Novel Mechanism of Negative Regulation of 1,25-Dihydroxyvitamin D₃-induced 25-Hydroxyvitamin D₃ 24-Hydroxylase (Cyp24a1) Transcription

Background: CYP24A1 is the principal enzyme involved in the catabolism of 1,25(OH)₂D₃.

Results: The SWI/SNF complex and PRMT5 converge at the transcriptional level to control 1,25(OH)₂D₃-induced Cyp24a1 gene expression.

Conclusion: PRMT5-mediated repression represents a novel mechanism of negative regulation of Cyp24a1.

Significance: Our study reveals key factors involved in the regulation of 1,25(OH)₂D₃ catabolism and therefore in the control of calcium homeostasis.

Abstract

The SWI/SNF chromatin remodeling complex facilitates gene transcription by remodeling chromatin using the energy of ATP hydrolysis. Recent studies have indicated an interplay between the SWI/SNF complex and protein-arginine methyltransferases (PRMTs). Little is known, however, about the role of SWI/SNF and PRMTs in vitamin D receptor (VDR)-mediated transcription. Using SWI/SNF-defective cells, we demonstrated that Brahma-related gene 1 (BRG1), an ATPase that is a component of the SWI/SNF complex, plays a fundamental role in induction by 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃) of the transcription of Cyp24a1 encoding the enzyme 25-hydroxyvitamin D₃ 24-hydroxylase involved in the catabolism of 1,25(OH)₂D₃. BRG1 was found to associate with CCAAT-enhancer-binding protein (C/EBP) β and cooperate with VDR and C/EBPβ in regulating Cyp24a1 transcription. PRMT5, a type II PRMT that interacts with BRG1, repressed Cyp24a1 transcription and mRNA expression. Our findings indicate the requirement of the C/EBP site for the inhibitory effect of PRMT5 via its methylation of H3R8 and H4R3. These findings indicate that the SWI/SNF complex and PRMT5 may be key factors involved in regulation of 1,25(OH)₂D₃ catabolism and therefore in the maintenance of calcium homeostasis by vitamin D. These studies also define epigenetic events linked to a novel mechanism of negative regulation of VDR-mediated transcription.

Introduction

Vitamin D is essential for mineral homeostasis and maintenance of bone mass (1, 2). 1,25-Dihydroxyvitamin D₃ (1,25(OH)₂D₃),² the hormonally active form of vitamin D, is produced by two sequential hydroxylations of vitamin D at C-25 in the liver by 25-hydroxylase and at C-1 by the enzyme 1α-hydroxylase (3). 1,25(OH)₂D₃ mediates its effects by binding to the vitamin D receptor (VDR), a member of the nuclear receptor family of transcription factors, which heterodimerizes with the retinoid X receptor and interacts with specific DNA sequences (vitamin D response elements) in target genes, resulting in activation or repression of transcription (1, 2, 4). The actions of 1,25(OH)₂D₃ and VDR are mediated by the recruitment of coregulatory complexes. These coregulatory complexes include the coactivator complex DRIP (VDR-interacting protein complex also known as Mediator complex) that functions at least in part through recruitment of RNA polymerase II and the p160 coactivators (steroid receptor coactivators 1, 2, and 3), which have histone acetyltransferase activity (1, 5).

For transcription to occur, chromatin structure is altered not only by covalent modification of histones but also by ATP-dependent chromatin-remodeling enzymes. One important ATP-dependent chromatin remodeling factor is the SWI/SNF multisubunit complex (6, 7). The SWI/SNF complex has been implicated in the regulation of differentiation as well as in the regulation of target genes stimulated by estrogen, glucocorticoid, and retinoic acid receptors (8–12). Each SWI/SNF complex contains one of two homologous ATPases, Brahma (Brm) or Brahma-related gene 1 (BRG1). BRG1-null mice are embryonic lethal, whereas Brm-null mice are viable (exhibiting a mild phenotype of increased body weight) (13, 14). C/EBPβ has been reported to recruit the SWI/SNF complex to specific gene promoters to promote tissue-specific transcription (15–17). In addition, it has been shown that ATP-dependent chromatin remodeling complexes can act together with histone-modifying enzymes to modulate transcription (18–20). Among the histone-modifying enzymes are the protein-arginine methyltransferases (PRMTs) that have been implicated in transcriptional activation or repression (21–24). Both type I and type II PRMTs catalyze the formation of monomethylarginine. Transfer of an additional methyl group results in the formation of asymmetrically dimethylated arginines (catalyzed by type I enzymes) or symmetrically dimethylated arginines (catalyzed by type II enzymes). PRMT4 (CARM1), a type I PRMT, methylates arginines 2, 17, and 26 of histone 3 and is associated with transcriptional activation, whereas PRMT5, a type II PRMT, has been linked to gene silencing through repressive histone marks including symmetrical dimethylation of H3R8 and H4R3 (21–24). Little is known about the role of SWI/SNF and PRMTs in VDR-mediated transcription.

One of the most pronounced effects of 1,25(OH)₂D₃ is increased synthesis of 25-hydroxyvitamin D₃ 24-hydroxylase (CYP24A1), the enzyme that accelerates the catabolism of 1,25(OH)₂D₃ (25). Thus, by inducing CYP24A1, 1,25(OH)₂D₃ regulates its own synthesis, protecting against hypercalcemia. We reported previously that C/EBPβ is induced by 1,25(OH)₂D₃ in kidney and osteoblastic cells and is a potent enhancer of VDR-mediated Cyp24a1 transcription (26). Here, we demonstrate that the SWI/SNF complex contributes to transcriptional activation by VDR. We found that BRG1 associates with C/EBPβ, and together they cooperate with VDR in the regulation of Cyp24a1. PRMT5, which interacts with BRG1, was found to be a negative regulator of 1,25(OH)₂D₃-induced Cyp24a1 transcription and mRNA expression. Our findings provide new insight into key factors and epigenetic events that regulate Cyp24a1 expression and thus affect the regulation of 1,25(OH)₂D₃ metabolism and the maintenance of calcium homeostasis.

EXPERIMENTAL PROCEDURES

Materials

Polyvinylidene difluoride (PVDF) membrane and the enhanced chemiluminescence (ECL) detection system were obtained from Bio-Rad. C/EBPβ antiserum (C-19), BRG1 antiserum (H-88), PRMT5 antiserum (A-11), and β-actin antiserum were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The mouse CYP24A1 antiserum was supplied by Dr. Harvey J. Armbrecht (St. Louis Veterans Affairs Hospital, St. Louis, MO). H3(Me2s)R8 antibodies were generated as described previously (27). H4(Me2s)R3 antibodies were obtained from Abcam. The secondary anti-mouse and anti-rabbit antibodies conjugated with horseradish peroxidase were purchased from Santa Cruz Biotechnology. 1,25(OH)₂D₃ was purchased from Cayman Chemical Co. (Ann Arbor, MI). Prestained protein markers were obtained from Bio-Rad. Protein A beads were obtained from Rockland Immunochemicals, Inc. (Gilbertsville, PA).

Cell Culture

COS-7 African green monkey kidney cells, MC3T3-E1 mouse osteoblastic cells, UMR-106 rat osteoblastic cells, SW-13 human adrenal gland cortex cells, and C33A human cervix carcinoma cells were obtained from American Type Culture Collection (ATCC, Manassas, VA). Mouse distal convoluted tubule (DCT) and mouse proximal convoluted tubule (PCT) cells were provided by Dr. P. Friedman (University of Pittsburgh School of Medicine). COS-7, SW-13, and C33A cells were cultured in DMEM (Mediatech, Inc., Manassas, VA) supplemented with 10% fetal bovine serum (FBS; Sigma-Aldrich) and 1% antibiotic mixture (penicillin, streptomycin, and neomycin; Invitrogen). UMR, DCT, and PCT cells were cultured in DMEM/F-12 (Invitrogen) supplemented with 5% FBS and 1% penicillin, streptomycin, and neomycin or 1% Geneticin, respectively. MC3T3-E1 cells were cultured in α-minimum Eagle's medium (Invitrogen) supplemented with 10% FBS and 1% penicillin, streptomycin, and neomycin. NIH-3T3 mouse fibroblast cells stably expressing antisense PRMT5 grown in DMEM with 10% FBS and 2.5 μg/μl puromycin have been described previously (27). Caco-2 cells, heterogeneous human epithelial colorectal adenocarcinoma cells, were grown in DMEM with l-glutamine from ATCC. Osteoblast-enriched bone cells were isolated from neonatal murine calvaria by serial collagenase digestion and cultured in α-minimum Eagle's medium supplemented with 10% FBS and 1% penicillin, streptomycin, and neomycin (26). All cells were cultured in a humidified atmosphere of 95% air and 5% CO₂ at 37 °C. Cells were seeded at 70–80% confluence 24 h before experiments. Treatments with 1,25(OH)₂D₃ were performed in medium supplemented with 2% charcoal-stripped fetal bovine serum. Cells were treated with vehicle (ethanol) or 1,25(OH)₂D₃ at the concentrations and times indicated.

Plasmids, Transfections, and Assays of Luciferase and Chloramphenicol Acetyltransferase (CAT) Activity

Luciferase reporter constructs of the rat Cyp24a1 promoter (−1367/+74 containing vitamin D response elements at positions −258/−244 and −151/−137, the intronic vitamin D response elements at +35 and +39, a C/EBP site at −395/−388, and deletion construct −298/+74) as well as a CAT reporter construct −671/+74 with and without the C/EBP site mutated (26) were used. pAV-hVDR was obtained from Dr. J. W. Pike (University of Wisconsin, Madison, WI), pBJ5-BRG1 and pBJ5-BRG1-DN (K785R) expression vectors were obtained from James DiRenzo and Myles Brown (Dana Farber Cancer Institute, Harvard Medical School, Boston, MA), and pMex-C/EBPβ was a gift from Dr. Simon Williams (Texas Tech University, Lubbock, TX). pBabe/Fl-PRMT5 and catalytically inactive PRMT5 (pBabe/Fl-PRMT5 (G367A/R368A) were described previously (28). Cells were seeded in a 24-well culture dish 24 h prior to transfection at 70% confluence. Cells in each well were transfected using Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer's instructions. To determine the involvement of the SWI/SNF complex, SWI/SNF-negative cells lines (SW-13 and C33A) were transfected with a component of the SWI/SNF complex, either BRG1 or Brm. COS-7 cells, which do not contain steroid receptors (including VDR), were used in transfection studies to assess the impact of VDR (using transfected VDR) on gene activation. Empty vectors were used to keep the total DNA concentration the same. 1,25(OH)₂D₃ (10⁻⁸ m) was added to cells in media supplemented with 2% charcoal-dextran-treated FBS 16 h post-transfection for another 24 h. Cells were harvested using 1× passive lysis buffer (Dual Luciferase reporter assay kit from Promega (Madison, WI)), and the assay for luciferase activity was performed according to the manufacturer's protocol. The efficiency of transfection was assessed by green fluorescent protein and PRMT5 co-transfection followed by visualization using a fluorescence microscope. The efficiency of transfection of MC3T3 and Caco-2 cells was estimated at 60–70% and was comparable with transfection efficiency in UMR cells (65–75%). For studies using promoter constructs linked to the CAT reporter gene, CAT assays were performed by standard protocols on cell extracts normalized to total protein. CAT activity was quantified by scanning TLC plates using the Packard Constant Imager System (Packard Instrument Co.).

RT-PCR Analysis and Western Blotting

For RT-PCR analysis, total RNA was extracted from cells using Ribozol from Amresco (Solon, OH). 2 μg of total RNA was used to make cDNA using Superscript III reverse transcriptase (Invitrogen) according to the manufacturer's protocol. For each primer set, PCR cycle numbers were chosen so that the amplification was in the linear range of amplification efficiency. Primers and annealing temperatures were as follows: Cyp24a1: forward, 5′-GTGCGGATTTCCTTTGTGAT-3′; reverse, 5′-ATTGTCTTCGCTAGAGCCCA-3′; T_a = 50 °C; TRPV6: forward, 5′-ACTGTCATTGGGGCTATCATC-3′; reverse, 5′-CAGCAGAATCGCATCAGGTC-3′; T_a = 60 °C; GAPDH: forward, 5′-TCACCATCTTCCAGGAGCG-3′; reverse, 5′-CTGCTTCACCACCTTCTTGA-3′; T_a = 60 °C; osteopontin (Opn): forward, 5′-CTTTCACTCCAATCGTCCCTA-3′; reverse, 5′-GCTCTCTTTGGAATGCTCAAGT-3′; T_a = 50 °C; Prmt5: forward, 5′-CTGGATGGAGCCCAGCAC-3′; reverse, 5′-GGCTCGGACCTCATTGTACA-3′; T_a = 52 °C.

For Western blotting, total cellular protein was prepared using radioimmune precipitation assay buffer, and protein concentration was measured by the Bradford method. 100 μg of protein was separated by SDS-PAGE and transferred to a PVDF membrane using a semidry transfer apparatus. The membrane was incubated overnight at 4 °C with primary antibody (1:500) diluted in phosphate-buffered saline (PBS) containing 5% nonfat milk. The membrane was washed with PBS and incubated for 2 h with the corresponding secondary antibody conjugated with horseradish peroxidase. The enhanced chemiluminescence Western blotting system was used to detect the antigen-antibody complex. The same blot was stripped and reprobed with β-actin polyclonal antibody to normalize for sample variation.

Co-immunoprecipitation Assay

Nuclear extracts were prepared, and protein concentration was determined by the Bradford method (29). 500 μg–1 mg of each preparation was used for immunoprecipitation with the addition of 4 μg of C/EBPβ, BRG1, or PRMT5 antiserum for 24 h at 4 °C. 30 μl of protein A-Sepharose 4 Fast Flow beads (Amersham Biosciences) was added to each sample, and after further incubation by rotating at 4 °C for 24 h, the immunoprecipitated complex was collected by centrifugation at 3,000 rpm for 5 min and washed with ice-cold PBS containing protease inhibitor. The complex was separated by 7.5 or 15% SDS-PAGE and analyzed by Western blot using BRG1 antibody, C/EBPβ antibody, or PRMT5 antibody.

Chromatin Immunoprecipitation (ChIP) Assay

Cells were treated with either vehicle or 10⁻⁸ m 1,25(OH)₂D₃ for 4 h followed by cross-linking with 1% formaldehyde for 15 min. Chromatin immunoprecipitation was performed as described previously (30). DNA fragments were purified using QIAquick PCR purification kits (Qiagen, Valencia, CA) and subjected to PCR using primers designed to amplify fragments of the Cyp24a1 promoter C/EBP motif at −395/−388 (forward, 5′-GAAATTCTGCAAACCGCATT-3′; reverse, 5′-CCAGACTTCCCTTGGATGAA-3′). PCR using the primers to amplify the upstream region of the Cyp24a1 promoter (−837/−567) was used as a negative control. PCR analysis was carried out in the linear range of DNA amplification. PCR products were resolved on a 2% agarose gel and visualized using ethidium bromide staining. DNA acquired prior to precipitation was collected and used as the input. 10% of input was used for PCR evaluation.

In re-chromatin immunoprecipitation (re-ChIP) experiments, complexes were eluted by incubation for 30 min at 37 °C in 60 μl of elution buffer containing 10 mm dithiothreitol. The eluted samples were diluted 50 times with ChIP dilution buffer and subjected again to the ChIP procedure with specific antibodies.

Statistical Analysis

Results are expressed as the mean ± S.E., and significance was determined by analysis with Student's t test for two group comparison or analysis of variance for multiple group comparison.

RESULTS

BRG1-containing SWI/SNF Complex, VDR, and C/EBPβ Cooperate in the Regulation of Cyp24a1 Transcription

Previous studies have shown that VDR-induced transcription is mediated by 1,25(OH)₂D₃-dependent recruitment of coregulatory complexes, which include p160 coactivators with histone acetyltransferase activity (1–4). For chromatin remodeling to occur, the effects of acetylation are complemented by structural modifications that are ATP-dependent (19). An important ATP-dependent chromatin remodeling factor is the SWI/SNF complex (6). Induction of Cyp24a1 transcription is one of the most pronounced effects of 1,25(OH)₂D₃. To determine whether SWI/SNF activity contributes to VDR-mediated Cyp24a1 transcription, we examined the BRG1- and Brm-deficient cell line SW-13. SW-13 cells were co-transfected with VDR and the rat Cyp24a1 promoter (−1367/+74). In the absence of BRG1 and Brm but under conditions of VDR overexpression, minimal activation of Cyp24a1 transcription by 1,25(OH)₂D₃ was observed (Fig. 1A). However, VDR-mediated activation of Cyp24a1 transcription in SW-13 cells was preferentially enhanced by BRG1 (Fig. 1A) compared with Brm (no significant induction compared with 1,25(OH)₂D₃ treatment of SW-13 cells at 0.1 and 0.3 μg of Brm (data not shown)). A similar low level of induction of Cyp24a1 transcription by 1,25(OH)₂D₃ and preferential enhancement by BRG1 were observed using the SWI/SNF-defective cell line C33A (Fig. 1A, inset). BRG1-DN (K798R), which has a mutation in the ATPase domain and is incapable of remodeling chromatin, was found to inhibit enhancement of Cyp24a1 transcription by BRG1 in SW-13 cells (Fig. 1A). BRG1-DN also inhibited 1,25(OH)₂D₃ induction of Cyp24a1 transcription in SWI/SNF-competent COS-7 cells in a dose-dependent manner, resulting in a maximum inhibition of 2.8 ± 0.3-fold at a concentration of 0.3 μg of BRG1-DN (p < 0.05 compared with 1,25(OH)₂D₃ induction in the absence of BRG1-DN) (Fig. 1B). There was no effect of BRG1-DN on basal levels of Cyp24a1 transcription (Fig. 1B). In additional studies in COS-7 cells, suboptimal 1,25(OH)₂D₃ Cyp24a1 transcription was significantly enhanced by BRG1 (2.3 ± 0.3-fold at a concentration of 0.1 μg of BRG1; p < 0.05 compared with 1,25(OH)₂D₃ induction in the absence of BRG1). Together these results indicate that BRG1 contributes to VDR-mediated transcriptional activation of Cyp24a1.

FIGURE 1.

BRG1 and the vitamin D receptor cooperate in the regulation of Cyp24a1 transcription. A, SW-13 or C33A (inset) cells were plated in a 24-well culture dish, and cells in each well were co-transfected with 0.3 μg of the rat Cyp24a1 promoter construct (−1367/+74) and 0.02 μg of hVDR expression plasmid. SW-13 and C33A cells were also transfected with increasing concentrations of BRG1 and/or BRG1-DN expression plasmids. Cells were treated with 1,25(OH)₂D₃ (10 nm) for 24 h and harvested, and luciferase activity was determined. The data were normalized based on protein content of the lysates, and Cyp24a1 promoter activity is represented as -fold induction (error bars represent S.E.; n = at least 3 observations per group) by comparison with basal levels. *, p < 0.05 BRG1-transfected, 1,25(OH)₂D₃-treated compared with vector-transfected, 1,25(OH)₂D₃-treated; +, p < 0.05 BRG1-DN + BRG1-transfected, 1,25(OH)₂D₃-treated compared with BRG1-transfected (0.3 μg), 1,25(OH)₂D₃-treated. A Western blot of BRG1 shows that similar levels of BRG1 and BRG1-DN are expressed when equal amounts of DNA are transfected. B, COS-7 cells were co-transfected with the rat Cyp24a1 promoter construct (−1367/+74) and VDR (0.02 μg) in the absence or presence of increasing concentrations of BRG1-DN and treated with vehicle or 1,25(OH)₂D₃ (10 nm) for 24 h. Cyp24a1 promoter activity was measured by firefly luciferase activity/protein concentration and is represented as -fold induction (error bars represent S.E.; n = 3–6 observations per group) by comparison with basal levels. *, p < 0.05 compared with vector-transfected, 1,25(OH)₂D₃-treated. For all transcription experiments, empty vectors were used to keep the total DNA concentration the same. VDRE, vitamin D response element; LUC, luciferase.

Previous studies from our laboratory showed that C/EBPβ is induced by 1,25(OH)₂D₃ in kidney and osteoblastic cells and is a potent enhancer of VDR-mediated Cyp24a1 transcription (26). C/EBPβ has also been reported to recruit the SWI/SNF complex to regulate cell type-specific genes (15–17). Hence, we next investigated the role of BRG1 in the C/EBPβ enhancement of 1,25(OH)₂D₃-induced Cyp24a1 transcription. C/EBPβ was cotransfected in COS-7 cells with VDR and the rat Cyp24a1 promoter construct (−1367/+74) in the presence or absence of BRG1-DN. Cells were treated with vehicle or 1,25(OH)₂D₃. BRG1-DN inhibited the stimulatory effect of C/EBPβ on VDR-mediated Cyp24a1 transcription in a dose-dependent manner (Fig. 2A). A similar inhibition by BRG1-DN was observed using MC3T3 osteoblastic cells, LLC-PK1 kidney cells and UMR osteoblastic cells (which contain endogenous VDR; Refs. 31–33) transfected with C/EBPβ and the Cyp24a1 promoter and treated with 1,25(OH)₂D₃ (data not shown). RT-PCR analysis also showed inhibition of the stimulatory effect of C/EBPβ on 1,25(OH)₂D₃-induced Cyp24a1 mRNA levels by BRG1-DN (Fig. 2B). In addition, Western blot analysis showed that in the presence of BRG1-DN 1,25(OH)₂D₃ induction and the stimulatory effect of C/EBPβ on CYP24A1 protein expression are repressed (Fig. 2C). When a rat Cyp24a1 promoter construct with the C/EBP site deleted (−298/+74) was used, 1,25(OH)₂D₃-mediated induction of Cyp24a1 transcription was neither enhanced by C/EBPβ nor inhibited by BRG1-DN (Fig. 2D). Similar results were observed using a mutated construct of the Cyp24a1 promoter in which the −395/−388 C/EBP binding site is mutated (data not shown). These findings indicate that both C/EBPβ and BRG1 contribute to 1,25(OH)₂D₃-mediated regulation of CYP24A1 expression.

FIGURE 2.

Cooperation between BRG1 and C/EBPβ in VDR-mediated Cyp24a1 transcription. A, COS-7 cells were co-transfected with the rat Cyp24a1 promoter construct (−1367/+74) and 0.02 μg of hVDR expression plasmid in the absence or presence of C/EBPβ expression plasmid and increasing concentrations of BRG1-DN plasmid. The cells were treated with vehicle or 10 nm 1,25(OH)₂D₃ for 24 h, and luciferase activity was determined. The data were normalized based on protein content of the lysates, and Cyp24a1 promoter activity is represented as -fold induction (error bars represent S.E.; n = 3–6 observations per group) by comparison with basal levels. +, p < 0.05 compared with 1,25(OH)₂D₃-treated; *, p < 0.05 compared with C/EBPβ-transfected, 1,25(OH)₂D₃-treated. A Western blot of C/EBPβ shows that similar levels of C/EBPβ are expressed in the presence and absence of BRG1-DN. B, RT-PCR analysis of Cyp24a1 mRNA was performed using total RNA from UMR cells. Cells were transfected with empty vector, C/EBPβ, or C/EBPβ and BRG1-DN expression vectors. 24 h post-transfection, cells were treated with vehicle (lane 1) or with 1,25(OH)₂D₃ (10 nm) (lanes 2–4) for another 24 h. Error bars represent S.E. C, CYP24A1 Western blotting was performed using total protein from UMR cells. Cells were transfected with empty vector, C/EBPβ, BRG1-DN, or C/EBPβ and BRG1-DN expression vectors. 24 h post-transfection, cells were treated with vehicle (lane 1) or with 1,25(OH)₂D₃ (10 nm) (lanes 2–5) for another 24 h. D, COS-7 cells were co-transfected with the rat Cyp24a1 promoter construct −298/+74 with the C/EBP site (−395/−388) deleted and VDR in the presence or absence of C/EBPβ or C/EBPβ with increasing concentrations of BRG1-DN followed by treatment with 1,25(OH)₂D₃ for 24 h. Cyp24a1 promoter activity was measured by firefly luciferase activity/protein concentration and is represented as -fold induction by comparison with basal levels (error bars represent S.E.; n = 3–6 observations per group). VDRE, vitamin D response element; LUC, luciferase.

To further understand the mechanisms involved in the cooperation between C/EBPβ and BRG1 in 1,25(OH)₂D₃ induction of CYP24A1 in vivo, we first assessed via co-immunoprecipitation whether BRG1 and C/EBPβ are components of the same nuclear complex. Nuclear extracts prepared from UMR cells, MC3T3 cells, mouse primary osteoblastic cells, and PCT cells were immunoprecipitated using C/EBPβ antibody followed by immunoblotting for BRG1 (Fig. 3A, left panel) or immunoprecipitated using BRG1 antibody followed by immunoblotting for C/EBPβ (Fig. 3A, right panel). We found that C/EBPβ and BRG1 interact and are components of the same nuclear complex. Next, we examined the recruitment of BRG1 and C/EBPβ to the Cyp24a1 promoter in response to 1,25(OH)₂D₃ using a ChIP assay. ChIP with C/EBPβ antibody and re-ChIP with BRG1 antibody showed that in vivo C/EBPβ and BRG1 bind simultaneously at the C/EBP site of the Cyp24a1 promoter in response to treatment with 1,25(OH)₂D₃ in UMR cells, MC3T3 cells, mouse primary osteoblastic cells, and PCT cells (Fig. 3B).

FIGURE 3.

C/EBPβ and BRG1 are components of the same nuclear complex and are recruited to the C/EBP site of the Cyp24a1 promoter. A, nuclear extracts were prepared from UMR cells, MC3T3 cells, mouse primary osteoblastic cells, and PCT cells and used for immunoprecipitation (IP) with C/EBPβ antibody, BRG1 antibody, or control rabbit IgG antibody. Similar results were observed in three independent experiments. B, ChIP analysis of C/EBPβ and re-ChIP analysis of BRG1 binding to the C/EBP site of the Cyp24a1 promoter. UMR cells, MC3T3 cells, mouse primary osteoblastic cells, and PCT cells were treated with vehicle or 1,25(OH)₂D₃ for 4 h and cross-linked by 1% formaldehyde for 15 min. Cross-linked cell lysates were subjected to immunoprecipitation first with C/EBPβ antibody (α-C/EBPβ) and then with BRG1 antibody (α-BRG1). DNA precipitates were isolated and then subjected to PCR using specific primers designed according to the C/EBP site on the Cyp24a1 promoter. Input DNA (10%) was collected before immunoprecipitation. Con, control.

PRMT5 Inhibits 1,25(OH)₂D₃-induced Cyp24a1 Transcription

Previous studies have shown an interplay between the SWI/SNF chromatin-remodeling enzymes and histone-modifying enzymes including PRMTs (18–20, 27, 28). Because PRMT5, which has been reported to be involved in transcriptional repression, has been shown to be associated with the BRG1-based SWI/SNF complex (28), we examined a possible role of PRMT5 in the regulation of Cyp24a1. We found that 1,25(OH)₂D₃-induced Cyp24a1 transcription is down-regulated in the presence of PRMT5 (Fig. 4A). A catalytically inactive form of PRMT5 with two point mutations (G367A/R368A) revealed no repressive influence on 1,25(OH)₂D₃-induced Cyp24a1 luciferase expression (Fig. 4A). 1,25(OH)₂D₃-induced Cyp24a1 mRNA was also repressed by PRMT5 (Fig. 4B). Unlike Cyp24a1 mRNA, 1,25(OH)₂D₃ induction of Vdr mRNA was unaffected by PRMT5 in UMR, DCT, and PCT cells (not shown). Using a PRMT5 antisense cell line (27), we found that Cyp24a1 mRNA is up-regulated when PRMT5 levels are reduced (Fig. 4C).

FIGURE 4.

PRMT5 suppresses 1,25(OH)₂D₃-induced Cyp24a1 transcription and mRNA. A, COS-7 cells were co-transfected with the rat Cyp24a1 promoter plasmid (−1367/+74) and 0.02 μg of pAV-hVDR expression vector in the presence or absence of PRMT5 or mutant PRMT5. Cells were treated with vehicle or 1,25(OH)₂D₃ for 24 h. Cyp24a1 promoter activity was measured by firefly luciferase activity/protein concentration and is represented as percent maximal response by comparison with basal levels (error bars represent S.E.; n = 3–6 observations per group). *, p < 0.05 compared with vector-transfected, 1,25(OH)₂D₃-treated. A Western blot of PRMT5 shows that similar levels of PRMT5 and mutant PRMT5 are expressed when equal amounts of DNA are transfected. B, RT-PCR analysis of Cyp24a1 mRNA. UMR, DCT, or PCT cells in 100-mm tissue culture dishes were transfected with PRMT5 or empty vector and treated with 1,25(OH)₂D₃ (D) (10 nm) or vehicle for 24 h. Data represent the mean of three independent experiments for each cell line, and error bars represent the S.E. *, p < 0.05 compared with vector-transfected, 1,25(OH)₂D₃-treated. A similar inhibition of 1,25(OH)₂D₃-induced Cyp24a1 mRNA by PRMT5 was also seen in MC3T3 cells (not shown). Note that there was no effect of PRMT5 on Opn mRNA in UMR cells. C, RT-PCR analysis of Cyp24a1 mRNA in NIH3T3 cells and NIH3T3 cells stably expressing antisense (AS) PRMT5. RT-PCR was performed using 2 μg of total RNA from either NIH3T3 cells (first two lanes) or antisense PRMT5 cells (last two lanes) treated with vehicle (−D) or 1,25(OH)₂D₃ (+D) for 24 h. *, p < 0.05 compared with vector-transfected, 1,25(OH)₂D₃-treated NIH3T3 cells. Data represent the mean of three independent experiments, and error bars represent the S.E. VDRE, vitamin D response element; LUC, luciferase.

It has been found previously that PRMT5 interacts with BRG1, and together they are part of a complex involved in transcriptional repression (28). Thus we examined the effect of PRMT5 on BRG1 enhancement of Cyp24a1 transcription. We found that PRMT5 inhibits the BRG1-mediated enhancement of 1,25(OH)₂D₃-induced Cyp24a1 transcription in a dose-dependent manner (Fig. 5A). A similar inhibition of C/EBPβ enhancement of VDR-mediated Cyp24a1 transcription by PRMT5 was also observed (data not shown). Furthermore, in SWI/SNF-defective SW-13 cells, PRMT5 (even at high concentrations; 0.1 and 0.2 μg) did not have a repressive effect on the low level of 1,25(OH)₂D₃-mediated activation of Cyp24a1 transcription observed in these cells (data not shown). Similar to what has been reported previously using HeLa cell nuclear extracts (27), co-immunoprecipitation experiments indicated that PRMT5 and BRG1 are components of the same nuclear complex in osteoblastic and renal cells (Fig. 5B; BRG1-deficient SW-13 cells were used as a negative control). When a rat Cyp24a1 promoter construct with the C/EBP site mutated (−671/+74) (Fig. 5C) or a deletion construct of the Cyp24a1 promoter (−298/+74), which does not include the −395/−388 C/EBP binding site (not shown), was used, neither enhanced transcription by C/EBPβ nor suppression of 1,25(OH)₂D₃-induced Cyp24a1 transcription by PRMT5 was observed. When COS-7 cells were transfected with a thymidine kinase-luciferase construct containing six consecutive vitamin D response elements, PRMT5 suppression of 1,25(OH)₂D₃-induced transcription was also not observed (not shown). These findings indicate a role for BRG1 and the requirement of the C/EBP element in the Cyp24a1 promoter for the inhibitory effect of PRMT5.

FIGURE 5.

PRMT5 suppresses BRG1 enhancement of 1,25(OH)₂D₃-induced Cyp24a1 transcription via their interaction at the C/EBP site. A, COS-7 cells were co-transfected with the rat Cyp24a1 promoter construct (−1367/+74) and 0.02 μg of pAV-hVDR in the presence or absence of BRG1 and increasing concentrations of PRMT5. Cells were treated with 1,25(OH)₂D₃ for 24 h. Cyp24a1 promoter activity was measured by firefly luciferase activity/protein concentration and is represented as percent maximal response as compared with basal levels (error bars represent S.E.; n = 3–6 observations per group). There was no significant effect of PRMT5 on basal levels of Cyp24a1 transcription. *, p < 0.05 compared with 1,25(OH)₂D₃-treated, BRG1 (0.1 μg)-transfected. B, PRMT5 and BRG1 are components of the same nuclear complex. Nuclear extracts were prepared from UMR cells, MC3T3 cells, PCT cells, and DCT cells and used for immunoprecipitation (IP) with PRMT5 antibody, BRG1 antibody, or control rabbit IgG antibody. SW-13 cells were used as a negative control. Similar results were observed in three independent experiments. C, COS-7 cells were transfected with the rat Cyp24a1 promoter CAT construct (−671/+74) wild type (WT) or with the C/EBP site mutated (MT) and 0.02 μg of pAV-hVDR in the presence or absence of C/EBPβ and/or PRMT5 followed by treatment with 1,25(OH)₂D₃ for 24 h. CAT activity is represented as percent maximal response by comparison with basal levels (error bars represent S.E.). Note the suppression by PRMT5 using the wild-type but not the mutant promoter.

To determine the specificity of inhibition by PRMT5 for Cyp24a1, we examined other targets of 1,25(OH)₂D₃ for inhibition by PRMT5. TRPV6 is an epithelial calcium channel involved in active intestinal calcium transport (34). TRPV6 mRNA was up-regulated when Caco-2 cells were treated with 1,25(OH)₂D₃. A suppressive effect of PRMT5 was not observed (Fig. 6A). OPN, a phosphoprotein that modulates both bone mineralization and resorption, is induced in the presence of 1,25(OH)₂D₃ in MC3T3 cells (31). 1,25(OH)₂D₃ induction of Opn mRNA was not repressed in cells transfected with PRMT5 (Fig. 6B). Similar results were observed using UMR cells (Fig. 4B).

FIGURE 6.

PRMT5 does not suppress 1,25(OH)₂D₃ induction of TRPV6 or Opn mRNA. A, RT-PCR analysis of TRPV6 mRNA in Caco-2 cells. Caco-2 cells in 100-mm tissue culture dishes were transfected with PRMT5 expression vector or empty vector and treated with 10 nm 1,25(OH)₂D₃ for 24 h. Data represent the mean of three independent experiments, and error bars represent the S.E. B, RT-PCR analysis of Opn mRNA in MC3T3 cells. MC3T3 cells in 100-mm tissue culture dishes were transfected with PRMT5 expression vector or empty vector and treated with 10 nm 1,25(OH)₂D₃ for 24 h. Data represent the mean of three independent experiments, and error bars represent the S.E. For A and B, p > 0.5 1,25(OH)₂D₃-treated versus PRMT5-transfected and 1,25(OH)₂D₃-treated.

H3R8 and H4R3 have been reported previously to be histone substrates for PRMT5 (27). Our findings as well as those of others have shown that PRMT5 binds to BRG1 (28). BRG1 is recruited to the C/EBP site on the Cyp24a1 promoter in the presence of 1,25(OH)₂D₃. Thus, to determine the mechanism involved in suppression of Cyp24a1 transcription by PRMT5 in vivo, ChIP assays were performed using primers for the C/EBP site on the Cyp24a1 promoter in 1,25(OH)₂D₃-treated cells. In cells treated with 1,25(OH)₂D₃, in response to PRMT5 transfection, there was increased symmetrical dimethylation of H3R8 and H4R3 as compared with 1,25(OH)₂D₃ treatment in cells transfected with vector alone (Fig. 7A). These data suggest that H3R8 and H4R3 methylations mediate at least in part the PRMT5 repression of VDR-mediated Cyp24a1 transcription as depicted in our mechanistic model (Fig. 7B).

FIGURE 7.

PRMT5 symmetrically dimethylates H3R8 and H4R3 at the C/EBP site of the Cyp24a1 promoter. A, ChIP analysis of PRMT5, H3R8Me2s, and H4R3Me2s binding to the C/EBP site of the Cyp24a1 promoter. UMR cells and PCT cells were treated with vehicle or 1,25(OH)₂D₃ for 4 h and cross-linked by 1% formaldehyde for 15 min. DNA precipitates were isolated and then subjected to PCR using specific primers designed according to the C/EBP site on the Cyp24a1 promoter. Input DNA (10%) was collected before immunoprecipitation. Error bars represent S.E. B, mechanistic model depicting induction and repression of Cyp24a1 transcription. 1,25(OH)₂D₃ induction of Cyp24a1 involves cooperation among C/EBPβ, BRG1, and VDR. PRMT5, which is recruited to the C/EBP site by BRG1, represses Cyp24a1 transcription via symmetrical dimethylation of H3R8 and H4R3. VDRE, vitamin D response element; RXR, retinoid X receptor.

DISCUSSION

Our findings show that PRMT5 is a negative regulator of Cyp24a1 transcription. Here, we provide evidence for the first time of cross-talk between protein arginine methylation and the SWI/SNF complex in the regulation of VDR-mediated transcription. We show that BRG1 associates with C/EBPβ and cooperates with VDR and C/EBPβ in regulating Cyp24a1 transcription. PRMT5, which interacts with BRG1, represses Cyp24a1 transcription. Our findings indicate the requirement of the C/EBP site for the inhibitory effect of PRMT5 via its methylation of H3R8 and H4R3. Our findings suggest that the SWI/SNF complex together with epigenetic modification by PRMT5 plays key roles in the regulation of Cyp24a1 and therefore in the maintenance of calcium homeostasis.

In addition to VDR-mediated transcription, BRG1 has also been shown to be a coregulator of transcription mediated by other steroid receptors. Estrogen receptor α fails to activate estrogen-responsive elements in SWI/SNF-defective cells (10). Similar to our studies, in SWI/SNF-defective cells, estrogen receptor α activity could be restored by expression of BRG1 (10). ChIP assays have shown that BRG1 binds to estrogen-responsive promoters in response to estrogen (10, 35). It has been suggested that estrogen receptor α recruits the BRG1 complex through the BRG1 complex subunit BAF57 (36). Transcriptional dependence for BRG1 activity has also been observed for GR-responsive promoters including mouse mammary tumor virus, p21, and 11β-hydroxysteroid dehydrogenase type 2 (11, 37). Studies examining hormone-mediated transcriptional activation of mouse mammary tumor virus have shown that the progesterone receptor can compete with GR for available BRG1, resulting in inhibition of GR-mediated transactivation (38). These findings further suggest a key role for BRG1 in the modulation of GR-mediated transcription. BRG1 was also shown to be required for androgen receptor activation of mouse mammary tumor virus transcription (39). However, Brm-containing complexes have been reported to be preferred in androgen receptor-mediated transcriptional activation of prostate-specific antigen and probasin (40). In the absence of SWI/SNF activity, androgen receptor-dependent activation of prostate-specific antigen transcription could not be restored by BRG1 but was strongly activated by Brm (40). Different functions for BRG1 and Brm have also been shown in gene knock-out experiments because loss of BRG1, but not Brm, is embryonic lethal (13, 14). In osteoblast differentiation, antagonistic roles for Brm and BRG1 have been reported (41). In our study, we found that VDR-mediated Cyp24a1 transcription is preferentially restored in SWI/SNF-deficient cells by BRG1. Whether there is a preference for specific components of the SWI/SNF complex in the regulation of vitamin D target genes, which is dependent on promoter context and mediated by specific protein interactions, remains to be determined.

BRG1 has been shown to direct different cellular processes by recruitment to cis elements through divergent transcription factors including hormone receptors, the erythroid factors erythroid Krüppel-like factor and GATA-1 (which activate the β-globin gene) and C/EBPβ (16, 17, 42, 43). Similar to our findings related to the regulation of Cyp24a1 transcription, C/EBPβ has been reported to recruit and cooperate with BRG1 to regulate the expression of myeloid genes (15), the osteocalcin gene in osteoblastic cells (16), and the mammary tissue-specific β- and γ-casein genes (17). We reported previously that C/EBPβ is a 1,25(OH)₂D₃ target gene in kidney and osteoblastic cells that can cooperate with CBP/p300 to augment VDR-mediated Cyp24a1 transcription (26). C/EBPβ is also an important factor in the control of the transcription of 25-hydroxyvitamin D₃ 1α-hydroxylase (CYP27B1), the enzyme involved in the synthesis of 1,25(OH)₂D₃ (44–46). We have reported previously that C/EBPβ and BRG1 also cooperate in the regulation of Cyp27b1 (44). These results indicate that C/EBPβ can recruit BRG1, resulting in chromatin remodeling and transcriptional regulation of specific vitamin D target genes.

In this study, we found that PRMT5, which interacts with BRG1, represses 1,25(OH)₂D₃-induced Cyp24a1 transcription and that the C/EBP site is required for the PRMT5-mediated repression. The BRG1-based SWI/SNF complex containing PRMT5 has been found in association with the mSin3A-HDAC2 complex and is implicated in the repression of the MYC target gene cad (carbamoyl-phosphate synthase-aspartate carbamoyltransferase-dihydroorotase) (28). The PRMT5 SWI/SNF complex is also involved in transcriptional repression of ST7, NM23, and retinoblastoma-like protein 2 (RBL2) tumor suppressor genes (27, 47). It has been suggested that SWI/SNF-associated PRMT5 is involved in silencing these genes through repressive histone marks including methylation of H3R8 and H4R3 (27, 47). In our study, we found that repression of 1,25(OH)₂D₃-induced Cyp24a1 transcription by PRMT5 involves recruitment of PRMT5 by BRG1, which is bound to C/EBPβ at the C/EBP site, and at least in part symmetrical dimethylation of H3R8 and H4R3. It should be noted that in addition to SWI/SNF other factors have been reported to be involved in PRMT5-mediated repression including COPR5, ZNF224, and the LIM protein AJUBA (48–51). COPR5 binds to PRMT5 and is thought to bridge PRMT5 to chromatin on a subset of target genes (48). ZNF224, a zinc finger protein, is involved together with PRMT5 in the repression of the aldolase genes (49). AJUBA has been shown to recruit PRMT5 to mediate suppression of SNAIL as well as retinoic acid receptor-dependent transcription (50, 51). A mechanism of gene silencing involving cooperation between histone methylation by PRMT5 and DNA methylation has also been reported (52). Further studies are needed to determine whether PRMT5-mediated repression of vitamin D target genes involves mechanisms in addition to cross-talk among C/EBPβ, SWI/SNF, and PRMT5. We found that, unlike Cyp24a1, 1,25(OH)₂D₃-induced TRPV6 and Opn mRNAs are not suppressed by PRMT5. A possible explanation for the lack of suppression of TRPV6 mRNA by 1,25(OH)₂D₃ is that C/EBPβ has not been reported to be involved in the regulation of TRPV6. Whether PRMT5 regulates other vitamin D target genes that have effects on cancer cell proliferation or the immune system remains a topic of future investigation.

Other studies relating to hormone-dependent regulation of transcription have noted, similar to our studies, that BRG1 can play a role in gene silencing as well as activation. Although SWI/SNF is a critical component required for transcriptional activation by GR of mouse mammary tumor virus, p21, and 11β-hydroxysteroid dehydrogenase type 2, repression of GR-mediated expression of liver tryptophan oxygenase and pituitary proopiomelanocortin, which is dependent on BRG1 and HDAC2 recruitment, has also been reported (53, 54). In addition, BRG1 has been found to be a coactivator and corepressor at the same estrogen receptor-responsive promoter in breast cancer cells depending on differential cooperation with and recruitment of HDAC, p300, BRG1-associated factors, and prohibitin (a potential tumor suppressor) (55). The ability of C/EBPβ to activate target genes has also been reported to be modulated depending on its association with coactivators or corepressors (56). Thus BRG1, which binds to C/EBPβ, may act as a scaffold that allows recruitment of BRG1-interacting proteins to the promoter in response to activating or repressing signals. The dual role of BRG1 may depend on hormonal context and intracellular environment including the presence and expression level of coactivators or corepressors and the expression level of the target gene.

In conclusion, these studies provide new insight into mechanisms involved in the regulation of Cyp24a1. We describe for the first time functional cooperation between the SWI/SNF complex and C/EBPβ in the induction of VDR-mediated Cyp24a1 transcription. In addition, we demonstrate a novel mechanism of negative regulation of CYP24A1 that includes epigenetic modification and cross-talk between PRMT5 and the SWI/SNF complex. This mechanism of negative regulation may be important to prevent catabolism of 1,25(OH)₂D₃ at times when protection against hypercalcemia is not needed. We and others have noted that renal Cyp24a1 increases with age and have suggested that increased catabolism of 1,25(OH)₂D₃ contributes to age-related bone loss (57, 58). Recent studies have shown an age-dependent decrease in renal PRMT5 (59). Thus it is possible that one factor involved in the age-related increase in renal Cyp24a1 may be decreased levels of renal PRMT5. Enhanced catabolism of 1,25(OH)₂D₃ by glucocorticoids in bone cells has been suggested as one mechanism that contributes to glucocorticoid-induced bone loss (30, 60). PRMT5, by inhibiting Cyp24a1 in bone cells, may also have a physiological role in protecting against glucocorticoid enhancement of 1,25(OH)₂D₃ catabolism in osteoblastic cells. In the regulation of Cyp24a1, PRMT5 and methylation of H3R4 or H3R8 may also be involved in the cyclical transcriptional process that requires both activating and repressive epigenetic mechanisms (35). Our findings indicate that the SWI/SNF complex and PRMT5 may be key factors involved in the regulation of 1,25(OH)₂D₃ catabolism and therefore in the maintenance of calcium homeostasis by vitamin D.