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Molecular Endocrinology, doi:10.1210/me.2005-0218
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Molecular Endocrinology 20 (11): 2695-2710
Copyright © 2006 by The Endocrine Society

The Pure Estrogen Receptor Antagonist ICI 182,780 Promotes a Novel Interaction of Estrogen Receptor-{alpha} with the 3',5'-Cyclic Adenosine Monophosphate Response Element-Binding Protein-Binding Protein/p300 Coactivators

Basem M. Jaber, Tong Gao, Luping Huang, Sudipan Karmakar and Carolyn L. Smith

Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas 77030

Address all correspondence and requests for reprints to: Carolyn L. Smith, Ph.D., Molecular and Cellular Biology, One Baylor Plaza, Houston, Texas 77030. E-mail: carolyns{at}bcm.tmc.edu.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Estrogen receptor-{alpha} (ER{alpha}) is a member of the nuclear receptor superfamily of ligand-activated transcription factors. Abundant evidence demonstrates that ER{alpha} agonists promote, whereas antagonists inhibit, receptor binding to coactivators. In this report we demonstrate that binding of the ICI 182,780 (ICI) pure antiestrogen to ER{alpha} promotes its interaction with the cAMP response element-binding protein-binding protein (CBP)/p300 but not the p160 family of coactivators, demonstrating the specificity of this interaction. Amino acid mutations within the coactivator binding surface of the ER{alpha} ligand-binding domain revealed that CBP binds to this region of the ICI-liganded receptor. The carboxy-terminal cysteine-histidine rich domain 3 of CBP, rather than its amino-terminal nuclear interacting domain, shown previously to mediate agonist-dependent interactions of CBP with nuclear receptors, is required for binding to ICI-liganded ER{alpha}. Chromatin immunoprecipitation assays revealed that ICI but not the partial agonist/antagonist 4-hydroxytamoxifen is able to recruit CBP to the pS2 promoter, and this distinguishes ICI from this class of antiestrogens. Chromatin immunoprecipitation assays for pS2 and cytochrome P450 1B1 promoter regions revealed that ICI-dependent recruitment of CBP, but not receptor, to ER{alpha} targets is gene specific. ICI treatment did not recruit the steroid receptor coactivator 1 to the pS2 promoter, and it failed to induce the expression of this gene. Taken together, these data indicate that recruitment of the CBP coactivator/cointegrator without steroid receptor coactivator 1 to ER{alpha} is insufficient to promote transcription of ER{alpha} target genes.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
ESTROGEN RECEPTOR-{alpha} (ER{alpha}) is a member of the nuclear receptor superfamily of ligand-dependent transcription factors that plays an important role in many biological processes, including growth, differentiation, and development (1). The transcriptional activity of ER{alpha} is dependent on the receptor’s two activation functions (AFs); AF-1 in the N terminus (A/B domain), and AF-2 in the C terminus (E domain). The AF-1 functions in a ligand-independent manner, whereas AF2 function is ligand dependent (2, 3). Although these two domains can function independently, maximal ER{alpha} transcriptional activity is achieved when the two AFs synergize (4). These domains are sites of interaction for a large number of coactivators: proteins that facilitate the interaction between ER{alpha} and the general transcriptional machinery (5, 6). Several coactivators also possess histone acetyltransferase, arginine methyltransferase, and ubiquitin ligase activities (6), and receptor recruitment of coactivators possessing these enzymatic activities contributes to chromatin remodeling and the dynamic occupancy of target gene promoters by receptors, coactivators, and RNA polymerase II (7, 8, 9).

The ligand binding domain (LBD) of ER{alpha} consists of 12 {alpha}-helices arranged as a three-layered antiparallel {alpha}-helical sandwich that forms a hydrophobic pocket to which ligands bind (10). Upon binding to an agonist, helix 12 is oriented over the ligand-binding pocket, allowing helices 3, 5, and 12 to generate a functional AF-2 domain consisting of a hydrophobic groove on the LBD surface (11, 12). This, in turn, enables the agonist-bound ER{alpha} to interact with coactivators, such as p160 [steroid receptor coactivator (SRC)] family coactivators, and ultimately stimulate target gene expression. Coactivator interaction with agonist-bound LBD is achieved through a highly conserved signature motif found within many coactivators termed the nuclear receptor box, which consists of the consensus sequence LXXLL (where L is leucine and X is any amino acid) (13). Extensive work has gone into characterizing the interaction of LXXLL-containing peptides with nuclear receptors, and although the affinities of the various p160 LXXLL motifs for ER{alpha} vary, they are greater than the ER{alpha}-binding affinity of the CBP LXXLL motifs, which are considered to be sufficiently low that CBP cannot bind to ER{alpha} without the assistance of other coregulators (13, 14, 15). Indeed, CBP/p300 binds to ER{alpha} as a component of a ternary complex consisting of CBP/p300, p160 coactivators, and ER{alpha} (16), and SRC-1 and CBP synergistically enhance ER{alpha} transcriptional activity (17, 18). Moreover, mutation of the CBP-binding region of SRC-1 blocks the ability of CBP to contribute to ligand-stimulated gene expression (14, 19, 20)

Because of the importance of ERs in the reproductive, skeletal, cardiovascular, and other systems, significant efforts have been extended to develop ligands that can positively or negatively regulate the transcriptional activity of the receptors. There are two types of antiestrogens: type I antiestrogens such as 4-hydroxytamoxifen (4HT) and raloxifene can function as partial agonists and antagonists of ER{alpha}, whereas type II antiestrogens, typified by ICI 164,384 and ICI 182,780 (ICI), are devoid of estrogenic activity. The partial agonist/antagonist activity of type I antiestrogens is derived from their ability to inhibit AF-2 but not AF-1 activity (6). x-Ray crystallographic studies of tamoxifen or raloxifene bound to the ER{alpha} LBD reveal that helix 12 adopts a distinct orientation in which it binds to the coactivator recognition groove of the LBD, thereby preventing coactivator binding to the receptor (12). In contrast, type I antiestrogens do not appear to block the interaction of coactivators, such as SRC-1 or CBP/p300 with the amino terminus of ER{alpha}, thereby enabling the receptor to exert AF-1-dependent transcriptional activity (21, 22, 23, 24). Consistent with the cell- and tissue-specific nature of AF-1 activity (4, 22, 25), type I antiestrogens exert agonist activity in a promoter-, cell-, and tissue-specific manner, and they have therefore been classified as selective ER modulators (SERMs).

Although a structure for ER{alpha} bound to pure ICI 164,384 and ICI antiestrogens is not yet available, the structure of the closely related ERß in the presence of ICI 164,384 reveals a distinct conformation in which the position of helix 12 cannot be determined (26). This is likely due to the long alkylamide side chain of ICI 164,384 that extends out of the ligand-binding pocket and toward the coactivator-binding groove (26). Neither AF-1 nor AF-2 activities are manifested for ER{alpha} in the presence of ICI compounds, and a variety of additional mechanisms have been proposed to account for the pure antiestrogen effects of these ligands, including inhibition of receptor dimerization and DNA binding, acceleration of ER{alpha} down-regulation, and nuclear export of ER{alpha} (27, 28, 29, 30, 31, 32). It also has been shown that ICI induces ER{alpha} interaction with the C terminus of the nuclear receptor corepressor and can promote histone deacetylase-mediated repression of an estrogen response element (ERE)-responsive reporter (33). In addition, ICI promotes an AF-2 independent interaction between ER{alpha} and CIA, a coregulator that possesses corepressor and coactivator functions (34), but inhibits interactions between ER and SRC family coactivators (35). The extent to which each of these mechanisms contributes to the antiestrogen activity of ICI has not been precisely defined.

In this study the ability of antiestrogens to regulate interactions between ER{alpha} and various coactivators was investigated, and our results reveal that pure antiestrogens promote ER{alpha} binding to CBP and p300, but not to p160 coactivators. An LXXLL-independent binding site within CBP responsible for interaction with ICI-bound ER{alpha} was defined, and ICI was shown to selectively recruit CBP/p300 to target gene promoters by chromatin immunoprecipitation (ChIP) assay. Taken together, our data provide evidence that CBP/p300 can bind to ER{alpha} independently of p160s in the presence of ICI and suggests that recruitment of CBP to target gene promoters is insufficient to promote transcription of ER{alpha} target genes.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
ICI Enhances the Interactions between CBP/p300 and the ER{alpha} LBD
Binding of the pure antiestrogen ICI to ER{alpha} is associated with loss of transcriptional activity by the receptor (36), and presumably is dependent on interactions of receptor with corepressors. Moreover, current hypotheses suggest that antiestrogens do not promote interactions of ER{alpha} with coactivators; this was investigated in the current study in which the effect of ICI on interactions between ER{alpha} and CBP were examined. To accomplish this, we employed a mammalian two-hybrid assay in which HeLa cells were cotransfected with a GAL4 responsive reporter and vectors for the GAL4 DNA-binding domain (DBD) (GAL) or chimeras of GAL fused to the amino termini of either full-length CBP or p300, in the presence of the VP16 activation domain alone (VP16) or VP16 fused to the amino terminus of the ER{alpha} LBD (VP16-LBD). Cells were treated with either vehicle, 10 nM 17ß-estradiol (E2) or 100 nM ICI for 20–24 h. As expected, E2 increased the ER{alpha} LBD-CBP interaction as well as the ER{alpha} LBD-p300 interaction (Fig. 1Go, A and B). Surprisingly, treatment with ICI increased the ER{alpha} LBD-CBP interaction to an extent even greater than for E2. Similar results were obtained for the ICI-induced interaction between ER{alpha} LBD and p300.


Figure 1
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  		Fig. 1. The Pure Antagonist ICI Enhances Interactions between CBP/p300 and the ER{alpha} LBD

HeLa cells were transfected with expression vectors for the GAL4 DBD alone (GAL) or GAL-CBP (panel A) or GAL-p300 (panel B) in the presence of expression vectors for the VP16 activation domain (VP16) or a chimera consisting of the VP16 activation domain fused to the amino terminus of the ER{alpha} LBD (VP16-ER{alpha}LBD) along with the pG5-Luc reporter plasmid. Cells were treated with 0.1% ethanol (vehicle), 10 nM E2, or 100 nM ICI. Values are normalized to those obtained for GAL-CBP (panel A) or GAL-p300 (panel B) and VP16 in the presence of vehicle, which was defined as 1. Bars represent the average ± SEM of six experiments.

 
Interaction of p160 Coactivators with the ER{alpha} LBD
To determine whether the ability of ICI to promote interactions with the ER{alpha} LBD was specific to CBP/p300, interactions between the ER{alpha}-LBD and each of the three p160 coactivators, SRC-1, transcription intermediary factor 2 (TIF2) and receptor-associated coactivator 3 (RAC3), were examined by mammalian two-hybrid assay in HeLa cells as described above. As shown in Fig. 2Go, the intrinsic transcriptional activities of GAL-SRC-1, GAL-TIF2, and GAL-RAC3 were modest in comparison to the GAL4 DNA-binding domain (DBD) alone. Moreover, control luciferase values obtained for cells transfected with expression vectors for the GAL4 DBD in the presence of VP16-LBD were low. Cotransfection of VP16-LBD with any of the GAL-p160s revealed increased luciferase activity indicative of a modest interaction between receptor and each coactivator in the absence of hormone. As expected, treatment with E2, but not ICI, significantly increased luciferase activity, indicating a ligand-dependent enhancement of the interaction of ER{alpha} LBD with SRC-1, TIF2, and RAC3. These data are consistent with previous reports showing that E2 promotes ER{alpha} interaction with p160 coactivators whereas ICI does not (15, 35, 37) and indicates that ICI-induced interactions between the ER{alpha} LBD and CBP/p300 are specific.


Figure 2
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  		Fig. 2. Mammalian Two-Hybrid Assay of SRC Family Coactivator Interactions with the ER{alpha} LBD

HeLa cells were transfected with expression vectors for the GAL4 DBD alone (GAL) or GAL-SRC-1 (SRC-1), GAL-TIF2 (TIF2), or GAL-RAC3 (RAC3) chimeras in the presence of an expression vector for the VP16 activation domain (VP16) or a VP16-LBD chimera along with the pG5-Luc reporter plasmid. Cells were treated with 0.1% ethanol (vehicle), 10 nM E2, or 100 nM ICI and subsequently assayed for luciferase activity. Values are normalized to that obtained for GAL-SRC-1 and VP16 in the presence of vehicle, which was defined as 1. Bars represent the average ± SEM of three experiments.

 
ICI Promotes CBP Interaction with Full-Length ER{alpha}
Our mammalian two-hybrid assays revealed that ICI promoted interactions between the ER{alpha} LBD and CBP/p300. To verify this finding through a distinct technical approach, we first employed a coimmunoprecipitation assay in which the ability of ICI to promote an interaction between full-length ER{alpha} and endogenous coactivators was assessed. For this, recombinant human ER{alpha} was preincubated with either ethanolic vehicle, E2, or ICI followed by incubation with HeLa cell extract and antibodies against SRC-1 or CBP. The ICI antiestrogen induced an interaction between ER{alpha} and CBP that was at least as strong as the interaction induced by the agonist, E2 (Fig. 3AGo, top panel). In contrast, only E2 increased ER{alpha} interaction with SRC-1, whereas ICI reduced the ER-SRC-1 binding below the level seen for vehicle treatment (Fig. 3AGo, bottom panel). The interaction between endogenous ER{alpha} and CBP in MCF-7 cells was also assessed. Treatment of cells with ICI or E2 promoted an interaction between ER{alpha} and CBP that could be detected by coimmunoprecipitation (Fig. 3BGo); no signal was obtained for lysates incubated with an irrelevant IgG antibody. Taken together, these data indicate that ICI promotes CBP interaction with full-length ER{alpha} and compliments our mammalian two-hybrid assessments of ER{alpha} LBD interactions with CBP.


Figure 3
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  		Fig. 3. CBP and SRC-1 Interaction with Full-Length ER{alpha}

A, Full-length recombinant ER{alpha} (100 ng) previously incubated with either ethanol [vehicle (V)], 10 nM E2, or 100 nM ICI was mixed with Hela cell lysates and subjected to coimmunoprecipitation with antibodies against CBP (top) or SRC-1 (bottom). The ER{alpha} content of the immunocomplex was assessed by Western blot with anti-ER{alpha} antibody (Cell Signaling Technology, Beverly, MA). The Western blot is a representative of two experiments. B, MCF-7 cells were treated with 0.1% ethanol (V), 10 nM E2, or 1 µM ICI. Western blot of CBP (top), ER{alpha} (middle), and actin (bottom) expression in total lysates of treated cells (lanes 1–3) or in complexes immunoprecipitated from cell lysates with an antibody to CBP (lanes 4–6) or IgG (lane 7). The blot is representative of five experiments. IB, Immunoblot; IP, immunoprecipitation.

 
ICI Promotes ER{alpha} LBD-CBP Interaction in a Dose-Dependent Manner and Is Antagonized by 4HT
We next tested whether the ICI-induced interaction between the ER{alpha} LBD and CBP was dose dependent. Mammalian two-hybrid assay was employed in which HeLa cells were transfected with GAL-CBP and VP16-LBD chimeras and treated with increasing concentrations of ICI (1 ->1000 nM) or 4HT (1 ->1000 nM). ICI was able to induce ER{alpha} LBD interactions with CBP in a dose-dependent manner (Fig. 4Go). In contrast, when cells were incubated with increasing concentrations of 4HT, no effect on ER{alpha} interaction with CBP was observed. Moreover, simultaneous treatment of cells with 100 nM ICI and increasing concentrations of 4HT demonstrated that the latter antiestrogen could antagonize the effects of ICI on ER{alpha}-CBP interactions consistent with ICI occupancy of the receptor’s LBD being required for promotion of ER{alpha}-CBP/p300 interactions.


Figure 4
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  		Fig. 4. Dose-Dependent Antagonism of ICI-Induced CBP-ER{alpha} LBD Interaction by 4HT

Cells were transfected with expression vectors for GAL-CBP and VP16-LBD chimeras along with the pG5-Luc reporter plasmid and treated with either 0.1% ethanol vehicle (lane 1), 10 nM E2 (lane 2), increasing concentration of 4HT (1–1000 nM; lanes 3–6), or ICI (1–1000 nM; lanes 7–10), or a combination of 100 nM ICI plus 1–1000 nM 4HT (lanes 11–14) for 20–24 h. The cells were subsequently harvested and assayed for luciferase activity. Values are normalized to those obtained for GAL-CBP and VP16 in the presence of vehicle, which was defined as 1 (not shown in the graph). Bars represent the average ± SEM of three experiments.

 
SERMs Do not Enhance ER{alpha}-CBP Interaction
To investigate the ability of other ER{alpha} ligands to modulate ER{alpha}-CBP interactions, expression vectors for VP16-LBD and GAL-CBP, and the pG5-Luc reporter were cotransfected into HeLa cells and treated with the SERMs, 4HT, raloxifene, or idoxifene, and the pure antiestrogens ICI 164,384 and ICI. None of the SERMs increased ER{alpha}-CBP interaction above the vehicle control (Fig. 5AGo). In contrast, the pure ER{alpha} antagonist, ICI 164,384, enhanced ER{alpha}-CBP interactions to an extent similar to that obtained for E2 or ICI. These results suggest that pure antiestrogens differ from SERMs in their effect on ER{alpha}-CBP interactions and suggest that structural features common to the ICI compounds may be important for promoting the interaction of CBP with the ER{alpha} LBD. This was investigated further through the use of two other pure antiestrogens, ZK-703 and ZK-253, able to inhibit the growth of tamoxifen-sensitive and tamoxifen-resistant breast cancer (38). Like the ICI compounds, both ZK-703 and ZK-253 are steroidal antiestrogens that possess long 7{alpha} side chains (38). As shown in Fig. 5BGo, neither ZK compound induced interaction between SRC-1 and the ER{alpha} LBD. In contrast, ZK-703, but not ZK-253, induced a strong interaction between CBP and ER{alpha}, suggesting that the nature of the 7{alpha} side chain may influence ER{alpha}-CBP interactions.


Figure 5
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  		Fig. 5. Effect of Pure Antagonists and SERMs on the Interactions between CBP and the ER{alpha} LBD

HeLa cells were transfected with expression vectors for the GAL4 DBD alone (GAL) or a GAL-CBP (panels A and B) or GAL-SRC-1 (panel B) chimera in the presence of an expression vector for VP16 alone or VP16-LBD along with the pG5-Luc reporter plasmid. Cells were treated with 0.1% ethanol (vehicle), 10 nM E2, or 100 nM of 4HT, raloxifene, idoxifene, ICI 164,380, ICI, ZK-703, or ZK-253 for 20–24 h, and subsequently harvested and assayed for luciferase activity. Values are normalized to those obtained for GAL-CBP and VP16 in the presence of vehicle which was defined as 1. Bars represent the average ± SEM of three experiments.

 
Mutation of the ER{alpha} LBD Blocks Interactions between CBP and ER{alpha}
The region of the ER{alpha} LBD to which coactivator LXXLL motifs bind is composed primarily of helices 3, 4, 5, and 12 and is referred to as the coactivator-binding groove (39). To determine whether this region of ER{alpha} was required for its ICI-induced interactions with CBP, the ability of mutations to affect ICI-induced ER{alpha}-CBP binding was assessed by mammalian two-hybrid assay. Mutations of isoleucine362 to aspartic acid362 (I362D), lysine366 to aspartic acid366 (K362D), valine380 to aspartic acid380 (V380D), and leucine543 to alanine543 (L543A) had been shown previously to inhibit the interaction of mouse ER{alpha} with p160 coactivators (39), and we therefore generated mutations in the corresponding positions of human ER{alpha} (I358D, K362D, V376D, L539A) and tested them to ensure that these mutations blocked human ER{alpha} from binding to p160 coactivators. As shown in Fig. 6AGo, these mutations blocked the interaction of TIF2 with each of the mutant forms of hER{alpha} LBD tested. These LBD mutants also interacted poorly with SRC-1 or RAC3 (data not shown).


Figure 6
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  		Fig. 6. Mutation of the ER{alpha} LBD Inhibits CBP-Receptor Interactions

HeLa cells were transfected with expression vectors for GAL-TIF2 (panel A) or GAL-CBP (panel B) along with plasmids for VP16 alone or VP16 fused to the amino termini of wild-type (WT) or mutant forms (I358D, K362D V376D or L539A) of the ER{alpha} LBD along with the pG5-Luc reporter plasmid. Cells were treated with either ethanolic vehicle, 10 nM E2, or 100 nM ICI for 20–24 h, and subsequently harvested and assayed for luciferase activity. Values are normalized to those obtained for GAL-CBP and VP16 in the presence of vehicle, which was defined as 1. Bars represent the average ± SEM of four independent experiments.

 
When these human ER{alpha} LBD mutants were used in CBP interaction assays, they revealed that the I358D, V376D, and L539A mutations in helices 3, 5 and 12, respectively, completely blocked CBP interaction with the ER{alpha} LBD induced by either E2 or ICI (Fig. 6BGo). These results are consistent with the coactivator binding groove being the surface that CBP utilizes to bind to ICI-liganded ER{alpha}. However, the K362D LBD mutant interacts with CBP in response to ICI but to a lesser extent than the wild-type LBD, suggesting that not all amino acids are equally important for ICI-dependent ER{alpha}-CBP interactions. An examination of the crystal structure of ERß bound to ICI 164,384 (26) shows that the ERß residue corresponding to ER{alpha} K362 is the furthest from where the 7{alpha}-alkylamide side chain of ICI 164,384 protrudes from the ligand-binding cavity, suggesting that amino acids located in the proximal portion of the coactivator-binding groove make a greater contribution to ICI-induced ER{alpha} LBD-CBP interaction.

To ensure that differences in ER mutant binding to ICI did not account for the lack of ICI-induced CBP-ER{alpha} LBD interaction, relative binding affinity (RBA) assays were conducted in which the ability of ICI to bind to wild-type ER{alpha} and each of the coactivator-binding groove mutants in comparison with E2 was determined. Scatchard plots had previously demonstrated that these mutations had little effect on the receptor’s binding affinity for E2 (39). As shown in Table 1Go, the RBA of the mutant ER{alpha}s varied little in comparison with the RBA of wild-type ER{alpha} for ICI. This indicates that the inability of CBP to bind to the ER{alpha} mutants is not due to lack of ICI binding to ER{alpha}(I358D), ER{alpha}(K362D), ER{alpha}(V376D), or ER{alpha}(L539A).


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  		Table 1. RBA of Wild-Type (WT) and Mutant ER{alpha} for ICI 182,780

 
A Novel Receptor-Interacting Domain of CBP
As a cointegrator of transcription (40), CBP/p300 has been demonstrated to bind to many different proteins, including specific transcription factors and other coactivators, through various domains including nuclear receptor-interacting domain (NID), cAMP response element binding protein (CREB)-binding domain (KIX), and each of its three zinc finger motifs [cysteine-histidine rich domains (CH1, CH2, and CH3); Fig. 7AGo]. Ligands promote nuclear receptor binding to CBP/p300 via the NID located in the first 101 amino acids of the coactivator (17). To determine which region was responsible for mediating the interaction between ER{alpha} and CBP in the presence of pure antiestrogens, a series of bait constructs consisting of the GAL DNA binding domain fused to either the NID or the KIX domains, or each of the three zinc finger domains, CH1, CH2 and CH3, was constructed and tested in our ER{alpha}-CBP mammalian two-hybrid interaction assay. As expected, the NID region was sufficient to promote interaction with the ER{alpha} LBD in the presence of E2, but no interaction with ICI-bound receptor was observed (Fig. 7BGo). Neither ligand promoted interaction between the ER{alpha} LBD and the KIX, CH1, or CH2 domains. However, both E2 and ICI induced strong ER{alpha} binding to the CH3 domain. To test whether the ER{alpha}-CH3 interaction is direct or indirect, glutathione-S-transferase (GST)-CH3 protein was incubated with in vitro expressed [35S]ER{alpha}-179C (amino acids 179–595) in the presence of either vehicle, E2, or ICI. The GST pull-down results showed that E2 promoted an interaction between [35S]ER{alpha}-179C and GST-CH3 in comparison with vehicle (Fig. 7CGo). The levels of pull-down complex were even higher in the presence of ICI. Taken together, these data show that ER{alpha} interacts directly with CBP via its CH3 domain in response to E2 and the pure antagonist ICI.


Figure 7
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  		Fig. 7. Mammalian Two-Hybrid Assay of CBP Domain Interactions with the ER{alpha} LBD

A, Schematic representation showing the major CBP interaction domains. B, HeLa cells were transfected with GAL chimeric expression vectors for full-length CBP (CBP) or the NID, KIX, CH1, CH2, or CH3 domains, in the presence of an expression vector for the VP16 activation domain or the VP16-LBD along with the pG5-Luc reporter plasmid. Cells were treated with 0.1% ethanol [vehicle (V)], 10 nM E2, or 100 nM ICI for 20–24 h. Values are normalized to those obtained for GAL-CBP and VP16 in the presence of vehicle, which were defined as 1. Bars represent the average ± SEM of three independent experiments. C, Interaction of in vitro translated [35S]ER{alpha}-179C with GST or GST-CH3 in the presence of ethanolic vehicle, 10 nM E2, or 100 nM ICI.

 
The LXXLL motif is a structure important for mediating interactions between coactivators and the coactivator-binding groove of members of the nuclear receptor superfamily (13). The CH3 domain does not encompass any LXXLL motifs, and we therefore sought to determine whether the overall structure of the zinc finger domain was important for CBP interaction with ER{alpha}. The structure of the CH3 domain of CBP has been determined by nuclear magnetic resonance analyses to be composed of 4 {alpha}-helices encompassing three histidine-cysteine rich regions (Fig. 8AGo) capable of forming loops surrounding three zinc atoms (41). Using this information as a guide, mutations of amino acids important for the coordinate binding of each zinc atom were generated in the context of the GAL-CH3 fragment, and the resulting GAL-CH3 zinc finger mutants (CH3-Zn-m1, CH3-Zn-m2, and CH3-Zn-m3) were tested for their ability to bind to ER{alpha} in response to E2 or ICI treatment. As shown in Fig. 8BGo, mutation of any of the amino acids important for zinc binding within the CH3 region blocked interaction with the ER{alpha} LBD, even though each of them were comparably expressed (Fig. 8CGo), suggesting that the overall structure of this region induced by zinc binding is important for ICI-induced interaction with ER{alpha}.


Figure 8
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  		Fig. 8. Mutation of CBP’s CH3 Domain Blocks Its Interaction with ER{alpha}

A, Amino acid sequence of CH3 domain showing the zinc finger-containing domains. Residues shown in bold are responsible for coordinate binding of zinc. Asterisks (*) indicate the amino acids that were mutated to alanine for each of the mutants, m1, m2, and m3. B, HeLa cells were transfected with expression vectors for the GAL-CH3 (CH3) or GAL-CH3 zinc finger mutants (m1, m2, and m3) along with the expression vector for the VP16 activation domain or VP16-ER{alpha} LBD along with the pG5-Luc reporter plasmid. Cells were treated with 0.1% ethanol (vehicle), 10 nM E2, or 100 nM ICI for 20–24 h. Values are normalized to those obtained for GAL-CH3 and VP16 in the presence of vehicle, which were defined as 1. Bars represent the average ± SEM of at least three independent experiments. C, HeLa cells were transfected with 2 µg of either GAL alone or GAL-CH3 or its corresponding mutants. Protein (30 µg) was resolved on 10% SDS-PAGE, and GAL4 chimeric proteins were detected by Western blot using HRP-conjugated antibody against the GAL4 DNA binding domain.

 
Selective Recruitment of CBP to Gene Promoters by ICI
To determine whether ICI could promote an interaction between ER{alpha} and CBP in the context of a target gene promoter, ChIP assays were performed. Consistent with published results (42), Western blot analyses of ER{alpha} expression in ICI-treated MCF-7 cells revealed a modest decline in ER{alpha} expression after 1 h of treatment and a more pronounced decrease with 2 h or greater exposure to ICI (Fig. 9Go). Based on this result, ChIP experiments were performed in cells treated with ER ligands for no longer than 60 min. Thus, for these experiments, MCF-7 cells were treated with {alpha}-amanitin for 1.5 h followed by treatment with either ethanol, E2, 4HT, or ICI for 30, 45, or 60 min and then subjected to ChIP assay using antibodies for ER{alpha} or SRC-1 or CBP. Protein-DNA cross-links were reversed, and DNA was quantitated using real-time PCR with the appropriate primers and probes for pS2 promoter region. Expression of the ER target gene, pS2, is stimulated by E2 and inhibited by ICI, especially after 24 h of ligand treatment (Fig. 10DGo and Ref. 43). As expected, ChIP assays demonstrated that E2 treatment promoted recruitment of ER{alpha}, CBP, and SRC-1 to the pS2 promoter at all three time points examined (Fig. 10Go, A–C). Although ICI induction of ER{alpha} binding to the promoter is weaker than for E2, it is clearly distinguishable from ER{alpha}-DNA interactions in the vehicle-treated cells. Although it has been reported that ICI-bound ER{alpha} is not detected by ChIP on the pS2 promoter (44), those studies were done after 3 h of antiestrogen treatment, and the difference between the two results is likely related to the duration of ICI treatment and possibly the extent of receptor down-regulation.


Figure 9
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  		Fig. 9. Down-Regulation of ER{alpha} Expression by ICI

MCF-7 cells were plated in phenol red-free DMEM containing 10% sFBS and treated with 100 nM ICI for 0–6 h. Cell lysates were analyzed by Western blot analysis for expression of ER{alpha} (top) or actin (bottom). Shown is a representative blot of four experiments.

 

Figure 10
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  		Fig. 10. ER{alpha} Selectively Recruits CBP to Gene Promoters

MCF-7 cells were treated with {alpha}-amanitin for 1.5 h followed by either ethanol, 10 nM E2, 100 nM 4HT, or 100 nM ICI for 30 min (panel A), 45 min (panel B), or 60 min (panels C and E). Cell lysates were subjected to immunoprecipitation with anti-ER or anti-CBP (A-22) or anti-SRC-1 antibodies. Immunoprecipitations were collected using protein A-sepharose beads, and purified DNA was quantified by real-time PCR analysis using specific primers for pS2 promoter (panels A–C) or CYP1B1 promoter (panel E). Values were normalized to input, and values for vehicle treatment were defined as 1. The values for ChIP experiments represent an average of two to four experiments (except n = 1 for 4HT in panel B). Levels of mRNAs for pS2 (panel D) or CYP1B1 (panel F) were measured for cells treated with the indicated hormones for 8 or 24 h and normalized to levels of 18S RNA. Values represent the average ± SEM of four experiments.

 
Recruitment of CBP and SRC-1 was assessed at three time points. In all cases, treatment of cells with either 4HT or ICI prevented recruitment of SRC-1 to the pS2 promoter, and this was consistent with our data demonstrating that these antiestrogens do not promote interaction of this coactivator with ER{alpha} (Fig. 2Go and Ref. 45). In contrast, CBP was found associated with the pS2 promoter after 45 and 60 min of ICI treatment (Fig. 10Go, B and C); no association was apparent with 15 min (data not shown) or 30 min (Fig. 10AGo) of ICI exposure. This recruitment of CBP at 45 and 60 min was comparable to that induced by E2 and was specific for the ICI antiestrogen because 4HT treatment did not produce CBP binding to the pS2 promoter. Thus, in agreement with our prior interaction assays, ICI had a specific effect on recruitment of CBP that was not observed for SRC-1 or for the mixed agonist/antagonist, 4HT.

This prompted us to examine whether ICI recruitment of CBP may be associated with a gene the expression of which is induced by ICI treatment. Recent microarray analyses indicated that ICI increases mRNA expression of the cytochrome P450 CYP1B1 gene (46), and, in our MCF-7 cells, 8 h of ICI treatment stimulates a significant induction of CYP1B1 mRNA (P < 0.05; Fig. 10FGo). Using primers that span the functional ERE within the CYP1B1 promoter (47, 48), ChIP assays demonstrated that both E2 and ICI recruited ER{alpha} to the CYP1B1 promoter (Fig. 10EGo). Consistent with our previous demonstration of SRC-3 recruitment to the CYP1B1 promoter (48), ligand-dependent interaction of CBP and SRC-1 was observed only after E2 treatment; no recruitment was observed for ICI-treated cells. Similar results were obtained for cells treated with ligands for 30 min (data not shown). Taken together, our results indicate that ICI-dependent recruitment of CBP to ER{alpha}-regulated genes is promoter specific. Moreover, the inability of ICI to stimulate pS2 gene expression indicates that recruitment of CBP was insufficient to initiate activation of this gene’s expression.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
It has been well established that interactions between coactivators and ER{alpha} are required for maximal transcriptional activation of target genes (5, 49). Although many studies have demonstrated the important contribution of the p160 and CBP/p300 coactivators to the ability of ER to activate target genes (18, 50, 51), the detailed mechanism by which these cofactors coordinate their function has not been elucidated. In the present study, we employed assays to observe interactions between ER{alpha} and CBP/p300 in the presence of agonist or antagonists. Surprisingly, the pure antagonist ICI, which blocks ER{alpha} transcriptional activity, increased ER{alpha}-CBP and ER{alpha}-p300 interactions to levels higher than those observed for E2. These interactions are unique because ICI was unable to promote ER{alpha} binding to any of the p160 coactivators. Although ICI has been demonstrated to enhance ER{alpha} binding to CIA [a cofactor containing LXXLL coactivator and {phi}XX{phi}{phi} corepressor motifs (34)], and promote the recruitment of silencing mediator of retinoid and thyroid hormone receptor and nuclear receptor corepressor to ER{alpha} (33), to the best of our knowledge, this is the first example of a pure antagonist strongly promoting the recruitment of a coactivator to ER{alpha}.

Numerous studies have demonstrated that LXXLL motifs are major determinants of coactivator interactions with agonist-bound nuclear receptors. There are two LXXLL motifs located within CBP; one in the NID (amino acids 68–75) and another in the CH1 (amino acids 356–366) domain (13, 14). As expected, E2-induced binding was observed for ER{alpha} with the NID, but not the CH1 domain. This is likely attributable to differences in the relative affinities of specific LXXLL motifs for binding to receptors (52, 53). In contrast to the CH1 domain, the CH3 domain, which does not contain any LXXLL motifs, showed strong interaction with the ER{alpha}-LBD in the presence of either E2 or ICI, as measured by mammalian two-hybrid, coimmunoprecipitation, and GST-pull-down assays. Our findings differ from a previous report on ER{alpha} interactions with the CH3 domains of CBP and p300 that were insensitive to either agonist or antagonist ligands (54). The reason for the apparent difference is unknown, but may relate to the previous report’s reliance on in vitro assays and/or experimental conditions. Mutation of residues important for binding each of the three zinc atoms in the CH3 domain disrupted ER{alpha} interaction with this domain, suggesting that its overall structure is important for supporting hormone-dependent interaction with the receptor.

Previous studies have demonstrated that p160 coactivators, as part of a ternary complex, are required for efficient, agonist-induced interaction of CBP with nuclear receptors (17, 55). The ability of the I358D, V376D, and L539A mutations to inhibit ER{alpha}-CBP interactions suggests that the ER{alpha} coactivator-binding groove is important for receptor binding to CBP/p300. However, the differing sensitivity of CBP vs. p160 coactivator binding to the K362D mutant form of ER{alpha}, and the ability of CBP/p300 to bind to the ICI-occupied ER{alpha} LBD independent of p160s suggest that CBP employs a distinct means of binding to receptor in the presence of ICI. The chemical structures of ICI and ICI 164,384 consist of a steroidal ring with a bulky alkylamide side chain protruding from the 7{alpha} position (30). Based on a structure determined for ICI 164,384 binding to ERß, the displacement of the H12 by the bulky side chain results in the enlargement of the ligand-binding pocket, thus creating an 11ß-channel, which can accommodate the 7{alpha}-side chain extending out of the ER{alpha} ligand-binding pocket toward the coactivator-binding surface (26). The inability of the SERMs, 4HT, raloxifene, and idoxifene, to promote CBP interaction with the ER{alpha} LBD indicates that these ligands do not induce an ER{alpha} conformation able to bind directly to CBP. Neither ligands possess a side chain similar in size to the 7{alpha}-side chains of the ICI compounds (11, 56), and the structures of ERs bound to 4HT or raloxifene indicate that in both cases, H12 binds to a portion of the receptor’s coactivator binding groove, which blocks coactivators from binding to this region of the receptor (11, 12). Based on these results, we propose that a ligand side chain of sufficient bulk that can disrupt H12 binding to the ER{alpha} LBD is important for promoting direct interactions between ER{alpha} and CBP/p300. In support of this concept, the ZK-703 compound, which belongs to a group of compounds referred to as specific ER destabilizers and also has a bulky 7{alpha}-side chain structure like ICI, can promote ER{alpha}LBD-CBP interaction to a similar extent as ICI. The inability of the ZK-253 compound to promote ER{alpha}-CBP binding is likely due to the extensive incorporation of the fluoride atoms into the 7{alpha}-side chain (five in ZK-703 and ICI vs. 9 in ZK-253) and possible steric interference. A detailed structural determination of ICI-occupied ER{alpha} bound to the CH3 domain of CBP will be required to determine whether the ICI side chain contributes directly to this interaction.

Previous in vivo footprinting studies demonstrated that ICI treatment of MCF-7 cells promoted the recruitment of multiple proteins to the pS2 promoter in a pattern distinct from that obtained for E2 or 4HT (57). Based on our results, as well as the ability of ICI to promote corepressor binding to ER{alpha} (33), it is likely that the ICI-specific footprinting pattern reflects recruitment of CBP/p300 as well as corepressor proteins to the pS2 promoter. We and others (43, 46, 48) have shown that ICI treatment suppresses pS2 gene expression, indicating that recruitment of CBP to this gene’s promoter does not support its transcription, and we recently demonstrated that RNA polymerase II is not recruited to the pS2 gene in ICI-treated MCF-7 cells (48). This is reminiscent of the experiment that demonstrated that addition of CBP before SRC-1 blocked progesterone receptor activity in in vitro transcription assays (58). In ChIP time course experiments, SRCs are recruited to the pS2 promoter before CBP (7, 9) and it is possible that ordered recruitment of SRCs before CBP is important for gene expression. A more active role for CBP/p300 relative to ICI inhibition of pS2 gene expression also may be possible based on the coactivator’s ubiquitin ligase activity (59) and the well-documented ability of ICI to promote rapid degradation of ER{alpha} through a proteasome-dependent pathway (42, 60). It has been demonstrated that p300, in conjunction with MDM2, serves as both coactivator and ubiquitin ligase for p53, and is required for polyubiquitination and subsequent proteasome-mediated degradation of this transcription factor (59). It is possible that a component of the ICI-dependent inhibition of pS2 gene expression is due to recruitment of the ubiquitin ligase activity of CBP/p300 and subsequent proteasome-dependent degradation of ubiquitinated ER{alpha} and/or other coregulators; this possibility is currently being investigated. Thus, in the context of pure antiestrogens, CBP/p300 does not serve as a coactivator of transcription and may potentially play a more active role in inhibiting gene expression.

Although the expression of pS2 and many other ER{alpha} target genes are inhibited by ICI treatment, other genes have been identified the expression of which is stimulated by this pure antiestrogen. One such gene is the monoxygenase, cytochrome P450 CYP1B1, which can modify E2 by 4-hydroxylation as well as metabolize a wide range of toxicants and carcinogenic chemicals (46, 61). Microarray analyses demonstrated that CYP1B1 expression also is increased by E2, 4HT, and raloxifene (46). Estrogen regulation of CYP1B1 mRNA is mediated via ER{alpha} binding to an imperfect ERE located in the gene’s promoter (47), and we have shown previously by ChIP assay that both ER{alpha} and SRC-3 are recruited to this region in response to E2 treatment (48). Although data in this report indicate that ER{alpha} is recruited to the promoter regardless of whether cells were treated with E2 or ICI, we detected binding of SRC-1 and CBP only in E2-treated cells. The difference in ICI-induced CBP binding to pS2 vs. CYP1B1 gene promoters indicates that this recruitment is promoter specific, perhaps due to differences within the ERE sequences or their context within the respective promoters.

At the present time, it is unclear how ICI increases CYP1B1 mRNA expression, and whether this is a transcriptional event. However, examination of other genes positively regulated by ICI suggests several possibilities. Expression of heat shock protein 27 is induced by ICI 164,384, and this is thought to be mediated through binding of ER{alpha} via Sp1 to a GC-rich region of the promoter (62). Conversely, induction of p21 mRNA expression after ICI treatment of MCF-7 cells is the result of release of ER{alpha} and histone deacetylase 1 from Sp1 sites located in the gene’s promoter (63). The ICI antiestrogen also has been associated with induction of gene expression via ER binding to AP-1 sites, and evidence suggests that this may be achieved via a squelching mechanism that removes repressors from binding to AP-1 (64, 65). Whether ICI recruitment of CBP plays a role in regulation of gene expression under any of these contexts remains to be determined.

In conclusion, our study demonstrates that CBP/p300 binding to ER{alpha} in the presence of the pure antagonist ICI is distinct from p160 interaction with the ER{alpha} ligand-binding domain and that this interaction depends on the nature of a long side chain protruding from the 7{alpha}-position of the steroidal backbone of these antiestrogens. Recruitment of CBP to the promoters of ER{alpha} target genes is gene specific, suggesting that binding of CBP to ICI-bound ER{alpha} contributes to the array of mechanisms that determine the gene- and cell-specific action of the ligands for this receptor. Finally, this work provides novel insight into the mechanistic differences between type I and II (partial and pure, respectively) antiestrogens that should help to clarify the distinct effects of these pharmacological agents critical for prevention and treatment of hormone-dependent cancers.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Plasmids
The pBIND vector encoding the GAL4-DNA binding domain (DBD; amino acids 1–147) and pACT plasmid encoding the herpes simplex virus VP16 activation domain (amino acids 411–456), as well as the pG5-Luc reporter gene containing five DNA binding sites for the GAL4 DBD, a TATA box and luciferase reporter gene were purchased from Promega Corp. (Madison, WI). The pBIND expression vectors for GAL-SRC-1e, GAL-TIF2, GAL-RAC3, and GAL-CBP have been described previously (43). An expression vector for GAL-p300 was obtained from Dr. Tso-Pang Yao (Duke University, Durham, NC). The VP16-ER{alpha} LBD chimera expression vector has been described previously (45). The mammalian expression vectors for the VP16-LBD point mutants (I358D, K362D, V376D, and L539A) were generated by PCR amplification. A portion of the LBD cDNA encoding amino acids 302–595 was amplified using the full-length hER{alpha} version of these mutants (60) as template with the 5'- and 3'-primers, 5'-CGGGATCCCTAAGAAGAACAGCCTGGCCT-3' and 5'-GCTCTAGATCAGACTGTGGCAGGGAAACC-3', respectively, and the resulting PCR products were subcloned into pCR3.1 vector using a TA Cloning kit (Invitrogen, Carlsbad, CA). From these plasmids, BamHI-NotI restriction enzyme fragments were purified and subsequently subcloned into the corresponding sites of pACT such that the LBDs are in frame with and downstream of the VP16 activation domain. To generate cDNAs of CBP domains for the mammalian two-hybrid assays, appropriate regions of the CBP cDNA were PCR amplified using primers listed in Table 2Go and subsequently subcloned into pBIND using PCR and the TA Cloning kit as described above. The CH3 domain mutants were generated by site-directed mutagenesis using the appropriate primers (Table 3Go). The GST-CH3 construct was made by a similar method as for pBIND-CH3 except with the use of the pGEX-4T-1 expression vector (Amersham Biosciences, Piscataway, NJ). The pCR3.1-hER{alpha}-179C construct consists of amino acids 179–595 of ER{alpha} and has been described previously (66). All constructs made through use of PCR were sequenced to verify that mutations did not occur during DNA amplification.


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  		Table 2. Primers Used to Generate CBP Domain cDNA by PCR

 

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  		Table 3. Primers Used to Amplify CH3 Domain Zinc Finger Mutants

 
Cell Culture and Transfection
HeLa cells were maintained in DMEM (Invitrogen) supplemented with 5% fetal bovine serum (FBS) obtained from Invitrogen. Cells were plated 24 h before transfections in six-well culture dishes at a density of 3 x 105 cells per well, in phenol-red free DMEM containing 5% charcoal-stripped fetal bovine serum (sFBS). Cells were transfected with the appropriate amount of DNA using Lipofectamine following the manufacturer’s protocols (Invitrogen). Serum-free medium was replaced 4–5 h later with phenol red-free DMEM and 5% sFBS, and 18–20 h, thereafter, cells were treated with hormones for 20–24 h. After the incubation, cells were harvested and cellular extracts were assayed for luciferase activity using the Luciferase Assay System kit and a Monolight 2010 Luminometer (Analytical Luminescence Laboratory, San Diego, CA); values were normalized to protein content measured with Bio-Rad Protein Assay reagent (Bio-Rad Laboratories, Hercules, CA).

Mammalian Two-Hybrid Assay
In most mammalian two-hybrid assays, HeLa cells were transfected with 100 ng of the expression vectors for GAL-p160 coactivators (pBIND-SRC-1e, pBIND-TIF2, pBIND-RAC3, or pBIND-CBP) or GAL-CBP domains. Along with pBIND expression vectors, 1000 ng of expression vector for VP16-ER{alpha}-LBD (pACT-hER{alpha}-LBD) and 1000 ng of pG5-Luc reporter plasmid were used. In p300-ER interaction assays, 50 ng of GAL-p300 was transfected along with 1000 ng of VP16-ER{alpha}-LBD and 1000 ng of pG5-Luc reporter. Control experiments employed equivalent amounts of the pACT and pBIND empty vectors. Cells were treated with either 0.1% ethanolic vehicle, 10–8 M E2 (Sigma-Aldrich, St. Louis, MO), 10–7 M 4HT (kindly supplied by D. Salin-Drouin, Laboratoires Besin Iscovesco, Paris, France), raloxifene, or idoxifene (kind gift of GlaxoSmithKline, King of Prussia, PA), ICI, or ICI 164,384 (kindly supplied by A. Wakeling, Zeneca Pharmaceuticals, London, UK), or ZK-703 or ZK-253 (kind gift from J. Hoffmann, Schering AG, Berlin, Germany) for 20–24 h before cell harvest and assay as described above.

RBA Assays
The RBA of wild-type and mutant ER{alpha}s for ICI was determined in vivo as previously described (67). Briefly, 2 µg of the expression plasmids for wild-type ER{alpha} (pCR3.1-hER{alpha}) or for the corresponding I358D, K362D, V376D, or L539A mutants (60) were transfected into HeLa cells using Lipofectamine Plus according to the manufacturer’s instructions. Media were aspirated from wells 16–18 h later and replaced with phenol-red free DMEM containing 5% sFBS, approximately 1.5 pmol [3H]estradiol (250 mCi) (PerkinElmer Life Sciences, Wellesley, MA), and increasing concentrations (from 10–10 to 10–3 M) of either unlabeled E2 or ICI. After incubation for 2 h at 37 C, media were aspirated, and cells were washed three times with ice-cold PBS and incubated in 100% ethanol for 10 min at room temperature to extract bound steroid. The amount of ER-bound [3H]E2 in the ethanol extract was quantified with a Beckman LS 6500 scintillation counter (Beckman Instruments, Fullerton, CA) and Biodegradable Counting Scintillant (Amersham Biosciences). The RBA was calculated as the ratio of the concentration of ICI in comparison with E2 required to reduce [3H]E2 binding by 50% times 100. The RBA value of nonradiolabeled E2 was defined as 100.

Coimmunoprecipitation
For studies employing recombinant ER{alpha}, HeLa cells (5 x 106) were harvested and incubated for 30–60 min on ice in lyses buffer [50 mM HEPES (pH 7.5), 100 mM KCl, 0.2 mM EDTA, and 0.1% Nonidet P-40 (NP40)] supplemented with Complete Mini-Tablets protease inhibitor tablets (Roche Applied Sciences, Indianapolis, IN). Thereafter, the cell lysate was centrifuged for 5 min at 21,000 x g, and its protein content was quantified using Bio-Rad Protein Assay reagent. Before immunocomplex preparation, 100 ng of recombinant ER{alpha} (PanVera, Madison, WI) was incubated with either ethanol or 270 ng of estradiol or ICI in 30 µl of buffer [50 mM Tris-HCl (pH 7.5) 10 mM MgCl2, 1 mM EGTA, and 2 mM dithiothreitol] for 30 min. The immunocomplex was prepared in a total volume of 1 ml containing 60 µl (50% slurry) of prewashed protein G+ agarose beads (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), 0.5 mg cell lysate, 100 ng of ligand-bound ER{alpha}, and either 1 µg of anti-CBP (C-1) antibody (Santa Cruz), or 1 µg of anti-SRC-1 antibody (GeneTex, San Antonio, TX). The immunoprecipitation reaction was incubated at 4 C for 1.5 h with constant rotation and then centrifuged and washed three times with lysis buffer. Subsequently, the immunocomplex was boiled for 5 min in 50 µl of 2x Laemmli solution and resolved by 7.5% SDS-PAGE. The Western blot was probed with H222 hER{alpha} antibody (gift of Abbot Laboratories, Abbot Park, IL) in 1% dry milk powder-Tris-buffered saline-Tween, followed by mouse antirat antibody conjugated to horseradish peroxidase (HRP; ICN Biochemicals, Inc., Aurora, OH). Protein bands were detected using ECL plus Western blotting detection system (Amersham Biosciences) and X-Omat film (Perkin Elmer).

For studies examining the interaction between endogenous ER{alpha} and CBP, MCF-7 cells (5 x 106 per 100-mm dish) were grown in phenol-red free DMEM containing 10% sFBS until they reached approximately 80% confluency, and were then treated with either vehicle (0.1% ethanol), 10–6 M ICI, or 10–8 M E2 for 15 min. Cells were harvested and incubated in lysis buffer [20 mM Tris (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 5% glycerol, 1 mM Na3VO4, and 1 mM NaF) supplemented with Complete Mini-Tablets protease inhibitor tablets at 4 C with rotation for 30–60 min. The cell lysates were centrifuged for 5 min at 20,000 x g, and protein contents were quantified using Bio-Rad protein assay reagent. After preclearing with prewashed protein G agarose beads (Amersham Biosciences) for 2 h at 4 C with rotation, the cell lysates (0.5 mg) were incubated with either a human CBP antibody (2 µg; BD Biosciences, San Jose CA) or normal mouse IgG (2 µg; Santa Cruz) at 4 C with rotation for 2 h. Thereafter, 25 µl of a 50% slurry of prewashed protein G agarose beads was added and the incubation continued for another 2 h at 4 C, followed by four washes with lysis buffer. Subsequently, the immunocomplex was boiled at 75 C for 10 min in 25 µl of 1x Laemmli buffer and resolved by 4–12% SDS-PAGE (Invitrogen), and transferred to nitrocellulose. The blot was probed with antibodies directed against human ER{alpha} (HC-20), mouse CBP-NT (Upstate Biotechnology, Inc., Charlottesville, VA) or ß-actin (Chemicon International, Temecula, CA) in PBS containing 5% skim milk powder (wt/vol) and 0.05% Tween 20, followed by the appropriate HRP-conjugated antimouse or antirabbit antibody (ICN). Protein bands were detected using ECL plus Western blotting detection system and X-Omat film.

ChIP Assay
MCF-7 cells (6 x 106) per 150-mm plate were grown for 4 d in DMEM supplemented with 10% sFBS. After that, cells were treated with 2.5 µM {alpha}-amanitin (Sigma-Aldrich, St. Louis, MO) for 1.5 h and then washed twice with serum-free DMEM media. Thereafter cells were treated with either 0.1% ethanol, 10 nM E2, or 100 nM ICI for 15–60 min. Cross-linking was done as follows: the media were replaced with PBS and then cells were treated with 1% formaldehyde for 10 min at 37 C. Cells were then washed twice with ice-cold PBS and harvested by scraping into 1 ml of ice-cold PBS supplemented with Complete Mini-Protease Inhibitor cocktail. Cells were centrifuged and then incubated in 500 µl of lysis buffer [1% sodium dodecyl sulfate (SDS), 10 mM EDTA, 50 mM Tris-HCl (pH 8.0)] for 30 min on ice or 1 ml of nuclei preparation buffer [5 mM piperazine-N,N'-bis (2-ethanesulfonic acid) (pH 8.0), 85 mM KCl, 0.5% NP-40 and Complete Mini-Protease Inhibitors] at 4 C for 30 min, followed by sonication pulses of 8 sec that were repeated four times. After centrifugation, 20 µl of the supernatants was retained as inputs, and the remainder was diluted 10-fold in dilution buffer [16.7 mM Tris-HCl (pH 8.1), 0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, 100 mM NaCl and a Complete Mini-Protease Inhibitor tablet]. The diluted lysate was precleared with 40 µl of a 50% slurry protein-A sepharose beads (Sigma-Aldrich) mixed with 4 µg of sheared salmon sperm DNA at 4 C for 30 min. This diluted fraction was subjected to immunoprecipitation overnight using the appropriate antibody, after which the immunocomplexes were recovered by 1 h incubation at 4 C with 60 µl of protein A-Sepharose and 4 µg of sheared salmon sperm DNA. Precipitates were washed serially with 1 ml Washing Buffer I [20 mM Tris-HCl (pH 8.0), 2 mM EDTA, 0.1% SDS, 1% Triton X-100, and 150 mM NaCl], Washing Buffer II [20 mM Tris-HCl (pH 8.0) and 2 mM EDTA], and Washing Buffer III [10 mM Tris-HCl (pH 8.0), 1 mM EDTA, 1% NP-40, 1% deoxycholate, and 0.25 M LiCl] and then twice with 1x TE [10 mM Tris-HCl (pH 8.0), 1 mM EDTA]. Precipitated chromatin complexes were eluted from the beads by two 15-min incubations with 100 µl of elution buffer (1% SDS and 0.1 M NaHCO3). Cross-linking was reversed by overnight incubation at 65 C, and DNA was purified with QIAquick columns (QIAGEN, Valencia, CA) and then subjected to quantification applying 40 cycles of amplification using pS2 primers and probe (Table 4Go) or CYP1B1 primers (48) and the Real Time PCR system protocol (Applied Biosystems, Foster City, CA).


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  		Table 4. Primers and Probe1 Used for Real Time PCR in pS2 ChIP Experiments

 
Reverse Transcription, Quantitative PCR
Total RNA was extracted from MCF-7 cells with Trizol (Invitrogen). RNA (2 µg) was reverse transcribed with SuperScript RNAseH– reverse transcriptase (Invitrogen) followed by real-time quantitative PCR using an ABI Prism 7700 detection system (Applied Biosystems) with SYBR Green as the fluorescent dye. Primer sequences for CYP1B1 are 5'-GCCCAACCTGCCCTATGTC-3' (forward) and 5'-GGGAATGTGGTAGCCCAAGA-3' (reverse); sequences for pS2 and 18S were defined previously (48). Cycling conditions were 50 C for 2 min and 95 C for 10 min followed by 40 cycles at 95 C for 15 sec and 60 C for 1 min. The threshold was set above the nontemplate control background and within the linear phase of target gene amplification to calculate the cycle number (CT) at which the transcript was detected. Dissociation curves were assessed to ensure that only one product was produced. Gene expression values were calculated based on the comparative {Delta}{Delta}CT method detailed in Applied Biosystems User Bulletin no. 2. Target genes were normalized to values obtained for 18S RNA.

In Vitro Protein-Protein Interaction by GST Pull-Down Assay
The GST-CH3 fusion protein was expressed in isopropyl-ß-D-thiogalactopyranoside-induced Escherichia coli BL21 cultures as recommended by the manufacturer (Amersham Biosciences). Bacteria were collected by centrifugation and resuspended in 5 ml sonication buffer [20 mM HEPES (pH 7.9), 80 mM KCl, 6 mM MgCl2, 1 mM dithiothreitol, 1 mM ATP, 1% Triton X-100, 1% NP-40 and 1% glycerol (33)], and sonicated four times with 10-sec pulses. The sonicated material was centrifuged and the supernatant was incubated with prewashed Glutathione Sepharose 4 Fast Flow beads (Amersham Biosciences) for 1 h with rotation at 4 C. After that, beads were centrifuged at 500 x g and washed three times with 1x TE. The GST-CH3-bound beads were resuspended in 100 µl binding buffer [20 mM HEPES (pH 7.9), 125 mM KCl, 10 mM MgCl2, 1 mM dithiothreitol, 1 mM EDTA (pH 8.0), 2 µg BSA, 0.08% TritonX-100, 0.08% NP40 and 1% glycerol]; CH3 protein was eluted from 10 µl of beads and examined on Coomassie-stained SDS-PAGE to evaluate relative protein expression. Three aliquots of 25 µl of CH3-bound beads were used for the pull-down assay. Each aliquot was added to a tube containing 400 µl of binding buffer, 5 µl of ER{alpha}-179C that was previously expressed using TNT in vitro expression kit (Promega), and labeled with [35S]methionine (Amersham Biosciences). Four microliters of either ethanol, estradiol (100 µM), or ICI (1 mM) was added to the mix. The pull-down mix was incubated at 4 C for 2 h with rotation. Thereafter the beads were washed three times with NETN buffer [20 mM Tris-HCl (pH 8.0), 1 mM EDTA, 0.5% NP40, and 0.5 M NaCl]. The pull-down complex was denatured by 1x reducing agent (Invitrogen) at 70 C for 10 min and resolved by 8% SDS-PAGE followed by electrotransferring to nitrocellulose membrane. The resulting membrane was dried at room temperature and then exposed to X-Omat Blue Film (PerkinElmer) for 16 h at –80 C.


   ACKNOWLEDGMENTS
 
We thank Dr. Martin Dutertre and Dr. Kevin Colman for making available several of the expression vectors used in this study. The assistance of Laura C. Savery for the RBA assays, and the technical support of Judy Roscoe, Cheryl Parker, and Estrella Foster are gratefully acknowledged.


   FOOTNOTES
 
This work was supported by grants from the National Institutes of Health (DK53002 and DK64038) and the Department of Defense (W81XWH-04–1-0424) (to C.L.S.).

Present address for B.M.J.: Department of Biological Sciences, The University of Jordan, Amman 11942, Jordan.

First Published Online July 13, 2006

Abbreviations: AF, Activation function; CBP, cAMP response element-binding protein (CREB)-binding protein; ChIP; chromatin immunoprecipitation assay; CH1, -2, and 3, cysteine-histidine rich domains 1–3; CYP1B1, cytochrome P450 1B1; DBD, DNA-binding domain; ER, estrogen receptor; ERE, estrogen response element; GST; glutathione-S-transferase; HRP, horseradish peroxidase; 4HT, 4-hydroxytamoxifen; KIX, CREB binding domain; LBD, ligand-binding domain; NID, nuclear receptor interacting domain; NP40, Nonidet P-40; RAC3, receptor-associated coactivator 3; RBA, relative binding affinity; SDS, sodium dodecyl sulfate; SERM, selective ER modulator; sFBS, charcoal-stripped fetal bovine serum; SRC-1, steroid receptor coactivator-1; TIF2, transcription intermediary factor 2.

Received for publication June 1, 2005. Accepted for publication July 6, 2006.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

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NURSA Molecule Pages Link:

Nuclear Receptors:   ERα  |  ERβ
Coregulators:   CBP  |  p300  |  SRC-1  |  GRIP1  |  AIB1
Ligands:   17β-Estradiol  |  4-Hydroxytamoxifen  |  Raloxifene  |  Fulvestrant



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