky. The pG5-Luc reporter was obtained from Promega Corp. (Madison, WI). The GST-ER{alpha} fusion was constructed by PCR amplification of HEGO using primers containing a BamHI site at the 5'-end and insertion into the BamHI site of pGEX-KG. The GST-ER{alpha}-A/B fusion was generated by introducing a stop codon after ER{alpha} amino acid 185 using the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA). His-tagged ER{alpha}-A/B was constructed by insertion into the pET-41 Ek/LIC expression vector (Novagen, Madison, WI) using ligation-independent cloning. VP16-ER{alpha} fusion constructs (ER{alpha} amino acids 1–188, 1–251, and 1–303) were generated by PCR amplification of HEGO using primers containing BamHI at the 5'-end and MluI at the 3'-end. PCR fragments were inserted into the BamHI and MluI sites of pACT (Promega). FLAG-tagged ER{alpha} and ERß were obtained by subcloning into pCMV-FLAG (Sigma Chemical Co., St. Louis, MO). FLAG-tagged HDAC4 was generated by PCR amplification of GAL4-HDAC4 using primers containing EcoRI at the 5'-end and XbaI on the 3'-ends and insertion into pCMV-FLAG.

Protein Expression and Purification
GST fusion proteins were expressed in Escherichia coli BL21(DE3) cells (Novagen) and purified under standard conditions (Amersham Pharmacia Biotech, Arlington Heights, IL). His-tagged ER{alpha}-A/B was produced in BL21(DE3) cells by induction of protein expression with 1 mM isopropyl-ß-D-thiogalactopyranoside at 30 C. Cells were harvested by centrifugation after 4 h, resuspended in extraction buffer (50 mM Tris, pH 8.0; 100 mM NaCl; 20 mM imidazole; 4 M urea) containing 1:1000 protease inhibitor cocktail (Calbiochem, La Jolla, CA), and sonicated three times for 30 sec on ice. Cellular debris was removed by centrifugation, and NaCl was added to a final concentration of 0.5 M. The extract was applied to a column preloaded with nickel beads equilibrated in extraction buffer. After washing twice with wash buffer (20 mM Tris, pH 8.0; 0.5 M NaCl; 40 mM imidazole), his tagged-ER{alpha}-A/B was eluted with imidazole buffer (20 mM Tris, pH 8.0; 0.5 M NaCl; 0.5 M imidazole).

T7 Phage Display
The ER{alpha} A/B domain was diluted in Tris-buffered saline (TBS) (5 ng/ul) and immobilized on an ELISA plate overnight at 4 C. The well was washed three times with TBS and blocked with 5% nonfat milk for 1 h. After washing, screening of the T7 phage human breast cDNA library (Novagen) was performed according to the manufacturer’s instructions with minor modifications. After incubation with ER{alpha}-A/B for 1 h, unbound phage were removed by washing five times with TBS-Tween 20. Bound phage were collected in elution buffer (Novagen) for 20 min and amplified by infection of a 50-ml culture of E. coli BLT5615 cells induced with 1 mM isopropyl-ß-D-thiogalactopyranoside. Amplified phage were collected by centrifugation after cell lysis and applied to the ER{alpha}-A/B-coated ELISA plate for a new round of selection. After a total of three rounds of selection, amplified phage were titered to obtain individual plaques on LB plates. Plaques were arbitrarily isolated and PCR amplified using T7 SelectUP and DOWN primers (Novagen). Purified PCR products were sequenced and compared with GenBank sequences using the BLAST search program.

GST Pull-Down Assay
Cellular extracts from COS-7 cells transfected with pFLAG-HDAC4 were precleared by incubation with glutathione-sepharose beads in binding buffer (20 mM Tris, pH 7.6; 50 mM NaCl; 0.2% Nonidet P-40; 1 mM dithiothreitol; 1:1000 protease inhibitor cocktail) for 30 min at 4 C. Precleared extract was subsequently incubated with GST-ER{alpha}-A/B bound to glutathione-sepharose beads for 1 h. Samples were washed three times with binding buffer, eluted with 2x SDS-PAGE sample buffer, and analyzed by Western blotting.

Cell Culture, Transient Transfection, and Luciferase Assays
COS-7 (monkey kidney), MDA-MB231 (human breast adenocarcinoma), SKBR3 (human breast adenocarcinoma), HeLa (human cervical adenocarcinoma), and Ishikawa (human endometrial adenocarcinoma) cells were routinely cultured in phenol red-free DMEM supplemented with 10% fetal bovine serum. MCF-7 (human breast adenocarcinoma) cells were additionally supplemented with essential amino acids and insulin. For transient transfections, cells were seeded at a density of 2.5 x 104 cells in 48-well plates. The next day, cells were transfected using Polyfect (QIAGEN, Chatsworth, CA) according to the manufacturer’s instructions in phenol red-free DMEM supplemented with 10% dextran-coated charcoal-stripped fetal bovine serum. Transfections contained 100–300 ng of reporters, 100 ng of the control ß-galactosidase expression plasmid, and, where indicated, 30–100 ng of expression or control vectors. For gene silencing experiments, cells were transfected with 100 nM nonspecific siRNA or HDAC4 siRNA (Dharmacon, Lafayette, CO) using Lipofectamine (Invitrogen) according to the manufacturer’s instructions. All treatments were reconstituted in ethanol and administered the day after transfection as a 1000-fold dilution in fresh media for 24 h. Cells were harvested and assayed for luciferase and ß-galactosidase activity. Luciferase activity was normalized for transfection using ß-galactosidase as an internal control and reported as a fold induction over control activity. All results are representative of at least three independent experiments and represent the average ± SD of triplicate samples.

Cellular Fractionation
Extraction of nuclear and cytosolic protein was performed as previously described (44). Briefly, COS-7 cells were washed with ice-cold PBS and lysed with TNM buffer (10 mM Tris-HCl, pH 7.4; 100 mM NaCl; 2 mM MgCl2; 300 mM sucrose; 1% thiodiglycol; 1:1000 protease inhibitor cocktail). After homogenization with a Teflon pestle on ice, samples were centrifuged at 4500 x g for 10 min at 4 C. The supernatant (cytosolic fraction) was retained, and the pellet (crude nuclear fraction) was extracted by the addition of 0.5% Triton X-100 (final concentration) in TNM buffer for 5 min on ice. After centrifugation, the pellet was reextracted in an equal volume of TNM buffer with 0.5% Triton X-100 to obtain nuclear matrix proteins.

Coimmunoprecipitation and Western Blot Analysis
Transfected COS-7 cells were washed with ice-cold PBS and lysed with extraction buffer (50 mM Tris, pH 7.4; 0.1% sodium dodecyl sulfate; 0.5% sodium deoxycholate; 0.1% Nonidet P-40; 150 mM NaCl; 1:1000 protease inhibitor cocktail). Whole-cell extracts were precleared for 1 h with 20 µl of protein G plus agarose beads (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) and incubated with 3 µg of the indicated antibody for 2 h in coimmunoprecipitation buffer (10 mM HEPES, pH 7.2; 144 mM KCl; 5 mM MgCl2; 1 mM EGTA) at 4 C. Coimmunoprecipitation was performed by incubation with 20 µl of fresh beads overnight at 4 C. Beads were washed three times and eluted with 2x SDS-PAGE sample buffer. Samples were fractionated by SDS-PAGE, transferred to a nitrocellulose membrane, and detected by immunoblotting with the indicated antibodies using the SuperSignal West Pico kit (Pierce Chemical Co., Rockford, IL). Equal protein loading was confirmed by Ponceau staining.

HDAC Assay
HDAC assays were performed on COS-7 immunoprecipitates using the HDAC assay kit (Upstate Biotechnology, Inc., Lake Placid, NY) according to the manufacturer’s instructions. Briefly, beads were washed twice with ice-cold PBS followed by HDAC assay buffer and incubated with fluorometric HDAC substrate in the absence or presence of 1 µM trichostatin A for 30 min at room temperature. Deacetylated substrate was detected using the Wallac Victor (2) 1420 Multilabel HTS counter (PerkinElmer, Norwalk, CT). All reported values are representative of at least three independent experiments and reflect the average ± SD of quadruple samples.

Immunofluorescence Staining
COS-7 cells were seeded at a density of 6 x 104 cells onto glass coverslips in six-well plates 1 d before transfection. Cells were transfected, treated for 30 min with the indicated compounds, and washed with PBS three times. Cells were subsequently fixed in 4% paraformaldehyde, permeabilized with 0.2% Triton X-100, and doubly stained with H226 and anti-HDAC4 (Santa Cruz) antibodies (1 µg/ml). Rhodamine- and fluorescein isothiocyanate-conjugated (Santa Cruz) secondary antibodies (1:1000) were applied with 4',6-diamidino-2-phenylindole (50 µg/ml). Coverslips were mounted on glass slides with 90% glycerol/2.3% 1,4-diazabicyclo(2,2,2)octane (DABCO) and examined with a Zeiss Axiovert 200M microscope (Carl Zeiss, Thornwood, NY).

Peptide Blot Analysis
Synthesis of ER{alpha} mapping peptides was performed with the Intavis AG MultiPep robot. Peptides (15 mer) spanning the ER{alpha} sequence were generated with a three-residue overlap and spotted sequentially on a polyvinylidine difluoride membrane. The membrane was washed with 100% ethanol and TBS and blocked overnight with 5% nonfat milk in TBS-Tween 20. Whole-cell extracts from pFLAG-HDAC4 transfected COS-7 cells were added and incubated for 2 h with rocking at room temperature. Protein interaction was detected by Western blotting using anti-HDAC4 antibody. Alanine scanning mutagenesis and amino acid substitution studies were performed similarly.

ChIP Assay
ChIP assays were performed using the ChIP assay kit (Upstate Biotechnology). SKBR3 cells were transfected and treated the next day for 1 h as indicated. Following treatment, ChIP was performed as previously described using primers for the promoter region of the human X box binding protein 1(XBP-1) gene: 5'-TTGATCACTGGTCACAAGCAGAAATGG GG-3', 5'-CCAGTTATGGCGTAATTCAAACCCTGCC-3' (Ref.28 and M. Brown, personal communication).


   ACKNOWLEDGMENTS
 
We thank Bernard Liu for technical support in the mapping studies.


   FOOTNOTES
 
This work was supported by the Virginia and D.K. Ludwig Fund for Cancer Research. H.L was supported by a postdoctoral National Institutes of Health Cancer Biology Training grant fellowship.

First Published Online July 28, 2005

Abbreviations: AF, Activation function; ChIP, chromatin immunoprecipitation; DBD, DNA-binding domain; E2, 17ßestradiol; ER, estrogen receptor; GST, glutathione S-transferase; ICI, ICI 182,780; HDAC, histone deacetylase; LBD, ligand-binding domain; NCoR, nuclear receptor corepressor; SERM, selective ER modulator; siRNA, small interfering RNA; SMRT, silencing mediator of retinoid and thyroid hormone receptor; SRC, steroid receptor coactivator; RTA, repressor of tamoxifen transcriptional activity; TBS, Tris-buffered saline; TSA, trichostatin A.

Received for publication May 3, 2005. Accepted for publication July 20, 2005.


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NURSA Molecule Pages Link:

Nuclear Receptors:   ERα  |  ERβ
Coregulators:   LCoR  |  HDAC4  |  REA  |  HDAC1  |  HDAC2  |  HDAC3  |  MTA1  |  NCOR  |  SMRT
Ligands:   17β-Estradiol  |  Diethylstilbestrol  |  Raloxifene



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Endocrinology Endocrine Reviews J. Clin. End. & Metab.
Molecular Endocrinology Recent Prog. Horm. Res. All Endocrine Journals