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and ß by coactivators and corepressors
Department of Biochemistry and Molecular Biology, Center for Genetics and Molecular Medicine, University of Louisville School of Medicine, Louisville, Kentucky 40292, USA
(Requests for offprints should be addressed to C M Klinge; Email: carolyn.klinge{at}louisville.edu)
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Abstract |
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 Top Abstract IntroductionMaterials and methodsResultsDiscussionReferences |
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and ß (ER and ERß) stimulate gene transcription is through direct ER interaction with specific DNA sequences, estrogen response elements (EREs). ERE-bound ER recruits coactivators that stimulate gene transcription. Binding of ER to natural and synthetic EREs with different nucleotide sequences alters ER binding affinity, conformation, and transcriptional activity, indicating that the ERE sequence is an allosteric effector of ER action. Here we tested the hypothesis that alterations in ER conformation induced by binding to different ERE sequences modulates ER interaction with coactivators and corepressors. CHO-K1 cells transfected with ER or ERß show ERE sequence-dependent differences in the functional interaction of ER and ERß with coactivators steroid receptor coativator 1 (SRC-1), SRC-2 (glucocorticoid receptor interacting protein 1 (GRIP1)), SRC-3 amplified in breast cancer 1 (AIB1) and ACTR, cyclic AMP binding protein (CBP), and steroid receptor RNA activator (SRA), corepressors nuclear receptor co-repressor (NCoR) and silencing mediator for retinoid and thyroid hormone recpetors (SMRT), and secondary coactivators coactivator associated arginine methyltransferase 1 (CARM1) and protein arginine methyltransferase 1 (PRMT1). We note both ligand-independent as well estradiol- and 4-hydroxytamoxifen-dependent differences in ER-coregulator activity. In vitro ER-ERE binding assays using receptor interaction domains of these coregulators failed to recapitulate the cell-based results, substantiating the importance of the full-length proteins in regulating ER activity. These data demonstrated that the ERE sequence impacts estradiol-and 4-hydroxytamoxifen-occupied ER and ERß interaction with coregulators as measured by transcriptional activity in mammalian cells.
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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and ß (ER and ERß) (Couse & Korach 1999). Stimulation of target gene expression in response to 17ß-estradiol (E2), or other agonists, is thought to be mediated by two mechanisms: (1) direct binding of E2-liganded ER (E2-ER) to a specific sequence called an estrogen response element (ERE) and (2) ‘tethering’, in which ER interacts with another DNA-bound transcription factor, e.g. Sp1 (Safe 2001) or AP-1 (Webb et al. 1995), in a way that stabilizes the DNA binding of that transcription factor in the absence of direct ER-DNA binding. Both processes result in recruitment of coactivators and components of the RNA polymerase II transcription initiation complex that enhances target gene transcription (Klinge 2000).
We recently reviewed studies on ER interaction with 38 estrogen-responsive genes whose promoters or 3' untranslated regions contain functional EREs (Klinge 2001). Most estrogen-regulated genes contain imperfect, non-palindromic EREs. One conclusion from our review is that the more nucleotide changes there are from the consensus within a half-site of the ERE palindrome, the lower the ER binding affinity and the lower the transcriptional activity (Klinge 2001). Further, EREs in which nucleotides are altered in both arms of the palindrome show lower transcriptional activity than those containing alterations in only one ERE half-site (Klinge 2001). These results have been confirmed experimentally (Driscoll et al. 1998, Tyulmenkov & Klinge 2000a, 2001a, Tyulmenkov & Klinge b, Tyulmenkov et al. 2000, Kulakosky et al. 2002).
Lefstin & Yamamoto (1998) proposed that response elements recognized by nuclear transcription factors, including members of the steroid/nuclear receptor superfamily, contain information that is interpreted by bound regulator factors. Our data fit the model of Lefstin and Yamamoto by showing that different EREs bound ER with different affinity and resulted in different amounts of transactivation of a reporter plasmid in transfected cells (Tyulmenkov et al. 2000, Klinge et al. 2001, Tyulmenkov & Klinge 2001b, Kulakosky et al. 2002). We and other investigators have proposed that DNA acts as an allosteric effector whose binding alters ER conformation such that different EREs would be hypothesized to regulate ER interaction with other proteins, e.g. coactivators or corepressors (reviewed in Klinge et al. 2001). In concordance with this hypothesis, we observed differences in ER conformation, assessed by -chymotrypsin or trypsin digestion, upon interaction with a palindromic ERE or non-palindromic EREs from the human pS2, progesterone receptor (PR), and c-fos genes and a direct repeat of the ERE half-site separated by 5 bp (Klinge et al. 2001, Ramsey & Klinge 2001). The ERE-mediated increase in ER sensitivity to protease digestion correlated with E2-stimulated transcription from the same EREs in transiently transfected cells and with the affinity of ER-ERE binding in vitro (Klinge et al. 2001, Ramsey & Klinge 2001, Tyulmenkov & Klinge 2001a,b, Kulakosky et al. 2002).
Similarly, other investigators have reported differential recognition of ER by select antibodies when ER was bound to the Xenopus laevis vitellogenin A2 (vit A2) ERE versus the non-palindromic ERE from the human pS2 gene in electrophoretic mobility shift assay (EMSA) experiments (Wood et al. 1998). Limited protease digestion of ER bound to vit A2 ERE or the non-palindromic pS2, vitellogenin B1, and oxytocin EREs revealed different sized [32P]DNA fragments in EMSAs (Wood et al. 2001). These data were interpreted as indicating that ER binding to different EREs changes ER conformation (Wood et al. 1998, 2001). Likewise, experiments using phage display revealed conformational differences in ER and ERß when bound to vit A2, pS2, lactoferrin, and complement 3 (C3) EREs in vitro (Hall et al. 2002).
Only a few investigators have examined how these ERE-induced alterations in ER conformation impact ER-coactivator interaction. One study reported that GRIP1 showed less interaction when ER bound to the pS2 ERE versus the vit A2, vitellogenin B1, or oxytocin EREs, indicating that the ERE sequence impacts ER-GRIP1 interaction in vitro (Wood et al. 2001). Similar conclusions based on EMSA and GST-pulldown assays were recently reported by another group of investigators (Yi et al. 2002). However, the functional significance of these observations of ERE-dependent differences in ER conformation and recognition by coactivator receptor interaction domains (RIDs) has not been tested by cell-based transcription assays.
Over the past 6 years, at least 28 different ER coactivator proteins have been identified (McKenna et al. 1999, Klinge 2000, McKenna & O’Malley 2002). Less information is available regarding ERß-coactivator interaction. In brief, coactivators function to (1) acetylate the N-terminal tails of lysine residues in histones H3 and H4 leading to ‘relaxed’ chromatin structure (reviewed in Struhl 1998), (2) acetylate other transcription factors and coactivators (Jiang et al. 2000), (3) recruit secondary coactivators including coactivator associated arginine methyltransferase 1 (CARM1) and protein arginine methyltransferase 1 (PRMT1) that methylate histones (Koh et al. 2001), (4) interact with components of various ATP-dependent chromatin-remodeling complexes (Hebbar & Archer 2003), and (5) direct interaction with and stabilization of basal transcription factor binding (reviewed in Hager et al. 1998). Once the transcription initiation complex is complete, RNA polymerase II is recruited to the transcription start site and begins transcription.
Most of the cell-based assays examining the effects of coactivators on ER-mediated transcription have used reporter genes containing two or three tandem copies of the vit A2 ERE (Norris et al. 1998, Lee et al. 1999, Mak et al. 1999, Delage-Mourroux et al. 2000). The non-palindromic EREs that have been used in transfection experiments are from the complement C3, transforming growth factor-ß3, and lactoferrin gene promoters that were used to examine the functional interaction of ER with the receptor of estrogen receptor activity (Delage-Mourroux et al. 2000) and SRC-2/GRIP1 (Norris et al. 1998).
Selective estrogen receptor modulators (SERMS) in vitro and in transfected cells (Metivier et al. 2002). Treatment of cells with the histone deacetylase (HDAC) inhibitor trichostatin A has been shown to enhance ligand-independent as well as E2-, tamoxifen-, and raloxifene-stimulated ER transcription of an ERE-reporter gene, indicating that repression of ER transcriptional activity is mediated by a corepressor-HDAC complex (Webb et al. 2003). To address the functional consequences of the ERE sequence-dependent alterations on ER- or ERß-coactivator interaction, we have performed transient transfection experiments and quantitated the impact of representative coactivators on E2-induced reporter gene expression from single copies of the vit A2, pS2, PR, and c-fos EREs. We report that different ERE sequences alter coactivator enhancement of E2-induced transcriptional activity by ER and ERß. Further, differences in coactivator interaction between ER and ERß were also apparent. These data demonstrated that alterations in ERE sequence functionally impact coregulatory protein interaction with ER and ERß and that ER and ERß show subtype-dependent coactivator interactions.
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Materials and methods |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Human ER and ERß1 were expressed from baculoviruses in Sf21 cells as described (Kulakosky et al. 2002). EMSAs were used to determine the affinity of E2-occupied ER and ERß for the following EREs: EREc13: 5'-CCGGTCAGAGTGACCAG-3'; EREc38 (which is identical to the commonly used Xenopus vit A2 ERE (Peale et al. 1989)): 5'-CCAGGTCAGAGTGACCTGAGCTAAAATAACACATT-3'; PR1148: 5'-AGCCCTCCCTCCTGCGAGGTCACCAGCTCTTGGTGCCTGTTT-3'; pS2: 5'-CTTCCCCCTGCAAGGTCAGCGTGGCCACCCCGTGAGCCACT-3'; and Fos-1211: 5'-AGCTTGGGCTGAGCCGGCAGCGTGACCCCGCATG-3'.
The underlined nucleotides correspond to the minimal core consensus ERE. The nucleotides in bold indicate an alteration in the consensus ERE. EREc38, PR1148, pS2, and Fos-1211 were cloned into the pGL3-promoter luciferase reporter vector (Promega, Madison, WI, USA) as previously described (Klinge et al. 1997). Details of the EMSA experiments to determine ER-ERE binding affinity are provided in Kulakosky et al.(2002), Tyulmenkov et al.(2000), and Tyulmenkov & Klinge (2001a,b).
Cell culture, transient transfection, and reporter assays
Chinese Hamster Ovary cells (CHO-K1) were purchased from ATCC (Manasas, VA, USA) and maintained in Iscove’s modified Dulbecco’s medium (IMDM) (Gibco/Invitrogen, Grand Island, NY, USA) supplemented with heat-treated, charcoal-stripped 10% fetal bovine serum (Atlanta Biologicals, Norcross, GA, USA). All other cell culture reagents were purchased from Gibco-BRL (Grand Island, NY, USA). For transient transfection, CHO-K1 cells were plated into 24-well plates at 1x105 cells/well with IMDM (without phenol red) supplemented with 10% charcoal-stripped fetal bovine serum. The cells were transfected when 80% confluent with 0.25 µg reporter construct containing the ERE, 5 ng pRL-CMV (RL=Renilla luciferase from Promega, Madison, WI, USA), 10 ng pCMV-human ER or pCMV-rat ERß, graciously provided by Drs B S Katzenellenbogen and J-A Gustafsson respectively. The expression plasmid for NCoR was provided by Dr M Bagchi (Zhang et al. 1998). The expression plasmid for SMRT was provided by Dr B G Rowan (Coleman et al. 2003). The expression plasmids for SRC-1 and SRA were provided by Dr B W O’Malley (Lanz et al. 1999). The expression plasmid for CBP was provided by Dr M Hadzopoulou-Cladaras (Dell & Hadzopoulou-Cladaras 1999). The expression plasmids for GRIP1, ACTR, CARM1, and PRMT1 were provided by Dr M Stallcup (Koh et al. 2001). The amounts of each of the coregulator plasmids used in transfection are indicated in the Figures and their legends. In experiments comparing the activity of a single coactivator with that achieved by co-transfecting two coactivators, coactivators were run alone or in combination within the same transfection to obviate differences between transient transfections.
The transient transfections were performed using Transfast (Promega) as previously described (Klinge et al. 1999, 2000, Tyulmenkov et al. 2000). The total amount of DNA transfected was kept constant using pcDNA3 (Invitrogen, Carlsbad, CA, USA) as ‘filler DNA’. Cells were treated, in triplicate, 24 h later with ethanol (EtOH, vehicle), 10 nM E2 (Sigma, St Louis, MO, USA), 100 nM 4-hydroxytamoxifen (4-OHT) (Research Biochemicals International, Natick, MA, USA), or both E2 and 4-OHT. Cells were harvested 30 h later and luciferase and Renilla-luciferase (RL-luc) activities assayed using the Promega dual luciferase reporter assay (Klinge et al. 1997, 1999, Tyulmenkov et al. 2000). All data for transient transfections were normalized by RL-luc to account for transfection efficiency.
Statistical analyses
Statistical analyses were performed on fold-induction data values from multiple experiments (minimally three) in which each treatment was run in triplicate using either two-tailed Student’s t-test or one-way ANOVA followed by Dunn’s multiple comparison or Dunnett’s post-hoc test using GraphPad Prism (GraphPad Software, Inc., San Diego, CA, USA).
CHO-K1 cells were transfected with expression vectors for coactivators, corepressors, ER, ERß, and reporter vectors as described above. Whole cell extracts were prepared as described previously (Klinge et al., 2000). Protein concentrations were determined by BioRad’s DCC (Lowry) assay (BioRad, Hercules, CA, USA) using BSA as a standard. Equal amounts of protein (75 µg/lane) were separated by electrophoresis on 10% SDS-PAGE gels. Proteins were transferred to polyvinylidene difluoride membranes (Pall Corporation, Ann Arbor, MI, USA). The blots were probed with primary antibodies: SRC-1 (GeneTex, San Antonio, TX, USA); GRIP1 and ACTR (both from US Biological, Swampscott, MA, USA); ß-actin (Amersham, Arlington Heights, IL, USA); glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Research Diagnostics, Inc., Flanders, NJ, USA). Immunoreactive complexes were visualized using SuperSignal West Pico Chemiluminescent substrate from Pierce (Rockford, IL, USA) on Eastman Kodak (Rochester, NY, USA) BioMax ML film. Data were quantitated from scanned films using Un-Scan-It software (Klinge et al. 2001).
GST-RID fusion protein preparation
The plasmids pGEX-2-TK-SRC-1 (219–399), pGEX-2TK-TIF2 (623–986), and pGEX-2TK-AIB1 (522–82) were graciously provided by Drs M Muylan and R Hilf of the University of Rochester (Yi et al. 2002). The plasmid pGEX-4T-1-NCoR was graciously provided by Dr A Hollenberg of Harvard University (Cohen et al. 2000). BL21 E. coli were used for expression of the GST-RID fusion proteins which were purified by GSH-Sepharose (Amersham Biosciences, Piscataway, NJ, USA) affinity chromatography as described previously (Klinge et al. 1997).
EMSA
Protein-DNA binding was measured by EMSA as previously reported (Klinge et al. 2001). Identical molar amounts of baculovirus-expressed human ER or ERß1, based on hydroxyapatite (HAP) assay results, were incubated with 150–500 fmol 32P-labeled 50 bp oligomers EREc38, EREc13, pS2, PR1148, and Fos-1211. Binding reactions included 40 mM Tris–HCl (pH 7.5), 10% glycerol, 0.75 µg/µl BSA, and 0.02 µg/µl poly d(I–C) (Midland Certified Reagents, Midland, TX, USA), 111 mM KCl, 1 mM EDTA, 1 mM dithiothreitol, and 0.5 mM phenylmethylsulfonyl fluoride. ER-and ERß-specific antibodies (G-20 and Y19 from Santa Cruz Biotechnology, Santa Cruz, CA, USA) were included in selected reactions. Dried EMSA gels were analyzed using a Packard Instruments InstantImager (Meridian, CT, USA) and associated software, Packard Imager for Windows v2.04 (Tyulmenkov & Klinge 2001b).
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Results |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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- and ERß-mediated transcriptional activity
Transient cotransfection assays were performed in CHO-K1 cells to determine how the nucleotide sequence of the ERE affects the enhancer activity of ER and ERß. This cell line was selected because it requires exogenous ER or ERß to activate ERE-driven reporter gene expression (McInerney et al. 1996) and thus allows evaluation of the transcriptional response of each ER subtype in isolation with each ERE. We selected five different ERE-driven luciferase reporter constructs to examine possible differences in transactivation potential between ER and ERß based on DNA sequence (see Materials and methods for sequences). EREc38 is a consensus ERE that includes the commonly used Xenopus vit A2 ERE palindrome (Klinge et al. 1992). EREc13 is the minimal consensus 13 bp palindromic ERE (Klein-Hitpass et al. 1988). The natural, non-consensus, imperfectly palindromic EREs were taken from the human pS2, c-fos, and PR genes (Kulakosky et al. 2002). The cells were treated with EtOH vehicle control, 10 nM E2, 100 nM 4-OHT, or both, and luciferase and RL-luc activities were assayed (Fig. 1
). For each of the four EREs tested, E2 increased luciferase expression above the basal level for both ER and ERß. Luciferase activity detected in the presence of 4-OHT was either at or below basal expression. Co-treatment with E2 and 4-OHT inhibited E2-induced reporter activity, indicating that the E2-induced activation is mediated specifically by ER and ERß. ER showed a greater E2-induced fold-induction of reporter gene activity with the EREc38, EREc13, and pS2 EREs compared with ERß. However, the fold-induction of luciferase by ER and ERß was similar on the Fos-1211 and PR1148 EREs. These results indicated that the ERE sequence differentially impacts the E2-dependent transcriptional activities of ER and ERß.
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We have previously reported that the ERE nucleotide sequence altered ER conformation as assessed by protease digestion studies (Klinge et al. 2001, Tyulmenkov & Klinge 2001a). Here we tested the hypothesis that the observed ERE sequence-induced alterations in ER conformation may alter ER-coregulator interactions in vivo. This is the first study to directly compare coregulator interactions with ER and ERß in response to single copy EREs in the context of their natural flanking sequence in a functional assay. Further, although coactivators have been shown to impact ER and ERß activity in transfected cell lines using different ERE reporters, most prominently multiple tandem copies of the vit A2 ERE, few investigators have separated the effect of coregulators on the basal activity of ER on the reporter gene assayed versus that stimulated by E2 (reviewed in Klinge 2003).
First, we examined the effect of coregulators on basal, ligand-independent ER activity by measuring luciferase expression from each ERE (Fig. 2). CHO-K1 contains endogenous SRC-1, GRIP1, ACTR, CBP, and SMRT (Western data not shown). To account for endogenous expression of coregulators, each ER-ERE combination tested was treated with ethanol in the absence of co-transfected coregulators. This control level of reporter activity was set to 1. SRC-1 and SRA increased basal ER activity in a concentration-dependent manner from EREc38 (Fig. 2A). CBP significantly stimulated basal activity only at 100 ng. PRMT1, CARM1, and NCoR stimulated basal ER transcriptional activity from EREc38 whereas SMRT repressed the basal ER activity in a concentration-dependent manner (Fig. 2B). Noteworthy is the fact that none of the coregulators affected the basal activity of ER or ERß on the parental pGL3-pro-luciferase vector without EREs (Fig. 2C and data not shown). None of the coregulators with the exception of CBP had any effect on either firefly luciferase activity from pGL3–1 EREc38 or the other reporter vectors or RL-luc activity. Since CBP did not stimulate luciferase activity from the pGL3-pro plasmid (Fig. 2C), despite the presence of one cyclic AMP response element binding protein (CREB) binding site in the luciferase gene and another in the ampicillain resistance gene (Promega; personal communication), the stimulation of luciferase activity from pGL3–1 EREc38 by CBP must be mediated by the inclusion of EREc38 which contains a cyclic AMP response element (CRE). Nonetheless, because our data are normalized for CBP stimulation on the ERE reporters in the absence of ER, the stimulation of ligand-independent ER basal activity by SRC-1, SRA, CBP, PRMT1, CARM1, and NCoR (Fig. 2A and B) is mediated by ERE-ER interaction. Based on the work of other investigators (Webb et al. 2003), we suggest that these effects of coactivators on unliganded ER may be mediated through the N-terminal activation domain (AF-1) which is ligand independent.
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and ERß with a fixed amount of each coregulator on EREc38 in transfected CHO-K1 cells (Fig. 2D
). SRC-1, SRA, PRMT1, CARM1, and NCoR stimulated the basal activity of ER and ERß. GRIP1 stimulated unliganded ERß but not ER and conversely CBP stimulated the activity of unliganded ER but not ERß. SMRT repressed the basal activity of both unliganded ER and ERß.
To address the role of the ERE nucleotide sequence in the effect of coregulators on ligand-independent ER and ERß, cells were transfected with EREc13, pS2, PR1148, and Fos-1211 EREs and a fixed amount of each coregulator (Fig. 2E–H). Each ERE showed both ER subtype- and coactivator-specific differences in transcriptional response. The most striking ligand-independent stimulation of ER and ERß activity was with SRA and SRC-1 on Fos-1211. ACTR did not stimulate either ER or ERß on any of the EREs. ER and ERß showed different responses to NCoR on EREc13 and pS2 and to PRMT1 and CARM1 on pS2.
To address the role of E2 in mediating functional interaction of ER and ERß with coregulators, in all subsequent experiments cells were transfected with each coregulator and treated with EtOH and 10 nM E2; the luciferase activity detected with E2 was normalized by that for EtOH. Thus, any effect of a coregulator on basal, ligand-independent ER transcription was excluded from subsequent analysis. This allows comparison of the effect of a coregulator on ligand-activated ER transcription independent of effect of that coregulator on ligand-independent (basal) transcription from the reporter.
ERE sequence impacts coactivator-mediated transactivation by E2-ER and E2-ERß
Next, we compared how different ERE sequences affected the ability of coregulators to affect E2-stimulated activity with ER or ERß. Figure 3A shows that of the tested coactivators, only SRC-1 stimulated E2-induced ER activity on the vit A2 ERE (EREc38). Singly, none of the coactivators stimulated ERß activity. The combination of CBP and SRC-1 synergistically activated E2-induced ERß but not ER activity. In contrast, both CARM1 and PRMT1 inhibited ER- and ERß-induced transcriptional activity (Fig. 3B). This result contrasts to the stimulation of basal activity of ER and ERß by CARM1 and PRMT1. Cotransfection of SRC-1 relieved PRMT1-mediated repression of ER, but not ERß activity. This result agrees with the ER-selective stimulation by SRC-1 shown in Fig. 3A. ACTR relieved PRMT1-mediated repression of both ER and ERß. GRIP1 did not relieve the PRMT1-mediated repression of either ER and ERß.
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and ERß with comparable affinity (Kuiper et al. 1997). However, 4-OHT only exhibits agonist activity with ER at EREs in certain cell types (Weatherman & Scanlan 2001). Our experiments show that 4-OHT had no agonist activity with either ER or ERß and inhibited E2-induced transcription by both ER and ERß in CHO-K1 cells (Fig. 3C). The corepressor SMRT decreased 4-OHT activity with ERß. In contrast, the corepressor NCoR further decreased 4-OHT-ER but not ERß activity. These results indicate that 4-OHT-occupied ER and ERß differ in their interactions with corepressors SMRT and NCoR. Our data are in agreement with phage display experiments showing differences in the conformation of 4-OHT-occupied ER and ERß in vitro (Paige et al. 1999).
Recently, the corepressors NCoR and SMRT were shown to interact directly with ACTR and facilitate thyroid receptor-ACTR binding in vitro and in a mammalian two-hybrid assay (Li et al. 2002). We tested the hypothesis that addition of SMRT or NCoR to cells transfected with ER or ERß and treated with E2 would result in increased reporter gene expression. NCoR increased E2-induced activity by ER only with the addition of 100 ng. In contrast, ERß activity was increased with 100 ng and higher amounts of NCoR (Fig. 3D). SMRT decreased E2-induced activity by ER or ERß in a concentration-dependent manner that appeared to saturate for ERß at 100 ng (Fig. 3E). These data indicated that under the assay conditions used NCoR acts as a coactivator whereas SMRT acts as a corepressor of ER and ERß.
EREc13, the minimal consensus ERE (Klein-Hitpass et al. 1988), binds ER and ERß with significantly lower affinity than ERE palindromes containing 15 bp or more, i.e. Kd 1.1 nM and 1.7 nM versus 0.11 nM and 0.13 nM for ER and ERß respectively (Table 1 and (Kulakosky et al. 2002)). GRIP1, ACTR, and SRA increased E2-ER activity on EREc13 (Fig. 4). In contrast, none of the coactivators stimulated E2-ERß activity. These results differ from stimulation of unliganded ERß by SRA (Fig. 2E). However, the combination of SRC-1 and CBP stimulated E2-induced ERß activity. NCoR stimulated E2-induced ERß but not ER activity. These data indicated that ER and ERß show functional differences in coactivator interaction on a minimal ERE reporter in E2-treated cells, a result concordant with differential interaction of small peptides mimicking coregulator interaction sequences with ER and ERß in vitro (Hall et al. 2000). Furthermore, comparison of the coactivator stimulation of ER and ERß on EREc38 versus EREc13 (Table 1) indicates that the length of the ERE palindrome differentially impacts ER and ERß interaction with coregulators, a result predicated by work using phage display to examine conformational differences between ER and ERß bound to different EREs (Hall et al. 2002).
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and ERß bound to natural, non-palindromic EREs from the human pS2, PR, and c-fos genes (Fig. 5). Notably, certain coactivators stimulated E2-ER transcription in an ER subtype- and ERE sequence-dependent manner. E2-induced ER activity was enhanced 30% by ACTR on pS2, but not on Fos-1211 or PR1148. E2-induced ERß activity on pS2 was enhanced by SRC-1, GRIP1, SRA, CBP, CARM1, and PRMT1 in the presence of GRIP1. In contrast, only CBP stimulated E2-induced ERß activity on PR1148. E2-induced ER activity on PR1148 was inhibited by cotransfection with SRA. ERß activity on PR1148 was reduced by CARM1 and PRMT1. PRMT1 was reported to have stronger coactivator activity when co-transfected with p160 coactivators compared with CARM1 (Koh et al. 2001). The inhibitory effect of PRMT1 on E2-induced ERß activity on PR1148 was relieved by cotransfection with ACTR, but not by SRC-1 or GRIP1.
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or ERß activity on Fos-1211 (Fig. 5C). This is in contrast to the high stimulation of unliganded ER or ERß activity on Fos-1211 (Fig. 2H). Only GRIP1 and SRA enhanced E2-induced ER and ERß activity respectively (Fig. 5C). SRC-1 inhibited E2-ER activity. CBP inhibited E2-induced ER and ERß activity. The lack of effect of coactivators on E2-induced reporter activity from the non-palindromic Fos-1211 ERE may be the result of the reduced ER binding affinity (Table 1) which would theoretically decrease ER-ERE occupancy (‘on time’) and therefore reduce assembly of the coactivator complex.
We also tested the effect of addition of NCoR or SMRT on E2-induced reporter activity from the non-palindromic EREs (Fig. 5D). As with the data on coactivators, NCoR and SMRT differed in their effect on transcription in an ER subtype- and ERE sequence-dependent manner. NCoR decreased E2-ER activity from pS2 and Fos-1211, but not PR1148. NCoR also decreased E2-ERß activity from Fos-1211. NCoR enhanced E2-ERß activity with PR1148. SMRT inhibited E2-induced ER activity from PR1148 and ERß activity from Fos-1211 and PR1148. We concluded that SMRT and NCoR differentially impact E2-induced transcriptional activity in an ER subtype- and ERE sequence-dependent manner.
A possible explanation for the differences detected between the effects of the coactivators on ER or ERß transcriptional activity is altered expression of the coactivators under different experimental conditions. To determine whether the expression of the coregulators was equal in transfected cells, Western blots were performed (Fig. 6). Densitometric analysis of the SRC-1, GRIP1, or ACTR/ß-actin ratio in repeated transient transfection experiments revealed no statistical differences in the amounts of coactivator expressed in cells transfected with either ER or ERß and that there was no effect of ERE sequence on the expression of transfected SRC-1, GRIP1, or ACTR in CHO-K1 cells. This obviates differences in coregulator expression as an explanation for the observed differences in transcriptional activity seen with the different ERE reporters and between ER or ERß.
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or ERß transcriptional activity with different EREs is that the if endogenous levels of a particular coregulator are already high, our experiments may not pick up any further effect of transfection of additional coregulator. Evidence that the effect of ER-coregulator interactions is not already saturated can be seen in Fig. 2 for SRC-1, SRA, CBP, CARM1, PRMT1, NCoR, and SMRT which stimulated unliganded ER and in some cases ERß transcriptional activity. Further, GRIP1 stimulated unliganded ER and ERß on PR1148 (Fig. 2G) and ACTR stimulated ER activity on EREc13 (Fig. 4) and pS2 (Fig. 5A). Thus, each of the coregulators studied stimulated ER and ERß activity on at least one transfected ERE reporter. If the response was already saturated for a particular coregulator, then no added stimulation would be seen under any circumstance.
Synergism of combinations of coactivators with ER and ERß varies with ERE sequence
Coactivators including CBP, SRA, CARM1, and PRMT1 are recruited to target gene promoters through their interaction with p160 family coactivators interacting directly with ER and other nuclear receptors (NR) (Teyssier et al. 2002). To test whether SRA synergized with p160 coactivators to stimulate E2-dependent transcription in an ERE sequence-dependent manner, CHO-K1 cells were transfected with either ER or ERß, plus SRA, alone or in combination with SRC-1, GRIP1, or ACTR with different ERE reporters (Fig. 7). SRA showed no synergism with ACTR (compare with SRA and ACTR alone in Figs 3A, 4, and 5). GRIP1 synergized with SRA for ER only on the Fos-1211 ERE (compare with GRIP1 and SRA alone in Fig. 5C). In contrast, SRA synergized with GRIP1 for ERß on EREc38, EREc13, and Fos-1211 (compare with data in Figs 3A, 4, and 5C). Lastly, SRA synergized with SRC-1 for ERß on EREc13 (compare with SRA and SRC-1 alone in Fig. 4).
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Since coregulators showed differential effects on ER and ERß transcriptional activity in an ERE sequence-dependent manner in transfected cells, we tested whether the RIDs of selected p160 coactivators showed ERE sequence-dependent interaction with ER and ERß in EMSAs. GST-fusion proteins containing the RIDs of SRC-1, GRIP1, ACTR, or NCoR were incubated with E2-occupied ER or ERß and different EREs in vitro. The sequence of the GST-RIDs and their location within the coregulator is shown in Table 2. As a negative control, each GST-RID was incubated with each ERE in the absence of ER. None of the GST-RIDs interacted with the EREs (Fig. 8 and data not shown). Figure 8 shows representative EMSAs for ER and ERß binding to the pS2 ERE with or without added GST-ACTR or GST-GRIP1.
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and ERß bound to pS2-ERE (Fig. 8) and EREc38 (Fig. 9A and B). In contrast, only the migration of ER-EREc13 was slowed by GST-ACTR (Fig. 9C and D). The amount of ‘supershifted’, i.e. complex with slowed migration, ACTR-ER-pS2 complex was greater than for ERß (Fig. 8C and D). The total amount of ER-bound EREc13 pS2, and EREc38 complexes was increased with the addition of GST-ACTR. For ERß, GST-ACTR increased the total amount of ERß-bound pS2 complex, but not ERß-bound EREc38 or EREc13 complexes. This indicated that GST-ACTR shows preferential interaction with ER rather than ERß bound to EREc13 and EREc38. Despite a previous report that the GST-SRC-1 and -GRIP1 constructs used here interacted stably with ER and ERß in EMSA (Yi et al. 2002), neither formed a complex with ER or ERß for any of the tested EREs and had no significant effect on ER-ERE binding (Figs 8C and 9). We concluded that the GRIP1 and SRC-1 RIDs used in the EMSAs do not stably bind ER under our experimental conditions. None of the GST-RIDs supershifted the ER- or ERß-Fos-1211 or PR1148 complexes (Table 3 and data not shown). Lastly, GST-NCoR RID showed no visible effect on the migration of the ERß-EREc38 complex (data not shown) and no effect on ERß-EREc13 binding (Fig. 9D).
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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and ERß conformation in vitro. Here we present the first cell-based assay, as a mimic of in vivo conditions, to address how differences in ERE sequence impact the functional interaction of ER and ERß with coactivator and corepressor proteins.
To separate the effects of ERE sequence on ER-coactivator interaction from the interaction of that coactivator with other transcription factors in a complex promoter, we used a simple model system in which each ERE, plus a relatively short stretch (6–20 bp) of nucleotides identical to those flanking that ERE in the natural human genes for pS2, c-Fos, and PR, was inserted in the same location in the multicloning region of a luciferase reporter gene. This obviates difficulties in data interpretation due to different distances between the ERE and transcription start site (Sathya et al. 1997, Nordeen et al. 1998) and focuses our study on the role of the ERE along with its natural flanking sequence in ER-coregulator interaction. Importantly, we evaluated ER-coregulator activity at a single copy of each ERE rather than multiple tandem copies of the vit A2 ERE. By using CHO-K1 cells that do not express ER or ERß (McInerney et al. 1996), we separated effects of coactivators and EREs on ER versus ERß. The importance of our study is that it is the first to examine ERß-ERE interactions in a functional assay in direct comparison with ER activity on the same single copy EREs in the context of their natural flanking sequence.
Our experiments have demonstrated that the sequence of the ERE impacts the functional interaction of ER and ERß with coactivators SRC-1, GRIP1, ACTR, CBP, and SRA, the coregulators CARM1 and PRMT1, and corepressors SMRT and NCoR in transiently transfected CHO-K1 cells. Notably, unliganded ER interacts with coregulators in an ERE sequence-dependent manner. Similar findings for CBP and SRC-1 were recently reported by others (Dutertre & Smith 2003). A brief summary model highlighting some of the ERE-specific differences between unliganded and E2-occupied ER interaction with coregulators detected in our experiments is presented in Fig. 10. Although the affinity of ER or ERß binding to an ERE correlates with E2-induced transcriptional activity (Klinge 2001), there is not a correlation between ERE occupancy (Kd) and the transcriptional response to the tested coactivators, coregulators, and corepressors in transfected CHO-K1 cells. Thus, the effects of coregulatory proteins on E2-activated transcription is not governed solely by the affinity of ER-ERE interaction, but we suggest reflects ERE-mediated alterations in ER conformation (Klinge et al. 2001, Ramsey & Klinge 2001, Yi et al. 2002) that we propose impact ER-coactivator interaction. Hence, although we are unable to compose a simple rule or set of rules that will predict how an ERE sequence impacts ER-coactivator interaction in transfected cells, our data clearly demonstrated that variations within the core ERE sequence regulate coactivator interaction with liganded ER. An important difference between our study and most previous studies (reviewed in Klinge 2003) is that we subtracted the effect of each coregulator on the ligand-independent (basal) activity of ER or ERß for each ERE. Thus, the findings in Table 1 summarize how coregulators impact E2-dependent ER and ERß transcriptional activity from different EREs. We suggest that small changes with individual coactivators may be physiologically significant when combined with the other coregulators in the cell. For example, a 50–100% increase in transcription of the transcription factors Fos or PR could effectively double the amount of Fos and PR proteins. Doubling PR expression subsequently could have a synergistic effect on downstream gene targets.
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and ERß. Of the EREs examined in this study, EREc38 has the longest (17 bp) perfectly palindromic ERE, binds ER and ERß with highest affinity (Table 1), gives the highest E2-induced transcription (Fig. 1), and shows the least effect of coactivators on E2-induced ER or ERß activity. SRC-1 was the only coactivator that stimulated E2-induced ER but not ERß transcription from EREc38. In contrast, SRC-1 had no effect on E2-ER activity on EREc13. Hence, the length of the ERE palindrome and the presence of the conserved 5' AT-rich region found in the vit A2 gene appear to enhance ER-SRC-1 functional interaction in this transient transfection assay.
The pS2 ERE showed the most ligand-dependent ER responsiveness to the coactivators tested. Based on a report showing low interaction of ER-pS2 with SRC-1 and ACTR-RID peptides in vitro (Hall et al. 2002), we predicted that SRC-1 and ACTR would have minimal impact on E2-ER activity on the pS2-ERE reporter. Indeed, SRC-1 enhanced E2-ERß, but not ER transcription from the pS2-ERE. On the other hand, ACTR stimulated E2-ER, but not ERß transcription from the pS2 ERE, despite the interaction of GST-ACTR with both ER and ERß bound to the pS2 ERE in vitro (Fig. 8). This difference from the aforementioned predication (Hall et al. 2002) suggests that amino acids in addition to the RID in ACTR impact transactivation by ER. This suggestion agrees with data showing that the three NR boxes from SRC-1, GRIP1, and ACTR have different affinities for E2-occupied ER and ERß in vitro (Wong et al. 2001). Our cotransfection data are also in agreement with the observation that less GRIP1 was retained by ER bound to the pS2 ERE versus the vit A2 ERE in vitro (Wood et al. 2001).
As anticipated from the discussion above, data from our EMSA experiments using GST-RIDs did not fully correlate with results from the transient transfection assays. These divergent results are most likely due to the use of a single RID domain in the EMSA experiments and the use of full-length proteins in the transient transfection assays. Our EMSA data are in agreement with time-resolved fluorescence resonance energy transfer (FRET) experiments showing no interaction between ER and the first RID of SRC-1 and minimal recruitment to ERß in vitro (Bramlett et al. 2001). Similarly, our EMSAs and the FRET studies (Bramlett et al. 2001) indicate that the first RID of ACTR interacts with both ER and ERß. Further experiments are necessary to determine how full-length coactivators influence ER-ERE binding affinity.
Results from scintillation proximity and mammalian two-hybrid assays showed that the ligand binding domain (LBD) of ERß interacts with the RIDs of p160 coactivators in the following order of affinity (high–low): GRIP1>ACTR>SRC-1>>>p300/CBP (Northrop et al. 2000). These data suggest that E2-ERß transcription would be expected to be stimulated most by GRIP1, less by ACTR and SRC-1, and the least with CBP. On the other hand, intact ERß interacted with comparable affinity with the immobilized NR boxes of SRC-1, GRIP1, and ACTR in vitro (Wong et al. 2001). Hence, the length of the ERß used influences coactivator interaction in vitro. In contrast to these in vitro results, we observed that GRIP1 stimulated E2-ERß transcription only from the pS2 ERE. One possible explanation of these results is that GRIP1 requires a cellular cofactor(s) for the other EREs that is not expressed in CHO-K1 cells. However, GRIP1 did not enhance E2-ERß activity on either EREc38 or pS2-ERE in HEC-1A human endome-trial cancer cells (C M Klinge, unpublished data), indicating that CHO-K1 are not unique in this response. Cells that express endogenous ERß should be tested to further evaluate the impact of GRIP1 on ERß activity.
ER interacts functionally with p300 and CBP (Shibata et al. 1997) which are ‘cointegrators’ because they form complexes with TBP and a variety of activator proteins (McKenna et al. 1999). CBP stimulated unliganded ER and ERß transcription at all EREs except PR1148. The finding that CBP enhanced E2-ER transcription only from the PR1148 ERE is consistent with experiments showing that CBP shows weaker interaction with the ER LBD than SRC-1 in vitro (Heery et al. 2001). In contrast to our ER findings, CBP enhanced E2-ERß transcription from pS2 and PR1148 EREs (Table 1). Ours is the first examination of the impact of CBP on ligand-dependent ERß-induced transcription. We conclude that CBP interacts differently with ER and ERß and that the ERE sequence impacts this interaction.
SRA is a unique ER coactivator because it functions as an RNA molecule in vivo and it enhances ER transactivation through AF-1 (Lanz et al. 1999). Ours is the first study to compare the activity of SRA with ER and ERß on natural EREs. Although a recent study reported that SRA enhanced the activities of E2-ER and -ERß on a two-tandem vit A2 ERE-luciferase reporter in COS-1 or HEK-293 cells without affecting basal activity (Deblois & Giguere 2003), we observed that SRA stimulated basal, unliganded ER and ERß activity on all EREs with the exception of ERß on the pS2 ERE. SRA showed the greatest activity with unliganded ER and ERß on the Fos-1211 ERE. This finding warrants further investigation in future studies. Data showing that SRA interacts with p72 and p68 coactivators, which bind GRIP1 but do not interact with or activate ERß (Watanabe et al. 2001), imply that SRA may not stimulate ERß transcription. Our data are in agreement with this prediction: SRA did not enhance E2-ERß transcription with the exception of the pS2 ERE, indicating that the ERE sequence impacts SRA-ERß interaction. Since SRA forms a complex with SRC-1 (Lanz et al. 1999), we evaluated the combined effect of the p160 coactivators and SRA on E2-induced ER and ERß activity on different EREs. Our results indicated that both ER subtype and DNA sequence impact SRA activity.
CARM1 and PRMT1 interact with p160 family coactivators and stimulate NR transcription (Chen et al. 2000, Koh et al. 2001). PRMT1 stimulated unliganded ER and ERß activity on all EREs. CARM1 differed from PRMT1 in that it did not stimulate unliganded ER and ERß on the PR1148 and Fos-1211 EREs. The only E2-dependent stimulatory activity of CARM1 was with E2-ERß on the pS2 ERE. It will be important to determine if the ERE sequence impacts the methyltransferase activity of PRMT1 and CARM1.
In GST pull-down assays, corepressors NCoR and SMRT interact with ER in a ligand-independent manner (Smith et al. 1997, Lavinsky et al. 1998). Chromatin immunoprecipitation experiments revealed that NCoR interacts with ER in the promoters of the c-Myc and cathepsin D genes in MCF-7 breast cancer cells treated with tamoxifen or raloxifene (Shang & Brown 2002), indicating that these corepressors play a role in transcriptional silencing. Here we noted that NCoR increased unliganded ER and ERß activity from all but the pS2 and PR1148 EREs. Differences between native chromatin and partial chromatin structure on transfected reporter constructs may be responsible for this difference. The mechanism of stimulation of ER basal activity by NCoR is unclear; however, NCoR and SMRT interacted directly with ACTR, SRC-1, and GRIP1 in vitro and in transfected cells (Li et al. 2002). Further, NCoR enhanced unliganded TRß-ACTR interaction (Li et al. 2002). Whether NCoR facilitates ER-endogenous ACTR interaction in the absence of ligand is unknown. Further studies are needed to address the role of NCoR in fine-tuning transcriptional repression versus activation by ER and ERß.
In mammalian two-hybrid assays, NCoR had no effect on E2-activated ER or ERß reporter expression (Zhang et al. 2000). In contrast, NCoR inhibited E2-induced ER transcription only from the pS2 and Fos-1211 EREs in our cell-based assays. Therefore, the ERE sequence impacts NCoR-mediated transcriptional repression from a transfected plasmid. SMRT reduced basal and 4-OHT-stimulated reporter gene activity in HepG2 cells transfected with ER, but had no effect on E2-activated reporter activity (Smith et al. 1997). Similarly, we found that SMRT inhibited basal transcription by ER or ERß, but SMRT inhibited E2-induced transcription for both ER and ERß, although the effect was more pronounced for ER than ERß. Notably, NCoR enhanced 4-OHT inhibition of ER activity, but not ERß activity whereas SMRT increased 4-OHT inhibition of ERß, not ER.
In summary, the data presented here have demonstrated for the first time that the nucleotide sequence of the ERE and its immediate flanking sequences from the native gene differentially impact ER and ERß transcription and the functional interaction of coregulatory proteins in transfected cells. In agreement with previous investigators we conclude that the ERE nucleotide sequence impacts physical (Wood et al. 1998, 2001, Loven et al. 2001a, Hall et al. 2002) and functional interaction of ER and ERß with coregulators. These data are in agreement with our previous studies and those of other investigators showing that DNA is an allosteric modulator of ER action (reviewed in Klinge 2003). This allows gene-specific recruitment of coregulators to ER and ERß which, along with cell-specific ratios of coregulator expression, results in tissue-specific gene transcriptional responses to E2 in vivo.
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References |
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Bramlett KS, Wu Y & Burris TP 2001 Ligands specify coactivator nuclear receptor (NR) box affinity for estrogen receptor subtypes. Molecular Endocrinology 15 909–922.
Chen D, Huang SM & Stallcup MR 2000 Synergistic, p160 coactivator-dependent enhancement of estrogen receptor function by CARM1 and p300. Journal of Biological Chemistry 275 40810–40816.
Cohen RN, Putney A, Wondisford FE & Hollenberg AN 2000 The nuclear corepressors recognize distinct nuclear receptor complexes. Molecular Endocrinology 14 900–914.
Coleman KM, Dutertre M, El-Gharbawy A, Rowan BG, Weigel NL & Smith CL 2003 Mechanistic differences in the activation of estrogen receptor-alpha (ERalpha )- and ERbeta-dependent gene expression by cAMP signaling pathway(s). Journal of Biological Chemistry 278 12834–12845.
Couse JF & Korach KS 1999 Estrogen receptor null mice: what have we learned and where will they lead us? Endocrine Reviews 20 358–417.
Deblois G & Giguere V 2003 Ligand-independent coactivation of ERalpha AF-1 by steroid receptor RNA activator (SRA) via MAPK activation. Journal of Steroid Biochemistry and Molecular Biology 85 123–131.[CrossRef][Web of Science][Medline]
Delage-Mourroux R, Martini PG, Choi I, Kraichely DM, Hoeksema J & Katzenellenbogen BS 2000 Analysis of estrogen receptor interaction with a repressor of estrogen receptor activity (REA) and the regulation of estrogen receptor transcriptional activity by REA. Journal of Biological Chemistry 275 35848–35856.
Dell H & Hadzopoulou-Cladaras M 1999 CREB-binding protein is a transcriptional coactivator for hepatocyte nuclear factor-4 and enhances apolipoprotein gene expression. Journal of Biological Chemistry 274 9013–9021.
Driscoll MD, Sathya G, Muyan M, Klinge CM, Hilf R & Bambara RA 1998 Sequence requirements for estrogen receptor binding to estrogen response elements. Journal of Biological Chemistry 273 29321–29330.
Dutertre M & Smith CL 2003 Ligand-independent interactions of p160/steroid receptor coactivators and CREB-binding protein (CBP) with estrogen receptor-: regulation by phosphorylation sites in the A/B region depends on other receptor domains. Molecular Endocrinology 17 1296–1314.
Hager GL, Smith CL, Fragoso G, Wolford R, Walker D, Barsony J & Htun H 1998 Intranuclear trafficking and gene targeting by members of the steroid/nuclear receptor superfamily. Journal of Steroid Biochemistry and Molecular Biology 65 125–132.[CrossRef][Web of Science][Medline]
Hall JM, Chang CY & McDonnell DP 2000 Development of peptide antagonists that target estrogen receptor beta-coactivator interactions. Molecular Endocrinology 14 2010–2023.
Hall JM, McDonnell DP & Korach KS 2002 Allosteric regulation of estrogen receptor structure, function, and coactivator recruitment by different estrogen response elements. Molecular Endocrinology 16 469–486.
Hebbar PB & Archer TK 2003 Chromatin remodeling by nuclear receptors. Chromosoma 111 495–504.[Web of Science][Medline]
Heery DM, Hoare S, Hussain S, Parker MG & Sheppard H 2001 Core LXXLL motif sequences in CREB-binding protein, SRC1, and RIP140 define affinity and selectivity for steroid and retinoid receptors. Journal of Biological Chemistry 276 6695–6702.
Jiang W, Nordeen SK & Kadonaga JT 2000 Transcriptional analysis of chromatin assembled with purified ACF and dNAP1 reveals that acetyl-CoA is required for preinitiation complex assembly. Journal of Biological Chemistry 275 39819–39822.
Klein-Hitpass L, Ryffel GU, Heitlinger E & Cato AC 1988 A 13 bp palindrome is a functional estrogen responsive element and interacts specifically with estrogen receptor. Nucleic Acids Research 16 647–663.
Klinge CM 1999 Estrogen receptor binding to estrogen response elements slows ligand dissociation and synergistically activates reporter gene expression. Molecular and Cellular Endocrinology 150 99–111.[CrossRef][Web of Science][Medline]
Klinge CM 2000 Estrogen receptor interaction with co-activators and co-repressors. Steroids 65 227–251.[CrossRef][Web of Science][Medline]
Klinge CM 2001 Estrogen receptor interaction with estrogen response elements. Nucleic Acids Research 29 2905–2919.
Klinge CM 2003 Estrogen response element sequence as an allosteric regulator of estrogen receptor action. CHEMTRACTS Biochemistry and Molecular Biology 16 1–18.
Klinge CM, Peale FV Jr, Hilf R, Bambara RA & Zain S 1992 Cooperative estrogen receptor interaction with consensus or variant estrogen responsive elements in vitro. Cancer 52 1073–1081.[CrossRef]
Klinge CM, Silver BF, Driscoll MD, Sathya G, Bambara RA & Hilf R 1997 COUP-TF interacts with estrogen receptor, binds to estrogen response elements and half-sites, and modulates estrogen-induced gene expression. Journal of Biological Chemistry 272 31465–31474.
Klinge CM, Bowers JL, Kulakosky PC, Kamboj KK & Swanson HI 1999 The aryl hydrocarbon receptor (AHR)/AHR nuclear translocator (ARNT) heterodimer interacts with naturally occurring estrogen response elements. Molecular and Cellular Endocrinology 157 105–119.[CrossRef][Web of Science][Medline]
Klinge CM, Kaur K & Swanson HI 2000 The aryl hydrocarbon receptor interacts with estrogen receptor alpha and orphan receptors COUP-TFI and ERR1. Archives of Biochemistry and Biophysics 373 163–174.[CrossRef][Web of Science][Medline]
Klinge CM, Jernigan SC, Smith SL, Tyulmenkov VV & Kulakosky PC 2001 Estrogen response element sequence impacts the conformation and transcriptional activity of estrogen receptor . Molecular and Cellular Endocrinology 174 151–166.[CrossRef][Web of Science][Medline]
Koh SS, Chen D, Lee YH & Stallcup MR 2001 Synergistic enhancement of nuclear receptor function by p160 coactivators and two coactivators with protein methyltransferase activities. Journal of Biological Chemistry 276 1089–1098.
Krieg AJ, Krieg SA, Ahn BS & Shapiro DJ 2004 Interplay between estrogen response element sequence and ligands controls in vivo binding of estrogen receptor to regulated genes. Journal of Biological Chemistry 279 5025–5034.
Kuiper GG, Carlsson B, Grandien J, Enmark E, Haggblad J, Nilsson S & Gustafsson J-A 1997 Comparison of the ligand binding specificity and transcript tissue distribution of estrogen receptors and ß. Endocrinology 138 863–870.
Kulakosky PC, Jernigan SC, McCarty MA & Klinge CM 2002 Response element sequence regulates estrogen receptor alpha and beta affinity and activity. Journal of Molecular Endocrinology 29 137–152.[Abstract]
Lanz RB, McKenna NJ, Onate SA, Albrecht U, Wong J, Tsai SY, Tsai MJ & 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][Web of Science][Medline]
Lavinsky R, Jepsen K, Heinzel T, Torchia J, Mullen TM, Schiff R, Del-Rio AL, Ricote M NS, Gemsch J, Hilsenbeck SG et al. 1998 Diverse signaling pathways modulate nuclear receptor recruitment of N-CoR and SMRT complexes. PNAS 95 2920–2925.
Lee SK, Anzick SL, Choi JE, Bubendorf L, Guan XY, Jung YK, Kallioniemi OP, Kononen J, Trent JM, Azorsa D et al. 1999 A nuclear factor, ASC-2, as a cancer-amplified transcriptional coactivator essential for ligand-dependent transactivation by nuclear receptors in vivo. Journal of Biological Chemistry 274 34283–34293.
Lefstin JA & Yamamoto KR 1998 Allosteric effects of DNA on transcriptional regulators. Nature 392 885–888.[CrossRef][Medline]
Li X, Kimbrel EA, Kenan DJ & McDonnell DP 2002 Direct interactions between corepressors and coactivators permit the integration of nuclear receptor-mediated repression and activation. Molecular Endocrinology 16 1482–1491.
Loven MA, Likhite VS, Choi I & Nardulli AM 2001a Estrogen response elements alter coactivator recruitment through allosteric modulation of estrogen receptor beta conformation. Journal of Biological Chemistry 276 45282–45288.
Loven MA, Wood JR & Nardulli AM 2001b Interaction of estrogen receptors alpha and beta with estrogen response elements. Molecular and Cellular Endocrinology 181 151–163.[CrossRef][Web of Science][Medline]
McInerney EM, Tsai M-J, O’Malley BW & Katzenellenbogen BS 1996 Analysis of estrogen receptor transcriptional enhancement by a nuclear receptor coactivator. PNAS 93 10069–10073.
McKenna NJ & O’Malley BW 2002 Minireview: nuclear receptor coactivators – an update. Endocrinology 143 2461–2465.
McKenna NJ, Lanz RB & O’Malley BW 1999 Nuclear receptor coregulators: cellular and molecular biology. Endocrine Reviews 20 321–344.
Mak H, Hoare S, Henttu PMS & Parker MG 1999 Molecular determinants of the estrogen receptor-coactivator interface. Molecular and Cellular Endocrinology 19 3895–3903.
Metivier R, Stark A, Flouriot G, Hubner MR, Brand H, Penot G, Manu D, Denger S, Reid G, Kos M et al. 2002 A dynamic structural model for estrogen receptor-alpha activation by ligands, emphasizing the role of interactions between distant A and E domains. Molecular Cell 10 1019–1032.[CrossRef][Web of Science][Medline]
Nordeen SK, Ogden CA, Taraseviciene L & Lieberman BA 1998 Extreme position dependence of a canonical hormone response element. Molecular Endocrinology 12 891–898.
Norris JD, Fan D, Stallcup MR & McDonnell DP 1998 Enhancement of estrogen receptor transcriptional activity by the coactivator GRIP-1 highlights the role of activation function 2 in determining estrogen receptor pharmacology. Journal of Biological Chemistry 273 6679–6688.
Northrop JP, Nguyen D, Piplani S, Olivan SE, Kwan ST, Go NF, Hart CP & Schatz PJ 2000 Selection of estrogen receptor beta-and thyroid hormone receptor beta-specific coactivator-mimetic peptides using recombinant peptide libraries. Molecular Endocrinology 14 605–622.
Paige LA, Christensen DJ, Gron H, Norris JD, Gottlin EB, Padilla KM, Chang C-Y, Ballas LM, Hamilton PT, McDonnell DP et al. 1999 Estrogen receptor (ER) modulators each induce distinct conformational changes in ER alpha and ER beta. PNAS 96 3999–4004.
Peale FV Jr, Ishibe Y, Klinge CM, Zain S, Hilf R & Bambara RA 1989 Rapid purification of the estrogen receptor by sequence-specific DNA affinity chromatography. Biochemistry 28 8671–8675.[CrossRef][Medline]
Ramsey TL & Klinge CM 2001 Estrogen response element binding induces alterations in estrogen receptor conformation as revealed by susceptibility to partial proteolysis. Journal of Molecular Endocrinology 27 275–292.[Abstract]
Safe S 2001 Transcriptional activation of genes by 17 beta-estradiol through estrogen receptor-Sp1 interactions. Vitamins and Hormones 62 231–252.[Web of Science][Medline]
Sathya G, Wenzhou L, Klinge CM, Anolik JH, Hilf R & Bambara RA 1997 Effects of multiple estrogen responsive elements, their spacing and location on estrogen response of reporter genes. Molecular Endocrinology 11 1994–2003.
Shang Y & Brown M 2002 Molecular determinants for the tissue specificity of SERMs. Science 295 2465–2468.
Shibata H, Spencer E, Onate SA, Jenster G, Tsai SY, Tsai MJ & O’Malley BW 1997 Role of co-activators and co-repressors in the mechanism of steroid/thyroid receptor action. Recent Progress in Hormone Research 52 141–164.
Smith CL, Nawaz Z & O’Malley BW 1997 Coactivator and corepressor regulation of the agonist/antagonist activity of the mixed antiestrogen, 4-hydroxytamoxifen. Molecular Endocrinology 11 657–666.
Sotiriou C, Neo SY, McShane LM, Korn EL, Long PM, Jazaeri A, Martiat P, Fox SB, Harris AL & Liu ET 2003 Breast cancer classification and prognosis based on gene expression profiles from a population-based study. PNAS 100 10393–10398.
Struhl K 1998 Histone acetylation and transcriptional regulatory mechanisms. Genes and Development 12 599–606.
Teyssier C, Chen D & Stallcup MR 2002 Requirement for multiple domains of the protein arginine methyltransferase CARM1 in its transcriptional coactivator function. Journal of Biological Chemistry 277 46066–46072.
Tyulmenkov VT & Klinge CM 2000a Interaction of tetrahydrocrysene ketone with estrogen receptors and ß indicates conformational differences in the receptor subtypes. Archives of Biochemistry and Biophysics 381 135–142.[CrossRef][Web of Science][Medline]
Tyulmenkov VT & Klinge CM 2000b Selectivity of detection of estrogen receptors and ß with cognate antibodies in electrophoretic mobility shift assay. Steroids 65 505–512.[CrossRef][Web of Science][Medline]
Tyulmenkov VV & Klinge CM 2001a Estrogen receptors alpha and beta exhibit different estradiol and estrogen response element binding in the presence of nonspecific DNA. Archives of Biochemistry and Biophysics 390 253–264.[CrossRef][Web of Science][Medline]
Tyulmenkov VV & Klinge CM 2001b A mathematical approach to predict the affinity of estrogen receptors alpha and beta binding to DNA. Molecular and Cellular Endocrinology 182 109–119.[CrossRef][Web of Science][Medline]
Tyulmenkov VT, Jernigan SC & Klinge CM 2000 Comparison of transcriptional synergy of estrogen receptors alpha and beta from multiple tandem estrogen response elements. Molecular and Cellular Endocrinology 165 151–161.[CrossRef][Web of Science][Medline]
Watanabe M, Yanagisawa J, Kitagawa H, Takeyama K, Ogawa S, Arao Y, Suzawa M, Kobayashi Y, Yano T, Yoshikawa H et al. 2001 A subfamily of RNA-binding DEAD-box proteins acts as an estrogen receptor alpha coactivator through the N-terminal activation domain (AF-1) with an RNA coactivator, SRA. EMBO Journal 20 1341–1352.[CrossRef][Web of Science][Medline]
Weatherman RV & Scanlan TS 2001 Unique protein determinants of the subtype-selective ligand responses of the estrogen receptors (ERalpha and ERbeta ) at AP-1 sites. Journal of Biological Chemistry 276 3827–3832.
Webb P, Lopez GN, Uht RM & Kushner PJ 1995 Tamoxifen activation of the estrogen receptor/AP-1 pathway. Molecular Endocrinology 9 443–456.
Webb P, Nguyen P & Kushner PJ 2003 Differential SERM effects on corepressor binding dictate ERalpha activity in vivo. Journal of Biological Chemistry 278 6912–6920.
Wong CW, Komm B & Cheskis BJ 2001 Structure–function evaluation of ER alpha and beta interplay with SRC family coactivators. ER selective ligands. Biochemistry 40 6756–6765.[CrossRef][Medline]
Wood JR, Greene GL & Nardulli AM 1998 Estrogen response elements function as allosteric modulators of estrogen receptor conformation. Molecular and Cellular Biology 18 1927–1934.
Wood JR, Likhite VS, Loven MA & Nardulli AM 2001 Allosteric modulation of estrogen receptor conformation by different estrogen response elements. Molecular Endocrinology 15 1114–1126.
Yi P, Driscoll MD, Huang J, Bhagat S, Hilf R, Bambara RA & Muyan M 2002 The effects of estrogen-responsive element- and ligand-induced structural changes on the recruitment of cofactors and transcriptional responses by ERalpha and ERbeta. Molecular Endocrinology 16 674–693.
Zhang H, Thomsen JS, Johansson L, Gustafsson JA & Treuter E 2000 DAX-1 functions as an LXXLL-containing corepressor for activated estrogen receptors. Journal of Biological Chemistry 275 39855–39859.
Zhang X, Jeyakumar M, Petukhov S & Bagchi MK 1998 A nuclear receptor corepressor modulates transcriptional activity of antagonist-occupied steroid hormone receptor. Molecular Endocrinology 12 513–524.
Received 24 February 2004
Accepted 11 June 2004
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S. Karmakar, E. A. Foster, and C. L. Smith Unique Roles of p160 Coactivators for Regulation of Breast Cancer Cell Proliferation and Estrogen Receptor-{alpha} Transcriptional Activity Endocrinology, April 1, 2009; 150(4): 1588 - 1596. [Abstract] [Full Text] [PDF]
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 M. Kouzu-Fujita, Y. Mezaki, S. Sawatsubashi, T. Matsumoto, I. Yamaoka, T. Yano, Y. Taketani, H. Kitagawa, and S. Kato Coactivation of Estrogen Receptor {beta} by Gonadotropin-Induced Cofactor GIOT-4 Mol. Cell. Biol., January 1, 2009; 29(1): 83 - 92. [Abstract] [Full Text] [PDF]
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 P. Hu, H. K. Kinyamu, L. Wang, J. Martin, T. K. Archer, and C. Teng Estrogen Induces Estrogen-related Receptor {alpha} Gene Expression and Chromatin Structural Changes in Estrogen Receptor (ER)-positive and ER-negative Breast Cancer Cells J. Biol. Chem., March 14, 2008; 283(11): 6752 - 6763. [Abstract] [Full Text] [PDF]
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 K. A. Mattingly, M. M. Ivanova, K. A. Riggs, N. S. Wickramasinghe, M. J. Barch, and C. M. Klinge Estradiol Stimulates Transcription of Nuclear Respiratory Factor-1 and Increases Mitochondrial Biogenesis Mol. Endocrinol., March 1, 2008; 22(3): 609 - 622. [Abstract] [Full Text] [PDF]
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A. D. Adamson, S. Friedrichsen, S. Semprini, C. V. Harper, J. J. Mullins, M. R. H. White, and J. R. E. Davis Human Prolactin Gene Promoter Regulation by Estrogen: Convergence with Tumor Necrosis Factor-{alpha} Signaling Endocrinology, February 1, 2008; 149(2): 687 - 694. [Abstract] [Full Text] [PDF]
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J. Cheng, C. Zhang, and D. J. Shapiro A Functional Serine 118 Phosphorylation Site in Estrogen Receptor-{alpha} Is Required for Down-Regulation of Gene Expression by 17{beta}-Estradiol and 4-Hydroxytamoxifen Endocrinology, October 1, 2007; 148(10): 4634 - 4641. [Abstract] [Full Text] [PDF]
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 J. R. Schultz-Norton, V. A. Gabisi, Y. S. Ziegler, I. X. McLeod, J. R. Yates, and A. M. Nardulli Interaction of estrogen receptor {alpha} with proliferating cell nuclear antigen Nucleic Acids Res., August 1, 2007; 35(15): 5028 - 5038. [Abstract] [Full Text] [PDF]
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N. Levy, X. Zhao, H. Tang, R. B. Jaffe, T. P. Speed, and D. C. Leitman Multiple Transcription Factor Elements Collaborate with Estrogen Receptor {alpha} to Activate an Inducible Estrogen Response Element in the NKG2E Gene Endocrinology, July 1, 2007; 148(7): 3449 - 3458. [Abstract] [Full Text] [PDF]
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 S. M Johnson, M. Maleki-Dizaji, J. A Styles, and I. N H White Ishikawa cells exhibit differential gene expression profiles in response to oestradiol or 4-hydroxytamoxifen Endocr. Relat. Cancer, June 1, 2007; 14(2): 337 - 350. [Abstract] [Full Text] [PDF]
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 Z. Lin, S. Reierstad, C.-C. Huang, and S. E. Bulun Novel Estrogen Receptor-{alpha} Binding Sites and Estradiol Target Genes Identified by Chromatin Immunoprecipitation Cloning in Breast Cancer Cancer Res., May 15, 2007; 67(10): 5017 - 5024. [Abstract] [Full Text] [PDF]
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J. Bao, C. Cao, X. Zhang, F. Jiang, S. V. Nicosia, and W. Bai Suppression of {beta}-Amyloid Precursor Protein Signaling into the Nucleus by Estrogens Mediated through Complex Formation between the Estrogen Receptor and Fe65 Mol. Cell. Biol., February 15, 2007; 27(4): 1321 - 1333. [Abstract] [Full Text] [PDF]
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S. Wang, C. Zhang, S. K. Nordeen, and D. J. Shapiro In Vitro Fluorescence Anisotropy Analysis of the Interaction of Full-length SRC1a with Estrogen Receptors {alpha} and beta Supports an Active Displacement Model for Coregulator Utilization J. Biol. Chem., February 2, 2007; 282(5): 2765 - 2775. [Abstract] [Full Text] [PDF]
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S. Rice and S. A Whitehead Phytoestrogens and breast cancer -promoters or protectors? Endocr. Relat. Cancer, December 1, 2006; 13(4): 995 - 1015. [Abstract] [Full Text] [PDF]
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S. Wagner, S. Weber, M. A. Kleinschmidt, K. Nagata, and U.-M. Bauer SET-mediated Promoter Hypoacetylation Is a Prerequisite for Coactivation of the Estrogen-responsive pS2 Gene by PRMT1 J. Biol. Chem., September 15, 2006; 281(37): 27242 - 27250. [Abstract] [Full Text] [PDF]
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J. R. Schultz-Norton, W. H. McDonald, J. R. Yates, and A. M. Nardulli Protein Disulfide Isomerase Serves as a Molecular Chaperone to Maintain Estrogen Receptor {alpha} Structure and Function Mol. Endocrinol., September 1, 2006; 20(9): 1982 - 1995. [Abstract] [Full Text] [PDF]
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Z. Zhang, K. Chen, J. C. Shih, and C. T. Teng Estrogen-Related Receptors-Stimulated Monoamine Oxidase B Promoter Activity Is Down-Regulated by Estrogen Receptors Mol. Endocrinol., July 1, 2006; 20(7): 1547 - 1561. [Abstract] [Full Text] [PDF]
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S. M Dougherty, W. Mazhawidza, A. R Bohn, K. A Robinson, K. A Mattingly, K. A Blankenship, M. O Huff, W. G McGregor, and C. M Klinge Gender difference in the activity but not expression of estrogen receptors {alpha} and {beta} in human lung adenocarcinoma cells. Endocr. Relat. Cancer, March 1, 2006; 13(1): 113 - 134. [Abstract] [Full Text] [PDF]
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 A. L. Moran, G. L. Warren, and D. A. Lowe Removal of ovarian hormones from mature mice detrimentally affects muscle contractile function and myosin structural distribution J Appl Physiol, February 1, 2006; 100(2): 548 - 559. [Abstract] [Full Text] [PDF]
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 G. Rizzo, B. Renga, E. Antonelli, D. Passeri, R. Pellicciari, and S. Fiorucci The Methyl Transferase PRMT1 Functions as Co-Activator of Farnesoid X Receptor (FXR)/9-cis Retinoid X Receptor and Regulates Transcription of FXR Responsive Genes Mol. Pharmacol., August 1, 2005; 68(2): 551 - 558. [Abstract] [Full Text] [PDF]
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 D. M. Ruden, L. Xiao, M. D. Garfinkel, and X. Lu Hsp90 and environmental impacts on epigenetic states: a model for the trans-generational effects of diethylstibesterol on uterine development and cancer Hum. Mol. Genet., April 15, 2005; 14(suppl_1): R149 - R155. [Abstract] [Full Text] [PDF]
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 P. Labhart, S. Karmakar, E. M. Salicru, B. S. Egan, V. Alexiadis, B. W. O'Malley, and C. L. Smith Identification of target genes in breast cancer cells directly regulated by the SRC-3/AIB1 coactivator PNAS, February 1, 2005; 102(5): 1339 - 1344. [Abstract] [Full Text] [PDF]
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P<0.05, values that are significantly different (Student’s t-test) between the 4-OHT activity without and with SMRT or NCoR.
