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Molecular and Cellular Biology, January 2009, p. 83-92, Vol. 29, No. 1
0270-7306/09/$08.00+0     doi:10.1128/MCB.00884-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Coactivation of Estrogen Receptor β by Gonadotropin-Induced Cofactor GIOT-4

Madoka Kouzu-Fujita,^1,2 Yoshihiro Mezaki,¹ Shun Sawatsubashi,^1,3 Takahiro Matsumoto,^1,3 Ikuko Yamaoka,^1,3 Tetsu Yano,² Yuji Taketani,² Hirochika Kitagawa,¹ and Shigeaki Kato^1,3*

Institute of Molecular and Cellular Biosciences, University of Tokyo, Yayoi 1-1-1, Bunkyo-ku, Tokyo 113-0032, Japan,¹ Department of Obstetrics and Gynecology, University of Tokyo, Hongo 7-3-1, Bunkyo-ku, Tokyo 113-0865, Japan,² ERATO, Japan Science and Technology Agency, Honcho 4-1-8, Kawaguchi, Saitama 332-0012, Japan³

Received 4 June 2008/ Returned for modification 17 July 2008/ Accepted 8 October 2008

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Estrogen exerts its diverse effects through two subtypes of estrogen receptors (ER), ER and ERβ. Each subtype has its own distinct function and expression pattern in its target tissues. Little, however, is known about the transcriptional regulatory mechanism of ERβ in the major ERβ-expressing tissues. Using biochemical methods, we identified and described a novel ERβ coactivator. This protein, designated GIOT-4, was biochemically purified from 293F cells. It coactivated ERβ in ovarian granulosa cells. GIOT-4 expression was induced by stimulation with follicle-stimulating hormone (FSH). GIOT-4 recruited an SWI/SNF-type complex in a ligand-independent manner to ERβ as an ER subtype-specific physical bridging factor and induced subsequent histone modifications in the ERβ target gene promoters in a human ovarian granulosa cell line (KGN). Indeed, two ERβ-specific target genes were upregulated by FSH at a specific stage of a normal ovulatory cycle in intact mice. These findings imply the presence of a novel regulatory convergence between the gonadotropin signaling cascade and ERβ-mediated transcription in the ovary.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Estrogen plays important roles in many target organs, including the female reproductive organs, the central nervous system, and bone. Estrogen exerts its diverse biological actions through binding to and activating one of two nuclear estrogen receptor (ER) subtypes (ER or ERβ) (12, 22, 35, 40). ERs are members of the nuclear receptor (NR) gene superfamily. ERs, bound to and activated by estrogen, bind to specific DNA sequences called estrogen-responsive elements (ERE) to induce target genes (14, 21).

Like the other NR members, the ER requires the cooperation of distinct classes of coregulators and multiprotein coregulator complexes in order to initiate estrogen-mediated chromatin reorganization (16, 46). These complexes appear to modify the chromatin configuration in a highly regulated manner by controlling nucleosomal rearrangement and enzyme-catalyzed modifications of histone tails. By altering chromatin structure, the coregulator complexes facilitate bridging between NRs and basal transcription factors, along with RNA polymerase II, thereby controlling transcription. As for the nucleosomal rearrangement, two major classes of chromatin-modifying complexes that coregulate NRs have been well-characterized. One class is the histone-modifying complexes, including discrete subfamilies of transcription coregulatory complexes (2, 29, 36). The best-characterized NR coregulator complexes possess either histone acetylase or histone deacetylase activities. Recently, histone methylases/demethylases have also been shown to be significant NR coregulators. The other class of coregulator complexes is ATP-dependent chromatin-remodeling complexes. These complexes use ATP hydrolysis to rearrange nucleosomal arrays in a noncovalent manner to facilitate or prevent the access of NRs to nucleosomal DNA (5, 17, 33). These ATP-dependent chromatin-remodeling complexes have been classified into three subfamilies based on the major catalytic components possessing DNA-dependent ATPase activity. BRG1/Brm is a core component of the SWI/SNF-type complexes, SNF2h is a major component of imitation SWI-type complexes, and Mi2 is a core component of NuRD-type complexes. Recently, several distinct complexes with spatiotemporally specific functions have been identified. Generally, these complexes have components that confer specificity for certain transcription factors, including NRs (11, 18, 26).

ERβ and ER have different distributions and biological functions in the target tissues. ER is expressed in the breast, uterus, and bone, while ERβ is expressed predominantly in the prostate, central nervous system, and intestinal tissues (23, 24, 31, 50, 62). Even within a single tissue, the expression pattern of each subtype is cell type specific. In the ovary, clear expression of ERβ is detectable in granulosa cells but ER is more abundant in theca cells than in granulosa cells (41). Reflecting the different subtype distribution patterns, ERβ knockout (KO) and ER KO mice show different phenotypes. ER KO mice are infertile and have a hypotrophic uterus and anovulatory, hemorrhagic ovaries (23, 32). In contrast, ERβ KO mice are subfertile, with reduced ovulation (34). Further analysis revealed previously that ERβ is essential for granulosa cell differentiation (9).

The ovary is an ER target tissue whose function and development are under control by ER-mediated estrogen actions. The ovulatory cycles are also regulated by hormones and cytokines through the hypothalamic-pituitary-ovarian axis. Gonadotropin-releasing hormone, produced in the hypothalamus, stimulates the secretion of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) from the pituitary. FSH then induces follicle maturation from preantral follicles to antral follicles via cyclic AMP signals (25). Though a number of hormones and cytokines coordinate to promote ovarian development and support ovarian function (13, 43-45), the molecular mechanisms underlying these complicated events in the ovary remain largely unknown.

In this study, we found that treatment with an FSH analogue (pregnant mare serum gonadotropin [PMSG]) stimulated ERβ function via protein kinase A (PKA) signaling in a human granulosa tumor cell line (KGN). We biochemically identified a novel coactivator of ERβ, designated GIOT-4. GIOT-4 expression was induced by PMSG in KGN cells and in the mouse ovary. GIOT-4 recruited an SWI/SNF complex to ERβ for further nucleosomal reorganization for gene activation. This GIOT-4-induced recruitment of SWI/SNF complex components was detected in the endogenous promoters of genes encoding aromatase and the activin βA precursor, both of which are essential for folliculogenesis. Thus, GIOT-4 is a novel ERβ coactivator that participates in a chromatin-remodeling complex to mediate gonadotropin actions in the ovary.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Plasmids. Glutathione S-transferase (GST)-ERβ AB domain and GST-ERβ DEF domain constructs were prepared as described previously (30). FLAG-tagged full-length cDNAs for ER and ERβ were inserted into the pcDNA3 vector (Invitrogen, Carlsbad, CA). Full-length cDNA for Myc-tagged GIOT-4 (GenBank accession no. AB021644) was cloned from 293F cells and inserted into the pcDNA3 vector. SRC-1 and BRG1 expression vectors were prepared as described previously (26). A BRG1 mutant construct (designated K798R) was made by site-directed mutagenesis as described previously (27, 57).

Reagents. Rabbit polyclonal anti-human GIOT-4 antibodies against human GIOT-4 and mouse GIOT-4 were made by Operon (Huntsville, AL). The following commercially available antibodies were used: anti-FLAG (Sigma, St Louis, MO); anti-BRG1 (catalog no. sc-17796 for immunofluorescence and catalog no. sc-10768 for immunoprecipitation and Western blotting), anti-ER (catalog no. sc-543), anti-INI1 (catalog no. sc-13058), anti-SRC-1 (catalog no. sc-8995), and anti-TRRAP (catalog no. sc-5405) (all from Santa Cruz Biotechnology, Santa Cruz, CA); anti-ERβ (catalog no. ab16813) and anti-BAF57 (catalog no. ab14764) (Abcam, Cambridge, United Kingdom); and anti-Myc (catalog no. 05724), anti-histone H4 (catalog no. 06-866), H3K9me3 (catalog no. 8898-100), and H3K9me2 (catalog no. 207-212) (Upstate Biotechnology, Lake Placid, NY). PMSG reagent and human chorionic gonadotropin (hCG) reagent were purchased from Teikokuzouki Co. Ltd. (Tokyo, Japan), and estradiol (E2) reagent and H89, a PKA inhibitor, were purchased from Sigma. Small interfering RNAs (siRNAs) for ERβ (catalog no. L-003402-00), GIOT-4 (catalog no. L-020805-01), and BAF57 (catalog no. L-017522-00) and the nonspecific control (catalog no. D-001810-01) were purchased as an ON-TARGETplus SMART pool from Dharmacon Inc. (Lafayette, CO).

Cell culture. 293F cells were maintained in Dulbecco's modified Eagle medium (Gibco BRL, Gaithersburg, MD) supplemented with antibiotics and 10% fetal bovine serum. KGN cells were maintained in Dulbecco's modified Eagle medium-Ham F-12 medium supplemented with antibiotics and 10% fetal bovine serum (8). For 72 h before transfection, the cells were cultured in phenol red-free medium with 10% charcoal-stripped serum.

Purification and characterization of the ERβ-interacting complex. The procedure to convert an adherent culture of 293 cells into a suspension culture of 293F cells was described previously (27, 54). Nuclear extracts from 293F cells, transformed with FLAG-ERβ in a suspension culture, were loaded onto an anti-FLAG M2 affinity resin column and washed extensively with washing buffer (20 mM Tris-HCl [pH 8.0], 300 mM KCl, 0.2 mM EDTA, 0.05% NP-40, 10% glycerol, 0.5 mM phenylmethylsulfonyl fluoride, and 1 mM dithiothreitol). Bound proteins were eluted from the column by incubation with 300 µg/ml FLAG peptide in washing buffer for 30 min at 4°C. For fractionation on glycerol gradients, eluates were layered on top of 13 ml of linear 10 to 40% glycerol gradients in washing buffer and centrifuged for 16 h at 4°C and 40,000 rpm in an SW40 rotor (Beckman Coulter, Fullerton, CA). After the collection of each fraction, Western blotting analysis of fractions 1 through 12 was performed with anti-ERβ antibody. Protein standards used were ovalbumin (44 kDa), beta globulin (158 kDa), and thyroglobulin (670 kDa). Each sample was applied to a NuPAGE bis-Tris 4 to 12% gradient gel (Invitrogen) (26, 27, 54).

RT-PCR and qRT-PCR. Total RNAs from KGN cells, 293F cells, and mouse ovary tissue were extracted using TRIzol reagent (Invitrogen), and cDNA was synthesized as described previously (27, 48). PCR was performed as previously described (27, 48). PCR products were visualized on 2% agarose-Tris-acetate-EDTA gels. Real-time quantitative reverse transcription-PCR (qRT-PCR) was performed using Sybr premix Ex Taq (TaKaRa Bio Inc., Tokyo, Japan) with the Dice real-time system TP800 thermal cycler (TaKaRa), and normalization and calculation steps were performed as reported previously (40, 54).

Specific primers for PCR were as follows: human GIOT-4 gene (GenBank accession no. NM_021030), 5'-CTGTGTGGGTGTCGAGAGCAAATG-3' and 5'-TGCCACTGTCATGGCTCAGCAATG-3'; mouse GIOT-4 gene (GenBank accession no. NM_145624), 5'-GGGCAGCACATCTTAGAAGC-3' and 5'-TTGCCAAAGCTGTTTCTCCT-3'; human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene, 5'-CGAGATCCCTCCAAAATCAA-3' and 5'-GTCTTCTGGGTGGCAGTGAT-3'; and mouse GAPDH gene, 5'-GGGTGTGAACCACGAGAAAT-3' and 5'-ACACATTGGGGGTAGGAACA-3'. Specific primers for qRT-PCR for human aromatase or activin βA were purchased from TaKaRa Bio Inc.

Immunoprecipitation and Western blotting. Immunoprecipitation was performed by following our standard protocol (26, 42). For 293F cells, 3 x 10⁷ cells were transfected with the FLAG-ERβ-expressing vector and the Myc-GIOT-4-expressing vector. Twenty-four hours after transfection, cells were harvested with 10 nM E2 for 1 h before collection. For KGN cells, 3 x 10⁸ cells were transfected with GIOT-4 siRNA or control siRNA and harvested, with or without 10 nM E2 and 500 mIU/ml PMSG. Anti-FLAG M2 affinity resin or anti-Myc affinity resin (Sigma) was used for the precipitation for 293F cells. ERβ antibody and protein A agarose were used for the KGN cells. The washed agarose was subjected to Western blotting.

GST pull-down assay. The GST pull-down assay was performed by following our standard protocol (26, 42). GST fusion proteins were expressed in Escherichia coli and bound to glutathione-Sepharose 4B beads (GE Healthcare, Buckinghamshire, England). In vitro-translated proteins were prepared by in vitro translation using the T7 promoter of the pcDNA3 vector. Proteins were labeled using [³⁵S]methionine (GE Healthcare), and in vitro translation was carried out using the TNT-coupled rabbit reticulocyte lysate system (Promega, Madison, WI). The in vitro-translated proteins were incubated with beads for 1 h at 4°C with or without 1 µM E2 (6).

Luciferase assay. Luciferase assays involving the ERs were performed by following our standard protocols (30, 42). For KGN cells, 80% confluent cells were transfected with plasmids and siRNA by using Lipofectamine 2000 reagent (Invitrogen). A 500-mIU/ml concentration of PMSG, 1,000-mIU/ml hCG, or 10 nM E2 was added 3 h after transfection, and the cells were incubated for 18 h at 37°C. Values were reported as the means ± standard deviations (SD) of results from at least three independent experiments. For the RNA interference (RNAi) experiment, KGN cells were transfected with 20 nM siRNA together with the DNA.

Chromatin immunoprecipitation (ChIP) assay. Samples of soluble chromatin from KGN cells treated with or without ligands (500 mIU/ml PMSG and 10 nM E2) and from mouse ovarian tissue were prepared with an immunoprecipitation assay kit (Upstate Biotechnology) and were immunoprecipitated with antibodies against the proteins indicated below. Specific primer pairs were designed to amplify the promoter region of the human aromatase gene; 5'-TTTGGCAATGACCAGAAATG-3' and 5'-AAGGACAACGGGACTCTGTG-3'), the human activin βA precursor gene; 5'-TGGGTCAGGGGTGAGTTTAG-3' and 5'-GTGTGGCTTAAGCAGGTTCC-3'), the mouse aromatase gene; 5'-GGTACGGGAGCCTTTTCCTG-3' and 5'-TGTGGCTCCTGTCACTTGGA-3'), or the mouse activin βA precursor gene; 5'-CCACAGGCTTTACTGGCTCAC-3' and 5'-TTCGGGTCCCTTCTGTTTTG-3') from genomic DNA. PCR products were visualized on 2% agarose-Tris-acetate-EDTA gels (18, 54, 61).

Immunohistochemistry. All mice were maintained according to the protocol approved by the animal care and use committee of the University of Tokyo. Immature 21-day-old female mice (CD-1) were injected intraperitoneally (i.p.) with 3.25 IU of PMSG or vehicle (saline) between 1300 and 1400 h. After 48 h, some of the animals were injected i.p. with 5 IU of hCG at 16 h before sampling (40). Anesthetized mice were perfused with 4% paraformaldehyde, and ovaries were sectioned after paraffin embedding. Immunohistochemistry was performed as described previously (48). Antigen retrieval was performed by incubating the slides in citric acid buffer (pH 6.0) at 95°C for 20 min. The sections were incubated with a mixture of mouse anti-BRG1 (1:100) and either rabbit anti-GIOT-4 (1:50) or anti-ERβ (1:50) at 4°C for 24 h. The sections were then incubated with a mixture of donkey anti-mouse immunoglobulin G labeled with Cy3 and anti-rabbit immunoglobulin G labeled with fluorescein isothiocyanate or Cy5 at room temperature for 1 h. Confocal microscopy was carried out with a Zeiss 510 confocal laser scanning system.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
FSH analogue treatment superactivates ERβ transcriptional activity through PKA signaling in KGN cells. Estrogen and gonadotropins are key hormones during folliculogenesis. Though their individual signaling pathways are well-described, cross talk between their signaling pathways has not been studied. To address this issue, we first tested possible gonadotropin effects on ER transactivation function. We used a luciferase assay that employed a luciferase vector plasmid with a consensus ERE in the promoter in KGN cells (Fig. 1A). We applied two commonly used gonadotropin compounds, PMSG and hCG, to provide FSH and LH stimulation, respectively. In this assay, clear activation of human ER and ERβ transactivation function by E2 was expectedly seen. PMSG stimulated ERβ, but not ER, in the presence or absence of E2. Such stimulation was not seen for hCG. A PKA inhibitor (H89) that blocks the downstream signaling cascade of gonadotropins (47, 49) inhibited this stimulation of ligand-bound ERβ by PMSG (Fig. 1A). In KGN cells, endogenous ERβ, but not ER, was expressed at significant levels (Fig. 1B) as previously reported (8). The expression of the known E2 target genes, including those for aromatase and the activin βA precursor (1, 49, 51, 58), was also detectable. Reflecting the observed hormonal actions, the additive induction of these genes by two hormones was detected. However, the knockdown of ERβ by RNAi (Fig. 1B) resulted in the loss of the hormonal effect on gene expression (Fig. 1C).

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FIG. 1. An FSH analogue superactivates ERβ transcriptional activity via PKA signaling in KGN cells. (A) PMSG potentiates the transcriptional activity of ERβ but not ER in KGN cells. KGN cells were transfected with ERs or a control vector. Without exogenous ERs, 10 nM E2 and 500m IU/ml PMSG, but not 1,000 mIU/ml hCG, increased transcriptional activity (lanes 1 and 2). Similar results were also obtained following transfection with ERβ. H89, a PKA inhibitor, suppressed the gonadotropin effect (lanes 1 to 4 and 9 to 12). Data are expressed as the mean ± SD of results from six independent experiments. Asterisks represent the findings of the statistical analysis, which showed that the results observed were significant (P < 0.05). ERE-TATA-LUC, construct containing ERE, TATA, and luciferase reporter sequences; Gn, gonadotropin; PKAi, PKA inhibitor H89; +, present; –, absent. (B) Expression of ERs in KGN cells. ERβ, but not ER, was detected in KGN cells by Western blot analysis. The expression level did not change with 10 nM E2 (E) or PMSG (P) stimulation. siRNA for ERβ suppressed the expression of ERβ. Cont., control; IB, immunoblotting. (C) Transcriptional regulation of ERβ target genes, those encoding aromatase and the activin βA precursor, by E2 and PMSG. KGN cells were treated with 10 nM E2 and 500 mIU/ml PMSG for 8 h, and total RNA was extracted. When ERβ RNAi was used, KGN cells were transfected with siRNA 12 h before stimulation. Real-time PCR analysis revealed that E2 and PMSG increased mRNA levels of target genes. The hormonal responses in gene expression were inhibited by ERβ RNAi. Data are expressed as the mean ± SD of results from six independent experiments. Asterisks represent the findings of the statistical analysis, which showed that the results observed were significant (P < 0.05).

Identification of GIOT-4 as a novel ERβ-interacting protein. ERβ is regulated by a different mechanism from that of ER (7, 28, 30, 31, 53, 55). This finding suggests that ERβ requires subtype-specific coregulators. To test this idea, we generated a stable transformant expressing FLAG-tagged ERβ in 293F cells in a suspension culture (Fig. 2A). We biochemically purified ERβ interactants from nuclear extracts by a standard column step purification as we have reported previously (Fig. 2B) (26, 54). The fractions, after elution with FLAG peptide off FLAG M2 resin, were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (Fig. 2C). SRC-1 interacted with ERβ in a ligand-dependent manner (Fig. 2C, lower panels), confirming our biochemical approach for identifying ERβ coregulators. By matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) analysis of the ERβ interactants, one interactant, designated GIOT-4, was identified in both the ligand-negative and -positive fractions (Fig. 2C). GIOT-4 has been recognized as a member of a Cys₂-His₂ (C2H2)-type zinc finger protein family, and the protein structure of GIOT-4 is closely related to those of GIOT-1 and GIOT-2 (38, 60). However, its physiological roles in gene regulation have remained to be studied (38, 52, 60). To examine whether GIOT-4 was a complex component, we then fractionated the ERβ interactants on a glycerol density gradient (Fig. 2D). GIOT-4 was detected in the fractions containing complexes of more than 670 kDa (Fig. 2D and E). Interestingly, in the same fractions, we identified BRG1 and INI1, both of which are components of mammalian SWI/SNF complexes (Fig. 2E). By Western blot analysis of the fractions, BRG1 was also seen together with GIOT-4 in the same fractions (Fig. 2D, lower panels), raising the possibility that GIOT-4 associates with SWI/SNF complex components interacting with ERβ.

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FIG. 2. Purification of ERβ interactants and identification of GIOT-4 as a novel ERβ-interacting protein. (A) Establishment of 293F cell lines stably expressing FLAG-tagged ERβ. FLAG-ERβ protein was detected in whole-cell lysates by Western blot analysis. Lanes: 1, mock-transfected 293F cells; 2, 293F cells stably expressing FLAG-ERβ. IB, immunoblotting; FLAG and ERβ, anti-FLAG and anti-ERβ antibodies. (B) Purification scheme for ERβ-interacting complexes. The cells were collected 12 h (12H) after the initiation of E2 stimulation. (C) Identification of the ERβ interactants. Nuclear extracts were loaded onto FLAG-M2 resin, and bound proteins were eluted with FLAG peptide solution. Each eluted solution was subjected to SDS-PAGE, followed by silver staining, the results of which are shown in the upper panel. ERβ and GIOT-4 were identified by MALDI-TOF MS analysis. Each solution was immunoblotted with the antibodies indicated in the lower panels. The known cofactors were detected in lanes 2 and 3. (D) Proteins interacting with FLAG-ERβ in stable transformant 293F cells were fractionated by molecular mass on a 10 to 40% glycerol density gradient following FLAG affinity purification. Each fraction was subjected to SDS-PAGE. The upper panels show the results of silver staining. The lower panels show Western blots with ERβ-containing fractions (fr.). BRG1 and GIOT-4 were present in the same fractions. –, absent; +, present. (E) Results of SDS-PAGE and silver staining of ERβ/GIOT-4-containing fractions (#8). ERβ, GIOT-4, BRG1, and INI1 were identified by MALDI-TOF MS analysis.

GIOT-4 physically interacts with ERβ as an SWI/SNF-type complex component. To confirm if ERβ, GIOT-4, and SWI/SNF complex components indeed form a complex, we performed the coimmunoprecipitation of ERs and GIOT-4 from 293F cells (Fig. 3A). ERβ, but not ER, was coimmunoprecipitated with GIOT-4 in a ligand-independent manner. This result was further confirmed by coimmunoprecipitation with endogenously expressed ERs from MCF7 cells (Fig. 3B). Additionally, in the immunoprecipitates containing ERβ, BRG1 and other SWI/SNF components, INI1 and BAF57 (20), were detected. In a GST pull-down assay, GIOT-4 physically interacted with the DEF region of ERβ (Fig. 3D). GIOT-4 and other GIOT family proteins are known to harbor a Kruppel-associated box (KRAB) domain and C2H2-type zinc finger motifs (Fig. 3C). As a GIOT deletion mutant protein (GIOT-4 del.) failed to associate with ERβ (Fig. 3D), the C-terminal C2H2 domain may be a domain for ERβ interaction.

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FIG. 3. GIOT-4 specifically interacts with ERβ in vivo and in vitro. (A) 293F cells were transfected with FLAG-tagged ER or ERβ and Myc-tagged GIOT-4 as indicated below the panels. Coimmunoprecipitation assays were performed as indicated. ERβ, but not ER, was immunoprecipitated with GIOT-4 in a ligand-independent manner. When cells were cotransfected GIOT-4 (lanes 6 to 10), BRG1, INI1, or BAF57 was precipitated with ERβ. IP, immunoprecipitation; IB, immunoblotting; Myc, FLAG, ER, ERβ, BRG1, INI1, and BAF57, anti-Myc, anti-FLAG, anti-ER, anti-ERβ, anti-BRG1, anti-INI1, and anti-BAF57 antibodies; +, present; –, absent. (B) ER subtype-specific association with GIOT-4. Myc-tagged GIOT-4 was detected in the immunoprecipitate from ERβ antibody but not in the immunoprecipitate from ER antibody. (C) Schematic illustration of GIOT family proteins. They have in common a KRAB domain in the N-terminal region and C2H2-type zinc finger motifs in the C-terminal region. GIOT-4 del. is a mutant protein in which most of the C-terminal region of GIOT-4 is deleted. a.a., amino acids. (D) GIOT-4 physically interacts with the DEF region of ERβ in a ligand-independent manner. GIOT-4 del. and BRG1 did not interact with ERβ. SRC-1 is shown as a positive control for the ligand-dependent interaction with ERβ. The GST pull-down assay was performed as explained in Materials and Methods.

The expression of GIOT-4 in KGN cells is PMSG dependent. Since the expression of GIOT-1, one of the GIOT family members (Fig. 3B), was inducible by gonadotropin (38, 52, 60), we examined whether the expression of GIOT-4 protein was also regulated by PMSG. The expression of the endogenous GIOT-4 gene in KGN cells was indeed induced by treatment with PMSG, but not E2, at both the mRNA and protein levels (Fig. 4A and B). However, such PMSG-dependent regulation was not seen in 293F cells, which do not express gonadotropin receptors (Fig. 4A). Moreover, in accordance with these observations, the association of GIOT-4 with ERβ was induced by PMSG (Fig. 4C). Additionally, GIOT-4 RNAi, but not BAF57 RNAi, was found to inhibit the recruitment of BRG1 onto ERβ following PMSG treatment (Fig. 4C). These results suggest that PMSG stimulation upregulates GIOT-4 and thereby induces the recruitment of the SWI/SNF complex to ERβ in a mode different from that of recruitment to ER (4, 7, 20).

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FIG. 4. The expression of GIOT-4 was induced by PMSG stimulation. (A) Semiquantitative RT-PCR analysis showing an increase in GIOT-4 mRNA in KGN cells but not in 293F cells following treatment with 500 mIU/ml PMSG. Cells were treated with (+) or without – 10 nM E2 for 6 h and 500 mIU/ml PMSG for the indicated times. (B) Western blot analysis showing the PMSG-dependent increases in GIOT-4 protein in KGN cells and 293F cells. Cells were stimulated as described in the legend to panel A. IB, immunoblotting; GIOT-4 and β-actin, anti-GIOT-4 and anti-β-actin antibodies. (C) PMSG-induced GIOT-4 recruits BRG1 onto endogenously expressed ERβ in a ligand-independent manner. KGN cells were transfected with 20 nM control siRNA, GIOT-4 siRNA, or BAF57 siRNA and stimulated by 10 nM E2 and 500 mIU/ml PMSG 12 h after siRNA transfection. IP, immunoprecipitation.

The transcriptional activity of ERβ is stimulated by the expression of GIOT-4 induced by PMSG treatment. Next, we examined whether GIOT-4 indeed activated the transcriptional function of ERβ through the recruitment of an SWI/SNF complex in KGN cells. In 293F cells, GIOT-4 and BRG1 cooperatively coactivated ERβ (Fig. 5A). In KGN cells, BRG1 hyperactivated the transcriptional property of ERβ in the presence of PMSG (Fig. 5B, compare lanes 1 and 2). This coactivation of BRG1 by PMSG was not seen when GIOT-4 was knocked down by RNAi (Fig. 5B, compare lanes 1 and 3 and lanes 2 and 4) or when cells were transfected with a BRG1 mutant form (K798R) lacking the ATPase activity (57) (Fig. 5A, compare lanes 2 and 5 and lanes 3 and 6, and B, compare lanes 2 and 5). These results suggest that the coactivation of ERβ by GIOT-4 is mediated through the ATPase activity of the BRG1-containing complex (56).

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FIG. 5. GIOT-4 and BRG1 cooperatively potentiate the transcriptional activity of ERβ through ATP-dependent chromatin remodeling. (A) Exogenous BRG1 and GIOT-4 cooperatively activate the ERβ transcriptional property. 293F cells were transfected with ERβ, BRG1, a BRG1 mutant form (K798R), and GIOT-4 as indicated. The transcriptional activity of ERβ was highest following the cotransfection of cells with ERβ and both BRG1 and GIOT-4 (lane 4). E2 concentration, 10 nM. ERE-TATA-LUC, construct containing ERE, TATA, and luciferase reporter sequences; ligand(–), without ligand; +, present, –, absent. (B) Endogenous GIOT-4, induced by PMSG, activates the ERβ transcriptional property with the recruitment of BRG1. KGN cells were transfected as described in the legend to panel A. The coactivation of BRG1 and PMSG was inhibited by transfection with GIOT-4 siRNA (lanes 3 and 4) or the replacement of BRG1 with the BRG1 mutant form (lanes 5 and 6). Concentrations: E2, 10 nM; PMSG, 500 mIU/ml.

GIOT-4 promotes histone modifications adjacent to the EREs in ERβ target gene promoters. We then confirmed that an SWI/SNF-type complex was associated with GIOT-4 and recruited to ERβ in the endogenous target gene promoters. For the ChIP assay, we chose the promoters for the aromatase and activin βA precursor genes. Both of these genes were expressed and were transcriptionally regulated by PMSG as well as E2 in KGN cells (Fig. 1C). As their promoters expectedly contained putative ERE-like sequences (Fig. 6A, upper panels), we first tested whether these putative ERE-like sequences indeed served as EREs by overexpressing ERβ and GIOT-4 in the presence or absence of E2 (Fig. 6A, lower panels). Clear E2-dependent recruitment of ERβ onto both promoters was detected, suggesting that these two genes were the direct target genes of ERβ. Endogenous ERβ was also detected on the putative EREs in an E2-dependent manner (Fig. 6B). Both PMSG treatment and the overexpression of GIOT-4 resulted in the recruitment of the SWI/SNF components BRG1 and INI1 and of GIOT-4 accompanied by ERβ (Fig. 6). Consistently, the demethylation of H3K9 (19), as well as the hyperacetylation of histone H4 adjacent to the ERβ binding sites in the aromatase gene promoter, was induced by PMSG treatment and was undetectable after the knockdown of either ERβ or GIOT-4 by RNAi. BAF57 RNAi had no effect on this observation (Fig. 6B). Thus, it is likely that the PMSG-induced expression of GIOT-4 triggers subsequent histone modifications of the adjacent chromatin domain of the ERβ binding site in certain ERβ target gene promoters. The histone modifications may account for the induction of the target genes following the treatment of KGN cells with PMSG or E2 (Fig. 1C).

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FIG. 6. GIOT-4 and human SWI/SNF complex components were recruited to consensus ERE-like elements in ERβ target gene promoters in a PMSG-dependent manner. (A, upper panels) Schematic diagrams of the aromatase promoter and the activin βA precursor promoter with putative EREs. (Lower panels) ERβ and GIOT-4 were overexpressed in KGN cells. Cells were then treated with E2 for 1 h and subjected to a ChIP assay. ERβ, GIOT-4, and BRG1 were recruited to promoters of the indicated genes. IgG, immunoglobulin G; +, present; –, absent. (B, upper panel) Protocol for PMSG treatment and transfection of KGN cells with siRNA. (Lower panels) ChIP assays were performed with or without siRNA against GIOT-4, ERβ, or BAF57. In the presence of PMSG, ERβ and components of the SWI/SNF complex were recruited to the aromatase promoter. The recruitment of SWI/SNF complex components and subsequent histone modifications, including the hyperacetylation of H4, were inhibited by the knockdown of GIOT-4 or ERβ but not by BAF57 knockdown. AcH4, acetylated H4.

PMSG-induced expression of ERβ target genes during murine folliculogenesis. As ERβ function appears to be indispensable for normal folliculogenesis, we tested if the observed FSH-induced regulation of ERβ target genes occurred during folliculogenesis in the intact ovary. To model the murine ovulatory cycle, we injected mice at the diestrous phase with PMSG and hCG and collected ovarian samples from immature ovaries (9, 15) (Fig. 7A).

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FIG. 7. GIOT-4 is expressed primarily in the growing follicles and functions with ERβ in a FSH-dependent manner in murine folliculogenesis. (A) Protocol for obtaining mouse ovary samples in the assays described herein. (1) Control sampling and pretreatment with an i.p. injection of 3.25 IU of PMSG. (2) Treatment with an i.p. injection of 3.25 IU of hCG 48 h after pretreatment. (3) Sampling 16 h after treatment. (B) Colocalization of ERβ (green) and GIOT-4 (blue) with Brg1 (red) in PMSG-treated mouse ovaries. Forty-eight hours after PMSG treatment (time point 2 as described in the legend to panel A), the expression of GIOT-4 was clearly induced in granulosa cells of various stages of follicles, including antral follicles (upper panel) and primary and secondary follicles (lower panel), but was undetectable in vehicle-treated ovaries. The colocalization of GIOT-4 and ERβ with Brg1 was detected in growing follicle granulosa cells at all developmental stages in PMSG-treated ovaries. GIOT-4, Brg1, and ERβ, anti-GIOT-4, anti-Brg1, and anti-ERβ antibodies. (C) Levels of expression of GIOT-4 and the ERβ target genes during the ovarian cycle. Semiquantitative RT-PCR was performed with follicles from mice at stages 1, 2, and 3, described in the legend to panel A. mRNA levels were the highest at a specific stage (preantral-antral follicle stage) during folliculogenesis and ovulation. (D) Recruitment of GIOT-4 and the SWI/SNF complex components to the aromatase promoter during an ovulatory cycle. An in vivo ChIP assay was performed as described in Materials and Methods. ERβ and BRG1 were recruited to the aromatase promoter at a specific stage (antral follicle formation) in folliculogenesis. IgG, immunoglobulin G; AcH4, acetylated H4.

By an immunofluorescence analysis, the expression of GIOT-4 in growing follicle granulosa cells at all developmental stages was detected in a PMSG-dependent manner. The colocalization of ERβ and GIOT-4 with Brg1 in the primary, secondary, and antral follicles after PMSG treatment was observed (Fig. 7B). Consistently, the expression levels of ERβ target genes were altered during ovulation (Fig. 7C). Moreover, by a ChIP analysis, PMSG-induced recruitment of these factors (GIOT-4, Brg1, and ERβ) to the ERβ target gene promoters was observed (Fig. 7D). These results suggest that the FSH-induced expression of GIOT-4 may potentiate ERβ function during an ovulatory cycle. This effect seems to result from the modulation of the chromatin structure by a recruited SWI/SNF-type chromatin-remodeling complex (Fig. 8).

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FIG. 8. Schematic representation of GIOT-4 function as a gonadotropin-induced cofactor of ERβ. At a specific antral stage during folliculogenesis, the expression of GIOT-4 in granulosa cells is induced by FSH stimulation. The association of GIOT-4 with ERβ recruits SWI/SNF-type complexes and other histone modifiers to ERβ on its target gene promoters and results in the potentiation of the genes. HAT, histone acetylase.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Stimulation of ERβ transcriptional activity by FSH. ER KO mice exhibit different phenotypic abnormalities in the female reproductive organs depending on which ER subtype is deleted (23, 32, 34, 41). For example, the ovarian function of ERβ, but not ER, appears to be controlled by gonadotropins (9). Distinct roles of LH and FSH in modifying ERβ function during follicle differentiation in granulosa cells have been proposed previously (9, 24, 41). The molecular mechanism behind the possible cross talk, however, has not yet been described. In the present study, the LH analogue (hCG) was not effective in coregulating the transcriptional activity of ERβ but FSH potently stimulated ERβ in KGN cells. Thus, the stimulation of ERβ activity by FSH may account for the FSH-specific biological actions at specific ovulatory stages in the granulosa cells of the ovary.

We used the promoters of two ERβ target genes, those for aromatase and the activin βA precursor, to model the potentiation of ERβ transcriptional activity by FSH (1, 49, 51, 58). These genes are responsible for regulating estrogen production by the granulosa cells via regulatory feedback by the pituitary. ERβ seems to participate in this regulatory axis through cross talk with the PKA-mediated gonadotropin cascade. The identification of other ERβ target genes will improve the understanding of the biological significance of this cross talk system (9).

The recruitment of an SWI/SNF-type chromatin-remodeling complex to ERβ through GIOT-4 is upregulated by gonadotropin at the gene expression level. Several ATP-dependent chromatin-remodeling complexes that coregulate the ligand-induced transactivation function of NRs exist (4, 37, 56). An SWI/SNF complex has already been reported to coactivate the function of ligand-bound ER (3). However, a more recent report showed that ERβ does not interact with BAF57, a bridging component for ER in the SWI/SNF complex (20). In the present study, GIOT-4 was a physical interactant with ERβ in vivo but not with ER. Moreover, BAF57 was also dispensable for the targeting of ERβ or the subsequent histone modifications (Fig. 3A, 4C, and 6B). Thus, it is likely that the modes and/or mechanisms of recruiting ATP-dependent chromatin-remodeling complexes differ between ER and ERβ subtypes.

In our ChIP assay, several histone modifications of adjacent chromatin areas in the ERβ target genes seem to have followed the recruitment of the SWI/SNF-type complex components (7, 37). These histone modifications occur through the anchoring of the other histone-modifying complexes, including a histone acetylase coactivator complex (19, 26, 59). In this respect, GIOT-4 may stabilize the assembly of coregulator complexes by inducing an association of an SWI/SNF-type complex with ERβ in the gene promoters. The targeting of ERβ by PMSG treatment in the absence of E2 (probably through GIOT-4 expression) may explain the E2-independent activation of ERβ transcriptional activity found in our luciferase assays (Fig. 1 and 5).

Functional regulation of GIOT family proteins as transcriptional cofactors. Certain GIOT family member proteins are induced by gonadotropins in both the male and female reproductive organs, but little is known about their functions (38, 52, 60). GIOT-1 serves as a corepressor for an orphan NR (SF-1) by recruiting histone deacetylase 2 through the GIOT-1 KRAB domain (52). Though GIOT-4 is a GIOT family protein, we have provided evidence that GIOT-4 is an ERβ coactivator in both cultured cells and the ovary. Nevertheless, we cannot exclude the possibility that GIOT-4 forms a distinct corepressor complex at another stage of folliculogenesis or in other tissues. It would be of interest to search for other transcription regulatory factors coregulated by GIOT-4. The identification of other stage-specific participants in GIOT-4 complexes in folliculogenesis would improve the understanding of the molecular mechanisms of gonadotropin action at each ovulation stage (10, 25, 39).

 
We thank Ichiro Takada, Sally Fujiyama, Mamoru Igarashi, Fumiaki Ohtake, Ryoji Fujiki, Takashi Nakamura, and Ken-ichi Takeyama for their technical assistance and helpful discussions. We also thank all the members of our laboratory for their invaluable assistance.

This work was supported in part by priority area grants from the Ministry of Education, Culture, Sports, Science and Technology (to H.K. and S.K.).

 
* Corresponding author. Mailing address: Institute of Molecular and Cellular Biosciences, University of Tokyo, Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan. Fax: 81-3-5841-8477. Phone: 81-3-5841-8478. E-mail: uskato{at}mail.ecc.u-tokyo.ac.jp

Published ahead of print on 3 November 2008.

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Molecular and Cellular Biology, January 2009, p. 83-92, Vol. 29, No. 1
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