Modulator Recognition Factor 1, an AT-Rich Interaction Domain Family Member, Is a Novel Corepressor for Estrogen Receptor {alpha}

Serban P. Georgescu, Joyce H. Li, Qing Lu, Richard H. Karas, Myles Brown and Michael E. Mendelsohn

Molecular Cardiology Research Institute (S.P.G., J.H.L., Q.L., R.H.K., M.E.M.), New England Medical Center and Department of Medicine, Tufts University School of Medicine, Boston, Massachusetts 02111; and Division of Molecular and Cellular Oncology (M.B.), Department of Medical Oncology, Dana-Farber Cancer Institute, and Brigham and Women’s Hospital and Harvard Medical School, Boston, Massachusetts 02115

Address all correspondence and requests for reprints to: Michael E. Mendelsohn, Molecular Cardiology Research Institute, New England Medical Center and Department of Medicine, Tufts University School of Medicine, Boston, Massachusetts 02111. E-mail: mmendelsohn{at}tufts-nemc.org.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Cardiovascular tissues are important targets of estrogen action. Vascular cells express the two known estrogen receptors (ERs), ER{alpha} and ERß, ligand-activated transcription factors that regulate gene transcription through interactions with both coactivator and corepressor molecules. To isolate ER{alpha} coregulators in vascular cells, we performed a yeast two-hybrid screen for ER{alpha}-interacting proteins using a human aorta library. Here we report the identification of modulator recognition factor 1 (MRF1) as an ER{alpha}-interacting corepressor protein. Full-length MRF1 binds to both the N terminus and the C terminus of ER{alpha}. ER{alpha} and MRF1 coimmunoprecipitate in an estradiol-independent manner, and recombinant ER{alpha} binds to both full-length and COOH-terminal MRF1 in the absence of estradiol. MRF1 also interacts in a ligand-dependent manner with thyroid receptor {alpha}, retinoid X receptor {alpha}, and androgen receptor, and in a ligand-independent manner with ERß and the retinoic acid receptor. MRF1 RNA is highly expressed in aorta, heart, skeletal muscle, and liver. MRF1 has intrinsic repressor activity in an in vitro GAL reporter assay. Transient transfection studies show that MRF1 represses transcription by ER{alpha} activated by estradiol in a dose-dependent manner, as well as by the selective ER modulators 4-hydroxy-tamoxifen and raloxifene. MRF1 repression is not influenced by pharmacological inhibition of histone deacetylase. These data identify MRF1 as a repressor of ER{alpha}-mediated transcriptional activation and support a role for MRF1 in regulating ER-dependent gene expression in cardiovascular and other cells.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
ESTROGENS EXERT COMPLEX actions in many tissues and organs, including the cardiovascular system. The effects of estrogen are mediated through two estrogen receptors (ERs), ER{alpha} and ERß, transcription factors that belong to a structurally similar superfamily of nuclear hormone receptors containing both an N-terminal ligand-independent transcription activation domain [activation function 1 (AF-1)], and a C-terminal ligand-dependent transcription activation domain (AF-2) (1, 2, 3, 4, 5, 6, 7, 8). Like other members of the nuclear receptor family, coregulatory proteins are important to efficient transcriptional regulation of target genes. To date, many nuclear hormone receptor-interacting proteins have been discovered, the majority of which are coactivators that facilitate receptor-dependent gene transcription (9, 10). Receptor-interacting proteins that mediate down-regulation of gene expression have also been identified. Of these corepressors, the best known and characterized are nuclear receptor corepressor (N-COR) (11), and silencing mediator of retinoid and thyroid receptors (SMRT) (12). Both of these corepressors can interact with and silence several nuclear receptors, including retinoic acid receptor (RAR) and thyroid hormone receptors.

One important target organ system for estrogen action is the cardiovascular system. In an effort to better understand the molecular mechanisms by which ERs regulate vascular cell biology, we sought to identify ER-interacting proteins expressed in the vasculature. In the present study, we report the identification of the modulator recognition factor 1 (MRF1) as an ER{alpha}-interacting protein. MRF1 was first described due to its ability to bind to and repress the activity of the human cytomegalovirus modulator domain (13). MRF1 belongs to the ARID (AT-rich interaction domain) family of proteins characterized by their ability to bind preferentially to AT-rich DNA sequences and known for their roles in regulating growth, differentiation, and development (14, 15). Here we characterized MRF1, which is expressed abundantly in cardiovascular tissues, as an ER{alpha}-interacting repressor of gene expression.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
ER{alpha}-MRF1 Interactions in Yeast Two-Hybrid Assays
An initial yeast two-hybrid screen was performed in the presence of estradiol (E2) with Gal4BD-hER{alpha} as bait in an aortic yeast two-hybrid library. Three hundred clones were identified, of which 27 proved subsequently to be E2 dependent. DNA sequence analysis identified two of the E2-dependent clones as the modular recognition factor protein, MRF1 (13). One clone included the entire coding sequence for MRF1, whereas the other clone encoded the C-terminal 341 aa of MRF1 (aa 254–594; MRF1ct). Yeast two-hybrid experiments confirmed the MRF1-ER{alpha} interaction and localized the interaction domain to the C-terminal portion of MRF1 (Fig. 1AGo). Additional yeast two-hybrid experiments demonstrated that full length MFR1 interacts with both the N-terminal 297 aa of ER{alpha} (ER{alpha}-NT) and the C terminus of ER{alpha} (aa 299–595; ER{alpha}-CT) (Fig. 1BGo). The interaction with ER{alpha}-NT, which includes AF-1, does not require E2, whereas the association with the ER{alpha}-CT, which contains the AF-2 domain, is dependent on E2 in this assay (Fig. 1BGo). Further mapping of ER{alpha} domains interacting with full-length MRF1 showed that ER{alpha} aa 1–82 interacted with MRF1, whereas ER{alpha} 51–167 did not, supporting the importance of the initial approximately 50 aa of ER{alpha} to the N-terminal ER{alpha} interaction with MRF1 (data not shown).



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Fig. 1. MRF1 Interacts with ER{alpha} and Other Nuclear Hormone Receptors in Yeast Two-Hybrid Assays

A, Interaction of MRF1 with ER{alpha} in yeast two-hybrid (Y2H) system. Only C-terminal MRF1 interacts with ER{alpha}. The lower panel shows a schematic diagram of the MRF1 deletion mutants used in the Y2H. B, N-terminal and C-terminal domains of ER{alpha} interact with MRF1 in Y2H system. C, MRF1 interacts with other nuclear hormone receptors. In addition to E2 for ER studies, the ligands used for the receptors shown included 5{alpha}-dihydrotestosterone for androgen receptor (AR), T3 for thyroid hormone receptor-{alpha} (TR{alpha}), 9-cis-retinoic acid for RXR{alpha}, or all trans-retinoic acid for RAR. F, F domain; H, hinge domain; Lig, ligand.

 
MRF1 Interactions with Other Nuclear Receptors
To determine whether MRF1 also interacts with other nuclear hormone receptors, additional yeast two-hybrid experiments were performed using several other members of this family as bait. As with full-length ER{alpha}, MRF1 interacted in a ligand-dependent manner with thyroid receptor {alpha} (TR{alpha}) and the retinoid X receptor {alpha} (RXR{alpha}) (Fig. 1DGo). MRF1 bound to ERß, the androgen receptor (AR), and the retinoic acid receptor (RAR) in a ligand-independent manner in these assays (Fig. 1CGo). We also tested the MRF1 interaction with several members of the type III orphan receptors, but no binding to chicken ovalbumin upstream promoter transcription factors (COUP-TF) 2 or 3 was detected (data not shown).

Human Tissue Distribution of MRF1
Northern blot analyses were used to determine the tissue distribution of MRF1. MRF1 was detected as a single transcript of 2.4 kb in most human tissues examined, with highest levels of expression in skeletal muscle, liver, and heart (Fig. 2AGo). The liver MRF1 transcript appears slightly smaller than 2.4 kb. Lung and peripheral blood leukocytes displayed the 2.4-kb transcript and an additional transcript of approximately 2.6 kb. A more detailed examination of MRF1 expression in the cardiovascular system demonstrated the highest level of expression was in aorta, with moderate levels in the cardiac ventricles and lower levels of expression in the cardiac atria (Fig. 2BGo).



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Fig. 2. Northern Blot Analysis of MRF1 Expression in Human Tissues

A, MRF1 is expressed in vascular and nonvascular human adult tissues. MRF1 message was detected as a single 2.4-kb transcript in most tissues, with highest levels in heart, skeletal muscle, and liver, although the hepatic transcript is smaller (~2 kb). In the lung and peripheral blood leukocytes an additional 2.6-kb MRF1 transcript was observed. B, MRF1 expression in human aorta and in the chambers of the adult heart. A single 2.4-kb MRF1 transcript was detected in the human cardiovascular system, with highest levels in aorta.

 
ER{alpha}-MRF1 Interaction Studies
In vitro binding assays were performed with recombinant ER{alpha} and MRF1 proteins. In pull-down assays, recombinant ER{alpha} bound both full-length MRF1 and the C-terminal 341 aa of MRF1 (aa 254–594) (MRF1ct), but not the N-terminal aa 1–256 of MRF1 (MRF1nt) (Fig. 3AGo). This pattern was identical in the absence or presence of E2 (Fig. 3AGo). Incubation of recombinant MRF1 protein with lysates from COS7 cells expressing ER{alpha}, followed by immunoprecipitation of ER{alpha}, gave identical results (data not shown). Full-length MRF1 interacted with the N-terminal 271 aa of ER{alpha} (hER{alpha} 1–271) in both pull-down assays (Fig. 3BGo). Incubation of recombinant MRF1 protein with lysates from COS7 cells expressing human (h)ER{alpha} 1–271, followed by immunoprecipitation of ER{alpha}, showed that this domain of ER{alpha} also bound MRF1 in this assay (Fig. 3CGo). In contrast to the yeast two-hybrid assay, the in vitro interaction between full-length ER{alpha} and MRF1 did not require the presence of E2 (Fig. 3Go).



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Fig. 3. Human ER{alpha} Associates with MRF1 in Vitro

A, Pull-down assay of MRF1 with ER{alpha}. Assays of MRF1 fusion proteins immobilized on beads and mixed with lysates of COS7 cells expressing hER{alpha} His-containing beads were used as negative controls. B, Pull-down assay of MRF1 with N-terminal ER{alpha} (aa 1–271). C, Immunoprecipitation of MRF1-ER{alpha} aa 1–271 in mixing studies. Incubation of recombinant MRF1 protein with lysates from COS7 cells expressing hER{alpha}, followed by immunoprecipitation of ER{alpha}, shows an interaction between MRF1 and the N-terminal domain of ER{alpha}. IB, Immunoblot; IP, immunoprecipitation; Ni, nonimmune.

 
Full-length hER{alpha} and epitope-tagged MRF1 were next coexpressed in COS7 cells in the absence or presence of E2 treatment, followed by immunoprecipitation of either ER{alpha} (Fig. 4AGo) or MRF1 (Fig. 4BGo). Coimmunoprecipitation of ER{alpha} and MRF1 occurred similarly in all of these experiments in the absence and presence of E2 (Fig. 4Go). Furthermore, in these COS7 immunoprecipitation studies, the ER{alpha} mutant lacking the A/B domain (ER{alpha} 176–595) interacted with full-length MRF1 (Fig. 4CGo).



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Fig. 4. Coimmunoprecipitation of ER{alpha} and MRF1

COS7 cells were cotransfected with full-length hER{alpha} and epitope-tagged MRF1, followed by E2 treatment and immunoprecipitation of ER{alpha} (A) or MRF1 (B). C, Coimmunoprecipitation of an ER{alpha} mutant lacking the A/B domain (ER{alpha} 176–595) with MRF-1 in the absence of E2. Full-length ER{alpha} was used as positive control. IB, Immunoblot; IP, immunoprecipitation.

 
MRF1 Is a Corepressor for ER{alpha}
The potential effect of MRF1 on ER{alpha} transcriptional activity was examined. Transient transfection studies using an estrogen-responsive element (ERE) reporter plasmid (ERE-Luc) were performed first in COS7 cells expressing ER{alpha} and varying amounts of MRF1. MRF1 repressed ER{alpha}-mediated activation of the ERE reporter in a dose-dependent manner (Fig. 5AGo). MRF1 significantly repressed both basal (white bars) and E2-induced (black bars) transcriptional activity. In control experiments for these studies, varying MRF1 expression levels had no detectable effect on the level of ER{alpha} protein expression (data not shown). The repressor activity of MRF1 was assessed next in vascular endothelial cells, in which MRF1 also was capable of repressing both basal and E2-induced transcriptional activity of the ERE-luc reporter by ER{alpha} (Fig. 5BGo). MRF1 also repressed ER{alpha}-mediated transactivation of a natural estrogen-responsive promoter, the complement C3-luciferase, both in the absence and the presence of E2 (Fig. 5CGo). Studies of the effect of MRF1 on endogenous ER{alpha}-mediated transactivation in MCF-7 cells demonstrated MRF1-mediated repression of ERE-luc activation in both the absence and presence of E2 (Fig. 5DGo). We next examined the effects of MRF1-mediated repression on the N-terminal ligand-independent activation domain, AF-1, and on the C-terminal ligand-dependent activation domain AF-2. Transactivation levels of both a ligand-independent AF-1-containing construct (1–271) and of a ligand-dependent AF-2-containing construct (176–595) were inhibited in a dose-dependent fashion by MRF1 (Fig. 6Go). Selective ER modulators like tamoxifen and raloxifene can induce the recruitment of corepressors to target gene promoters in mammary cells (16). Transient transfection studies were performed to determine the effects of MRF1 transcriptional regulation of ER{alpha} in the presence of the selective ER modulators 4-hydroxy-tamoxifen and raloxifene. MRF1 also repressed 4-hydroxy-tamoxifen-mediated (Fig. 7Go, left panel) and raloxifene-mediated (Fig. 7Go, right panel) ER{alpha} transcriptional activation in a dose-dependent manner.



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Fig. 5. MRF1 Represses ER{alpha}-Mediated Transactivation in Vivo

A, Dose-dependent inhibition of transcriptional activity of ER{alpha} by MRF1. COS7 cells were cotransfected with pCMV-ER{alpha}, ERE-Luc, L7RH-ß-galactosidase control, and the indicated amounts of pcDNA3.1His-MRF1. Cells were stimulated for 9 h with either vehicle (white bars) or 10 nM E2 (black bars). The fold activation is calculated relative to the reporter activity of the receptor in the absence of E2, which was set to 1. *, P < 0.05 vs. ER{alpha} alone in the absence of E2; and **, P < 0.001 vs. ER{alpha} alone in the presence of E2. B, MRF1 functions as a corepressor for ER{alpha} in BAECs. BAECs cotransfected with ER{alpha}, MRF1, and ERE-Luc reporter were stimulated with vehicle of 10 nM E2 for 24 h. *, P < 0.001 vs. ER{alpha}-E2; and **, P < 0.001 vs. ER{alpha} + E2. C, Dose-dependent inhibition of ER{alpha}-mediated transcriptional activation of C3-Luc promoter in COS7 cells by MRF1. Transfected cells were treated with either vehicle or 10 nM E2 for 9 h. Fold activation is calculated relative to ER{alpha} activity in the absence of E2, which was set to 1. *, P < 0.0001 vs. ER{alpha} alone without E2; and **, P < 0.0005 vs. ER{alpha} alone with E2. D, MRF1 represses transcriptional activity of endogenous ER{alpha} in MCF-7 cells. MRF1- and ERE-Luc-transfected MCF-7 cells were treated with vehicle or 10 nM E2 for 24 h, as described in Materials and Methods. *, P < 0.001 vs. ER{alpha}-E2; and **, P < 0.001 vs. ER{alpha} + E2.

 


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Fig. 6. MRF1 Represses Both AF1- and AF2-Mediated Transcriptional Activity of ER{alpha}

COS7 cells were transiently transfected with ERE-Luc reporter plasmid, various ER{alpha} constructs (100 ng), and two different amounts of MRF1 (100 ng or 200 ng), as indicated. hER{alpha} constructs used were aa 1–595 (full-length receptor), aa 1–271 (AF-1 and DBD), and aa 176–595 (lacking the A/B domain). Cells were incubated in the absence of ligand (white bars) or in the presence of 10 nM E2 (black bars). The fold activation is calculated relative to the reporter activity of the full-length receptor (aa 1–595) in the absence of ligand, which was set at 1. Bars represent the means ± SE from three independent experiments. *, P < 0.01; and **, P < 0.003 vs. ER{alpha} alone.

 


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Fig. 7. MRF1 Represses 4-Hydroxytamoxifen- and Raloxifene-Mediated ER{alpha} Transactivation

Dose-dependent repression of 4-hydroxytamoxifen- and raloxifene-induced transcriptional activity of ER{alpha} by MRF1. COS7 cells cotransfected with plasmids expressing ER{alpha}, ERE-Luc, L7RH-ß-galactosidase control, and the indicated amounts of MRF1 were stimulated for 9 h with either 10 nM OHT (left graph) or RAL (right graph). The fold activation is calculated relative to the reporter activity of empty vector, which was set to 1. Note the different scaling for the two graphs. *, P < 0.0001 vs. ER{alpha} alone in the absence of ligand; and **, P < 0.0001 vs. ER{alpha} alone in the presence of ligand. OHT, 4-Hydroxytamoxifen; Ral, raloxifene.

 
MRF1 Is a Corepressor for Other Nuclear Hormone Receptors
We also examined whether MRF1 can inhibit the transcriptional activity of other nuclear hormone receptors with which it interacts (cf. Fig. 1CGo). MRF1 coexpression with ERß led to a dose-dependent reduction of both ligand-independent and -dependent ERß-mediated transactivation (Fig. 8AGo). MRF1 also repressed androgen receptor-mediated transactivation of a natural testosterone-responsive promoter, the mouse mammary tumor virus (MMTV)-luciferase reporter, both in the absence and the presence of 5{alpha}-dihydrotestosterone (Fig. 8BGo). In separate studies, MRF1 had no effect on transactivation of a myocardin-responsive SM22 reporter by the smooth and cardiac muscle transcription factor myocardin (data not shown), suggesting that the repressor effects are somewhat specific for nuclear hormone receptors. These results suggest that MRF1 may be a more general corepressor for the steroid hormone receptors.



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Fig. 8. MRF1 Is a Corepressor for Other Nuclear Hormone Receptors

A, Dose-dependent inhibition of transcriptional activity of ERß by MRF1. COS7 cells transfected with vectors for ERß, ERE-Luc, ß-galactosidase control, and increasing amounts of MRF1 were stimulated for 9 h with either vehicle (white bars) or 10 nM E2 (black bars). The fold activation is determined relative to the reporter activity of ERß in the absence of E2, which was set to 1. *, P < 0.0001 vs. ERß alone without E2; and **, P < 0.0001 vs. ERß alone with E2. B, MRF1 represses androgen receptor-mediated transcriptional activation of MMTV-Luc promoter in COS7 cells. Transfected cells were treated for 9 h with either vehicle or 10 nM 5{alpha}-dihydrotestosterone. Fold activation is calculated relative to androgen receptor activity in the absence of ligand, which was set to 1. *, P < 0.0001 vs. androgen receptor alone in the absence of 5{alpha}-dihydrotestosterone; and **, P < 0.05 vs. androgen receptor alone in the presence of 5{alpha}-dihydrotestosterone. AR, Androgen receptor; DHT, 5{alpha}-dihydrotestosterone.

 
MRF1-Induced Repression Activity
To further characterize the effects of MRF1 on transcription, we first fused full-length MRF1, as well as MRF1nt and MRF1ct fragments in frame to Gal-DNA-binding domain (DBD) and tested each for the ability to repress the Gal4-dependent reporter construct MH100tk-luciferase in COS7 cells. Full-length, N-terminal, and C-terminal MRF1 constructs each displayed dose-dependent repression activity. Maximal repression by MRF1nt and MRF1ct was 3- to 4-fold, whereas maximal repression by the full-length MRF1 protein was significantly greater (>12-fold) (Fig. 9AGo). Similar results were obtained in human embryonic kidney HEK 293 cells (not shown).



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Fig. 9. Effect of HDAC Inhibition on MRF1-Mediated Repression

A, COS7 cells were transiently transfected with mammalian expression vectors for Gal-MRF1, Gal-MRF1nt, and Gal-MRF1ct fusion proteins or Gal-DBD alone, a Gal4-responsive luciferase reporter construct (MH100tk-Luc), and L7RH-ß-galactosidase plasmid as internal control. The amounts of each construct used were 1 ng, 10 ng, 50 ng, or 100 ng. Values are presented as fold repression of Gal-DBD basal transcriptional activity, which was set arbitrarily to 1. *, P < 0.005 vs. Gal-DBD. Full-lengthMRF1 and N-terminal and C-terminal constructs each show dose-dependent repression of transcription. B, N-terminal repression domain of MRF1 colocalizes with ARID domain in the GAL assay. COS7 cells, transiently transfected with various deletion constructs of MRF1 fused to Gal-DBD, were analyzed for the luciferase activity. Values are calculated as fold repression of Gal-DBD basal transcriptional activity, which was set to 1. *, P < 0.005 vs. Gal-DBD alone. C, Effect of full-length, N-terminal, and C-terminal MRF1 on E{alpha}-mediated transcriptional activity, demonstrating a repressive effect of only full-length MRF1 in this assay. ER{alpha} and Gal-DBD or Gal-DBD-MRF1, Gal-DBD-MRF1nt, and Gal-DBD-MRF1ct fusion constructs, together with ERE-Luc reporter plasmid, were transfected into COS7. Cells were stimulated for 9 h with vehicle (white bars) or 10 nM E2 (black bars). Fold activation was calculated relative to unstimulated ER{alpha} alone, which was set at 1. *, P < 0.0001 vs. ER{alpha}-E2; and **, P < 0.0001 vs. ER{alpha} + E2. D, Lack of effect of the HDAC inhibitor TSA on Gal-MRF1, Gal-MRF1nt, or Gal-MRF1ct repression activities. HEK293 cells were transfected with 100 ng Gal-DBD, Gal-MRF1, Gal-MRF1nt, or Gal-MRF1ct. TSA (0, 10 nM, or 100 nM) was added to cultures 24 h after transfection, and cells were incubated for an additional 12 h before harvesting. Fold repression was determined relative to Gal-DBD alone in the absence of TSA treatment, which was set at 1. *, P < 0.05 vs. Gal-DBD-MRF1 in the absence of TSA. E, TSA effect on MRF1-mediated repression of ER{alpha} transactivation. COS7 cells cotransfected with vectors encoding for ER{alpha} (100 ng), MRF1 (150 ng), and ERE-Luc were stimulated for 9 h with either vehicle (left panel) or 10 nM E2 (right panel) in the absence or presence of 50 nM TSA for 1 h. For each group, fold change was calculated relative to the reporter activity in the absence of ER{alpha} and TSA, which was set to 1. Bars represent the mean ± SE from at least three independent assays performed in triplicate. BD, Binding domain.

 
These studies confirm the presence of repression activity in each half of MRF1 protein. To further characterize the N-terminal repression domain and its possible relation to MRF1’s known ARID domain (cf. Fig. 1Go), we used gal4-MRF1 N-terminal deletion mutants in the MH100tk-Luc reporter assay to test for repressor activity (Fig. 9BGo). These studies demonstrate that the repressor activity in the MRF1 N terminus is contained in aa 61–153, the ARID domain. These results colocalize the N-terminal repression domain of MRF1 with the ARID domain of the protein. Thus, both N and C termini of MRF1 contain a repression domain. Next, to determine the effect of these MRF1 domains on ER{alpha} transcriptional activity, the Gal-DBD-MRF1nt and Gal-DBD-MRF1ct constructs were each expressed with ER{alpha} and the ERE-Luc reporter in COS7 cells. Neither MRF1nt nor MRF1ct alone repressed ER{alpha} transcriptional activation in this assay (Fig. 9CGo).

To determine whether MRF1-induced repression involves histone deacetylase (HDAC) activity, we first transfected HEK293 cells with expression plasmids for Gal4-DBD alone or fused to full-length, N-terminal, or C-terminal parts of MRF1. The cells were treated with vehicle alone or with the HDAC inhibitor trichostatin A (TSA). TSA had no effect on the repression induced by the N-terminal or C-terminal MRF1 in these studies. At the highest TSA dose, a modest but significant loss of repression by full-length MRF1 was observed, although the majority of MRF1 repressor activity remained (Fig. 9DGo). As expected, TSA fully reversed repression induced by DAXX (17), a repressor with known HDAC activity, which was used as a positive control for TSA activity in these studies (data not shown). We next investigated the effect of TSA on the repressive effects of MRF1 on ER{alpha} transcription. TSA significantly increased both basal and E2-induced activity of ER{alpha}. However, MRF1-dependent repression of ER{alpha} -mediated gene activation was not blocked by TSA, either in the absence or in the presence of E2 (Fig. 9EGo). In the absence of E2, the degree of repression due to MRF1 in the absence and presence of TSA did not differ [40% vs. 38%, P = NS (P>0.72)]. In the presence of E2, however, MRF1-dependent repression of gene activation was significantly greater in the presence of TSA as compared with in the absence of TSA (57% repression vs. 40% repression; P < 0.05). These data support that MRF1 repression of gene activation by ER{alpha} is not due principally to HDAC activity of MRF1.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The studies presented here support that MRF1 is a repressor molecule that binds to ER{alpha}, which has at least two MRF1 interaction domains: an N-terminal domain that in two hybrid assays appears to be contained in ER{alpha} aa 1–50, and a C-terminal domain that involves the ligand-binding domain of ER{alpha}. MRF1 binds full-length ER{alpha} via its C terminus in several in vitro assays. MRF1 is expressed in cardiovascular tissues, as well as other muscle lineages and in liver, lung, and peripheral leukocytes, although the MRF1 mRNAs in these noncardiovascular and nonmuscle cells appear distinct in size from the 2.4-kb transcript found in heart, skeletal muscle, and vascular cells. MRF1 has intrinsic repressor activity in the in vitro GAL reporter assay, in which both N-terminal and C-terminal domains of MRF1 demonstrate intrinsic repressor activity. However, full-length MRF1 shows a degree of repression in this assay that is far greater than the sum of the effects of the N-terminal and C-terminal domains alone (synergistic, not additive). In transactivation assays with ER{alpha}, however, only full-length MRF1 is able to repress ER{alpha}-mediated gene activation. Finally, MRF1 was able to repress several other members of the nuclear hormone receptor family, but not the transcription factor myocardin. Taken together, these data support a model in which MRF1 is brought to the ERE by ER{alpha} and the ARID domain of MRF1 mediates repression. It is also possible that the C terminus of MRF1, which binds ER{alpha} and thus should be in the proximity of the promoter in the transient transfection studies, is unable alone to repress the ERE reporter, perhaps because its effect is too weak for us to detect in the assay being used and/or because it is sterically hindered from access to the transcriptional complex in isolation from the full-length MRF1 molecule.

MRF1 was originally identified as a repression activity present in the nuclear extracts from undifferentiated human embryonal carcinoma cells Tera-2 (13) and belongs to a diverse family of proteins characterized by the presence of the ARID DBD. ARID family proteins play important roles in the control of growth, differentiation, and development (14, 15). Although the ARID protein MRF1 is identified as a repressor of ER{alpha} transcriptional activity in these studies, other members of the ARID family, hOsa1/p270 and hOsa2, the largest subunits of the human SWI/SNF chromatin-remodeling complex, are involved in transcriptional activation. A recent report showed that both of the ARID proteins, hOsa1/p270 and hOsa2, stimulate transcription by the estrogen, androgen, and glucocorticoid receptors in mammalian cells (18). At least one other ARID family member, PLU-1, has been reported to function as a corepressor (19).

Corepressor proteins inhibit activation of steroid hormone receptors by various mechanisms, including competition with coactivators for DNA binding, sequestration of coactivators, interaction with the core transcriptional machinery, DNA methylation, and recruitment of complexes with histone deacetylase (HDAC) activity (20). Our data indicate the presence of two repression domains in MRF1 protein, neither of which seems to be influenced by pharmacological inhibition of HDAC. The corepressor proteins silencing mediator of retinoid (SMRT) and thyroid hormone receptor/nuclear receptor corepressor (N-CoR) (11, 12) bind to the hormone-binding domains of unliganded or antagonist-bound (in the case of ER) nuclear hormone receptors. Ligand binding triggers a structural change in the receptor, releasing the corepressor and recruiting coactivators into the transcriptional initiation complex. The data here support that MRF1 acts differently, because it can bind to and repress receptors both in the absence and presence of ligand.

In summary, we have identified MRF1, which is expressed abundantly in human blood vessels and heart, as a corepressor for ER{alpha}. MRF1 interacts with ER{alpha} in a ligand-independent manner in vitro and in vivo and represses ER{alpha}-mediated transcriptional activation. MRF1 is not a cardiovascular-specific or an ER{alpha}-specific corepressor, but its identification in vascular two-hybrid library screening, its abundance in cardiovascular tissues, and its repressor activity in vascular endothelial cells all suggest that further studies of MRF1 repression of ERs in vascular cells are worth pursuing. Subsequent studies will need to address the mechanism of MRF1 repression in more detail in an endogenous cell expressing both ER and MRF1, so that the relative roles of intrinsic MRF1 repression activity vs. coactivators blockade (or other mechanisms) in MRF1-mediated repression of ER action can be better understood. Further understanding of MRF1 actions in cardiovascular biology have the potential to refine our understanding of the cardiovascular actions of estrogen and advance the development of selective nuclear hormone receptor modulators with beneficial cardiovascular effects.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Reagents
3-Amino-triazole, E2, ICI 182 780, 4-hydroxytamoxifen, raloxifene hydrochloride, 5{alpha}-dihydrotestosterone, T3, 9-cis-retinoic acid and all trans-retinoic acid were from Sigma Chemical Co. (St. Louis, MO), as were all other chemicals, unless otherwise specified.

Yeast Two-Hybrid Screening and Assays
A yeast two-hybrid screening was performed using the BD Matchmaker Two-Hybrid System 3, according to the manufacturer’s instructions (CLONTECH Laboratories, Inc., Palo Alto, CA). The full-length hER{alpha} cloned into pGBKT7 vector (CLONTECH) was used as bait to screen 7 x 106 clones from a human aortic BD Matchmaker cDNA library (CLONTECH) in Saccharomyces cerevisiae strain AH109. The initial screen was done in selection plates (Trp/Leu/Ade/His-deficient, 5 mM 3-amino-triazole) containing 500 nM E2. Growing colonies were picked and patched on similar interaction plates in the absence of the hormone. Only the colonies showing estrogen-dependent growth were selected for further analysis. They were assayed for ß-galactosidase activity, after which plasmids were isolated and cDNA inserts were sequenced. The hormone-dependent interaction with ER{alpha} was reconfirmed by cotransforming the positive clone and bait plasmids into AH109 yeast. Interactions between MRF1 and other nuclear receptors (hERß, AR, TR{alpha}, RXR{alpha}, RAR) were carried out by cotransforming yeast with the respective plasmids, selecting transformants on Trp/Leu-deficient plates and patching them on interaction plates in the absence or presence of the appropriate ligands. Controls containing empty vectors were negative in all cases.

Plasmids
Deletion mutants of hER{alpha} were constructed by restriction digestions of the receptor followed by in-frame cloning into the yeast expression vector pGBKT7-GalBD. The PCR-amplified coding sequences of full-length MRF1 (aa 2–594) or of C-terminal MRF1 (aa 256–594; MRF1ct) were subcloned into the EcoRI/XhoI sites of the pcDNA3.1/HisA vector (Invitrogen, San Diego, CA), generating pcDNA3.1/HisA-MRF1 and pcDNA3.1/HisA-MRF1ct. These plasmids contain the His6 and Xpress tags. N-terminal MRF1 (aa 1–259; MRF1nt) was obtained by digesting the full-length clone with MscI. MRF1 full-length, MRF1nt, and MRF1ct were subcloned in frame into the pCMX-Gal vector [previously described (22)] to generate fusion proteins pCMX-Gal-MRF1, pCMX-Gal-MRF1nt, and pCMX-Gal-MRF1ct. Construction of the expression vectors for the full-length, wild-type hER{alpha} (pCMV3-ER{alpha}-NTF), as well as for the ER{alpha} deletion mutant pCMV3-ER{alpha}-271 (aa 1–271) was previously described (23). The expression plasmid pCMV4-{Delta}ABER{alpha}, driving expression of the hER{alpha} deletion mutant (aa 176–595), was the kind gift of Dr. B. S. Katzenellenbogen (24). pCMX-hER{alpha} (aa 320–595) was constructed by PCR amplification of hER{alpha} LBD and subcloned into the CMX vector for mammalian expression. The plasmids F-RXRa-pGBT9, hRARf-pAS, and MH100tk-Luc were described previously (25). The human androgen receptor cDNA was PCR amplified and subcloned into the pCMX-HA mammalian expression vector (26). Control luciferase reporter pLd40-Luc was constructed by cloning the 40-bp H-2Ld promoter into the pGL basic luciferase plasmid (Promega Corp., Madison, WI) (21), and the ERE-luciferase reporter, constructed by cloning of 3 vitellogenin ERE into this vector, was the kind gift of Dr. Ming-Jer Tsai (Baylor College of Medicine, Houston, TX). The complement C3-Luc reporter plasmid, kindly provided by Dr. Donald McDonnell, has been described previously (27). MMTV-Luc containing the luciferase gene regulated by the testosterone-inducible hormone response element from the MMTV also has been described (28). All constructs were verified by restriction enzyme digestion and DNA sequencing.

Cell Culture
COS7 and HEK293 cells were maintained in phenol red-free DMEM containing 10% fetal bovine serum (FBS), penicillin, streptomycin, and L-glutamine. Hormone-depleted FBS was prepared by stripping FBS with activated charcoal and Dextran 70, as previously described (23). For experiments, cells were cultured in phenol red-free DMEM containing 10% stripped FBS. Bovine aortic endothelial cells (BAECs) passage 4–6, as well as human breast cancer cell line MCF-7 were maintained in DMEM with 10% FBS. After transfection, they were cultured for 6 h in phenol red-free DMEM containing 10% stripped FBS, followed by overnight arrest in serum-free media in the presence of 1 nM ER antagonist ICI 182780, as previously described (23). Cells were then washed and stimulated with vehicle or 10 nM E2 in serum-free media for 24 h.

Northern Blot Analysis
A [32P]dCTP-labeled C-terminal 720-bp MRF1 cDNA was used as a probe for hybridization of an Adult Human Multiple Tissue and a Human Cardiovascular Northern blot (CLONTECH). Fifty nanograms of the 720-bp MRF1 cDNA fragment were labeled by random priming with [32P]dCTP, using Rediprime II Random Prime Labeling System, according to the manufacturer’s instruction (Amersham Pharmacia Biotech, Arlington Heights, IL).

Recombinant Protein Expression and Purification
The PCR-amplified coding sequences of full-length MRF1 (aa 2–594), of N-terminal MRF1 (aa 2–259) or of C-terminal MRF1 (aa 256–594) were subcloned into the vector pET28c(+) (Novagen, San Diego, CA). The His6-tagged fusion proteins were expressed in Escherichia coli strain BL21(DE3) (Novagen) and purified under native conditions using cobalt sepharose beads [BD Talon Metal Affinity Resin (BD Biosciences, Palo Alto, CA)]. Coomassie blue staining and Western blot analysis [with an anti-T7 tag monoclonal antibody (Novagen) of the eluted recombinant protein showed a prominent band corresponding to the expected size.

In Vitro Pull-Down Assays
COS7 cells overexpressing full-length or the indicated ER{alpha} deletion mutants were lysed in a Triton lysis buffer (20 mM Tris-Cl, pH 7.5; 137 mM NaCl; 1% Triton-X; 10% glycerol; 1 mM EDTA; 25 mM ß-glycerol-phosphate; and protease inhibitors). Lysates were incubated with His beads or His-fusion MRF1 protein beads in the absence or presence of 10–6 M E2 for 4 h at 4 C. For the reverse pull downs, lysates were mixed with recombinant His-fusion MRF1 proteins eluted from the beads, followed by immunoprecipitation with an anti-ERa antibody (HC-20, H-184, or Ab-7) or nonimmune IgG (Sigma). Beads were washed five times in wash buffer (50 mM Tris-Cl, pH 7.5; 7 mM MgCl2; 2 mM EDTA; 1 mM phenylmethylsulfonylfluoride) and boiled in sodium dodecyl sulfate-sample buffer. Proteins were resolved by SDS-PAGE, and immunoblotting was performed with anti-T7 tag antibody for MRF1 proteins or anti-ER{alpha} antibody.

Immunoblots and Coimmunoprecipitations
Total lysates were prepared by lysing transfected COS7 cells in a high-salt lysis buffer [450 mM NaCl, 0.4 mg/ml cholesteryl hemisuccinate, 2 mg/ml n-dodecyl-ß-D-maltoside (Calbiochem, La Jolla, CA), 20 mM HEPES (pH 7.5), 0.5 mM EDTA, 10% glycerol, 7 mM MgCl2, 10 mM Na2MoO4] with Protease Inhibitors Cocktail Mix III (Calbiochem). Proteins were resolved on 10% SDS-PAGE and electrophoretically transferred to nitrocellulose.

For immunoprecipitation studies, transfected COS7 cells were washed in PBS and lysed in a modified RIPA buffer [450 mM NaCl, 1% Triton X-100, 0.25% sodium deoxycholate, 50 mM Tris (pH 7.5), 10% glycerol, 1 mM ß-glycerol phosphate] containing Protease Inhibitors Cocktail Mix III. The final concentration of NaCl was then adjusted to 150 mM by the addition of buffer without salt. Cleared cell lysates were split into two; one half was incubated with an anti-ER{alpha} HC-20 polyclonal antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), and the other half was incubated with an anti-Xpress monoclonal antibody recognizing the leader sequence Asp-Leu-Tyr-Asp-Asp-Asp-Asp-Lys (Invitrogen), followed by incubation with a mixture of protein G+A-agarose beads (Amersham). After three washes, the beads were boiled in 2x Laemmli sample buffer and subjected to SDS-PAGE and immunoblot analysis. Antibodies used for immunoblots include the anti-Xpress tag monoclonal antibody, anti-ER{alpha} Ab-7 (AER320) monoclonal antibody (LabVision, Freemont, CA), and anti-ER{alpha} H-184 polyclonal antibody (Santa Cruz). Immunoreactive proteins were detected by enhanced chemiluminescence (Amersham) using one of the primary antibodies in combination with a secondary donkey antirabbit antibody or a sheep antimouse antibody linked to horseradish peroxidase (Amersham).

Transient Transfections and Reporter Assays
All transfections were carried out using PolyFect Transfection Reagent (QIAGEN, Chatsworth, CA), according to the manufacturer’s instructions. For ERE-luciferase assays, COS7, HEK293, BAEC, and MCF-7 cells were set up in 12-well plates. Typically, transfections contained 300 ng reporter (ERE-Luc, C3-Luc, or MMTV-Luc), 10 ng L7RH-ß-galactosidase control, 100 ng receptor (ER{alpha}, ERß, or AR expressing vectors), and the indicated amounts of the MRF1 plasmid. To keep the total DNA amount constant at 600 ng in every well, empty vectors were used. One day after transfection, the cells were stimulated with vehicle or 10 nM E2 or the indicated ligand for 9 h or as noted in the text. For Gal4-luciferase assays, COS7 and HEK293 cells were seeded into 12-well plates. Typically, 400 ng MH100tk-Luc reporter plasmid, 10 ng of L7RH-ß-gal internal control, 100 ng CMX-Gal fusion protein or CMX-Gal-DBD (or otherwise indicated), and carrier plasmid (pcDNA3.1, Invitrogen) in a total amount of 700 ng/well were used for transfection. Cells were harvested 36 h post transfection. Cells were lysed in Reporter Lysis Buffer (Promega) and assayed for luciferase [Luciferase Assay System (Promega)] and ß-galactosidase [Chemiluminescent Reporter Gene Assay System (Applied Biosystems)] activity. The measured luciferase units were normalized to ß-gal activity, which served as an internal control for transfection efficiency. If not indicated otherwise, fold activation was calculated relative to the reporter activity of the empty vector, which was set to 1. All assays were performed in triplicate, and data in all figures represent the average of at least three independent experiments.

Statistical Analysis
All values are expressed as mean ± SE. Statistical differences between mean values were calculated by ANOVA, followed by Scheffe’s test for pairwise comparisons where appropriate. P ≤ 0.05 was considered significant.


    ACKNOWLEDGMENTS
 
We thank Wendy Baur and Jennifer Brown for expert cell culture assistance, Steven Swift for help with the yeast two-hybrid studies, and Ulla Hansen for helpful discussions.


    FOOTNOTES
 
This work was supported in part by National Institutes of Health Grants 5P50HL063494 and 5R01HL056069 (to M.E.M.).

First Published Online June 7, 2005

Abbreviations: aa, Amino acids; AF-1, activation function 1; ARID, AT-rich interaction domain; BAEC, bovine aortic endothelial cell; DBD, DNA-binding domain; E2, estradiol; ER, estrogen receptor; ERE, estrogen-responsive element; FBS, fetal bovine serum; HDAC, histone deacetylase; HEK, human embryonic kidney; MMTV, mouse mammary tumor virus; MRF1, modulator recognition factor 1; RAR, retinoic acid receptor; RXR, retinoid X receptor; TSA, trichostatin.

Received for publication August 3, 2004. Accepted for publication May 27, 2005.


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