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Molecular Endocrinology 18 (11): 2649-2659
Copyright © 2004 by The Endocrine Society

A Novel Estrogen Receptor {alpha}-Associated Protein Alters Receptor-Deoxyribonucleic Acid Interactions and Represses Receptor-Mediated Transcription

Margaret A. Loven, Roger E. Davis, Carol D. Curtis, Nemone Muster, John R. Yates and Ann M. Nardulli

Department of Molecular and Integrative Physiology (M.A.L., R.E.D., C.D.C., A.M.N.), University of Illinois, Urbana, Illinois 61801; Department of Biological Sciences (N.M.), University of California, Irvine, California 92697; and Department of Cell Biology (J.R.Y.), The Scripps Research Institute, La Jolla, California 92037

Address all correspondence and requests for reprints to: Ann M. Nardulli, Department of Molecular and Integrative Physiology, University of Illinois at Urbana-Champaign, 524 Burrill Hall, 407 South Goodwin Avenue, Urbana, Illinois 61801. E-mail: anardull{at}life.uiuc.edu.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Estrogen receptor {alpha} (ER{alpha}) serves as a ligand-activated transcription factor, turning on transcription of estrogen-responsive genes in target cells. Numerous regulatory proteins interact with the receptor to influence ER{alpha}-mediated transactivation. In this study, we have identified pp32, which interacts with the DNA binding domain of ER{alpha} when the receptor is free, but not when it is bound to an estrogen response element. Coimmunoprecipitation experiments demonstrate that endogenously expressed pp32 and ER{alpha} from MCF-7 breast cancer cells interact. Although pp32 substantially enhances the association of the receptor with estrogen response element-containing DNA, overexpression of pp32 in MCF-7 cells decreases transcription of an estrogen-responsive reporter plasmid. pp32 Represses p300-mediated acetylation of ER{alpha} and histones in vitro and inhibits acetylation of ER{alpha} in vivo. pp32 Also binds to other nuclear receptors and inhibits thyroid hormone receptor ß-mediated transcription. Taken together, our studies provide evidence that pp32 plays a role in regulating transcription of estrogen-responsive genes by modulating acetylation of histones and ER{alpha} and also influences transcription of other hormone-responsive genes as well.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
ESTROGEN RECEPTOR {alpha} (ER{alpha}) belongs to a large superfamily of ligand-inducible transcription factors. Each of these nuclear receptor superfamily members is comprised of six evolutionarily conserved functional domains A–F (Refs.1 and 2 and references therein). The most highly conserved region of these receptors, the DNA binding domain (domain C), is necessary and sufficient for specific binding of the receptor to its cognate recognition sequence in DNA. The hormone binding domain (domain E) is also highly conserved and is required for the interaction of the receptor with a variety of ligands. In addition to these two highly conserved domains are regions with considerable variation in amino acid sequence including the amino-terminal A/B domain, the carboxy-terminal F domain, and the centrally located hinge region, domain D. Within the receptor domains A–F are discrete amino acid sequences that are important in maintaining receptor function. Sequence analysis of ER{alpha} from different species in combination with functional studies of receptor mutants have identified two ER{alpha} regions that are important in enhancing transcription of estrogen-responsive genes (3, 4). The ligand-independent activation function 1, AF-1, is located in the amino terminal A/B domain of the receptor and the hormone-inducible activation function 2, AF-2, is present in the hormone binding domain (5, 6, 7).

ER{alpha} is subject to post translational modifications including acetylation and phosphorylation. Acetylation of the ER{alpha} hinge domain has been reported to occur in vitro and in cells and has been linked to changes in transactivation (8, 9). Phosphorylation of amino acid residues in the A/B domain of the receptor is increased upon exposure of target cells to hormone and is associated with enhanced estrogen-mediated transactivation (10, 11, 12, 13).

Upon binding to ligand, ER{alpha} undergoes a ligand-induced conformational change and interacts with estrogen response elements (EREs) in the promoter regions of estrogen-responsive genes. Interaction of the receptor with the ERE causes changes in receptor structure, which influences recruitment of coregulatory proteins and ultimately alters transcription (14, 15, 16, 17). The interaction of ER{alpha} with coregulatory proteins can increase or decrease transcription of estrogen-responsive genes. Coregulatory proteins that interact with ER{alpha} and enhance estrogen-mediated transcription include the p160 proteins steroid receptor coactivator 1, amplified in breast cancer 1 (AIB1/RAC3/ACTR), and transcription intermediary factor 2 (TIF- 2/GRIP1, Refs.18, 19, 20, 21, 22, 23, 24, 25). Steroid receptor coactivator 1 and AIB1/RAC3/ACTR as well as cAMP binding protein-binding protein (CBP/p300) and p300/CBP-associated factor (PCAF) contain intrinsic histone acetyltransferase activity that enhances gene expression by modifying chromatin structure (26, 27, 28, 29, 30). Acting in opposition to these coactivator proteins are the nuclear corepressors silencing mediator for retinoid X receptor (RXR) and thyroid hormone receptor (TR) and nuclear receptor corepressor. These proteins bind to antiestrogen-occupied ER{alpha} and inhibit transcription by recruiting protein complexes containing Sin3 and histone deacetylases (31, 32). Thus, ER{alpha}-associated coregulatory proteins can have positive or negative effects on the ability of the receptor to activate transcription.

Coregulatory proteins have most often been identified on the basis of their abilities to interact with specific nuclear receptor domains, most commonly the LBD (33). However, we have previously demonstrated that conformational changes induced in one region of the receptor can be translated into conformational changes in another region of the receptor and that these structural changes can alter recruitment of the coactivator proteins (14, 15, 16, 17). In this study, we have examined the recruitment of proteins to full-length ER{alpha} and identified a protein previously cloned and designated pp32. This protein has been implicated in regulating acetylation, phosphorylation, cell transformation, and nucleocytoplasmic shuttling (34, 35, 36, 37).

We demonstrate here that pp32 is associated with ER{alpha} in cells and enhances ER{alpha}-ERE complex formation but decreases ER{alpha}-mediated transcription. Our studies suggest that the ability of pp32 to decrease acetylation of ER{alpha} and histones may play a role in limiting ER{alpha}-mediated transcription.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Isolation and Identification of ER{alpha}-Associated Proteins
The ability of immobilized, full-length ER{alpha} to recruit HeLa nuclear proteins was examined using in vitro pull-down assays. Full-length, flag-tagged ER{alpha} was adsorbed to anti-flag agarose beads and incubated with HeLa nuclear extracts in the absence or in the presence of oligos containing either a nonspecific DNA sequence or the vitellogenin A2 ERE (38). After extensive washing, ER{alpha} and its associated proteins were eluted, fractionated on SDS acrylamide gels, and silver stained. A prominent 66-kDa protein was present on the silver-stained gel (Fig. 1Go), which was identified through Western analysis as ER{alpha} (data not shown). Interestingly, another silver-stained band with an apparent molecular mass of 32 kDa was eluted with ER{alpha} when no DNA (–) or oligos containing a nonspecific DNA sequence (NS) were present in the binding reaction, but not when the oligos containing the A2 ERE (A2) were included. Neither the 66 nor the 32 kDa silver-stained band was present when ER{alpha} was omitted from the reaction (data not shown). A 45-kDa silver-stained band, which we previously identified as template-activating factor I{alpha} (TAF-I{alpha}) and TAF-Iß (8) was also observed.



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  		Fig. 1. HeLa Nuclear Proteins Are Associated with ER{alpha} in the Absence of ERE-Containing DNA

Purified, flag-tagged ER{alpha} was immobilized on flag-affinity resin and incubated without oligos (–) or with oligos containing a NS or the vitellogenin A2 ERE (A2). HeLa nuclear extract was added and unbound protein was washed from the resin. ER{alpha}-DNA-protein complexes were eluted, separated by SDS-PAGE and silver stained. The molecular masses (kDa) of the isolated proteins are indicated on the left.

 
The 32-kDa silver-stained band was excised from the gel and subjected to mass spectrometry analysis. Three peptides from the trypsin-digested protein contained amino acid sequence that was identical to amino acid sequence in the acidic nuclear phosphoprotein pp32 suggesting that pp32 was associated with ER{alpha}. pp32 has also been referred to as the putative HLA class II associated protein I (PHAPI; Ref.39) and the inhibitor of protein phosphatase 2A (I1PP2A; Ref.40).

To confirm the identity of the 32-kDa protein, Western blot analysis was performed using a pp32-specific antibody. As seen in Fig. 2AGo, HeLa nuclear extracts contained substantial amounts of pp32 (lane 1), but the purified ER{alpha} used in our pull-down assays did not (lane 2). pp32 Was present in eluates from our ER{alpha} pull-down assays when HeLa nuclear extracts and ER{alpha} were incubated in the absence and in the presence of 17ß-estradiol (E2, lanes 3 and 4). However, pp32 was not detected in our pull-down assays when A2 ERE-containing oligos were included in the binding reaction regardless of whether E2 was added (lanes 5 and 6). The consensus A2 ERE sequence was not the only ERE that caused dissociation of pp32. Oligos that contained the imperfect pS2 ERE also disrupted the pp32-ER{alpha} interaction (data not shown).



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  		Fig. 2. pp32 Competes with the A2 ERE for Binding to ER{alpha}

A, Flag-tagged ER{alpha} was immobilized on flag-affinity resin (lanes 2–6) and HeLa nuclear extract (HeLa NX, lanes 3–6), E2 (lanes 4 and 6), or oligos containing the vitellogenin A2 ERE (A2 oligo, lanes 5 and 6) were added as indicated. ER{alpha}-associated proteins were separated by SDS-PAGE. Western analysis was carried out with a pp32-specific antibody. Ten percent of the HeLa nuclear extract input was included for reference (lane 1). B, E. coli-expressed, purified GST-pp32 was combined with in vitro translated 35S-labeled ER{alpha}. The resin was washed and pp32-bound ER{alpha} was incubated without (lane 1) or with 0.075, 0.75, or 7.5 pmol of oligos containing the vitellogenin A2 ERE (A2 oligo, lanes 2–4, upper panel) or NS oligo (lanes 2–4, lower panel). Unbound proteins were washed from the resin and the pp32-associated proteins were eluted with SDS sample buffer and separated by SDS-PAGE. The 35S-labeled ER{alpha} was visualized by autoradiography. C, Data from four independent experiments was quantitated by PhosphorImager analysis and analyzed using ImageQuant software. Each data point represents the mean ± SEM. Samples containing oligos with a nonspecific DNA sequence or with the A2 ERE are indicated by open and closed circles, respectively.

 
These initial experiments suggested that the ERE-containing oligos disrupted the interaction of pp32 with ER{alpha}. To determine whether pp32 and the A2 ERE compete for ER{alpha} binding, immobilized glutathione-S-transferase (GST)-pp32 was combined with in vitro transcribed and translated 35S-labeled ER{alpha}. Oligos that contained the A2 ERE or a nonspecific DNA sequence were not (Fig. 2BGo, lane 1) or were added to the reaction (lanes 2–4). The amount of 35S-labeled receptor associated with pp32 was less when oligos containing the A2 ERE were included in the binding reaction (Fig. 2BGo, upper panel) than when oligos containing a nonspecific DNA sequence were included (lower panel). Compiled data from four independent experiments demonstrated that the A2 ERE-containing oligos (panel C, closed circles) decreased the pp32-ER{alpha} interaction to a greater extent than oligos containing a nonspecific DNA sequence (open circles). Taken together, these experiments demonstrate that pp32 was associated with ER{alpha}, that the in vitro association of pp32 with ER{alpha} was not affected by E2, and that pp32 and the A2 ERE compete for ER{alpha} binding.

Interaction of ER{alpha} with pp32
We had demonstrated that pp32 and ER{alpha} interacted in our pull-down assays, but we wanted to determine whether ER{alpha} and pp32 interact in cells where they are normally expressed. We first assessed the level of pp32 in MCF-7, HeLa, and COS cells in Western blot assays using an antibody against pp32. All three cell lines expressed substantial levels of pp32 (Fig. 3AGo). To determine whether endogenously expressed pp32 and ER{alpha} interact, coimmunoprecipitation assays were carried out with extracts from MCF-7 cells, which express significant levels of endogenous ER{alpha} (41) and pp32. Nuclear extracts were prepared from MCF-7 cells that had been exposed to ethanol or E2 and incubated with an immobilized, ER{alpha}-specific antibody. ER{alpha} and its associated proteins were eluted, separated on a denaturing acrylamide gel, and subjected to Western blot analysis with pp32- and ER{alpha}-specific antibodies. When an ER{alpha}-specific antibody was used for immunoprecipitation, comparable levels of ER{alpha} (Fig. 3BGo, lanes 5 and 6, lower panel) and pp32 (lanes 5 and 6, upper panel) were recovered. When similar experiments were carried out using an unrelated antibody (IgG), neither pp32 nor ER{alpha} was detected (lanes 3 and 4). Similar levels of pp32 and ER{alpha} were also present in the nuclear extracts from ethanol- and E2-treated MCF-7 cells (10% input, lanes 1 and 2). These experiments provide evidence that endogenously expressed pp32 and ER{alpha} interact in MCF-7 cells and that this interaction is biologically relevant.



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  		Fig. 3. Endogenous ER{alpha} and pp32 Are Associated in MCF-7 Breast Cancer Cells

A, Nuclear extracts from MCF-7, HeLa, or COS cells were separated by SDS-PAGE, transferred to nitrocellulose and pp32 was detected with a pp32-specific antibody. B, MCF-7 nuclear extracts were incubated with an ER{alpha}-specific antibody or with a nonspecific antibody (IgG). Immunoprecipitated proteins were separated by SDS-PAGE and subjected to Western analysis with pp32- and ER{alpha}-specific antibodies.

 
Domains Required for ER{alpha}-pp32 Interaction
To identify the regions of ER{alpha} required for interaction with pp32, an immobilized GST-pp32 fusion protein was incubated with in vitro translated, 35S-labeled full-length or truncated ER{alpha}. Although the full-length ER{alpha} interacted with full-length pp32 in the absence and in the presence of E2 (Fig. 4Go), pp32 did not interact with the amino terminus (AB) of ER{alpha}. However, addition of the DNA binding domain to the amino-terminal domain (ABC) resulted in binding of the 35S-labeled ABC to pp32. The 35S-labeled protein containing the DNA binding domain and the hinge region (CD) also interacted with pp32. Neither the ligand binding domain combined with the carboxyl-terminal domain (EF) nor the ligand binding domain combined with the carboxyl-terminal and hinge regions (DEF) was retained by pp32. Binding of pp32 was not detected when the translation reaction was carried out in absence of a DNA template (unprogrammed lysate, UPL) or with the GST resin alone (lane 2). These experiments demonstrate that the DNA binding domain is the sole region of the receptor required for interaction with pp32.



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  		Fig. 4. ER{alpha} and pp32 Interact through the DNA Binding Domain of ER{alpha}

GST-affinity resin alone (lanes 2) or E. coli-expressed, purified GST-pp32 (lanes 3 and 4) was combined with in vitro translated 35S-labeled full-length or truncated ER{alpha} or UPL. E2 was added as indicated. Ten percent of the input was included for reference (lanes 1). Assays were carried out three times, and a representative experiment is shown.

 
pp32 Inhibition of ER{alpha}-Mediated Transcription
To test the functional consequence of the ER{alpha}-pp32 interaction, transient transfection assays were carried out in MCF-7 cells, which contain endogenously expressed ER{alpha} (41) and pp32 (Fig. 3AGo). These experiments relied on the endogenously expressed ER{alpha}, not overexpressed ER{alpha}, to activate transcription of a luciferase reporter plasmid containing two EREs. Increasing concentrations of the pp32 expression vector elicited a dose-dependent reduction in E2-mediated transcription (Fig. 5Go). In contrast, the activity of constitutively active simian virus 40 (SV40) and cytomegalovirus (CMV) promoters were not altered by addition of the pp32 expression vector (data not shown). Thus, pp32 effectively inhibited E2-mediated transactivation. Unfortunately, we were unable to knock down expression of pp32 in MCF-7 cells using RNA interference without adversely affecting cell viability. Difficulty in knocking down pp32 expression has been reported previously (42).



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  		Fig. 5. pp32 Inhibits E2-Induced Transcription Activation in Transient Transfection Assays

Transient transfection assays were carried out in MCF-7 cells with the luciferase reporter plasmid 2EREtkLUC and 0, 1, 10, 50, or 100 ng of the pp32 expression vector. Luciferase units were measured and corrected for transfection efficiency. Assays were carried out in duplicate and repeated three times. Normalized data are expressed as the mean ± SEM.

 
Effect of pp32 on Acetylation
Previous studies have demonstrated that pp32 inhibits acetylation of histones (34) and that acetylation of ER{alpha} is also associated with changes in E2-mediated transcription (8, 9). Thus, we reasoned that pp32 might alter acetylation of ER{alpha} and thereby modulate estrogen responsiveness. To determine whether this was the case, acetylation assays were carried out with 3H-acetyl coenzyme A (CoA) and p300, which acetylates histones and interacts with ER{alpha} (34, 43). Purified ER{alpha}, histones, and pp32 were included in the incubation as indicated in Fig. 6Go, and the proteins were eluted and separated on a denaturing gel. When p300 and histones were included in the reaction, acetylation of histones and auto-acetylation of p300 was observed (Fig. 6AGo, lane 1). The identity of the acetylated histones was confirmed by immunoblotting (data not shown). A nonspecific acetylated product that ran between the histones and ER{alpha} was also present. Inclusion of purified pp32 in the reaction significantly reduced histone acetylation (lane 2). Baculovirus-expressed, purified ER{alpha} was acetylated in the presence of p300 (lane 3), but ER{alpha} acetylation was decreased when pp32 was present (lane 4). Inclusion of equal amounts of ovalbumin in lieu of pp32 had no effect on p300-mediated acetylation of ER{alpha} (data not shown). Quantitation of data from three independent assays indicated that pp32 decreased acetylation of histones by nearly 75% and reduced acetylation of ER{alpha} by 15% (Fig. 6BGo). In contrast, pp32 did not significantly affect p300 autoacetylation. Thus, pp32 modulates acetylation of histones and ER{alpha} in vitro.



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  		Fig. 6. pp32 Inhibits Acetylation of Histones and ER{alpha} in Vitro and Acetylation of ER{alpha} in Cells

A, Acetyltransferase assays were carried out using baculovirus-expressed, purified p300, and 3H-acetyl CoA. Purified histones, ER{alpha}, and pp32 were included as indicated. Reactions were separated by SDS-PAGE and visualized by autoradiography. B, Data from four independent acetylation assays were combined and quantitated. The decrease in acetylation in the presence of pp32 compared with the absence of pp32 is expressed as mean ± SEM. C, COS cells were transfected with an ER{alpha} expression vector without (–) or with (+) a pp32 expression vector. ER{alpha} was immunoprecipitated from cell lysates with an ER{alpha}-specific antibody, separated by SDS-PAGE and detected by immunoblotting (IB) with an antibody directed against acetylated lysine residues (upper panel) or ER{alpha} (lower panel).

 
To determine whether pp32 alters acetylation of ER{alpha} in cells, an ER{alpha} expression vector was transfected into COS cells in the absence or in the presence of a pp32 expression vector. ER{alpha} was immunoprecipitated from cell lysates with an ER{alpha}-specific antibody, and Western blots were carried out with an antibody to acetylated lysine (Fig. 6CGo, top panel) or ER{alpha} (bottom panel). The level of acetylated ER{alpha} was decreased when the pp32 expression vector was included. It is possible that because the levels of pp32 are already relatively high in COS cells (Fig. 3AGo), endogenous pp32 may inhibit ER{alpha} acetylation even in the absence of the transfected pp32 expression vector, resulting in the relatively modest decrease in ER{alpha} acetylation observed when the pp32 expression vector was included. Differences in ER{alpha} acetylation levels with pp32 coexpression were not due to variation in the amount of ER{alpha} present because the levels of immunoprecipitated ER{alpha} were similar (bottom panel). Thus, pp32 not only decreases ER{alpha} acetylation in vitro, but also decreases ER{alpha} acetylation in a cellular environment.

Because pp32 is a phosphatase inhibitor, we also monitored the phosphorylation of ER{alpha} in MCF-7 cells. Although we failed to detect any differences when pp32 was overexpressed, phosphorylation of serine 118 was increased when another phosphatase inhibitor, TAF-Iß, was overexpressed (data not shown).

Effect of pp32 on the ER{alpha}-ERE Interaction
Another way that pp32 might decrease transcription is by decreasing the ability of the receptor to bind to DNA. Thus, we tested the ability of ER{alpha} to bind to ERE-containing DNA in the absence and in the presence of pp32. Surprisingly, when increasing amounts of purified pp32 were added to the binding reaction, there was a striking increase in ER{alpha}-ERE complex formation, both in the absence and in the presence of E2 (Fig. 7AGo). This increase in complex formation was not due to the presence of additional protein because protein levels were held constant in all reactions by the addition of BSA. Interestingly, the migration of the receptor-DNA complex was not altered by the presence of pp32, suggesting that pp32 enhanced ER{alpha} binding but was not part of the protein-DNA complex. These findings are reminiscent of previous studies with the high-mobility group protein 1, which increases the interaction of the ER{alpha} and a number of steroid hormone receptors with their cognate recognition sequences without altering the migration of the receptor-DNA complexes (44, 45, 46). Other ER{alpha}-associated proteins including TAF-Iß and 3-methyladenine DNA glycosylase also enhance the ER{alpha}-ERE interaction but decrease estrogen-mediated transcription (8, 47).



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  		Fig. 7. pp32 Enhances the ER{alpha}-ERE Interaction

Baculovirus expressed, purified ER{alpha} was incubated with 32P-labeled DNA fragments containing a consensus ERE. Purified pp32 and E2 were added to the binding reaction as indicated. Binding reactions were incubated for 15 (A) or 0.25, 1 or 2 (B) min, loaded onto nondenaturing acrylamide gels, and separated. C, Four independent gel mobility shift assays were carried out in the absence or in the presence of pp32 and E2 (–E2 –pp32, closed squares; –E2 +pp32, open squares; +E2 –pp32, closed circles; +E2 +pp32 open circles) as shown in panel B, quantitated by PhosphorImager analysis, and analyzed using ImageQuant software. Data are expressed as mean ± SEM.

 
To more carefully examine ER{alpha}-ERE complex formation, incubation times were decreased to 0.25, 1, or 2 min. In the absence of pp32, lengthening the incubation time from 0.25–2 min slightly increased formation of the receptor-DNA complex (panel B, lanes 1–3 and 7–9; and panel C). When pp32 was present, extending the incubation time increased formation of the receptor-DNA complex nearly 2-fold (panel B, lanes 4–6 and 10–12; and panel C). Furthermore, when both E2 and pp32 were present in the incubation, the level of ER{alpha}-ERE complex formation was further enhanced. Thus, rather than decreasing the association of ER{alpha} with DNA, pp32 substantially enhanced the association of the receptor with ERE-containing DNA and hormone further enhanced ER{alpha}-ERE complex formation.

Interaction of pp32 with Other Nuclear Receptors
ER{alpha} is a member of the closely related nuclear receptor superfamily. To determine whether pp32 might also interact with other nuclear receptors, pp32 pull-down experiments were carried out. Immobilized GST-pp32 was incubated with in vitro transcribed and translated 35S-labeled progesterone receptor B (PR-B), TRß, peroxisome proliferator-activated receptor {gamma} (PPAR{gamma}) or RXR{alpha}. Although PR-B, TRß, and PPAR{gamma} bound to pp32 in the absence and in the presence of their respective hormones, RXR{alpha} was unable to interact with pp32 (Fig. 8AGo). More importantly, when MCF-7 cells were cotransfected with a TRß expression vector and a chloramphenicol acetyl transferase (CAT) reporter plasmid containing two thyroid hormone response elements (TREs) in the absence and in the presence of a pp32 expression vector, pp32 inhibited thyroid hormone-induced transcription (Fig. 8BGo). These findings suggest that pp32 may be involved in regulation of other hormone-responsive genes as well.



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  		Fig. 8. Immobilized, Partially Purified pp32 Interacts with Other Nuclear Receptors

A, GST affinity resin containing no protein (–) or purified GST-pp32 (+pp32) was combined with in vitro translated 35S-labeled PR-B, TRß, RXR{alpha}, PPAR{gamma}, or UPL and appropriate hormone was not (–) or was (+) added. 10% of the input was included for reference (lane 1). B, Transient cotransfection assays were carried out in MCF-7 cells using a TRß expression vector, the CAT reporter plasmid TRE2tkCAT and a ß-gal internal control vector in the absence (–) or in the presence (+) of 100 ng of a pp32 expression vector. CAT assays were carried out and normalized to ß-galactosidase activity. Data from three independent experiments were combined and are expressed as the mean ± SEM.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
We have identified a novel ER{alpha}-associated protein, pp32, which interacts with the receptor in in vitro pull-down assays, increases ER{alpha}-ERE complex formation and inhibits ER{alpha}-mediated transcription. Importantly, endogenously expressed ER{alpha} and pp32 interact in a cell background where these two proteins are routinely expressed.

pp32 Appears in the literature in a wide variety of contexts. It has been identified as an acetylation and phosphatase inhibitor and is thought to play a role in differentiation of cerebellar neurons (34, 36, 48). pp32 is expressed in both normal and neoplastic cells, it interacts with the nuclear export receptor, chromosomal region maintenance protein 1, and it modulates the activity of an mRNA-stabilizing protein, HuR (37, 49). Thus, the involvement of pp32 in ER{alpha}-mediated transcription adds yet another role to this diverse collection of pp32 functions.

Role of pp32 in Regulating Estrogen-Responsive Gene Expression
Our initial pull-down experiments, which demonstrated that pp32 was not associated with the ERE-bound receptor, combined with our gel shift assays, which demonstrated that pp32 was not present in the ER{alpha}-ERE complex and that it competed with the ERE for receptor binding, suggest that the receptor can bind to either pp32 or to ERE-containing DNA, but that it does not bind simultaneously to both pp32 and the ERE. A plausible explanation for these findings is that the DNA binding domain, which is required for interaction with pp32 and with the ERE, may only be able to accommodate one of these binding partners at a time. Alternatively, an ERE-induced change in ER{alpha} conformation could play a role in pp32 dissociation. Previous work in our laboratory has demonstrated that subtle changes in the ERE sequence alter receptor conformation, recruitment of coregulatory proteins, and transcription activation (15, 16, 17). As seen in our in vitro binding assays and immunoprecipitation experiments with endogenously expressed pp32 and ER{alpha} from MCF-7 cells, addition of hormone does not affect the interaction of ER{alpha} with pp32. Rather, it is the binding of the receptor to the ERE that disrupts the pp32-ER{alpha} interaction. These findings contrast with those of many coregulatory proteins, whose interactions with nuclear receptors are altered by hormone binding. In the case of pp32, it is DNA rather than hormone that affects binding of pp32 to the receptor.

We envision three ways in which pp32 could decrease ER{alpha}-mediated transactivation. First, pp32 may alter estrogen-mediated transcription by decreasing ER{alpha} acetylation. It has been suggested that pp32 may mask lysine residues on histones and thereby decrease histone acetylation and limit transcription (34). It seems possible that pp32 may also mask the DNA binding domain of the receptor and help to maintain the receptor in a more quiescent state. Second, the pp32-enhanced binding of less acetylated ER{alpha} to estrogen-responsive genes could alter the interaction of the DNA-bound receptor with other coregulatory proteins. It is also possible, though we have not tested at this point, that pp32 could also regulate ER{alpha}-mediated transcription through modifying the acetylation state of other coregulatory proteins. Third, the dissociation of pp32 that occurs when ER{alpha} binds to DNA could foster pp32 binding to nearby histones in promoter regions containing EREs. This targeting of pp32 to ERE-containing genes could decrease histone acetylation and limit ER{alpha}-mediated transactivation. Through these combined actions, pp32 could decrease estrogen-mediated gene expression, which is consistent with pp32’s role as a tumor suppressor (50).

Actions of Novel Coregulatory Proteins
Recently, we identified another novel coregulatory protein, template activating factor Iß (TAF-Iß), which interacts with ER{alpha} (8) and with pp32 (34, 39, 51). TAF-Iß and pp32 are similar in a number of ways. Both proteins 1) interact with free ER{alpha} but not with the ERE-bound receptor, 2) decrease acetylation of ER{alpha} in vitro and in cells, and 3) inhibit ER{alpha}-mediated transcription (8). Studies from other laboratories have reported that TAF-Iß and pp32 are components of an approximately 150- to 170-kDa nuclear complex that inhibits histone acetylation (34) and that they interact with granzyme A, a protein involved in apoptosis (51), and HuR, a protein involved in mRNA stability (37).

Despite these similarities, TAF-Iß and pp32 also have distinct properties. For example, TAF-Iß is associated with ER{alpha} in MCF-7 breast cancer cells only in the absence of E2, but pp32 is associated with ER{alpha} both in the absence and in the presence of ligand. Although TAF-Iß and pp32 stabilize ER{alpha}-ERE complex formation in vitro, only pp32 causes an increase in ER{alpha}-ERE complex formation in the presence of E2 and only TAF-Iß alters phosphorylation of ER{alpha} (Fig. 8Go, Ref.8 and data not shown). Thus, although both pp32 and TAF-Iß affect ER{alpha}-mediated signaling pathways, the exact mechanism by which they exert their effects may differ in subtle ways.

Role of pp32 in Cells
Interestingly, Pasternack and co-workers (50, 52, 53, 54) have demonstrated that two pp32 variants, pp32r1 and pp32r2, are expressed in prostate and mammary tumors, but that only pp32 is expressed in adjacent normal tissue. This raises the intriguing possibility that the ability of pp32 to inhibit estrogen-mediated gene expression may decrease hormone responsiveness of target cells and help to maintain normal cell biology. Moreover, our studies indicate that pp32’s actions are not restricted to ER{alpha}-mediated transcription. Given the wide distribution of nuclear receptors, it seems possible that pp32 may modulate expression of numerous hormone-responsive genes in a variety of cellular contexts.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
HeLa Pull-Down Assay
The isolation of ER{alpha}-associated proteins was carried out as described previously (8). Briefly, baculovirus-expressed ER{alpha} (15) was immobilized on M2 agarose (Sigma, St. Louis, MO) and resuspended in HeLa binding reaction buffer [15 mM Tris (pH 7.9), 2 mM EDTA, 20 mM KCl, 4 mM dithiothreitol (DTT), and 0.5 mM ZnCl2] containing 2 µg/ml poly(deoxyinosine/deoxycytosine) in the presence or absence of 1 µM 17ß-estradiol (E2). Annealed oligos containing a nonspecific sequence or consensus ERE were allowed to bind to immobilized ER{alpha} for 45 min at 4 C and unbound DNA was washed from the resin. HeLa nuclear extracts were prepared as described previously (15), added to the immobilized ER{alpha} in HeLa binding reaction buffer, incubated for 1 h at 4 C, and the resin was washed extensively in wash buffer [20 mM Tris (pH 7.5), 50 mM NaCl, 0.2 mM EDTA, 10% glycerol, 0.1% Nonidet P-40, and 2 mM DTT]. The DNA-bound ER{alpha} and its associated proteins were eluted from the resin in wash buffer containing 0.2 mg/ml flag peptide (University of Illinois Biotechnology Center, Urbana, IL). Eluted proteins were separated by SDS-PAGE.

Cell Culture and Transfections
For MCF-7 breast cancer cell transfections, cells were maintained in phenol red-containing MEM (Sigma, St. Louis, MO) with 5% calf serum. Two days before plating, cells were transferred to phenol red-free MEM supplemented with 5% charcoal dextran-treated calf serum. Cells were then seeded in 24-well plates and transfected using Lipofectin as previously described (16) with 0.25 ng of the Renilla expression vector pRLSV40 (Promega, Madison, WI) and 2 µg of the firefly luciferase reporter vector 2EREtkLUC (kindly provided by Benita Katzenellenbogen, University of Illinois, Urbana, IL). For TRß studies, MCF-7 cells were transfected in 24-well plates with 5 ng of the TR-ß expression vector pCI-TRß, 2 µg of the firefly luciferase reporter vector pGL3-TRE (both kindly provided by Milan Bagchi, University of Illinois, Urbana, IL) and 0.25 ng of the renilla expression vector pRLSV40. Increasing concentrations of the pp32 expression vector (50) kindly provided by Gary Pasternack (Johns Hopkins, Baltimore, MD) were added as indicated. Luciferase assays were carried out as previously described (8, 16). At least three independent experiments were carried out in duplicate. To allow for variation in luciferase readings between experiments, the global mean for all measurements was compared with the mean for each individual experimental. Data were normalized by a factor reflecting the variation between the global mean and the experimental mean. Student’s t tests were used to determine whether statistical difference existed in the luciferase activity when cells had been transfected with or without the pp32 expression vector.

Micro-Liquid Chromatography-Electrospray Ionization Tandem Mass Spectrometry
Silver-stained protein bands were excised from acrylamide gels and analyzed as previously described (8). Three peptides, VSGGLEVLAEKCPNLTHLNLSGNK, SLDLFNCEVTNLNDYR, and LLPQLTYLDGYDR, which comprise approximately 16% of the full protein sequence, were recovered from the gel slices and used to identify pp32.

Plasmid Construction
The GST-pp32 expression vector pGEX2T-pp32 was created by subcloning the BamHI-XbaI fragment of pcDNA3 pp32flag vector (37), kindly provided by Joan Steitz (Yale University, New Haven, CT) into SmaI-cut pGEX2T (Amersham Bioscience, Piscataway, NJ). The integrity of the DNA junctions was verified by DNA sequencing.

Pull-Down Assay Using in Vitro-Translated Proteins
GST-pp32 was expressed in Escherichia coli using the expression vector pGEX2T-pp32. Pull-down experiments were carried out as previously described (8). Briefly, pp32 was immobilized on GST-Sepharose (Amersham Pharmacia Biotech, Piscataway, NJ). 35S-labeled proteins were synthesized using the TNT T7 Quick coupled transcription/translation system (Promega, Madison, WI) and incubated for 1 h at 4 C with the immobilized GST-pp32 in the presence or absence of 1 µM E2, the PPAR{gamma} agonist BRL49653, 9-cis retinoic acid, progesterone or thyroid hormone as appropriate. Beads were washed and bound proteins were eluted with SDS sample buffer and separated by SDS-PAGE. Dried gels were subjected to autoradiography for 1–2 d.

For DNA competition assays, GST-pp32 was immobilized on GST-Sepharose and combined with in vitro-translated 35S-labeled ER{alpha} as described above. The resin was washed and the pp32-bound ER{alpha} was incubated in the presence of 0, 0.075, 0.75, or 7.5 pmol of oligos containing a nonspecific DNA sequence or the vitellogenin A2 ERE. Unbound proteins were washed from the resin and the retained proteins were eluted with SDS sample buffer and separated by SDS-PAGE. Four independent experiments were performed and autoradiograms were scanned and quantitated using ImageQuant 5.0 (Molecular Dynamics, Inc., Sunnyvale, CA).

Gel Mobility Shift Assays
Gel mobility shift assays were carried out essentially as described previously (16) with the following modifications. In Fig. 7AGo, purified ER{alpha} and the 32P-labeled, 34-bp ERE-containing oligos were incubated for 5 min in binding reaction buffer [15 mM Tris (pH 7.9), 0.2 mM EDTA, 50 mM KCl, 7.5 mM NaCl, 10% glycerol, 4 mM DTT, and 50 µM ZnCl2] in the absence or in the presence of 50 nM E2 as indicated. Purified pp32 was added to the reaction as indicated and incubations were continued for 10 min. Total protein and salt concentrations were held constant in all reactions by the addition of BSA (Roche Diagnostics Corp., Indianapolis, IN) and KCl. In Fig. 7BGo, ER{alpha} and pp32 were incubated for 5 min before the addition of 32P-labeled, 34-bp ERE-containing oligos. Incubations were extended for the times indicated. The ER{alpha}-ERE complexes and free DNA were fractionated on nondenaturing acrylamide gels. The level of bound and free 32P-labeled DNA was quantitated using a Molecular Dynamics PhosphorImager and ImageQuant 5.0.

SDS-PAGE, Silver Staining, and Western Analysis
For Western analysis, samples were fractionated on 15% SDS-PAGE gels and transferred to a nitrocellulose membrane. pp32-specific antibody, generously provided by Gary Pasternack (55) was used to detect pp32. ER{alpha} was detected with an ER{alpha}-specific antibody (sc-8005 or sc-543, Santa Cruz Biotechnology, Inc., Santa Cruz, CA). Acetylation of ER{alpha} was detected using a rabbit polyclonal antibody specific to acetylated lysine residues (06-933, Upstate Biotechnology, Lake Placid, NY). Blots were probed with horseradish peroxidase-conjugated secondary antibody (Zymed, South San Francisco, CA) and the SuperSignal West Femto Maximum Sensitivity Substrate chemiluminescent detection kit (Pierce Chemical Co., Rockford, IL) was used to visualize the proteins as per the manufacturer’s instructions.

Acetylation Assay
Baculovirus-expressed p300 was prepared using viral stock kindly provided by W. Lee Kraus (Cornell University, Ithaca, NY) as described (43). Histones were isolated from MCF-7 cells as described (56), and acetyl transferase assays were carried out using a protocol modified from Brownell and Allis (57). Purified p300 was incubated with 0.2 µl 3H-acetyl CoA (4.7 Ci/mmol, Amersham Pharmacia Biotech). Histones, ER{alpha}, and pp32 were added as indicated in 15 mM Tris (pH 7.8), 0.25 mM EDTA, 0.05% Tween 20, 0.25 mM DTT, and 5% glycerol, and samples were incubated for 30 min at 30 C. Proteins were separated by SDS-PAGE and gels were treated with En3hance (NEN-DuPont, Boston, MA) before exposure to film at –80 C for 1–5 d. Three independent experiments were performed and autoradiograms were scanned and quantitated using ImageQuant 5.0 (Molecular Dynamics, Inc.). Student’s t tests were used to determine whether statistical differences existed between samples that had been incubated with or without pp32.

Coimmunoprecipitation Assay
To examine ER{alpha}-associated proteins, endogenously expressed ER{alpha} and its associated proteins were coimmunoprecipitated from MCF-7 breast cancer cells. MCF-7 cells were incubated with ethanol or 100 nM E2 for 20 min and nuclear extracts were prepared as previously described (15). To reduce nonspecific background, 0.5 mg of MCF-7 nuclear extract and 15 µl protein A agarose slurry were incubated in NaCl-TE [10 mM Tris (pH 8.0), 1 mM EDTA, and 285 mM NaCl] in the absence or presence of E2 for 1.5 h at 4 C. Meanwhile, 5 µg of the ER{alpha}-specific antibody sc-8005 (Santa Cruz Biotechnology, Inc.) and 15 µl of protein A agarose slurry (Santa Cruz Biotechnology, Inc.) were incubated in NaCl-TE for 1.5 h at 4 C. The nuclear extract slurry and the antibody slurry were pelleted. Precleared nuclear extract solution was added to the antibody-agarose pellet and incubated for 2.5 h at 4 C. The agarose beads were washed four times with 24 mM Tris (pH 7.4), 2 mM KCl, and 163 mM NaCl before boiling for 10' in sample buffer. The resulting eluates were separated by SDS-PAGE and transferred to a nitrocellulose membrane for Western analysis.

To examine ER{alpha} acetylation in vivo, COS cells were transfected as described above, except that cells were plated in six-well plates and 750 ng of the human ER{alpha} expression vector CMV5hER (58) and either 5 µg of the pp32 expression vector pp32 CMV (50) or 5 µg of the parental expression vector pCMV were included. Following transfection and incubation for 24 h, cells were resuspended in lysis buffer containing 20 mM Tris (pH 8.0), 200 mM NaCl, 1 mM EDTA, and 0.2% Nonidet P-40. ER{alpha} was immunoprecipitated using half the cell lysate from each well as described above.


   ACKNOWLEDGMENTS
 
We are grateful to J. Steitz and G. Pasternack for providing pp32 expression vectors and antibodies and to B. Katzenellenbogen, W. L. Kraus, and M. Bagchi for providing additional reagents used in these studies.


   FOOTNOTES
 
This work was supported by National Institutes of Health (NIH) Grant DK-53884 (to A.M.N.). M.A.L. received support from NIH Training Grants 2 T32 HD07028 and T32 ES07326, and N.M. was supported by NIH Grant NCRR RR11823-05.

Abbreviations: AF, Activation function; CAT, chloramphenicol acetyl transferase; CBP, cAMP binding protein-binding protein; CMV, cytomegalovirus; CoA, coenzyme A; DTT, dithiothreitol; ER, estrogen receptor; ERE, estrogen response element; GST, glutathione-S-transferase; NS, nonspecific DNA sequence; PPAR{gamma}, peroxisome proliferator-activated receptor {gamma}; PR-B, progesterone receptor B; RXR, retinoid X receptor; SV40, simian virus 40; TAF, template-activating factor; TR, thyroid hormone receptor; TREs, thyroid hormone response elements; UPL, unprogrammed lysate.

Received for publication May 27, 2003. Accepted for publication July 29, 2004.


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TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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NURSA Molecule Pages Link:

Nuclear Receptors:   TRβ  |  PPARγ  |  RXRα  |  ERα  |  PR
Coregulators:   pp32  |  p300
Ligands:   17β-Estradiol



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