help button home button Endocrine Society Molecular Endocrinology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Norris, J. D.
Right arrow Articles by McDonnell, D. P.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Norris, J. D.
Right arrow Articles by McDonnell, D. P.
Molecular Endocrinology 16 (3): 459-468
Copyright © 2002 by The Endocrine Society

A Negative Coregulator for the Human ER

John D. Norris, Daju Fan, Andrea Sherk and Donald P. McDonnell

Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, North Carolina 27710

Address all correspondence and requests for reprints to: Dr. Donald McDonnell, Department of Pharmacology and Cancer Biology, Duke University Medical Center, Box 3813, Durham, North Carolina 27710. E-mail: mcdon016{at}acpub.duke.edu.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
ER{alpha} is a ligand-activated transcription factor and a key regulator of the processes involved in cellular proliferation and differentiation. In addition, aberrant ER{alpha} activity is linked to several pathological conditions including breast cancer. A complex network of coregulatory proteins is largely believed to determine the transcriptional activity of ER{alpha}. We report here the isolation of a protein, denoted RTA for repressor of tamoxifen transcriptional activity, which contains an RNA recognition motif and interacts with the receptor N-terminal activation domain. RTA interacts with RNA in vitro, and its overexpression inhibits the partial agonist activity manifest by the antiestrogen tamoxifen while minimally affecting E2-activated transcription. Mutation of the RNA recognition motif alters RNA binding specificity and results in a dominant negative form of RTA that leads to derepression of ER{alpha} transcriptional activity, allowing all classes of antiestrogens to manifest partial agonist activity and enhancing agonist efficacy. These findings suggest a role for RNA binding proteins as coregulatory factors of the nuclear receptor family and reveal a novel mechanism by which antiestrogens can manifest agonist activities in some tissues.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
THE BIOLOGICAL ACTIONS of estrogens and antiestrogens are manifest through two distinct high-affinity receptors, ER{alpha} and ERß (1, 2, 3). Estrogens are potent activators of both receptor subtypes and, with respect to ER{alpha}, are known to play a critical role in the development and progression of ER{alpha}-positive breast cancers (4). In this regard, antiestrogens such as tamoxifen, which compete with estrogen for binding to the receptor, effectively inhibit the growth of breast tumor cells (5). However, these cells ultimately become refractory to antiestrogen therapy, an event that is believed to stem from the ability of tamoxifen and other antiestrogens to display tissue-selective agonist activity (6, 7, 8). In particular, tamoxifen, which is an effective antagonist of ER{alpha} action in the breast, is a partial ER{alpha} agonist in the uterus, bone, and cardiovascular systems (6, 7, 8). These findings have led to the reclassification of tamoxifen and other antiestrogens as selective ER modulators (SERMs) (9). The molecular mechanism underlying the tissue-restricted agonist activity of SERMs remains elusive. However, it is widely believed that a complex network of tissue-specific coregulatory proteins determines this specificity.

A large number of proteins have been identified recently that interact with and regulate the transcriptional activity of ER{alpha} by modifying the activity of a ligand-inducible activation domain (activation function 2; AF-2) located within the carboxy terminus of the receptor (10, 11, 12). AF-2 is formed by a conformational change in the ligand-binding domain that repositions several {alpha}-helices within this domain (13). Receptor agonists, but not antagonists, induce this conformational change. Once repositioned, these {alpha}-helices form a hydrophobic groove in the receptor that allows it to interact with a canonical LXXLL motif, a structure that constitutes the receptor interaction domain of most known coactivators (14). These proteins are believed to facilitate transcription by remodeling the chromatin structure and stabilizing the transcription preinitiation apparatus at target genes (15, 16). All antagonists to date are AF-2 antagonists and do not allow coactivators to bind the ligand-binding domain. Therefore, the ability of these ligands to behave as cell-specific agonists is likely to reflect their ability to function as activators of the N-terminal activation function, AF-1 (17, 18). AF-1 functions in a tissue-specific manner and is a constitutive activator; little is known about how this domain signals. We report here the isolation of a novel ER{alpha} coregulator that interacts with the ER{alpha} N-terminal domain. Expression of this protein, denoted RTA for repressor of tamoxifen transcriptional activity, in cells completely inhibits tamoxifen-mediated partial agonist activity and partially inhibits estrogen-mediated transcription. RTA contains a consensus RNA recognition motif (RRM) commonly found in heterogeneous nuclear ribonucleoproteins, and this RRM is required for the repressor function of RTA. Furthermore, RTA variants in which the RRM domain is mutated function as dominant negative repressors, allowing ER antagonists to manifest agonist activity, and significantly enhance agonist efficacy, suggesting that this pathway may be a key determinant of the pharmacology of ER ligands and their tissue-specific agonist activity.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Identification of an ER{alpha} N-Terminal Interacting Protein, RTA
The ER{alpha} N-terminal activation domain, AF-1, is an important element in both estrogen and antiestrogen signaling (17, 18). To identify potential coregulators that interact with the ER{alpha} N-terminal domain, we used a fragment of ER{alpha} containing amino acids 51–149 as bait in a yeast two-hybrid screen. Two cDNAs from the same gene were identified, the corresponding proteins of which interact with the ER{alpha} N-terminal bait. The larger clone, RTA (accession no. AY072786), was sequenced and was found to contain a large open reading frame encoding a protein of 390 amino acids (Fig. 1AGo). This sequence is highly homologous to a putative human cDNA clone (accession no. NM014309) generated by the Sanger Centre chromosome 22 mapping and sequencing groups and likely represents an alternatively spliced variant of that cDNA. Several homologs of RTA have been identified and include the human ataxin-2 binding protein 1 and the Caenorhabditis elegans protein FOX-1 (19, 20). The most notable sequence feature of RTA is the identification of a well characterized RRM or RNA-binding domain (21, 22). This domain is characterized by the presence of two short stretches of six and eight amino acids, respectively, termed RNP-2 and RNP-1 (Fig. 1AGo). The sequence spanning the RNP domains includes residues that are critical for proper protein folding. Other notable features of RTA are the presence of a glutamine-rich N-terminal domain and an alanine-rich C-terminal domain. Unusual amino acid distributions are common structural features of RNA-binding proteins and are proposed to be involved in protein-protein interactions. RTA also contains several arginine/glycine-rich (RGG) boxes, sequences thought to be involved in binding RNA (21) (Fig. 1AGo).



View larger version (40K):
[in this window]
[in a new window]
  		Figure 1. Amino Acid Sequence of RTA

A, RTA comprises a 390-amino acid protein. It contains an RRM, which consists of two ribonucleoprotein (RNP) domains and the sequences spanning them. RNP-2 (six amino acids) and RNP-1 (eight amino acids) are shown boxed. These domains along with the RGG sequences (underlined) are common structural features of RNA-binding proteins. B, Analysis of RTA mRNA. Multiple-tissue blot (CLONTECH Laboratories, Inc.) was hybridized with 32P-labeled RTA cDNA and subjected to autoradiography.

 
The tissue distribution of RTA was analyzed using a multiple-tissue Northern blot (CLONTECH Laboratories, Inc., Palo Alto, CA) (Fig. 1BGo). Several transcripts were identified ranging in size from 1.4 to 9.5 kDa. Interestingly, many RNA-binding proteins have been shown to exist in multiple forms due to alternative splicing of mRNA. The existence of multiple RTA transcripts suggests that there may be several isoforms of RTA. The most abundant RTA expression was found in the heart, liver, and pancreas. Minimal RTA expression was seen in the brain, lung, and placenta. These results demonstrate that RTA is not a ubiquitous factor, and its expression appears to be tissue restricted.

A direct interaction between ER{alpha} and RTA was verified in vitro using a protein-protein interaction assay. As expected for an AF-1 interacting protein, RTA was found to interact with ER{alpha} in a ligand-independent manner (Fig. 2AGo). A marginal but reproducible increase in binding was found between RTA and the tamoxifen-ER complex. ER{alpha} did not interact with the control protein [glutathione-S-transferase (GST) alone]. Similar to ER{alpha}, RTA demonstrates ligand-independent interactions with ERß (Fig. 2AGo). To determine which domains of ER{alpha} were responsible for binding RTA, we performed interaction assays between RTA and either ER{alpha} N-terminal or ER{alpha} hormone binding domain (HBD) GST fusion proteins. Consistent with the yeast two-hybrid results, RTA interacts with the N-terminal fragment of ER{alpha} in vitro (Fig. 2BGo). We could not detect an interaction between RTA and the ER-HBD under any conditions tested. Coimmunoprecipitation experiments were performed to determine whether an interaction could be detected between ER{alpha} and c-Myc-tagged RTA in cells. ER{alpha} was efficiently immunoprecipitated as a complex with RTA only in the presence of an antibody specific for c-Myc and not mouse IgG alone (Fig. 2CGo). Furthermore, transfection of either ER{alpha} or c-Myc-tagged RTA alone resulted in the failure to detect ER{alpha} in the immunoprecipitates. These data demonstrate that ER{alpha} and RTA form a complex within the environment of the cell.



View larger version (67K):
[in this window]
[in a new window]
  		Figure 2. RTA Interacts with ER{alpha} and ERß in Vitro

A, GST alone or GST fusion protein, GST-RTA, was incubated with in vitro translated [35S]methionine-labeled ER{alpha} or ERß. NH, No hormone; E2, 17ß-E2 (100 nM); OT, 4-hydroxy-tamoxifen (100 nM). Bound proteins were analyzed by SDS-PAGE. One tenth input [35S]methionine-labeled protein is included as control. B, GST alone, GST-ER-N-term (amino acids 1–182), or GST-ER-HBD (amino acids 282–595) proteins were incubated with in vitro translated RTA, and bound proteins were analyzed as above. C, HeLa cells were transfected with expression vectors for ER{alpha} and c-Myc-tagged RTA together (lanes 1–2) or alone (lanes 3–4). After transfection, cells were lysed and immunoprecipitated with mouse IgG (lane 1) or anti c-Myc monoclonal antibody (lanes 2–4). Reactions were washed six times with lysis buffer, subjected to SDS-PAGE, and immunoblotted with anti-ER{alpha} monoclonal antibody H222. Mouse IgG heavy chain cross-reacts with secondary antirat IgG and is indicated in the figure.

 
RTA Is a Coregulator of ER Transcriptional Activity
We were next interested in determining whether RTA was a coregulator of ER{alpha} transcriptional activity. To address this question, we examined the effect of RTA expression on ER{alpha}-mediated transcriptional activity. Both estrogen and tamoxifen activate the 1X-ERE-tata-Luc reporter gene in HeLa cells transfected with ER{alpha} (Fig. 3AGo, top panels). At low levels of expression (200 ng input expression plasmid), RTA had a minimal effect on E2-mediated transcription, inhibiting this activity by less than 10%. At high levels of RTA expression (1,500 ng input expression plasmid), we did observe a 50% reduction in estrogen-activated transcription (data not shown). However, RTA completely inhibits tamoxifen-mediated transcription indicating that RTA is a novel corepressor of ER{alpha} signaling. Concordant with the results obtained in HeLa cells, RTA was found to be a potent repressor of tamoxifen-mediated transcription in HepG2 cells (Fig. 3AGo, lower panels).



View larger version (27K):
[in this window]
[in a new window]
  		Figure 3. RTA Is a Novel Coregulator of ER Signaling

A, HeLa cells (top panels) or HepG2 cells (lower panels) were transfected with either the 1X-ERE-tata-Luc or C3-Luc reporter genes along with an ER{alpha} expression plasmid. RTA expression plasmid (pCDNA-5XM-RTA) or control plasmid (PCDNA-5XM) was transfected as indicated. pCMV-ß-Gal was included as internal control. Transfections contained 2,200 ng reporter gene, 500 ng ER{alpha} expression plasmid, 100 ng pCMV-ß-Gal, and 200 ng of either RTA or control plasmid. Cells were induced with ligand as indicated in the figure. Data for HeLa cells are presented as normalized response, which was obtained by dividing the luciferase activity by ß-galactosidase activity. Data for HepG2 is presented as fold induction, which was obtained by dividing the normalized response in the presence of ligand by that in the absence of ligand. Transfections were performed in triplicate, and error bars are presented as SEM. B, HeLa cells were transfected as above with either ERß, PR, or GR expression plasmids, and transcriptional activity was analyzed using either the 1X-ERE-tata-Luc for ERß, or MMTV-Luc for PR and GR. Dex, 10 nM dexamethasone; R5020, 10 nM synthetic progestin. C, HeLa cells were transfected with either TR or RAR expression plasmids and analyzed using either TRE-PAL or RARE-Luc. T3, 10 nM thyroid hormone; 9-cis RA, 1 µM 9-cis-RA. For CMV promoter activity, 100 ng pCMV-ß-Gal in addition to either RTA expression or control plasmid (200 ng) was transfected. ß-Galactosidase activity was measured after 48 h and normalized to protein concentration. D, RTA contains autonomous repressor activity. HepG2 cells were transfected with either pM (control), pM-NCoR, or pM-RTA (500 ng) in addition to either 5X-Gal4-tata-Luc or TK-Gal4-Luc.

 
The results of the in vitro interaction assays indicate that RTA interacts with ERß in a manner similar to ER{alpha}, suggesting that RTA may have an effect on ERß signaling. Accordingly, RTA expression inhibits E2-mediated ERß transcriptional activity by greater than 50% (Fig. 3BGo). We have shown previously that ERß contains a repressor domain in the N terminus of the receptor (23). A strong association between RTA and ERß may account for this repression. RTA was found to be a potent repressor of agonist-activated GR and PR, suggesting that RTA is a corepressor for the steroid-receptor family (Fig. 3BGo). These data are particularly surprising considering that the N-terminal domains of ERß, PR, and GR share little sequence homology with the N-terminal domain of ER{alpha}. It may suggest that some structural features are conserved within the N-terminal domains of these receptors although their sequences are divergent. To confirm the functional specificity of RTA repression, we tested the ability of RTA to inhibit the transcriptional activity of several nonsteroid nuclear receptors as well as the cytomegalovirus (CMV) viral gene promotor. The failure of RTA to repress both TR and RAR transcriptional activity as well as CMV promotor activity demonstrates that RTA is not a general repressor of transcription (Fig. 3CGo).

Our results indicate that RTA is a potent repressor of steroid receptor-mediated transcriptional activity. However, RTA does not induce receptor turnover nor does it interfere with the DNA binding activity of the receptor (data not shown). Therefore, we considered that RTA may function as a repressor in a manner similar to REA (repressor of ER activity) by competing with coactivator binding to the receptor (24) or, alternatively, may contain an autonomous repressor domain. To determine whether RTA contains an autonomous transcriptional repressor domain, we fused RTA to the Gal4 DNA-binding domain and tested the transcriptional activity of this modified protein using two distinct Gal4-responsive reporter genes (Fig. 3DGo). For comparison, we also analyzed the transcriptional activity of a known transcriptional repressor, nuclear receptor corepressor (NCoR) (25). RTA and NCoR inhibit the transcriptional activity of both reporter genes. Interestingly, RTA was at least as efficacious as NCoR under these conditions. These data suggest that the repressor function of RTA can be ascribed, at least in part, to the presence of an autonomous repressor domain.

The RRM Is Required for the Repressor Function of RTA
To investigate the domains or sequences within RTA required for repressor activity, we created several RTA mutants and tested their ability to inhibit ER{alpha}-mediated transcriptional activity on the 1X-ERE-tata-Luc (Fig. 4AGo) or 3X-ERE-tata-Luc (Fig. 4BGo) reporter genes. As expected, RTA inhibits the partial agonist activity of tamoxifen manifest on the 1X-ERE-tata-Luc-reporter gene (Fig. 4AGo). A C-terminal deletion mutant of RTA (RTA-N) containing amino acids 1–223 has no effect on ER{alpha} signaling in this context, demonstrating that the C-terminal domain of RTA is required for repressor activity. Because the C-terminal fragment of RTA was isolated in the yeast two-hybrid screen, this domain likely mediates the interaction between ER{alpha} and RTA. Removal of the RRM (RTA-C) or deletion of either RNP domain (RTA-{Delta}2 and RTA-{Delta}1) leads to loss of repressor function. These results clearly demonstrate the importance of the RRM in mediating the repressor activity of RTA. Expression of RTA-C enhances both E2- and tamoxifen-mediated transcription, suggesting that this mutant acts in a dominant negative manner to relieve the repressive activity of endogenous RTA (Fig. 4AGo). Remarkably, expression of RTA-{Delta}2 converts the pure antagonist ICI 182,780 into an agonist. Similar results were obtained when the repressor function of the RTA mutants was analyzed using the 3X-ERE-tata-Luc-reporter gene (Fig. 4BGo). Expression of RTA-C converts tamoxifen into a powerful agonist, whereas expression of RTA-{Delta}2 converts both tamoxifen and ICI 182,780 into agonists (Fig. 4BGo). We also analyzed the activity of dominant negative RTA on endogenous ER{alpha} levels in both breast (MCF-7) and ovarian (OVCA432) carcinoma cells and found similar results (data not shown). The ability of RTA-{Delta}2 to alter the pharmacology of ER ligands was not limited to tamoxifen and ICI 182,780, as all SERMs demonstrate agonist activity when this mutant is expressed (Fig. 4CGo). Taken together, these results suggest that RTA acts to repress ER{alpha} transcriptional activity. Mutant forms of RTA, which lack a functional RRM, operate to block the repressive activity of endogenous RTA. It seems likely that differences in the expression of RTA or a related protein may explain the tissue-selective agonist/antagonist activity of SERMs.



View larger version (26K):
[in this window]
[in a new window]
  		Figure 4. RTA Repressor Activity Is Mediated by the RNA Recognition Motif

HeLa cells were transfected with either 1X-ERE-tata-Luc (panel A) or 3X-ERE-tata-Luc (2200 ng) (panel B) along with pRST7-ER{alpha} (500 ng) and the indicated RTA expression plasmid (200 ng). Shown in panel A is schematic representation of RTA mutants. Cells were induced with ligand (10 nM) as indicated in the figure. ICI, ICI 182,780. Transfections were performed in triplicate and error is presented as SEM. C, HeLa cells were transfected with the 3X-ERE-tata-Luc reporter gene along with the RTA-{Delta}2 expression plasmid. Cells were induced with ligand (10 nM) as indicated in the figure. 5638, GW5638; Ralox, raloxifene; Nafox, nafoxidene; Idox, idoxifene. D, Derepression of AF-1 by RTA-{Delta}2. HeLa cells were transfected with the 3X-ERE-tata-Luc reporter plasmid (2,200 ng) along with RTA-{Delta}2 (200 ng) and the indicated ER{alpha} expression plasmid (500 ng). TAF1–3X contains amino acid substitutions of D538N, E542Q, and D545N. ER-351 contains amino acids 1–351. Cells were induced with ligand (10 nM) as indicated in the figure.

 
We were next interested in determining how RTA-{Delta}2 alters the pharmacology of antiestrogens on ER{alpha} signaling. To accomplish this, the transcriptional activity of several ER{alpha} mutants was analyzed in the presence and absence of RTA-{Delta}2 (Fig. 4DGo). ER{alpha} contains two primary activation domains, AF-1 and AF-2, the transcriptional activity of which is manifest in a cell- and promoter-specific manner (17, 18). Although they function independently, full ER{alpha} transcriptional activity typically requires the presence of both AFs. Mutants either lacking AF-2 (ER-351) or containing point mutations that destabilize AF-2 (TAF1–3X) are transcriptionally inactive in most environments (Fig. 4DGo) (18, 26, 27). Both receptor mutants contain functional AF-1 domains, the activity of which is somehow silenced. Remarkably, expression of RTA-{Delta}2 restores the transcriptional activity of these mutants and allows antiestrogens to be recognized as ER{alpha} agonists when TAF1-3X activity is analyzed (Fig. 4DGo). It should be noted that ER-351 does not bind ligand, and thus this mutant is activated by RTA-{Delta}2 in a ligand-independent manner (Fig. 4DGo). These results demonstrate that when the repressor function of RTA is blocked, the need for a functional AF-2 is bypassed. When bound by antagonist, RTA functions to suppress the constitutive activity of the AF-1 activator. RTA-{Delta}2 acts in a dominant negative manner to derepress AF-1 activity. When bound by agonist, AF-2 functions to overcome the repressive effect of RTA. Therefore, RTA overexpression has only a marginal effect on E2-activated ER{alpha} transcriptional activity. The ability of RTA to suppress agonist-activated ERß -, GR-, and PR-mediated transcription suggests that the AF-2 domains of these receptors are less efficient at overcoming the repressive function of RTA. These results are similar to previous studies from our laboratory that identified SSN6/TUP1 as a negative regulator of AF-1 function in yeast (28). This complex is believed to function at the level of chromatin to suppress transcription (29). Sequence comparisons between RTA and SSN6/TUP1 did not reveal any similarities; however, their functional characteristics suggest that they may operate in a similar manner.

RTA Interacts with RNA in Vitro
Our results suggest that RNA binding is an important factor in the repressor function of RTA. Using an RNA homopolymer binding assay, we wished to determine whether RTA interacts directly with RNA. RTA interacts strongly with p(G) and p(U) RNAs but not with p(A) or p(C) (Fig. 5Go). Dominant negative RTA, RTA-{Delta}2, demonstrates a decrease in binding to p(G) and an increase in binding to p(A). RTA-C interacts with p(U) but only weakly binds p(G). The ability of RTA-C to interact with RNA in the absence of the RRM suggests that the RGG sequences contained within the C-terminal domain of RTA may also function to bind RNA. Taken together, these results suggest that the ability of RTA to interact with RNA is an important determinant of the repressor function of RTA. Furthermore, there appears to be a correlation between the dominant negative transcriptional phenotype of the RTA mutants and disruption or alteration of RNA binding specificity.



View larger version (63K):
[in this window]
[in a new window]
  		Figure 5. RNA Homopolymer Binding Assay

Binding of p(A), p(C), p(G), and p(U) ribonucleic acids to in vitro translated RTA, RTA-{Delta}2, RTA-C, and luciferase (Luc) is shown. Ten percent input is included for reference.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
Using the ER{alpha} N terminus as bait in a yeast two-hybrid screen, we have identified a novel coregulator of nuclear steroid receptor signaling. This protein, denoted RTA, is a potent repressor of tamoxifen-mediated ER{alpha} transcriptional activity as well as the agonist activities of ERß, PR, and GR. RTA does not structurally resemble any of the known nuclear receptor corepressors including NCoR, SMRT (silencing mediator of retinoic and thyroid hormone), and small unique nuclear receptor corepressor (25, 30, 31). These factors participate in the formation of large multiprotein complexes, the repressor function of which is related to their ability to deacetylate histones and induce chromatin condensation (25, 32). Interestingly, a novel RRM containing protein, SMRT/histone deacetylase 1-associated repressor protein (SHARP), was recently identified by its ability to interact with SMRT (33). SHARP mediates repression presumably by sequestering the RNA coactivator SRA (34) and/or the recruitment of histone deacetylase activity. SHARP contains three RRMs that were shown to be essential for its ability to repress SRA-potentiated transcription. Functionally, RTA can be distinguished from this class of corepressors as its activity is unaffected by histone deacetylase inhibitors (data not shown). It is not yet known whether RTA can interact with SRA through its RRM. However, this method of transcriptional repression remains an intriguing possibility.

The mechanism by which RTA mediates transcriptional repression is not known. Based on the current available data, we propose the following model as detailed in Fig. 6Go. RTA interacts with the N-terminal domain of ER{alpha} and recruits a corepressor or corepressor complex to the receptor. This interaction may involve RNA, as mutation of the RRM within RTA compromises its ability to repress transcription. Of significant interest is the finding that these RNA binding domains are also capable of directly interacting with other proteins (35). It is therefore possible that mutation of the RRM destabilizes both protein-protein and protein-RNA interactions. RTA interacts with the receptor in a ligand-independent manner and may therefore function to silence the constitutive transcriptional activity of AF-1. When bound by agonist, ER{alpha} is known to recruit several classes of p160 coactivating proteins (14). These coactivators appear to overcome the repressor function of RTA. In support of this role, we have been able to show that ER mutants that do not interact with the p160 coactivators (TAF1-3X, ER-351) are inactive until the presumed activity of endogenous RTA is compromised by overexpression of dominant negative RTA (RTA-{Delta}2). This implies that RTA is an effective inhibitor of tamoxifen-mediated transcription because tamoxifen-activated ER does not recruit p160 coactivating proteins.



View larger version (51K):
[in this window]
[in a new window]
  		Figure 6. Model for Repression Mediated by RTA

Detailed in text.

 
The mechanism by which SERMs exhibit tissue-specific agonist activity is, to date, poorly understood, although evidence has been presented to suggest that ER{alpha} phosphorylation, differential expression of coactivators and corepressors, receptor mutations, and tissue-specific expression of the ERß isoform may be involved (36, 37). Our results define RTA as a novel coregulator of ER signaling. We find that RTA mRNA is expressed in most tissues but appears to be down-regulated in some, suggesting that its differential expression may be an important determinant of the resultant pharmacology of a particular ligand. RTA contains a well characterized RRM and several other structural features found in RNA binding proteins. RTA interacts with RNA in vitro and is likely to be involved in some aspect of RNA metabolism. In this regard, the link between nuclear receptors and RNA processing events has recently become apparent. Interestingly, RNA binding proteins have been implicated in both nuclear receptor activation and repression. An RRM-containing coactivator activator (CoAA) was recently identified which interacts with the TR binding protein (TRBP), itself a coactivator (38). The PPAR{gamma} coactivator PGC-1 is an RNA-binding protein recently found to colocalize with splicing factors (39). Interestingly, PGC-1 can alter the splicing pattern of a test minigene when loaded onto the promoter. The orphan nuclear receptor DAX-1, a transcriptional repressor, is a shuttling RNA binding protein that is associated with polyribosomes (40), and polypyrimidine tract-binding protein-associated splicing factor (PSF) was recently identified as a repressor of TR (41). We find that RTA localizes to nuclear speckles; however, RTA does not colocalize with the splicing factor SC35, suggesting that RTA functions in some aspect of RNA metabolism other than splicing (data not shown). An interesting characteristic of many RNA binding proteins is that they can exist in multiple forms within target cells. Northern blot analysis revealed that there were several distinct RTA transcript sizes. Our results with RTA-{Delta}2 indicate that isoforms lacking a functional RRM will have a significant impact on signaling by both estrogen- and antiestrogen-activated ER{alpha}. Whether such proteins occur in nature awaits the generation of antibodies to RTA.

In summary, these studies suggest a role for RNA binding proteins and RNA as important determinants of the tissue-specific agonist activities of SERMs as well as define a novel pathway by which breast cancer cells can acquire resistance to tamoxifen.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Plasmids and Chemicals
Yeast bait expression plasmid (pGBT9-ER-N-term) was created by PCR using ER{alpha} cDNA as template. Resultant DNA was subcloned into the EcoRI/SalI site of the parent vector (pGBT9, CLONTECH Laboratories, Inc.). pRST7-ER{alpha} and pRST7-ERß were described elsewhere (23). pCDNA3-RTA was created by PCR using RTA cDNA. Resultant DNA was subcloned into the BamHI/XhoI site of pCDNA3 (Stratagene, La Jolla, CA). Digesting pCDNA3-RTA with BamHI/XhoI and subsequent subcloning of resultant DNA into pGEX-5X-1 created GST-RTA. GST-ER-N-term was created by PCR using ER{alpha} cDNA as template. Resultant DNA was subcloned into the EcoRI/XhoI site of pGEX-5X-1. GST-ER-HBD (HE14G) was described elsewhere (42). pCDNA-5XM-RTA was created by PCR using RTA cDNA as template. Resultant DNA was subcloned into pCDNA-5XM digested with BamHI/XbaI. 1X-ERE-tata-Luc, C3-Luc, and pCMV-ß-gal were described elsewhere (43). Digesting pCDNA-5XM-RTA with BamHI/XbaI and subsequent subcloning of resultant DNA into pM (CLONTECH Laboratories, Inc.) created pM-RTA. 5X-Gal4-tata-Luc is described elsewhere (43). pCDNA-5XM-RTA-N (amino acids 1–223), pCDNA-5XM-RTA-C (amino acids 224–390), pCDNA-5XM-RTA-{Delta}2 (deletion amino acids 123–128), and pCDNA-5XM-RTA-{Delta}1 (deletion amino acids 160–167) were created by PCR using RTA cDNA as template. Resultant DNA was subcloned into pCDNA-5XM. ER{alpha} expression vectors TAF1-3X, TAF-2, ER-LL, and ER-351 are described elsewhere (18, 26, 27). SV40-hPR-B, pRST7-GR, and MMTV-Luc were described elsewhere (44). 17ß-E2, 4-hydroxy-tamoxifen, and dexamethasone were purchased from Sigma (St. Louis, MO). R5020 was purchased from NEN Life Science Products (Boston, MA).

Yeast Two-Hybrid
The yeast strain HF7C (CLONTECH Laboratories, Inc.) was transformed with pGBT9-ER N-term, which expresses ER amino acids 51–149 fused to the Gal4 DNA-binding domain along with a HeLa cell cDNA library fused to the Gal4 activation domain cloned in pACT2. HF7C contains two inducible reporter genes, His3 and ß-galactosidase, both under control of a Gal4 upstream enhancer sequence. Cells were plated on yeast minimal media lacking histidine plus 5 mM aminotriazole. ER N-terminal interacting clones were identified based on their ability to induce HIS3 expression. HIS3+ colonies were selected and analyzed for ß-galactosidase expression using a filter lift assay.

GST Interaction Assays
[35S]Methionine-labeled ER{alpha} and ERß were translated from pRST7-ER{alpha} and pRST7-ERß, respectively, using in vitro TNT rabbit reticulocyte lysate kit (Promega Corp., Madison, WI) as described in the manufacturer’s protocol. PGEX-5X-1 (GST alone) (Amersham Pharmacia Biotech, Arlington Heights, IL), and pGEX-5X-RTA (GST-RTA) were transformed into bacterial strain BL-21 (Stratagene, La Jolla, CA), and proteins were purified with glutathione-Sepharose beads (Amersham Pharmacia Biotech). Resultant labeled proteins were combined with GST fusion proteins in 1 ml NETN binding buffer (50 mM NaCl, 1 mM EDTA pH 8.0, 20 mM Tris pH 8.0, 0.5% NP-40) and incubated overnight at 4 C. Beads were washed in washing buffer (NETN, 100 mM NaCl) five times, and binding proteins were eluted by boiling in SDS sample buffer and analyzed by SDS-PAGE.

Cell Culture, Transfection, and Immunoprecipitations
HepG2 or HeLa cells were cultured in 24-well plates overnight in phenol red free MEM (Life Technologies, Inc., Gaithersburg, MD) plus 10% charcoal/dextran-treated FBS (HyClone Laboratories, Inc., Logan, UT). Cells were transfected as described previously (26). For immunoprecipitation, HeLa cells were cultured in tissue culture dishes at 2.0 x 106 cells per plate in MEM containing 10% FBS (Life Technologies, Inc.). Cells were transfected with pRST7-ER{alpha} and pCDNA-5XM-RTA (c-Myc-tagged RTA) either individually or in combination. After transfection, cells were cultured in MEM/FBS containing 0.1 µM 4-hydroxy-tamoxifen for 24 h. Cells were washed with PBS and 0.5 ml lysis buffer (PBS, 0.5% Triton-X, protease inhibitors) was added to the plates. Lysate was precleared with 50 µl protein A-Sepharose beads (Zymed Laboratories, Inc., South San Francisco, CA) and mouse IgG for 2 h at 4 C. Either anti-c-Myc monoclonal antibody 9E10 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) or antimouse IgG was then added for 2 h at 4 C. Forty microliters of protein A beads were then added to the antibody/lysate mixture for an additional hour at 4 C. Beads were washed six times with lysis buffer and resuspended in SDS-PAGE sample buffer. Proteins were resolved on 10% SDS-PAGE and transferred to nitrocellulose membranes and probed with monoclonal antibody H222. Complexes were detected using enhanced luminescence following the manufacturer’s protocol (Amersham Pharmacia Biotech).

RNA Homopolymer Binding Assays
Assays were performed as described previously (40). 35S-labeled RTA, RTA-{Delta}2, RTA-C, and luciferase were generated using TNT rabbit reticulocyte lysate kit (Promega Corp.). Labeled proteins were mixed with p(G)- or p(C)-conjugated agarose (Sigma) or p(A)- or p(U)-conjugated Sepharose (Pharmacia Biotech, Piscataway, NJ) in binding buffer (10 mM Tris, pH 8.0, 2.5 mM MgCl2, 0.5% Triton, and 100 mM NaCl). Reactions were incubated at 4 C for 15 min and washed six times in binding buffer. Bound proteins were analyzed by SDS-PAGE.

Northern Blot Analysis
32P-Labeled RTA probe was generated by random prime labeling. Multiple-tissue northern blot was then probed according to manufacturers protocol (CLONTECH Laboratories, Inc.)


   ACKNOWLEDGMENTS
 
ICI 182,780 was a gift from Dr. Alan Wakeling (Zeneca Pharmaceuticals, Macclesfield, UK). Raloxifene was a gift from Dr. Eric Larson (Pfizer Pharmaceuticals, Groton, CT). Idoxifene was a gift from Dr. Mark Nuttall (SmithKline Beecham Pharmaceuticals, King of Prussia, PA.). GW 5638 was a gift from Dr. Tim Willson (Glaxo SmithKline, Research Triangle Park, NC). pCDNA-5XM was a gift from Dr. Xiao-Fan Wang (Duke University Medical Center, Durham, NC). Anti-ER{alpha} monoclonal antibody H222 was a gift from Dr. Geoffrey Greene (Ben May Institute, Chicago, IL). TK-Gal4-Luc, pM-NCoR, and pCDNA-TR were gifts from Dr. Xiaolin Li (Duke University Medical Center, Durham, NC). HE14G was a gift from Dr. Peter Kushner (University of California San Francisco, San Francisco, CA). pRST7-RAR, LUC-24J (RARE-LUC), and 9-cis-RA) were gifts from Dr. Rich Heyman (X-Ceptor Pharmaceuticals, San Diego, CA). TRE-PAL was a gift from Dr. Christopher Glass (University of California at San Diego, La Jolla, CA).


   FOOTNOTES
 
This work was supported by NIH Grant DK-48807 (to D.P.M.).

Abbreviations: AF-1 and -2, Activation functions 1 and 2; CMV, cytomegalovirus; GST, glutathione-S-transferase; HBD, hormone-binding domain; NCoR, nuclear receptor co-repressor; RRM, RNA recognition motif; RTA, repressor of tamoxifen transcriptional activity; SERM, selective estrogen receptor modulator; RGG, arginine/glycine-rich box; SHARP, SMRT/histone deacetylase-1-associated repressor protein; SMRT, silencing mediator of retinoic and thyroid hormone.

Received for publication September 27, 2001. Accepted for publication November 14, 2001.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. Evans RM 1988 The steroid and thyroid hormone receptor superfamily. Science 340:889–895
  2. Kuiper GGJM, Enmark E, Pelto-Huikko M, Nilsson S, Gustafsson J-A 1996 Cloning of a novel estrogen receptor expressed in rat prostate and ovary. Proc Natl Acad Sci USA 93:5925–5930[Abstract/Free Full Text]
  3. Mosselman S, Polman J, Dijkema R 1996 ERß: identification and characterization of a novel human estrogen receptor. FEBS Lett 392:49–53[CrossRef][Medline]
  4. Jordan VC 1992 The strategic use of antiestrogen to control the development and growth of breast cancer. Cancer 70:977–982[Medline]
  5. Katzenellenbogen BS, Montano MM, Ekena K, Herman ME, McInerney EM 1997 William L. McGuire Memorial Lecture. Antiestrogens: mechanisms of action and resistance in breast cancer. Breast Cancer Res Treat 44:23–38[CrossRef][Medline]
  6. Bertelli G, Pronzato P, Amoroso D, Cusimano MP, Conte PF, Montagna G, Bertolini S, Rosso R 1988 Adjuvant tamoxifen in primary breast cancer: influence on plasma lipids and antithrombin III levels. Breast Cancer Res Treat 12:307–310[CrossRef][Medline]
  7. Love RR, Mazess RB, Barden HS, Epstein S, Newcomb PA, Jordan VC, Carbone PP, DeMets DL 1992 Effects of tamoxifen on bone mineral density in postmenopausal women with breast cancer. N Engl J Med 326:852–856[Abstract]
  8. Kedar RP, Bourne TH, Powles TJ, Collins WP, Ashley SE, Cosgrove DO, Cambell S 1994 Effects of tamoxifen on uterus and ovaries of postmenopausal women in a randomised breast cancer prevention trial. Lancet 343:1318–1321[CrossRef][Medline]
  9. Sato M, Rippy MK, Bryant HU 1996 Raloxifene, tamoxifen, nafoxidine, or estrogen effects on reproductive and nonreproductive tissues in ovariectomized rats. FASEB J 10:905–912[Abstract]
  10. Onate SA, Tsai S, Tsai M-J, O’Malley BW 1995 Sequence and characterization of a coactivator for the steroid hormone receptor superfamily. Science 270:1354–1357[Abstract/Free Full Text]
  11. Anzick SL, Kononen J, Walker RL, Azorsa DO, Tanner MM, Guan X-Y, Sauter G, Kallioniemi O-P, Trent JM, Meltzer PS 1997 AIB1, a steroid receptor coactivator amplified in breast and ovarian cancer. Science 277:965–968[Abstract/Free Full Text]
  12. Robyr D, Wolffe AP, Wahli W 2000 Nuclear hormone receptor coregulators in action: diversity for shared tasks. Mol Endocrinol 14:329–347[Free Full Text]
  13. Brzozowski AM, Pike AC, Dauter Z, Hubbard RE, Bonn T, Engstrom O, Ohman L, Greene GL, Gustafsson JA, Carlquist M 1997 Molecular basis of agonism and antagonism in the oestrogen receptor. Nature 389:753–758[CrossRef][Medline]
  14. Heery DM, Kalkhoven E, Hoare S, Parker MG 1997 A signature motif in transcriptional co-activators mediates binding to nuclear receptors. Nature 387:733–736[CrossRef][Medline]
  15. Spencer TE, Jenster G, Burcin MM, Allis CD, Zhou J, Mizzen CA, McKenna NJ, Onate SA, Tsai SY, Tsai M-J, O’Malley BW 1997 Steroid receptor coactivator-1 is a histone acetyltransferase. Nature 389:194–198[CrossRef][Medline]
  16. Ogryzko VV, Schiltz RL, Russanova V, Howard BH, Nakatani Y 1996 The transcriptional coactivators p300 and CBP are histone acetyltransferases. Cell 87:953–959[CrossRef][Medline]
  17. Berry M, Metzger D, Chambon P 1990 Role of the two activating domains of the oestrogen receptor in the cell-type and promoter-context dependent agonistic activity of the anti-oestrogen 4-hydroxytamoxifen. EMBO J 9:2811–2818[Medline]
  18. Tzukerman MT, Esty A, Santiso-Mere D, Danielian P, Parker MG, Stein RB, Pike JW, McDonnell DP 1994 Human estrogen receptor transactivational capacity is determined by both cellular and promoter context and mediated by two functionally distinct intramolecular regions. Mol Endocrinol 8:21–30[Abstract]
  19. Skipper M, Milne CA, Hodgkin J 1999 Genetic and molecular analysis of fox-1, a numerator element involved in Caenorhabditis elegans primary sex determination. Genetics 151:617–631[Abstract/Free Full Text]
  20. Shibata H, Huynh DP, Pulst SM 2000 A novel protein with RNA-binding motifs interacts with ataxin-2. Hum Mol Genet 9:1303–1313[Abstract/Free Full Text]
  21. Krecic AM, Swanson MS 1999 hnRNP complexes: composition, structure, and function. Curr Opin Cell Biol 11:363–371[CrossRef][Medline]
  22. Burd CG, Dreyfuss G 1994 RNA binding specificity of hnRNP A1: significance of hnRNP A1 high-affinity binding sites in pre-mRNA splicing. EMBO J 13:1197–1204[Medline]
  23. Hall JM, McDonnell DP 1999 The estrogen receptor ß-isoform (ERß) of the human estrogen receptor modulates ER{alpha} transcriptional activity and is a key regulator of the cellular response to estrogens and antiestrogens. Endocrinology 140:5566–5578[Abstract/Free Full Text]
  24. Montano MM, Ekena K, Delage-Mourroux R, Chang W, Martini P, Katzenellenbogen BS 1999 An estrogen receptor-selective coregulator that potentiates the effectiveness of antiestrogens and represses the activity of estrogens. Proc Natl Acad Sci USA 96:6947–6952[Abstract/Free Full Text]
  25. Alland L, Muhle R, Hou H, Potes J, Chin L, Schreiber-Agus N, DePinho RA 1997 Role for NCoR and histone deacetylase in Sin3-mediated transcription repression. Nature 387:49–55[CrossRef][Medline]
  26. Norris JD, Fan D, Kerner SA, McDonnell DP 1997 Identification of a third autonomous activation domain within the human estrogen receptor. Mol Endocrinol 11:747–754[Abstract/Free Full Text]
  27. Norris JD, Fan D, Stallcup MR, McDonnell DP 1998 Enhancement of estrogen receptor transcriptionl activity by the coactivator GRIP-1 highlights the role of activation function 2 in determining estrogen receptor pharmacology. J Biol Chem 273:6679–6688[Abstract/Free Full Text]
  28. McDonnell DP, Vegeto E, O’Malley BW 1992 Identification of a negative regulatory function for steroid receptors. Proc Natl Acad Sci USA 89:10563–10567[Abstract/Free Full Text]
  29. Watson AD, Edmondson DG, Bone JR, Mukai Y, Yu Y, Du W, Stillman DJ, Roth SY 2000 Ssn6-Tup1 interacts with class I histone deacetylases required for repression. Genes Dev 14:2737–2744[Abstract/Free Full Text]
  30. Chen JD, Evans RM 1995 A transcriptional co-repressor that interacts with nuclear hormone receptors. Nature 377:454–457[CrossRef][Medline]
  31. Zamir I, Dawson J, Lavinsky RM, Glass CK, Rosenfeld MG, Lazar MA 1997 Cloning and characterization of a corepressor and potential component of the nuclear hormone receptor repression complex. Proc Natl Acad Sci USA 94:14400–14405[Abstract/Free Full Text]
  32. Heinzel T, Lavinsky RM, Mullen T-M, Söderström M, Laherty CD, Torchia J, Yang W-M, Brard G, Ngo SD, Davie JR, Seto E, Eisenman RN, Rose DW, Glass CK, Rosenfeld MG 1997 A complex containing N-CoR, mSin3 and histone deacetylase mediates transcriptional repression. Nature 387:43–48[CrossRef][Medline]
  33. Shi Y, Downes M, Xie W, Kao HY, Ordentlich P, Tsai CC, Hon M, Evans RM 2001 Sharp, an inducible cofactor that integrates nuclear receptor repression and activation. Genes Dev 15:1140–1151[Abstract/Free Full Text]
  34. Lanz RB, McKenna NJ, Onate SA, Albrecht U, Tsai SY, Tsai M-J, O’Malley BW 1999 A steroid receptor coactivator, SRA, functions as an RNA and is present in an SRC-1 complex. Cell 97:17–27[CrossRef][Medline]
  35. Sakashita E, Sakamoto H 1996 Protein-RNA and protein-protein interactions of the Drosophila sex-lethal mediated by its RNA-binding domains. J Biochem (Tokyo) 120:1028–1033[Abstract/Free Full Text]
  36. Katzenellenbogen BS, Korach KS 1997 Editorial: A new actor in the estrogen receptor drama—enter ER-ß. Endocrinology 138:861–862[Free Full Text]
  37. McDonnell DP 1999 The molecular pharmacology of SERMs. Trends Endocrinol Metab 10:301–311[CrossRef][Medline]
  38. Iwasaki T, Chin WW, Ko L 2001 Identification and characterization of a RRM-containing coactivator activator (CoAA) as TRBP-interacting protein, and its splice variant as a coactivator modulator (CoAM). J Biol Chem 229:33375–33383
  39. Monsalve M, Wu Z, Adelmant G, Puigserver P, Fan M, Spiegelman BM 2000 Direct coupling of transcription and mRNA processing through the thermogenic coactivator PGC-1. Mol Cell 6:307–316[CrossRef][Medline]
  40. Lalli E, Ohe K, Hindelang C, Sassone-Corsi P 2000 Orphan receptor DAX-1 is a shuttling RNA binding protein associated with polyribosomes via mRNA. Mol Cell Biol 20:4910–4921[Abstract/Free Full Text]
  41. Mathur M, Tucker PW, Samuels HH 2001 PSF is a novel corepressor that mediates its effect through Sin3A and the DNA binding domain of nuclear hormone receptors. Mol Cell Biol 21:2298–2311[Abstract/Free Full Text]
  42. Sadovsky Y, Webb P, Lopez G, Baxter JD, Fitzpatrick PM, Gizang-Ginsberg E, Cavailles V, Parker MG, Kushner PJ 1995 Transcriptional activators differ in their responses to overexpression of TATA-box-binding protein. Mol Cell Biol 15:1554–1563[Abstract]
  43. Norris JD, Paige LA, Christensen DJ, Chang C-Y, Huacani MR, Fan D, Hamilton PT, Fowlkes DM, McDonnell DP 1999 Peptide antagonists of the human estrogen receptor. Science 285:744–746[Abstract/Free Full Text]
  44. Wagner BL, Norris JD, Knotts TA, Weigel NL, McDonnell DP 1998 The nuclear corepressors NCoR and SMRT are key regulators of both ligand- and 8-bromo-cyclic AMP-dependent transcriptional activity of the human progesterone receptor. Mol Cell Biol 18:1369–1378[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 BloodHome page
G. Yang, S.-C. Huang, J. Y. Wu, and E. J. Benz Jr
Regulated Fox-2 isoform expression mediates protein 4.1R splicing during erythroid differentiation
Blood, January 1, 2008; 111(1): 392 - 401.
[Abstract] [Full Text] [PDF]

Home page
 Nucleic Acids ResHome page
K. Fukumura, A. Kato, Y. Jin, T. Ideue, T. Hirose, N. Kataoka, T. Fujiwara, H. Sakamoto, and K. Inoue
Tissue-specific splicing regulator Fox-1 induces exon skipping by interfering E complex formation on the downstream intron of human F1{gamma} gene
Nucleic Acids Res., August 13, 2007; 35(16): 5303 - 5311.
[Abstract] [Full Text] [PDF]

Home page
 Physiol. Rev.Home page
N. Heldring, A. Pike, S. Andersson, J. Matthews, G. Cheng, J. Hartman, M. Tujague, A. Strom, E. Treuter, M. Warner, et al.
Estrogen Receptors: How Do They Signal and What Are Their Targets
Physiol Rev, July 1, 2007; 87(3): 905 - 931.
[Abstract] [Full Text] [PDF]

Home page
 Nucleic Acids ResHome page
J. Yan, Y.-S. Kim, X.-P. Yang, M. Albers, M. Koegl, and A. M. Jetten
Ubiquitin-interaction motifs of RAP80 are critical in its regulation of estrogen receptor {alpha}
Nucleic Acids Res., March 12, 2007; 35(5): 1673 - 1686.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
N. J. Camp, J. M. Farnham, and L. A. Cannon-Albright
Localization of a Prostate Cancer Predisposition Gene to an 880-kb Region on Chromosome 22q12.3 in Utah High-Risk Pedigrees.
Cancer Res., October 15, 2006; 66(20): 10205 - 10212.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
J. L. Ponthier, C. Schluepen, W. Chen, R. A. Lersch, S. L. Gee, V. C. Hou, A. J. Lo, S. A. Short, J. A. Chasis, J. C. Winkelmann, et al.
Fox-2 Splicing Factor Binds to a Conserved Intron Motif to Promote Inclusion of Protein 4.1R Alternative Exon 16
J. Biol. Chem., May 5, 2006; 281(18): 12468 - 12474.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Cell. Biol.Home page
A. P. Baraniak, J. R. Chen, and M. A. Garcia-Blanco
Fox-2 Mediates Epithelial Cell-Specific Fibroblast Growth Factor Receptor 2 Exon Choice
Mol. Cell. Biol., February 15, 2006; 26(4): 1209 - 1222.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
C. Li, M. Kato, L. Shiue, J. E. Shively, M. Ares Jr., and R.-J. Lin
Cell Type and Culture Condition-Dependent Alternative Splicing in Human Breast Cancer Cells Revealed by Splicing-Sensitive Microarrays
Cancer Res., February 15, 2006; 66(4): 1990 - 1999.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Interv.Home page
J. M. Hall and D. P. McDonnell
Coregulators in Nuclear Estrogen Receptor Action: From Concept to Therapeutic Targeting
Mol. Interv., December 1, 2005; 5(6): 343 - 357.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Endocrinol.Home page
H. Leong, J. R. Sloan, P. D. Nash, and G. L. Greene
Recruitment of Histone Deacetylase 4 to the N-Terminal Region of Estrogen Receptor {alpha}
Mol. Endocrinol., December 1, 2005; 19(12): 2930 - 2942.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Cell. Biol.Home page
J. G. Underwood, P. L. Boutz, J. D. Dougherty, P. Stoilov, and D. L. Black
Homologues of the Caenorhabditis elegans Fox-1 Protein Are Neuronal Splicing Regulators in Mammals
Mol. Cell. Biol., November 15, 2005; 25(22): 10005 - 10016.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Cell. Biol.Home page
D. Auboeuf, D. H. Dowhan, M. Dutertre, N. Martin, S. M. Berget, and B. W. O'Malley
A Subset of Nuclear Receptor Coregulators Act as Coupling Proteins during Synthesis and Maturation of RNA Transcripts
Mol. Cell. Biol., July 1, 2005; 25(13): 5307 - 5316.
[Full Text] [PDF]

Home page
 Mol. Endocrinol.Home page
D. G. Monroe, F. J. Secreto, M. Subramaniam, B. J. Getz, S. Khosla, and T. C. Spelsberg
Estrogen Receptor {alpha} and {beta} Heterodimers Exert Unique Effects on Estrogen- and Tamoxifen-Dependent Gene Expression in Human U2OS Osteosarcoma Cells
Mol. Endocrinol., June 1, 2005; 19(6): 1555 - 1568.
[Abstract] [Full Text] [PDF]

Home page
 Physiol. GenomicsHome page
P. G. Giresi, E. J. Stevenson, J. Theilhaber, A. Koncarevic, J. Parkington, R. A. Fielding, and S. C. Kandarian
Identification of a molecular signature of sarcopenia
Physiol Genomics, April 14, 2005; 21(2): 253 - 263.
[Abstract] [Full Text] [PDF]

Home page
 Nucleic Acids ResHome page
S. Nakahata and S. Kawamoto
Tissue-dependent isoforms of mammalian Fox-1 homologs are associated with tissue-specific splicing activities
Nucleic Acids Res., April 11, 2005; 33(7): 2078 - 2089.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
X. Dong, O. Shylnova, J. R. G. Challis, and S. J. Lye
Identification and Characterization of the Protein-associated Splicing Factor as a Negative Co-regulator of the Progesterone Receptor
J. Biol. Chem., April 8, 2005; 280(14): 13329 - 13340.
[Abstract] [Full Text] [PDF]