- Research article
- Open Access
Arginine methyltransferase CARM1/PRMT4 regulates endochondral ossification
© Ito et al; licensee BioMed Central Ltd. 2009
Received: 17 December 2008
Accepted: 2 September 2009
Published: 2 September 2009
Chondrogenesis and subsequent endochondral ossification are processes tightly regulated by the transcription factor Sox9 (SRY-related high mobility group-Box gene 9), but molecular mechanisms underlying this activity remain unclear. Here we report that coactivator-associated arginine methyltransferase 1 (CARM1) regulates chondrocyte proliferation via arginine methylation of Sox9.
CARM1-null mice display delayed endochondral ossification and decreased chondrocyte proliferation. Conversely, cartilage development of CARM1 transgenic mice was accelerated. CARM1 specifically methylates Sox9 at its HMG domain in vivo and in vitro. Arg-methylation of Sox9 by CARM1 disrupts interaction of Sox9 with beta-catenin, regulating Cyclin D1 expression and cell cycle progression of chondrocytes.
These results establish a role for CARM1 as an important regulator of chondrocyte proliferation during embryogenesis.
The precise patterning of the developing skeletal framework relies on the appropriate control of chondrogenesis, a multistep process during which mesenchymal cells differentiate into chondrocytes[1, 2]. This process is tightly regulated by transcription factors, including Sox9 [3–6]. Mice lacking Sox9 display distortion of numerous cartilage-derived skeletal structures. In addition, mice overexpressing Sox9 in chondrocytes show dwarfism with decreased chondrocyte proliferation and delayed endochondral bone formation. However, the precise mechanism how Sox9 regulates chondrogenesis both spatially and temporally is still largely unknown.
CARM1 belongs to a family of arginine-specific protein methyltransferases (PRMTs), which includes at least eight members (PRMT1-8) . All PRMT family members share a core arginine methyltransferase region composed of a conserved Ado-Met binding domain and a more divergent C-terminal domain. CARM1 has been shown to synergistically activate transcription with nuclear receptors in combination with other coactivators, such as p160 family, p300/CBP and SRC-2/TIF2/GRIP1[9, 10]. After recruitment to promoters of steroid-responsive genes, CARM1 methylates specific residues (Arg17 and Arg26) at the N-terminus of histone H3 resulting in transcriptional activation[11, 12].
Results and Discussion
Skeletal phenotype of CARM1 null mice
Bone development of CARM1-transgenic mice
For gain-of-function analysis, we generated transgenic mice in which CARM1 expression is driven by the ubiquitously expressed beta-actin promoter (Additional file 2). In contrast to null mutants, CARM1-transgenic mice analyzed at E18.5 were larger than controls (Figure 1E: Double stain, Tg). Alizarin red positive regions appeared at the shaft of humerus in E14.5 CARM1-transgenic mice but were not visible in wild type mice (Figure 1E: Double stain, Tg limb, arrow heads). von Kossa staining showed absorption of calcified regions was also accelerated in E14.5 transgenic compared with wild type mice (Figure 1F: von Kossa), while SafraninO staining was unchanged and chondrocyte differentiation marker, Col2a1 and Col10a1 expression was not significantly altered in cartilage in E14.5 transgenic compared to wild type mice (Figure 1F: SafraninO, Tg, Additional file 1). These data indicate that endochondral bone formation in CARM1 transgenic mice is accelerated relative to wild type mice, although we could not exclude the possibility that CARM1 may also directly regulate osteoblasts differentiation.
CARM1 expression in growth plates
Sox9 and CARM1 interaction
We next asked whether CARM1 and Sox9 interact. In a GST-pull down assay, full length GST-Sox9 protein bound to in vitro translated CARM1 protein, suggesting direct interaction (Figure 2B: GST-pull down). To define the interaction domain of Sox9 with CARM1, we performed the assay using in vitro translated CARM1 and bacterially expressed GST-Sox9 fragments. Full length GST-Sox9 (1-507aa) and 328-508aa and 423-508aa fragments interacted with CARM1, while 1-327aa and 1-422aa fragments did not (Figure 2B GST-pull down). Taken together, CARM1 likely interacts with Sox9 via the Sox9 carboxy-terminal domain, which was previously reported to be a transcription activation domain.
CARM1 methylates Sox9
Since CARM1 methylates some non-histone proteins, such as CBP/p300 and splicing factors, we asked whether Sox9 can serve as a substrate for CARM1. Recombinant Flag-Sox9 was incubated with recombinant CARM1/PRMT4 or PRMT1 in the presence of 3H-labeled S-adenosyl-L-methionine ([3H]AdoMet) as a methyl donor. Histone H3 was assayed as a positive control because H3 can be methylated by CARM1 and PRMT1. We observed that Sox9 could be methylated by CARM1/PRMT4, but not by PRMT1 (Figure 2C). Arginine methylation was also seen in endogenous Sox9 immunoprecipitated from mouse (Figure 2D, left) or human (data not shown) primary cultured chondrocytes. A recent report showing that the HMG domain of HMGA1a can be arginine-methylated prompted us to determine whether arginine residues in the HMG domain of Sox9 (74-179aa) are CARM1 targets. Based on in vitro methylation assays, simultaneous mutation of five arginine residues within the HMG domain (mt(b)) completely abolished methylation signals, whereas mutants exhibiting R177K, R178K and R179K (mt(a)) were methylated similarly to wild type (Figure 2D, right &2E). These data indicate that CARM1 methylates multiple arginine residues within the HMG domain of Sox9.
CARM1 regulates chondrocytes proliferation
CARM1 regulates Cyclin D1expression
Cyclin D1 expressed in proliferating chondrocytes of growth plates is required for normal chondrocyte proliferation[22, 23]. In growth plates of E16.5 CARM1-null mutants, Cyclin D1 mRNA levels were reduced (Figure 3B: ISH of Cyclin D1 in CARM1-null), whereas Col2a1 was expressed in the limb bud of CARM1-null mutants (Figure 3B: RT-PCR). By contrast, Cyclin D1 mRNA levels were remarkably increased in E14.5 CARM1-transgenic mice (Figure 3B: ISH of Cyclin D1 in CARM1-Tg). These data suggest that CARM1 promote Cyclin D1 gene expression. To determine how CARM1 regulates Cyclin D1 expression, we performed Cyclin D1 promoter assays in human embryonic kidney 293 cells, which express endogenous Lef1. On the -963CD1 Cyclin D1 promoter reporter plasmid and on a Tcf mt reporter plasmid with a mutation in the Tcf binding site, Sox9 inhibited basal promoter activity, as previously reported (Figure 3C). CARM1 overexpression rescued inhibition by Sox9 in this assay, suggesting that CARM1 may inhibit Sox9 and beta-catenin complex formation and thus increase Cyclin D1 expression. In contrast CARM1 and Sox9 did not activate transcription using the Tcf mt reporter plasmid, which lacks beta-catenin reactive sites. With the mutation at all 74,152,177,178,179 shows strong suppression at CyclinD1 promoter, however, this suppression did not relieved by overexpression of CARM1 (data not shown). This suggests that CARM1 and Sox9 may cooperatively regulate CyclinD1 promoter activity.
Sox9 function is likely regulated by post-translational modifications such as phosphorylation, ubiquitination or sumoylation [24–26]. Here we demonstrate that Sox9 is Arg-methylated by CARM1. CARM1 is involved in the epigenetic programming of early embryo development, and also early T cell development. Our study shows that CARM1 also plays a key role in cartilage development. Further analysis of the role of CARM1 on chromatinized and non-chromatinized substrates during different developmental stages should indicate the physiological role of arginine methylation signals.
Tissue Culture Methods and Transfections
The human chondrosarcoma cell line (SW1353) was grown in Dulbecco's modified Eagle's medium (Cellgro, Mediatech) supplemented with 10% fetal calf serum and penicillin/streptomycin (Sigma). Cells were transfected using GeneJammer (Stratagene) according to the manufacturer. Amounts of transfected plasmids were as follows: for Sox9-dependent activation, 50 ng of the Cyclin D1 promoter plasmids, -963CD1, Tcf mt, and 100 ng of pCDNA3-beta-catenin, 100 ng of Flag-tagged Sox9 plasmids and pCMX-CARM1 expression plasmid. Luciferase activity was normalized using 1 ng of pcDNA-LacZ as an internal control for transfection efficiency.
Cells were washed once in ice-cold phosphate-buffered saline before scraping them at 4°C in 1 ml of phosphate-buffered saline. Cells were resuspended in radioimmune precipitation assay buffer (RIPA) buffer (50 mM Tris-HCl, pH 7.5, 200 mM NaCl, 1% Nonidet P-40, 1 mM EDTA, 2.5 mM EGTA, 10% glycerol, and the phosphatase inhibitors P-5726 and P-2850 (Sigma)). Cell extracts were sonicated and centrifuged at 14,000 rpm for 10 min. Supernatants were used as crude extracts for immunoprecipitations. Nonspecific binding was reduced by preincubation of extracts with protein G-Sepharose (P-4691; Sigma) for 30 min. Pellets were discarded, and extracts incubated with immune sera or controls for 2-4 h. Immunoprecipitations were performed with 5 μl of anti-Flag anti-antibody body (M2; Sigma).
Glutathione S-Transferase (GST) Pull-downs
GST-Sox9 fusion proteins were produced in Escherichia coli and purified. Binding of proteins to glutathione-Sepharose was done in 20 mM Hepes, pH 7.4, 50 mM NaCl, 1 mM MgCl2, 0.2 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, 20 μg/ml leupeptin, 20 μg/ml aprotinin, and 0.05% Tween 20. Bound proteins were resolved on SDS-PAGE and visualized by autoradiography.
Poly(A)+ and total RNA were extracted from homogenized mouse embryo limbs and mouse chondrocyte cells using the Fast Track 2.0 (Invitrogen) or the RNeasy (Qiagen) kit. RNA samples were treated with DNase I (Promega), and RNA quality was assessed by gel electrophoresis. cDNA was prepared by reverse transcription of 500 ng of total RNA using the Superscript II enzyme and oligo (dT) primer (Invitrogen). Resulting cDNAs were amplified using the QuantiTect SYBR Green PCR kit (Qiagen) and the iCycler iQ Real Time PCR detection system (Bio-Rad). All mRNA expression data from quantitative PCR with reverse transcription was normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression in the corresponding sample.
Whole-mount alcian blue staining of mouse embryos and alcian blue and alizarin red staining of skeletons were performed as described. For histological analysis, embryos were fixed with 4% paraformaldehyde and embedded in paraffin. Sections of 4 μm were stained with SafraninO, or with the von Kossa reaction and nuclear fast red. Immunohistochemical staining was performed using peroxidase chromogens (Zymed Laboratories) and TrueBlue substrate (KPL). The following antibodies were used: goat polyclonal anti-Sox9 (1:100, Santa Cruz); rabbit polyclonal anti-Sox9 (1:100); rabbit polyclonal anti-Cyclin D1 (1:100, Santa Cruz); and mouse monoclonal anti-beta-catenin (1:100, BD Transduction Lab). Cell proliferation analysis was performed on paraffin-embedded sections using the PCNA Staining Kit (Zymed Laboratories) following the manufacturer's protocol. Cell proliferation was also evaluated by BrdU pulse labeling. BrdU was injected intraperitoneally into pregnant mice 4 h before death and detected using the Zymed BrdU staining kit (Zymed Laboratories) following the manufacturer's protocol.
In VivoMethylation Assay
Subconfluent 293T cells seeded on 100 mm diameter TC dishes were transfected with indicated vectors. Protein synthesis inhibition and in vivo methylation were carried out as described. Cells were washed with ice-cold phosphate-buffered saline and harvested and lysed in 1 ml of ice-cold lysis buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 0.1% Nonidet P-40, 1 mM phenylmethylsulfonyl fluoride, 5 mM NaF, 1 mM Na3VO4) supplemented with protease inhibitors (Sigma). Cell lysates were quantified using the standard Bedford method( and 1 mg incubated with 8 mg of A-Sepharose (Amersham Biosciences). Beads were then washed three times in ice-cold lysis buffer, and bound proteins were solubilized by addition of SDS sample buffer. Proteins were separated by SDS-PAGE and transferred to nitrocellulose membranes. Western blot analyses were performed by standard procedures with an anti-Flag antibody body (M2; Sigma) and enhanced chemiluminescence visualization. Radioactivity (methyl-3H) was visualized by fluorography; membranes were soaked in NAMP100 Amplify (Amersham Biosciences), air dried, and exposed to films at -80°C for 1 month.
In vitro methyltransferase assays and fluorography of methylation reactions were carried out as described. Experiments were performed as described using the Histone Methyltransferase Assay Protocol for active CARM1/PRMT4 protein (Upstate). Recombinant wild type and mutant Sox9 proteins (1 μg) were added to HMT reaction buffer containing 1 mM PMSF, 50 mM Tris-HCl (pH 9.0), 0.5 mM DTT and 1 mM [3H]AdoMet (0.5 Ci/mmol), and incubated with 700 ng of recombinant CARM1 protein (Upstate) at 30°C for 30 min in a total volume of 50 μl.
Purification of Recombinant Proteins
Sox9 was synthesized using the BaculoDirect system, according to the manufacturer's protocol (Invitrogen). Recombinant Flag-tagged Sox9 was immunoprecipitated by anti-Flag M2 affinity gel with immunoprecipitation buffer 0 (50 mM Tris-HCl, pH 7.5, 100 mM NaCl, 0.05% Nonidet P-40, 1 mM EDTA, 2.5 mM EGTA, 10% glycerol, protease inhibitors) after indicated treatments. Purified proteins were assessed by silver staining (Bio-Rad) and Western blotting analyses. Western blotting were performed using anti-Flag M2 (Sigma) or anti-Sox9 (Chemicon) antibodies as described previously.
E14.5 to E18.5 embryos and neonatal P1 mice were fixed in 10% formalin for 24 h or more (depending on size). After dehydration with increasing concentrations of ethanol, embryos were embedded in paraffin, sectioned at 3 to 4 -μm, and placed on silane-treated slides. Probes were labeled with 35S-rUTP to a specific activity of 105 cpm/μl. ISH were performed with the following modifications. Sections were post-fixed in 4% formaldehyde, treated with 10 μg/ml proteinase K for 7.5 min, and treated with 0.1 M triethanolamine containing 0.25% acetic anhydride for 10 min. Slides were heated at 75°C and cooled on ice before hybridization. The hybridization mix containing 50% formamide, 0.3 M NaCl, 1× Denhardt's solution, 5 mM EDTA, 10 mM Tris, 10% dextran, 10 μM DTT, 10 mM NaHPO4, 500 μg/ml tRNA, ~200 g/ml calf thymus DNA, and the denatured probe was added to each slide and incubated at 65°C overnight. Sections were treated with RNAse at 37°C using 20 μg/ml RNAse A and 1 μg/ml RNAse T1. After washing, slides were dipped in a 50% mixture of NTB-2 (Eastman Kodak) and 0.6 M NH4Ac, further exposed at 4°C for 3-4 day for autoradiography, and subsequently developed. Sections were counter-stained with hematoxylin. The Cyclin D1 probe was amplified by PCR and inserted into the PCRII topo-vector (Invitrogen) (data not shown).
We are grateful to Dr. Kazuhisa Nakashima (Department of Molecular Pharmacology, Medical Research Institute, Tokyo Medical and Dental University), Dr. Hidesato Ogawa (Department of Developmental Biology, National Institute for Basic Biology), Dr. Kerri Mowen (Department of Department of Immunology, The Scripps Research Institute), and Dr. Tetsu Osamu (Department of Pathology, School of Medicine, University of California, San Francisco) for providing materials. We also thank Dr. Donghang Cheng (Department of Carcinogenesis, University of Texas M. D. Anderson Cancer Center), Dr. Yasuhiko Kawakami (Gene Expression Laboratory, The Salk Institute for Biological Studies), Dr. Hirohito Shimizu, Dr. Teruyuki Tanaka (National Research Institute for Child Health and Development) and Diana C. Brinson (Department of Molecular and Experimental Medicine, The Scripps Research Institute) for technical assistance and discussion. We thank Dr. Marc Ladanyi (Memorial Sloan-Kettering Cancer Center) for reading and comments. We also thank Lilo Creighton and Jacque Quach for staining and FACS scan assays. This work was supported in part by the National Institutes of Health Grant R01 AR050631 (to HA), National Institutes of Health Fellowship F32 AR053442-01A1 (to TI), and National Institutes of Health Grant M01 RR00833 (to ML), National Institutes of Health Grant DK62248 (to MTB)
- Cancedda R, Descalzi Cancedda F, Castagnola P: Chondrocyte differentiation. Int Rev Cytol. 1995, 159: 265-358. 10.1016/S0074-7696(08)62109-9.View ArticlePubMedGoogle Scholar
- de Crombrugghe B, Lefebvre V, Behringer RR, Bi W, Murakami S, Huang W: Transcriptional mechanisms of chondrocyte differentiation. Matrix Biol. 2000, 19: 389-94. 10.1016/S0945-053X(00)00094-9.View ArticlePubMedGoogle Scholar
- Tanaka K, Matsumoto Y, Nakatani F, Iwamoto Y, Yamada Y: A zinc finger transcription factor, alphaA-crystallin binding protein 1, is a negative regulator of the chondrocyte-specific enhancer of the alpha1(II) collagen gene. Mol Cell Biol. 2000, 20: 4428-35. 10.1128/MCB.20.12.4428-4435.2000.PubMed CentralView ArticlePubMedGoogle Scholar
- Chimal-Monroy J, Rodriguez-Leon J, Montero JA, Ganan Y, Macias D, Merino R, Hurle JM: Analysis of the molecular cascade responsible for mesodermal limb chondrogenesis: Sox genes and BMP signaling. Dev Biol. 2003, 257: 292-301. 10.1016/S0012-1606(03)00066-6.View ArticlePubMedGoogle Scholar
- Sekiya I, Vuoristo JT, Larson BL, Prockop DJ: In vitro cartilage formation by human adult stem cells from bone marrow stroma defines the sequence of cellular and molecular events during chondrogenesis. Proc Natl Acad Sci USA. 2002, 99: 4397-402. 10.1073/pnas.052716199.PubMed CentralView ArticlePubMedGoogle Scholar
- Kawakami Y, Rodriguez-Leon J, Belmonte JC: The role of TGFbetas and Sox9 during limb chondrogenesis. Curr Opin Cell Biol. 2006, 18: 723-9. 10.1016/j.ceb.2006.10.007.View ArticlePubMedGoogle Scholar
- Akiyama H, Chaboissier MC, Martin JF, Schedl A, de Crombrugghe B: The transcription factor Sox9 has essential roles in successive steps of the chondrocyte differentiation pathway and is required for expression of Sox5 and Sox6. Genes Dev. 2002, 16: 2813-28. 10.1101/gad.1017802.PubMed CentralView ArticlePubMedGoogle Scholar
- Bedford MT, Richard S: Arginine methylation an emerging regulator of protein function. Mol Cell. 2005, 18: 263-72. 10.1016/j.molcel.2005.04.003.View ArticlePubMedGoogle Scholar
- Koh SS, Chen D, Lee YH, Stallcup MR: Synergistic enhancement of nuclear receptor function by p160 coactivators and two coactivators with protein methyltransferase activities. J Biol Chem. 2001, 276: 1089-98. 10.1074/jbc.M004228200.View ArticlePubMedGoogle Scholar
- Lee YH, Koh SS, Zhang X, Cheng X, Stallcup MR: Synergy among nuclear receptor coactivators: selective requirement for protein methyltransferase and acetyltransferase activities. Mol Cell Biol. 2002, 22: 3621-32. 10.1128/MCB.22.11.3621-3632.2002.PubMed CentralView ArticlePubMedGoogle Scholar
- Bauer UM, Daujat S, Nielsen SJ, Nightingale K, Kouzarides T: Methylation at arginine 17 of histone H3 is linked to gene activation. EMBO Rep. 2002, 3: 39-44. 10.1093/embo-reports/kvf013.PubMed CentralView ArticlePubMedGoogle Scholar
- Daujat S, Bauer UM, Shah V, Turner B, Berger S, Kouzarides T: Crosstalk between CARM1 methylation and CBP acetylation on histone H3. Curr Biol. 2002, 12: 2090-7. 10.1016/S0960-9822(02)01387-8.View ArticlePubMedGoogle Scholar
- Yadav N, Lee J, Kim J, Shen J, Hu MC, Aldaz CM, Bedford MT: Specific protein methylation defects and gene expression perturbations in coactivator-associated arginine methyltransferase 1-deficient mice. Proc Natl Acad Sci USA. 2003, 100: 6464-8. 10.1073/pnas.1232272100.PubMed CentralView ArticlePubMedGoogle Scholar
- Lefebvre V, Li P, de Crombrugghe B: A new long form of Sox5 (L-Sox5), Sox6 and Sox9 are coexpressed in chondrogenesis and cooperatively activate the type II collagen gene. Embo J. 1998, 17: 5718-33. 10.1093/emboj/17.19.5718.PubMed CentralView ArticlePubMedGoogle Scholar
- Tsuda M, Takahashi S, Takahashi Y, Asahara H: Transcriptional co-activators CREB-binding protein and p300 regulate chondrocyte-specific gene expression via association with Sox9. J Biol Chem. 2003, 278: 27224-9. 10.1074/jbc.M303471200.View ArticlePubMedGoogle Scholar
- Xu W, Chen H, Du K, Asahara H, Tini M, Emerson BM, Montminy M, Evans RM: A transcriptional switch mediated by cofactor methylation. Science. 2001, 294: 2507-11. 10.1126/science.1065961.View ArticlePubMedGoogle Scholar
- Cheng D, Cote J, Shaaban S, Bedford MT: The arginine methyltransferase CARM1 regulates the coupling of transcription and mRNA processing. Mol Cell. 2007, 25: 71-83. 10.1016/j.molcel.2006.11.019.View ArticlePubMedGoogle Scholar
- Chen D, Ma H, Hong H, Koh SS, Huang SM, Schurter BT, Aswad DW, Stallcup MR: Regulation of transcription by a protein methyltransferase. Science. 1999, 284: 2174-7. 10.1126/science.284.5423.2174.View ArticlePubMedGoogle Scholar
- Sgarra R, Lee J, Tessari MA, Altamura S, Spolaore B, Giancotti V, Bedford MT, Manfioletti G: The AT-hook of the chromatin architectural transcription factor high mobility group A1a is arginine-methylated by protein arginine methyltransferase 6. J Biol Chem. 2006, 281: 3764-72. 10.1074/jbc.M510231200.View ArticlePubMedGoogle Scholar
- Tetsu O, McCormick F: Beta-catenin regulates expression of cyclin D1 in colon carcinoma cells. Nature. 1999, 398: 422-6. 10.1038/18884.View ArticlePubMedGoogle Scholar
- Akiyama H, Lyons JP, Mori-Akiyama Y, Yang X, Zhang R, Zhang Z, Deng JM, Taketo MM, Nakamura T, Behringer RR, et al: Interactions between Sox9 and beta-catenin control chondrocyte differentiation. Genes Dev. 2004, 18: 1072-87. 10.1101/gad.1171104.PubMed CentralView ArticlePubMedGoogle Scholar
- Beier F, Ali Z, Mok D, Taylor AC, Leask T, Albanese C, Pestell RG, LuValle P: TGFbeta and PTHrP control chondrocyte proliferation by activating cyclin D1 expression. Mol Biol Cell. 2001, 12: 3852-63.PubMed CentralView ArticlePubMedGoogle Scholar
- Long F, Zhang XM, Karp S, Yang Y, McMahon AP: Genetic manipulation of hedgehog signaling in the endochondral skeleton reveals a direct role in the regulation of chondrocyte proliferation. Development. 2001, 128: 5099-108.PubMedGoogle Scholar
- Huang W, Zhou X, Lefebvre V, de Crombrugghe B: Phosphorylation of SOX9 by cyclic AMP-dependent protein kinase A enhances SOX9's ability to transactivate a Col2a1 chondrocyte-specific enhancer. Mol Cell Biol. 2000, 20: 4149-58. 10.1128/MCB.20.11.4149-4158.2000.PubMed CentralView ArticlePubMedGoogle Scholar
- Akiyama H, Kamitani T, Yang X, Kandyil R, Bridgewater LC, Fellous M, Mori-Akiyama Y, de Crombrugghe B: The transcription factor Sox9 is degraded by the ubiquitin-proteasome system and stabilized by a mutation in a ubiquitin-target site. Matrix Biol. 2005, 23: 499-505. 10.1016/j.matbio.2004.10.002.View ArticlePubMedGoogle Scholar
- Komatsu T, Mizusaki H, Mukai T, Ogawa H, Baba D, Shirakawa M, Hatakeyama S, Nakayama KI, Yamamoto H, Kikuchi A, et al: Small ubiquitin-like modifier 1 (SUMO-1) modification of the synergy control motif of Ad4 binding protein/steroidogenic factor 1 (Ad4BP/SF-1) regulates synergistic transcription between Ad4BP/SF-1 and Sox9. Mol Endocrinol. 2004, 18: 2451-62. 10.1210/me.2004-0173.View ArticlePubMedGoogle Scholar
- Torres-Padilla ME, Parfitt DE, Kouzarides T, Zernicka-Goetz M: Histone arginine methylation regulates pluripotency in the early mouse embryo. Nature. 2007, 445: 214-8. 10.1038/nature05458.PubMed CentralView ArticlePubMedGoogle Scholar
- Kim J, Lee J, Yadav N, Wu Q, Carter C, Richard S, Richie E, Bedford MT: Loss of CARM1 results in hypomethylation of thymocyte cyclic AMP-regulated phosphoprotein and deregulated early T cell development. J Biol Chem. 2004, 279: 25339-44. 10.1074/jbc.M402544200.View ArticlePubMedGoogle Scholar
- Lee J, Cheng D, Bedford MT: Techniques in protein methylation. Methods Mol Biol. 2004, 284: 195-208.PubMedGoogle Scholar
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