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Molecular Endocrinology, doi:10.1210/me.2002-0280
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Molecular Endocrinology 17 (1): 67-78
Copyright © 2003 by The Endocrine Society

A Novel Estrogen Receptor {alpha}-Associated Protein, Template-Activating Factor Iß, Inhibits Acetylation and Transactivation

Margaret A. Loven, Nemone Muster, John R. Yates and Ann M. Nardulli

Department of Molecular and Integrative Physiology (M.L., A.M.N.), University of Illinois, Urbana, Illinois 61801; and Department of Cell Biology (N.M., 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}) functions as a ligand-activated transcription factor that alters expression of estrogen-responsive genes in target cells. Numerous regulatory proteins interact with ER{alpha} to influence estrogen-mediated transactivation. We have identified a novel coregulatory protein, template-activating factor-Iß (TAF-Iß), which binds to ER{alpha} in vitro when the receptor is not complexed with an estrogen response element. The central region of TAF-Iß interacts with both the DNA-binding domain and the carboxy-terminal region of ER{alpha}. Coimmunoprecipitation experiments demonstrate that TAF-Iß is associated with the unoccupied, but not the estrogen-occupied, ER{alpha} in MCF-7 breast cancer cells. Overexpression of TAF-Iß inhibits ER{alpha}-mediated transcription in a dose- dependent manner. TAF-Iß represses p300-mediated acetylation of histones and ER{alpha} in vitro and decreases ER{alpha} acetylation in vivo. TAF-Iß also binds to other nuclear receptor superfamily members and represses thyroid hormone receptor ß- induced transcription in transient transfection assays. Taken together, these data provide evidence that TAF-Iß regulates transcription of estrogen- responsive genes by modulating acetylation of histones and ER{alpha} and that the effects of TAF-Iß extend to other nuclear receptor superfamily members as well.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
ESTROGEN RECEPTOR {alpha} (ER{alpha}) belongs to a large superfamily of nuclear receptors that function as ligand-inducible transcription factors. Each of these nuclear receptor superfamily members is comprised of six functional domains (A–F) that have been evolutionarily conserved (Refs. 1 and 2 and references therein). The most highly conserved region is the DNA-binding domain, domain C, which is necessary and sufficient for specific binding of the receptor to its cognate recognition sequence. The hormone-binding domain, domain E, is also highly conserved and directs the specific interaction of the receptor with hormone. 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-dependent activation function 1 is localized in the amino-terminal A/B domain of the receptor, and the hormone-inducible activation function 2, is present in the hormone-binding domain (5, 6, 7).

ER{alpha} is subject to posttranslational modifications including phosphorylation and acetylation. Phosphorylation of serine 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 (8, 9, 10, 11). Acetylation of the ER{alpha} hinge region has been reported to occur in vitro and in vivo and has been linked to an increase in hormone sensitivity (12).

Upon binding hormone, ER{alpha} undergoes a conformational change, binds to estrogen response elements (EREs) that reside in estrogen-responsive genes, and initiates changes in transcription of these genes. Numerous coregulatory proteins associate with ER{alpha} and enhance estrogen-mediated transcription including the p160 proteins steroid receptor coactivator 1, amplified in breast cancer 1, and transcription intermediary factor 2 (reviewed in Refs. 13 and 14). Both steroid receptor coactivator 1 and amplified in breast cancer 1, as well as cAMP response element binding protein/p300 and p300/cAMP response element binding protein-associated factor, possess intrinsic histone acetyltransferase activity that has been implicated in enhancing gene expression by modifying chromatin structure. A large protein complex identified on the basis of its interaction with the thyroid hormone and vitamin D receptors has been designated as the thyroid hormone receptor-associated protein (TRAP) or vitamin D receptor interacting protein (DRIP) complex. DRIP205/TRAP 220, which anchors the DRIP/TRAP complex to nuclear receptors, interacts with ER{alpha} in a ligand-dependent manner and enhances transcription (15, 16). The nuclear corepressors SMRT [silencing mediator for retinoid X receptor (RXR) and thyroid hormone receptor (TR)] and NCoR (nuclear receptor corepressor) bind to the antiestrogen-occupied ER and unoccupied nuclear receptors and inhibit transcription by recruiting protein complexes containing Sin3 and histone deacetylases (reviewed in Refs. 13 and 14). Thus, ER{alpha}-associated coregulatory proteins can have positive or negative effects on the ability of the receptor to activate transcription.

The majority of these ER{alpha}-associated coregulatory proteins have been isolated on the basis of their ability to interact with specific nuclear receptor domains, most commonly the ligand-binding domain (LBD) (17). 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 alter recruitment of the coactivator proteins (18, 19). In this study we have examined the recruitment of proteins to full-length ER{alpha} and identified a protein previously implicated in inhibiting acetylation of histones and modulating chromatin structure (20, 21, 22). We demonstrate that this ER{alpha}-associated protein, template-activating factor Iß [TAF-Iß (21)], inhibits p300-mediated acetylation of ER{alpha} in vitro and is associated with the unliganded receptor in MCF-7 cells. Overexpression of TAF-Iß in transfected cells decreases ER{alpha}-mediated transcription and ER{alpha} acetylation. We propose that TAF-Iß inhibits ER{alpha}-mediated transcription and maintains cells in a quiescent state by binding to the receptor and to histones and decreasing acetylation.


   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 (23). After extensive washing, ER{alpha} and its associated proteins were eluted, fractionated on sodium dodecyl sulfate acrylamide gels, and silver stained. A prominent 66-kDa protein was present on the silver-stained gel (Fig. 1AGo), which was identified through Western analysis (Fig. 1BGo) as ER{alpha}. Interestingly, another silver-stained band with an apparent molecular mass of approximately 45 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 contained the A2 ERE (A2). In contrast, neither of these silver-stained bands was present when the flag resin was used in the absence of ER{alpha} (data not shown).



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  		Figure 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 nonspecific DNA sequence (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 (panel A) or immunoblotted using antibody against ER{alpha} (panel B).

 
The approximately 45-kDa silver-stained band was excised from the gel and subjected to mass spectrometry analysis. Six peptides from the trypsin-digested protein contained amino acid sequence that was identical to amino acid sequence in template activating factor I (TAF-I) {alpha} and ß (24) suggesting that TAF-I{alpha} and/or TAF-Iß was associated with ER{alpha}. TAF-I{alpha} and TAF-Iß have 37 and 24 unique amino acids, respectively, at their amino termini followed by 253 identical amino acids (24). Both proteins have been implicated in modulating DNA replication, histone acetylation, and chromatin remodeling (20, 21, 22, 25, 26). TAF-Iß has also been referred to as the putative histocompatability leukocyte antigen class II-associated protein II and the inhibitor of protein phosphatase 2A [I2PP2A (27, 28)].

To determine whether one or both of these proteins was present, Western blot analysis was performed using antibody to either TAF-I{alpha} (Fig. 2Go, upper panel), TAF-Iß (middle panel), or TAF-I{alpha} and -ß (lower panel). The HeLa nuclear extracts, but not purified ER{alpha}, used in our pull-down assays contained substantial amounts of TAF-I{alpha} and TAF-Iß (lanes 1 and 2), with the TAF-I{alpha} migrating more slowly than TAF-Iß. Both TAF-I{alpha} and TAF-Iß were detected in eluates from our ER{alpha} pull-down assays with HeLa nuclear extracts and ER{alpha} in the absence and in the presence of 17ß-estradiol (E2, lanes 3 and 4). However, neither TAF-I{alpha} nor TAF-Iß was detected in pull-down assays when A2 ERE-containing oligos were used, regardless of E2 inclusion (lanes 5 and 6). In addition, oligos containing the nonconsensus pS2 ERE also disrupted the TAF-I-ER{alpha} interaction (data not shown). Taken together, these data indicated that TAF-I{alpha} and TAF-Iß were associated with ER{alpha}, that ERE-containing oligos disrupted the TAF-I-ER{alpha} interaction, and that this in vitro association of TAF-I with ER{alpha} was not affected by E2.



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  		Figure 2. Antibodies to TAF-I{alpha} and TAF-Iß Confirm the Identity of the ER{alpha}-Associated Proteins

Purified flag-tagged ER{alpha} was immobilized on flag-affinity resin (lanes 2–6) and HeLa nuclear extract (HeLa NX, lanes 1 and 3–6), 17ß estradiol (E2 lanes 4 and 6), and DNA containing the vitellogenin A2 ERE (A2 oligo, lanes 5 and 6) were added as indicated. ER{alpha}-DNA-protein complexes were eluted and separated by SDS-PAGE. Immunoblotting was carried out using antibodies to TAF-I{alpha}, TAF-Iß, or to a region common to both proteins. Ten percent of the HeLa nuclear extract input was included for reference (lane 1).

 
Interaction of ER{alpha} with TAF-Iß
We had demonstrated that TAF-Iß and ER{alpha} interacted in our pull-down assays, but we wanted to determine whether ER{alpha} and TAF-Iß interact in a cell environment. We first assessed the level of TAF-I{alpha} and TAF-Iß in MCF-7, HeLa, and U2 osteosarcoma (U2-OS) cells in Western blot assays using an antibody that recognizes amino acids common to TAF-I{alpha} and TAF-Iß. Whereas HeLa cells express both TAF-I{alpha} (Fig. 3AGo, upper band) and TAF-Iß (lower band), MCF-7 cells express only TAF-Iß, and U2-OS cells express neither TAF-I{alpha} nor TAF-Iß. To determine whether endogenously expressed TAF-Iß and ER{alpha} interact, coimmunoprecipitation assays were carried out with extracts from MCF-7 cells, which express significant levels of endogenous ER{alpha} (29) and TAF-Iß (panel A). MCF-7 nuclear extracts were prepared from cells that had been exposed to either ethanol or E2 and incubated with an immobilized ER{alpha}-specific antibody. ER{alpha} and its associated proteins were eluted, separated on denaturing acrylamide gels, and subjected to Western blot analysis with the TAF-I antibody. TAF-Iß was present when nuclear extracts from ethanol-treated MCF-7 cells were used (panel B, lane 3), but not when nuclear extracts from E2-treated MCF-7 cells were used (lane 4). The relatively weak signal observed for TAF-Iß in the ER{alpha} coimmunoprecipitation was expected because ER{alpha} is likely to be associated with numerous protein complexes, a few of which may contain TAF-Iß. When MCF-7 proteins were coimmunoprecipitated with TAF-I antibody, ER{alpha} was detected when nuclear extracts were prepared from ethanol-treated but not E2-treated MCF-7 cells (Fig. 3CGo, lanes 3 and 4). When similar experiments were carried out using an unrelated antibody, neither TAF-Iß nor ER{alpha} was detected (data not shown). Thus, in contrast to our in vitro pull-down assays, interaction of endogenously expressed ER{alpha} and TAF-Iß from MCF-7 cells occurred in the absence, but not in the presence, of E2. Our data provide support for the idea that endogenously expressed TAF-Iß and ER{alpha} do interact in MCF-7 cells and that this interaction is physiologically relevant. With this demonstration that endogenous ER{alpha} and TAF-Iß do interact in an estrogen-responsive cell line, we focused the remainder of our studies on the interaction of TAF-Iß and ER{alpha}.



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  		Figure 3. Endogenous ER{alpha} and TAF-Iß Are Associated in MCF-7 Breast Cancer Cells in the Absence of E2

A, Extracts from MCF-7, HeLa, or U2-OS cells were separated by SDS-PAGE, transferred to nitrocellulose, and TAF-I{alpha} (upper band) or TAF-Iß (lower band) was detected with TAF-I-specific antibody. B, ER{alpha} and associated proteins were coimmunoprecipitated from MCF-7 nuclear extracts with an ER{alpha}-specific antibody, separated by SDS-PAGE, and subjected to Western analysis (ER{alpha} IP, lanes 3 and 4). TAF-Iß was detected with a TAF-I-specific antibody. C, TAF-Iß and associated proteins were coimmunoprecipitated from MCF-7 nuclear extracts with TAF-I-specific antibody and subjected to Western analysis (TAF-I IP, lanes 3 and 4). ER{alpha} was detected with an ER{alpha}-specific antibody. E2 was included as indicated. Ten percent of the MCF-7 nuclear extract input was included for reference (lanes 1 and 2).

 
Domains Required for ER{alpha}-TAF-Iß Interaction
To identify the region(s) of TAF-Iß required for interaction with ER{alpha}, we tested the ability of immobilized, baculovirus-expressed ER{alpha} to interact with in vitro translated 35S-labeled full-length or truncated TAF-Iß. When full-length TAF-Iß was incubated with immobilized ER{alpha}, TAF-Iß bound to the receptor in the absence and in the presence of E2 (Fig. 4Go, -E2 and +E2). A carboxy-terminal deletion mutant of TAF-Iß (1–225), in which an extended acidic domain was deleted, was still retained by ER{alpha}. Similarly, two amino-terminal deletion mutants of TAF-Iß (25–277 and 133–277) also bound to the immobilized ER{alpha}. However, a fragment of TAF-Iß containing only amino acids 1–119 was unable to interact with ER{alpha}, either in the presence or in the absence of E2. No binding was detected with unprogrammed lysate (UPL) in which the translation reaction was carried out without a DNA template. None of the TAF-Iß proteins bound to the resin when ER{alpha} was omitted from the binding reaction (lanes 2). These studies indicated that neither the amino-terminal (1–133) nor the carboxy-terminal (225–277) portion of TAF-Iß interacts with ER{alpha}, but that amino acids in the central portion of TAF-Iß (133–225) are required.



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  		Figure 4. ER{alpha} and TAF-Iß Interact through Amino Acids 133–225 of TAF-Iß and the C and F Domains of ER{alpha}

A, Flag-affinity resin alone (lane 2) or complexed with purified flag-tagged ER{alpha} (lanes 3 and 4) was combined with in vitro translated 35S-labeled full-length or truncated TAF-Iß or UPL, which was processed with no DNA template. B, GST-affinity resin alone (lane 2) or E. coli-expressed, purified GST-TAF-Iß (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 input is included for reference (lanes 1). Assays were carried out three times and a representative experiment is shown.

 
To identify the region(s) of ER{alpha} required for interaction with TAF-Iß, an immobilized glutathione-S-transferase (GST)-TAF-Iß fusion protein was incubated with in vitro translated 35S-labeled full-length or truncated ER{alpha}. The full-length receptor bound to TAF-Iß both in the absence and in the presence of E2 (Fig. 4BGo). The amino terminus of ER{alpha} (AB) was unable to bind to TAF-Iß. However, when the amino-terminal region of ER{alpha} was combined with the DNA-binding domain, the 35S-labeled ABC was retained by TAF-Iß. Likewise, the hinge region, LBD, and carboxy terminus (DEF) bound to TAF-Iß, as did the carboxy-terminal deletion mutant 1–530 and a deletion mutant containing only the DNA-binding domain and hinge region (CD). Surprisingly, the LBD (E), which contains activation function 2 and interacts with numerous coregulatory proteins (30, 31, 32, 33), was unable to interact with TAF-Iß in the absence or in the presence of E2. However, when the F domain of ER{alpha} was expressed with the LBD, a weak ligand-dependent interaction with TAF-Iß was observed. The E domain, which contains the ligand-binding function of ER{alpha}, undergoes a conformational change upon binding to ligand (34, 35, 36) and may affect recruitment of TAF-Iß. No binding was detected with UPL, or with the resin alone (-). Thus, the DNA binding domain and the carboxy terminus of the receptor are involved in interacting with TAF-Iß.

TAF-Iß Inhibition of ER{alpha}-Mediated Transcription
To test the functional consequence of the ER{alpha}-TAFIß interaction, transient transfection assays were carried out in U2-OS cells, which do not express detectable levels of endogenous TAF-Iß (Fig. 3AGo), but have been used extensively to characterize estrogen action (19, 37, 38). An ER{alpha} expression vector and a luciferase reporter plasmid containing two EREs were used to cotransfect U2-OS cells. When increasing concentrations of a TAF-Iß expression vector were included, TAF-Iß inhibited E2-induced transcription of the estrogen-responsive reporter plasmid in a dose-dependent manner (Fig. 5Go), but did not alter the activity of constituitively active Simian virus 40 or cytomegalovirus (CMV) promoters (data not shown).



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  		Figure 5. TAF-Iß Inhibits E2-Induced Transcription Activation by ER{alpha} in Transient Transfection Assays

Transient cotransfection assays were carried out in U2-OS cells using an ER{alpha} expression vector and the luciferase reporter plasmid 2EREtkLUC. Increasing concentrations of TAF-Iß expression vector (0, 5, and 50 ng) were included and luciferase units were measured. Assays were carried out in duplicate and repeated three times. Combined data are expressed as the mean ± SEM.

 
Effect of TAF-Iß on Acetylation
Previous studies have demonstrated that TAF-Iß inhibits acetylation of histones (20) and that acetylation of ER{alpha} enhances E2-mediated transcription (12). Thus we reasoned that TAF-Iß might alter acetylation of ER{alpha} and thereby modulate receptor-mediated transactivation. To determine whether this was the case, acetylation assays were carried out with p300, which acetylates histones and interacts with ER{alpha} (20, 39), and 3H-acetyl coenzyme A in the absence or in the presence of either purified ER{alpha} or acid-precipitated histones. The proteins were separated on a 12.5% denaturing gel so that all the proteins of interest could be observed on the same gel. When only p300 was included in the reaction, autoacetylation of p300 was observed (Fig. 6AGo). When acid-precipitated histones from MCF-7 cells were included in the reaction, acetylation of histones was observed as well as a nonspecific acetylated product that ran between the histones and TAF-Iß. The identity of the acetylated histones was confirmed by immunoblotting (data not shown). Inclusion of purified TAF-Iß in the reaction reduced acetylation of histones as reported (20) and produced a 3H-labeled product around 45 kDa, which indicated that TAF-Iß was also acetylated. ER{alpha} was acetylated when baculovirus-expressed, purified ER{alpha} was incubated with p300 as reported (12). Inclusion of TAF-Iß decreased the level of ER{alpha} acetylation. Quantitation of four independent assays indicated that TAF-Iß decreased acetylation of histones (Fig. 6BGo) by nearly 90% and reduced acetylation of ER{alpha} by 30%. In contrast, TAF-Iß did not affect p300 autoacetylation. Furthermore, inclusion of equal amounts of ovalbumin in lieu of TAF-Iß had no effect on p300 mediated-acetylation of ER{alpha} (data not shown). These data demonstrate that TAF-Iß modulates acetylation of histones and ER{alpha} in vitro.



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  		Figure 6. TAF-Iß Inhibits Acetylation of Histones and ER{alpha} in Vitro and Acetylation of ER{alpha} in Cells

A, Liquid acetyltransferase assays were carried out using baculovirus-expressed, purified p300 and 3H-acetyl coenzyme A. Histones, ER{alpha}, and TAF-Iß were included as indicated. Reactions were separated by SDS-PAGE and visualized by autoradiography. B, Four independent acetylation assays were quantitated and counts are expressed as mean ± SEM. Statistical differences were measured by Student’s t test and are indicated by an asterisk (P < 0.01). The effect of TAF-Iß on p300 acetylation (p300 + TAF-Iß) was determined from lanes 3 and 5 in panel A. C, U2-OS cells were transfected with an ER{alpha} expression vector without (-) or with (+) a TAF-Iß expression vector. ER{alpha} was immunoprecipitated from cell lysates with an ER{alpha}-specific antibody, separated by SDS-PAGE, and detected with an antibody against acetylated lysine residues (upper panel) or ER{alpha} (lower panel).

 
To determine whether TAF-Iß alters acetylation of ER{alpha} in cells, an ER{alpha} expression vector was transfected into U2-OS cells in the absence or in the presence of a TAF-Iß 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 residues (Fig. 6CGo, top panel) or an ER{alpha}-specific antibody (bottom panel). The level of acetylated ER{alpha} was decreased when the TAF-Iß expression vector was included. Differences in ER{alpha} acetylation levels with TAF-Iß 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, TAF-Iß not only decreases ER{alpha} acetylation in vitro, but also decreases ER{alpha} acetylation in a cellular environment.

Effect of TAF-Iß on the ER{alpha}-ERE Interaction
Another way that TAF-Iß 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 TAF-Iß. Surprisingly, when increasing amounts of purified TAF-Iß were added to the binding reaction, there was a striking increase in ER{alpha}-ERE complex formation (Fig. 7AGo, lanes 1–5). 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. Furthermore, the ability of an ERE-containing oligo, but not an oligo containing a nonspecific sequence, to compete for ER{alpha} binding (lanes 6 and 7) demonstrated the specificity of the ER{alpha}-ERE interaction in the presence of TAF-ß. The migration of the receptor-DNA complex was not altered by the presence of TAF-Iß, suggesting that TAF-Iß enhanced ER{alpha} binding but was not part of the stable protein-DNA complex. These findings are reminiscent of previous studies with the high-mobility group protein 1 (HMG1), which increased the interaction of the ER{alpha} and a number of steroid hormone receptors with their cognate recognitions sequences without altering the migration of the receptor-DNA complexes (40, 41, 42). In the absence of TAF-Iß, extending the incubation time from 0.25 to 2 min slightly increased formation of the receptor-DNA complex (Fig. 7Go, B and C). However, when TAF-Iß was present, extending the incubation time from 0.25–2 min increased formation of the receptor-DNA complex more than 2-fold. Thus, rather than decreasing the association of ER{alpha} with DNA, TAF-Iß substantially enhanced the association of the receptor with ERE-containing DNA.



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  		Figure 7. TAF-Iß Enhances the ER{alpha}-ERE Interaction

Baculovirus-expressed, purified ER{alpha} was incubated with 32P-labeled DNA fragments containing a consensus ERE. Purified TAF-Iß and unlabeled competitor DNA containing a consensus ERE (ERE) or a nonspecific sequence (NS) were added to the binding reaction as indicated. Binding reactions were incubated for 15 (panel A) or 0.25, 1, or 2 (panel B) minutes, loaded onto nondenaturing acrylamide gels, and separated. C, Four independent gel mobility shift assays were carried out in the absence (closed circles) or in the presence (open circles) of TAF-Iß, as shown in panel B, quantitated by PhosphorImager analysis, and analyzed using ImageQuant software (Molecular Dynamics, Inc.). Data are expressed as the percentage of probe bound vs. the time of incubation.

 
Interaction of TAF-Iß with Other Nuclear Receptors
ER{alpha} is a member of the closely related nuclear receptor superfamily. To determine whether TAF-Iß might also interact with other nuclear receptors, TAF-Iß pull-down experiments were carried out. Immobilized GST-TAF-Iß was incubated with in vitro transcribed and translated 35S-labeled progesterone receptor B (PR-B), thyroid hormone receptor ß (TRß), or RXR{alpha}. All three nuclear receptors bound to TAF-Iß in the absence and in the presence of their respective hormones (Fig. 8AGo). More importantly, when U2-OS cells were cotransfected with a TRß expression vector and a chloramphenicol acetyltransferase (CAT) reporter plasmid containing two thyroid hormone response elements in the absence and in the presence of a TAF-Iß expression vector (Fig. 8BGo), TAF-Iß was able to inhibit T3-induced transcription. These findings suggest that TAF-Iß may be involved in regulation of other hormone-responsive genes as well.



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  		Figure 8. Immobilized, Partially Purified TAF-Iß Associates with Other Nuclear Receptors

A, GST-affinity resin containing no protein (-) or purified GST-TAF-Iß (+TAF-Iß) was combined with in vitro translated 35S-labeled PR-B, TRß, RXR{alpha}, or UPL. Appropriate hormone was not (-) or was (+) added. Ten percent of input is included for reference (lane 1). B, Transient cotransfection assays were carried out with U2-OS cells using a TRß expression vector, the CAT reporter plasmid TRE2tkCAT, and a ß-gal internal control vector. TAF-Iß expression vector (50 ng) was included, and CAT assays were carried out and normalized to ß-gal 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, TAF-Iß, which interacts with the receptor in in vitro pull down assays. More importantly, we have demonstrated that TAF-Iß inhibits ER{alpha}-mediated transcription, that TAF-Iß increases ER{alpha} binding to DNA, and that the endogenously expressed ER{alpha} and TAF-Iß interact in a normal cell background where these proteins are routinely expressed.

Effect of TAF-Iß on Acetylation
TAF-Iß has been implicated in regulating transcription, adenoviral replication, histone acetylation, nucleosome remodeling, chromatin decondensation, and phosphatase 2A activity (20, 22, 25, 26, 28, 43). Thus, TAF-Iß is a multifunctional protein that could influence transcription of estrogen-responsive genes in a variety of ways. We have demonstrated that TAF-Iß inhibits histone acetylation and decreases acetylation of ER{alpha} in vitro and in vivo. Hyperacetylation of histones is correlated with transcriptionally active genes, whereas hypoacetylation is generally restricted to transcriptionally silent genes (44, 45). Likewise, increased acetylation of ER{alpha} has been correlated with increased hormone sensitivity (12). Taken together, our findings provide evidence that TAF-Iß may play a role in limiting ER{alpha}-mediated transcription by 1) modulating the acetylation of ER{alpha} and histones and 2) fostering the association of the less acetylated ER{alpha} to the promoters of target genes. It is also possible, although we have not yet tested at this point, that TAF-Iß might also influence acetylation of other transcription factors or coregulatory proteins involved in modulating estrogen-responsive gene expression.

Role of TAF-Iß in Cells
A number of studies have demonstrated that the hormone sensitivity that occurs in intact cells in vivo is not necessarily recapitulated in in vitro binding and transcription assays (46, 47, 48, 49). Likewise, we found that the TAF-Iß-ER{alpha} interaction was unaffected by hormone in our in vitro binding assays but displayed hormone sensitivity in intact MCF-7 cells, suggesting that the cellular environment is required for full TAF-Iß function.

The effect of TAF-Iß on the ER{alpha}-ERE interaction is similar to that of HMG1 (40, 41, 42, 50). Both TAF-Iß and HMG1 dramatically enhance binding of the ER{alpha} to ERE-containing DNA but are not associated with the ER{alpha}-ERE complex. It is thought that HMG1 serves as a chaperone, which transiently associates with the nucleoprotein complex in the process of complex formation (51). Similarly, TAF-Iß might either transiently associate with the ER{alpha}-ERE complex or be displaced as the ER{alpha} binds to DNA and thereby serve as a molecular chaperone in the assembly of the ER{alpha}-ERE nucleoprotein complex.

It has been proposed that TAF-Iß inhibits histone acetylation by binding to histone tails and masking lysine residues that are modified by histone acetyltransferases (20, 22, 52). We propose that the interaction of TAF-Iß with ER{alpha} may be similar. In this model, TAF-Iß binds to the ER{alpha} DNA binding domain in the absence of hormone and decreases transcription by 1) decreasing ER{alpha} acetylation and thereby rendering the receptor less sensitive to any low level of circulating hormone and 2) masking the region of the receptor required for DNA interaction. When hormone is present, ER{alpha} undergoes a conformational change, TAF-Iß enhances binding of this less acetylated ER{alpha} with ERE-containing DNA and dissociates from the receptor. The dissociated TAF-Iß might interact with adjacent histones or coregulatory proteins, thereby inhibiting additional histone acetylation and limiting transcription of estrogen-responsive genes. In this manner, TAF-Iß could not only help to maintain estrogen-responsive genes in a quiescent state in the absence of ligand, but could also foster the binding of less acetylated ER{alpha} to DNA and thereby provide a graded response to hormone and help to moderate estrogen-activated gene expression.

Interestingly, the mode of action of TAF-Iß differs from that of other nuclear corepressors. NCoR associates with the receptor as part of a complex that contains Sin3 and histone deacetylase (reviewed in Refs. 13 and 14). Thus the NCoR-associated proteins are responsible for histone modification and transcriptional silencing. In contrast, our studies demonstrate that rather than relying on the activities of its associated proteins, TAF-Iß itself is able to repress histone acetylation.

Effect of TAF-I Proteins in Regulating Hormone-Responsive Gene Expression
The effects of TAF-Iß are not limited to ER{alpha}, but extend to other members of the nuclear receptor superfamily. We have shown that TAF-Iß inhibits estrogen- and thyroid hormone-mediated transactivation. Thus, TAF-Iß interacts with both type I and type II nuclear receptors. Given the wide distribution of nuclear receptors, it seems probable that TAF-Iß may modulate expression of numerous hormone-responsive genes.

TAF-I{alpha} and TAF-Iß are differentially expressed in a number of tissues and cultured cell lines (53). Interestingly, only TAF-Iß is expressed in MCF-7 cells, which also express ER{alpha} and numerous coregulatory proteins required for estrogen action (29, 54). Although TAF-Iß is a more potent activator of DNA replication than TAF-I{alpha} (24), both TAF-I{alpha} and TAF-Iß inhibit histone acetylation and retinoid acid receptor signaling (20). We have examined only the effect of TAF-Iß on ER{alpha}-mediated transcription. However, because the region of TAF-Iß that interacts with ER{alpha} is completely conserved in TAF-I{alpha}, TAF-I{alpha} may also have the potential to alter ER{alpha}-mediated transcription. The distinct differences in their amino termini could influence the abilities of TAF-I{alpha} and TAF-Iß to modulate histone acetylation, chromatin remodeling, and transcription activation (20, 22, 25, 26). Thus, the tissue-specific distribution of TAF-I{alpha} and TAF-Iß (53) combined with the different abilities of these two proteins to influence transcription provide substantial versatility in regulating hormone-responsive gene expression in different cell types.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
HeLa Pull-Down Assay
Baculovirus-expressed ER{alpha} (18) was immobilized on M2 agarose (Sigma, St. Louis, MO) and resuspended in binding reaction buffer [15 mM Tris, pH 7.9; 2 mM EDTA; 20 mM KCl; 4 mM dithiothreitol (DTT), 0.5 mM ZnCl2] containing 2 µg/ml polydIdC in the presence or absence of 1 µM 17ß-estradiol (E2). Annealed oligos containing a nonspecific sequence (5'-GATCTGAGTACATGAAACACGAGAAGTAATCTAG-3' and 5'-CTAGATTACTTCTCGTGTTTCATGTACTCA-3') or consensus ERE (5'-CTAGATTACAGGTCACAGTGACCTTACT CA-3' and 5'-GATCTGAGTAAGGTCACTGTGACCTGTAATCTAG-3') were allowed to bind to the immobilized ER{alpha} for 45 min at 4 C, and unbound DNA was washed from the resin. HeLa nuclear extract was prepared as described previously (18) and added to the immobilized ER{alpha} in binding reaction buffer. Binding reactions were incubated for 1 h at 4 C, and 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 (NP-40); 2 mM DTT). ER{alpha} and associated proteins and oligos 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
U2 osteosarcoma (U2-OS) cells were transfected using Lipofectin (Life Technologies, Inc., Gaithersburg, MD) as previously described (55) with 0.25 ng of the human (h) ER{alpha} expression vector CMV5 hER (56), 500 ng of the firefly luciferase reporter vector 2EREtkLUC (generously provided by Dr. Benita Katzenellenbogen, University of Illinois, Urbana-Champaign), and 0.5 ng of the Renilla expression vector pRLSV40 (Promega Corp., Madison, WI) or 5 ng of the TRß expression vector pCI-TRß, 500 ng of the CAT reporter vector TRE2tkCAT (Ref.57 ; both kindly provided by Dr. Milan Bagchi, University of Illinois, Urbana, IL) and 150 ng of the ß-galactosidase (ß-gal) expression vector CMV-ß-gal (CLONTECH Laboratories, Inc., Palo Alto, CA). Increasing concentrations of the TAF-Iß expression vector pCHATAF1ß (53), kindly provided by Dr. Kyosuke Nagata (Tokyo Institute of Technology, Yokohama, Japan) were added as indicated. CAT and ß-gal assays were carried out as previously described (55). Firefly and and Renilla luciferase assays were carried out with 50 µl of Luciferase Assay Reagent II (Promega Corp.) and 50 µl of Stop and Glo solution (Promega Corp.) per reaction and a Monolight 2010 (Analytical Luminescence Laboratory, San Diego, CA). Firefly luciferase assays were normalized to Renilla fluorescence to adjust for transfection efficiency. At least three independent experiments were carried out in duplicate.

Micro-Liquid Chromatography (LC)-Electrospray Ionization Tandem Mass Spectrometry (MS)
Silver-stained protein bands were excised from 10% acrylamide gels, and the proteins were digested in-gel with trypsin (sequence-grade trypsin, 12.5 ng/µl; Promega Corp.). The peptides were eluted from the gel pieces by extracting three times, first with equal parts of 25 mM ammonium bicarbonate and acetonitrile and then twice with equal parts of 5% (vol/vol) formic acid and acetonitrile. Tryptic peptides extracted from the gel spots were used directly for µ-LC-Electrospray Ionization-MS/MS without further purification.

Tryptic digests were loaded onto a 365 x 100 µm fused silica capillary column packed with 5-µm Zorbax XDB packing material (PE Applied Biosystems, Foster City, CA) at a length of 10–15 cm. The tryptic peptides were separated with a 30-min linear gradient of 0–60% solvent B (80% acetonitrile/0.5% acetic acid) and then loaded into an LCQ ion trap mass spectrometer (Finnigan, San Jose, CA). Tandem mass spectra were automatically collected under computer control during the 30-min LC-MS runs. Obtained MS/MS spectra were then subjected directly to SEQUEST database searches (58) without need of manual interpretation. SEQUEST was used to identify proteins in a spot by correlating experimental MS/MS spectra to protein sequences in the human sequence database. Six peptides, KEQQEAIEHIDEVQNEIDRL, KIPNFWVTTFVNHPQVSALLGEEDEEALHYLTRV, RIDFYFDENPYFENKV, RLNEQASEEILKV, RVEVTEFEDIKS, and SALLGEEDEEALHYLTRV, were recovered from the gel slices and used to identify TAF-I{alpha} and TAF-Iß constituting approximately 30% of the full protein sequences.

Pull-Down Assay Using in Vitro Translated Proteins
GST-TAF-Iß was expressed in E. coli using the expression vectors pGEX2tk-TAF-Iß (26), kindly provided by Dr. Ken Matsumoto [The Institute of Physical and Chemical Research (RIKEN), Saitama, Japan] as previously described (26). Expressed protein was immobilized on GST-Sepharose (Amersham Pharmacia Biotech, Piscataway, NJ), washed in buffer A (20 mM Tris, 50 mM NaCl, 0.2%–0.4% NP-40, 1 mM DTT), and resuspended in buffer A containing protease inhibitors (50 µg/ml leupeptin, 5 µg/ml phenylmethylsulfonylfluoride, 1 µg/ml pepstatin, and 5 µg/ml aprotinin). The ER{alpha} expression vectors pBSK-ER{alpha} (59), pBSK-ER{alpha}(1–530), pBSK-ER{alpha}(ABC), pBSK-ER{alpha}(AB), pBSK-ER{alpha}(DEF) (60), pET15b-ER{alpha}(304–554), and pET15b-ER{alpha}(304–595)(61)were kindly provided by Dr. Benita Katzenellenbogen (University of Illinois, Urbana, IL) and used to synthesize 35S-labeled ER{alpha} and ER{alpha} fragments AB, ABC, DEF, E, EF, and 1–530 in vitro. The vector p21b+flagERCD (62), kindly provided by Dr. David Shapiro (University of Illinois, Urbana, IL) was used to synthesize 35S-labeled ER{alpha} fragment CD. Vectors pCMX-RXR{alpha} (kindly provided by Dr. Ronald Evans, Salk Institute, La Jolla, CA), pT7ßhPR-B (35), and pT7pLINKhTRß (both kindly provided by Dr. Milan Bagchi, University of Illinois, Urbana, IL) were used to synthesize 35S-labeled RXR{alpha}, PR-B, and TRß, respectively. Labeled proteins were synthesized using the TNT T7 Quick coupled transcription/translation system (Promega Corp.) and incubated at 4 C for 1 h with the immobilized GST-TAF-Iß in the presence or absence of E2, 9-cis retinoic acid, progesterone, or thyroid hormone as appropriate. After four washes with buffer A, and one wash with 50 mM Tris (pH 8.0), proteins were eluted with 10 mM glutathione in 50 mM Tris, pH 8.0. Eluted proteins were separated by SDS-PAGE, and autoradiograms were exposed for 1–2 d.

To examine interactions of TAF-Iß domains with full-length ER{alpha}, baculovirus-expressed flag-tagged ER{alpha} (18) was immobilized on M2 Agarose (Sigma) and resuspended in buffer A. 35S-labeled full-length and truncated TAF-Iß containing amino acids 1–225, 25–277, 133–277, or 1–119 were translated using the vectors pET14b-rTAF-Iß(1–277), pET14b-rTAF-Iß(1–225)(24), pET14b-rTAF-Iß(26–277)pET14b-rTAF-Iß(133–277) (25), and pET14b-rTAF-Iß(1–119), kindly provided by Dr. Kyosuke Nagata (Tokyo Institute of Technology, Yokohama, Japan). 35S-labeled TAF-Iß proteins were incubated with immobilized ER{alpha}. Resin was washed four times in buffer A and proteins were eluted with 20 mM Tris, pH 7.5; 50 mM NaCl; 0.2 mM EDTA; 10% glycerol; 0.1% NP-40; 2 mM DTT; and 2 mg/ml M2 peptide (University of Illinois Biotechnology Center, Urbana, IL). Eluted proteins were separated by SDS-PAGE and autoradiograms were exposed for 1–2 d.

Gel Mobility Shift Assays
Gel mobility shift assays were carried out essentially as described previously (55) with the following modifications. Purified ER{alpha} and TAF-Iß were incubated as indicated for 10 min in binding reaction buffer (50 mM KCl; 7.5 mM NaCl; 15 mM Tris; pH 7.9; 0.2 mM EDTA; 10% glycerol; 4 mM DTT; 50 µM ZnCl2) in the presence of 50 nM E2. Unlabeled 34-bp DNA fragments containing an ERE or a nonspecific sequence were added as indicated. Total protein concentrations were held constant in all reactions by the addition of BSA (Roche Diagnostics Corp., Indianapolis, IN). 32P-labeled, 34-bp ERE-containing DNA fragments were added to the binding reactions and incubated at 4 C for the times indicated. The ER{alpha}-ERE complexes and free DNA were fractionated on a nondenaturing acrylamide gel. The level of bound and free 32P-labeled DNA was quantitated using a Molecular Dynamics, Inc. PhosphorImager and ImageQuant 5.0 software (Molecular Dynamics, Inc., Sunnyvale CA).

SDS-PAGE, Silver Staining, and Western Analysis
For Western analysis, samples were fractionated on 15% SDS-PAGE gels and transferred to nitrocellulose membrane. Antibodies generously provided by Kyosuke Nagata (Tokyo Institute of Technology, Yokohama, Japan) that recognize TAF-I{alpha}, TAF-Iß, or a region common to both TAF-I{alpha} and TAF-Iß (53) were used to detect TAF-I proteins. ER{alpha} was detected using an antibody specific to ER{alpha} (sc-8002, 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 (Upstate Biotechnology, Inc. Lake Placid, NY). Blots were probed with horseradish peroxidase-conjugated secondary antibody (Zymed Laboratories, Inc., 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 Dr. W. Lee Kraus (Cornell University, Ithaca, NY) as previously described (39). Histones were isolated from MCF-7 cells as described (63), and liquid acetyl transferase assays were carried out using a protocol modified from Brownell and Allis (64). Purified p300 was incubated with 0.2 µl 3H-acetyl coenzyme A (4.7 Ci/mmol, Amersham Pharmacia Biotech) and histones or ER{alpha} and TAF-Iß was 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 for 30 min at 30 C. Proteins were separated by SDS-PAGE and gels were treated with En3hance (NEN Life Science Products-DuPont, Boston, MA) before exposure to film at -80 C for 1–5 d. Four 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 difference existed between samples incubated with or without TAF-Iß.

Coimmunoprecipitation Assay
To isolate endogenous ER{alpha}-associated proteins, MCF-7 cells were incubated in the absence or presence of 100 nM E2 for 20 min, and nuclear extracts were prepared as previously described (18). MCF-7 nuclear extract (0.5 mg) was precleared with 15 µl protein A agarose slurry (Santa Cruz Biotechnology, Inc.) in 10 mM Tris, 1 mM EDTA buffer (TE) with NaCl adjusted to 285 mM in the absence or presence of E2 for 1.5 h at 4 C. Five micrograms of the ER{alpha}-specific antibody sc-543 or sc-8005 (Santa Cruz Biotechnology, Inc.) were incubated in TE with salt adjusted to 285 mM with 15 µl of protein A agarose slurry (Santa Cruz Biotechnology, Inc.) for 1.5 h at 4 C. After incubation, precleared nuclear extract was added to the antibody-agarose mixture and incubated for 2.5 h at 4 C. The agarose beads were then washed four times with 24 mM Tris (pH 7.4), 2 mM KCl, and 163 mM NaCl before boiling for 10 min in 1x sodium dodecyl sulfate sample buffer. The resulting eluates were separated on SDS-PAGE gels and transferred to nitrocellulose membranes for Western analysis.

To isolate endogenous TAF-I-associated proteins, TAF-I antibody was conjugated to agarose resin using the Seize Immunoprecipitation kit (Pierce Chemical Co.) according to the manufacturer’s instructions and incubated with MCF-7 nuclear extracts in the absence or presence of 1 µM E2 for 3 h at 4 C. Resin was then washed four times with TE containing 50 mM NaCl before elution with Gentle Elution Buffer (Pierce Chemical Co.). The eluted proteins were separated on SDS-PAGE gels and transferred to nitrocellulose membranes for Western analysis.

U2-OS cells were transfected for immunoprecipitation of ER{alpha} as described above, except cells were seeded in six-well plates and 750 ng of the human ER{alpha} expression vector CMV5 hER (56) and 15 µg of the TAF-Iß expression vector pCHATAF1ß (53) were included as indicated. Transfected cells were resuspended in lysis buffer containing 20 mM Tris, pH 8.0; 200 mM NaCl; 1 mM EDTA; and 0.2% NP-40. ER{alpha} was immunoprecipitated from half the cell lysate from each well of a six-well plate as described for immunoprecipitation of ER{alpha} above.


   ACKNOWLEDGMENTS
 
We are indebted to K. Nagata and K. Matsumoto for their generosity in providing TAF-I expression vectors and antibodies. We thank W. L. Kraus, M. Bagchi, B. Katzenellenbogen, R. Evans, D. Shapiro, R. Pestell, and J. Kadonaga for generously providing additional reagents; P. Cheung, M. Barton, and K. Hines for protocols; J. Wood, V. Likhite, J. Schultz, R. Rajendran, and P. Martini for technical assistance; and L. Petz and J. Kim for helpful comments during the preparation of this manuscript.


   FOOTNOTES
 
This work was supported by NIH Grant DK-53884 (to A.M.N.). M.A.L. received predoctoral support from the NIH Reproductive Biology Training Program Grant PHS 5T32 HD07028, and N.M. was supported by NIH Grant NCRR RR11823-05.

Abbreviations: CAT, Chloramphenicol acetyltransferase; CMV, cytomegalovirus; DRIP, vitamin D receptor-interacting protein; DTT, dithiothreitol; E2, 17ß-estradiol; ER, estrogen receptor; ERE, estrogen response element; ß-gal, ß-galactosidase; GST, glutathione-S-transferase; HMGI, high-mobility group protein I; LBD, ligand-binding domain; LC, liquid chromatography; MS, mass spectrometry; NCoR, nuclear receptor corepressor; NP-40, Nonidet P-40; PR-B, progesterone receptor B; RXR, retinoid X receptor; TAF-I{alpha}, template-activating factor I {alpha}; TAF-Iß, template activating factor I ß; TR, thyroid hormone receptor; TRAP, TR-associated protein; TRE, thyroid response element; U2-OS, U2 osteosarcoma; UPL, unprogrammed lysate.

Received for publication August 13, 2002. Accepted for publication October 7, 2002.


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 DISCUSSION
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NURSA Molecule Pages Link:

Nuclear Receptors:   TRβ  |  RXRα  |  ERα  |  PR
Coregulators:   p300
Ligands:   17β-Estradiol  |  9-cis-Retinoic acid  |  Progesterone  |  Thyroid hormone



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