Abstract
Plant homeodomain finger protein 2 (PHF2), which contains a plant homeodomain and a Jumonji-C domain, is an epigenetic regulator that demethylates lysine 9 in histone 3 (H3K9me2). On the other hand, runt-related transcription factor 2 (Runx2) plays essential roles in bone development and regeneration. Given previous reports that the PHF2 mutation can cause dwarfism in mice and that PHF2 expression is correlated with that of Runx2 in differentiating thymocytes, we investigated whether PHF2 regulates Runx2-mediated bone formation. Overexpression of PHF2 facilitated bone development in newborn mice, and viral shRNA-mediated knockdown of PHF2 delayed calvarial bone regeneration in adult rats. In primary osteoblasts and C2C12 precursor cells, PHF2 enhances osteoblast differentiation by demethylating Runx2, while suppressor of variegation 3-9 homolog 1 (SUV39H1) inhibits bone formation by methylating it. The PHF2-Runx2 interaction is mediated by the Jumonji-C and Runt domains of the two proteins, respectively. The interaction between Runx2 and osteocalcin promoter is regulated by the methylation status of Runx2, i.e., the interaction is augmented when Runx2 is demethylated. Our results suggest that SUV39H1 and PHF2 reciprocally regulate osteoblast differentiation by modulating Runx2-driven transcription at the post-translational level. This study may provide a theoretical basis for the development of new therapeutic modalities for patients with impaired bone development or delayed fracture healing.
Similar content being viewed by others
Log in or create a free account to read this content
Gain free access to this article, as well as selected content from this journal and more on nature.com
or
References
Fortschegger K, Shiekhattar R . Plant homeodomain fingers form a helping hand for transcription. Epigenetics 2011; 6:4–8.
Nakayama J, Rice JC, Strahl BD, Allis CD, Grewal SI . Role of histone H3 lysine 9 methylation in epigenetic control of heterochromatin assembly. Science 2001; 292:110–113.
Vakoc CR, Mandat SA, Olenchock BA, Blobel GA . Histone H3 lysine 9 methylation and HP1gamma are associated with transcription elongation through mammalian chromatin. Mol Cell 2005; 19:381–391.
Baba A, Ohtake F, Okuno Y, et al. PKA-dependent regulation of the histone lysine demethylase complex PHF2-ARID5B. Nat Cell Biol 2011; 13:668–675.
Okuno Y, Ohtake F, Igarashi K, et al. Epigenetic regulation of adipogenesis by phf2 histone demethylase. Diabetes 2013; 62:1426–1434.
Stender Joshua D, Pascual G, et al. Control of proinflammatory gene programs by regulated trimethylation and demethylation of histone H4K20. Mol Cell 2012; 48:28–38.
Schroeder TM, Jensen ED, Westendorf JJ . Runx2: a master organizer of gene transcription in developing and maturing osteoblasts. Birth Defects Res C Embryo Today 2005; 75:213–225.
Long F . Building strong bones: molecular regulation of the osteoblast lineage. Nat Rev Mol Cell Biol 2012; 13: 27–38.
Kugimiya F, Kawaguchi H, Ohba S, et al. GSK-3beta controls osteogenesis through regulating Runx2 activity. PLoS One 2007; 2:e837.
Jonason JH, Xiao G, Zhang M, Xing L, Chen D . Post-translational regulation of Runx2 in bone and cartilage. J Dent Res 2009; 88:693–703.
Jun JH, Yoon WJ, Seo SB, et al. BMP2-activated Erk/MAP kinase stabilizes Runx2 by increasing p300 levels and histone acetyltransferase activity. J Biol Chem 2010; 285:36410–36419.
Zhao M, Qiao M, Oyajobi BO, Mundy GR, Chen D . E3 ubiquitin ligase Smurf1 mediates core-binding factor alpha1/Runx2 degradation and plays a specific role in osteoblast differentiation. J Biol Chem 2003; 278:27939–27944.
Schroeder TM, Kahler RA, Li X, Westendorf JJ . Histone deacetylase 3 interacts with runx2 to repress the osteocalcin promoter and regulate osteoblast differentiation. J Biol Chem 2004; 279:41998–42007.
Bradley EW, McGee-Lawrence ME, Westendorf JJ . Hdac-mediated control of endochondral and intramembranous ossification. Crit Rev Eukaryot Gene Expr 2011; 21:101–113.
Yang D, Okamura H, Nakashima Y, Haneji T . Histone demethylase Jmjd3 regulates osteoblast differentiation via transcription factors Runx2 and Osterix. J Biol Chem 2013; 288:33530–33541.
Hansen J, Floss T, Van Sloun P, et al. A large-scale, gene-driven mutagenesis approach for the functional analysis of the mouse genome. Proc Natl Acad Sci USA 2003; 100:9918–9922.
Zhao FQ, Sheng ZM, Tsai MM, et al. Serial analysis of gene expression in murine fetal thymocyte cell lines. Int Immunol 2002; 14:1383–1395.
Huang J, Berger SL . The emerging field of dynamic lysine methylation of non-histone proteins. Curr Opin Genet Dev 2008; 18:152–158.
Shinkai Y, Tachibana M . H3K9 methyltransferase G9a and the related molecule GLP. Genes Dev 2011; 25:781–788.
Rathert P, Dhayalan A, Murakami M, et al. Protein lysine methyltransferase G9a acts on non-histone targets. Nat Chem Biol 2008; 4:344–346.
Jonason JH, Xiao G, Zhang M, Xing L, Chen D . Post-translational regulation of Runx2 in bone and cartilage. J Dent Res 2009; 88:693–703.
Kim BG, Kim HJ, Park HJ, et al. Runx2 phosphorylation induced by fibroblast growth factor-2/protein kinase C pathways. Proteomics 2006; 6:1166–1174.
Selvamurugan N, Pulumati MR, Tyson DR, Partridge NC . Parathyroid hormone regulation of the rat collagenase-3 promoter by protein kinase A-dependent transactivation of core binding factor alpha1. J Biol Chem 2000; 275:5037–5042.
Wee HJ, Huang G, Shigesada K, Ito Y . Serine phosphorylation of RUNX2 with novel potential functions as negative regulatory mechanisms. EMBO Rep 2002; 3:967–974.
Jeon EJ, Lee KY, Choi NS, et al. Bone morphogenetic protein-2 stimulates Runx2 acetylation. J Biol Chem 2006; 281:16502–16511.
Huang J, Dorsey J, Chuikov S, et al. G9a and Glp methylate lysine 373 in the tumor suppressor p53. J Biol Chem 2010; 285:9636–9641.
Lee JS, Kim Y, Kim IS, et al. Negative regulation of hypoxic responses via induced Reptin methylation. Mol Cell 2010; 39:71–85.
Chakraborty S, Sinha KK, Senyuk V, Nucifora G . SUV39H1 interacts with AML1 and abrogates AML1 transactivity. AML1 is methylated in vivo. Oncogene 2003; 22:5229–5237.
Reed-Inderbitzin E, Moreno-Miralles I, Vanden-Eynden SK, et al. RUNX1 associates with histone deacetylases and SUV39H1 to repress transcription. Oncogene 2006; 25:5777–5786.
Tandon M, Gokul K, Ali SA, et al. Runx2 mediates epigenetic silencing of the bone morphogenetic protein-3B (BMP-3B/GDF10) in lung cancer cells. Mol Cancer 2012; 11:27.
Acknowledgements
This work was supported by grants from the Bone Metabolism Research Center (SRC; 2011-0001026), the Korean Health Technology R&D (A121106 and A120476), and the National Research Foundation (2009-0090188, 2011-0030737 and 2013R1A2A1A01015228) supported by Korean government.
Author information
Authors and Affiliations
Corresponding authors
Additional information
( Supplementary information is linked to the online version of the paper on the Cell Research website.)
Supplementary information
Supplementary information, Figure S1
Based on micro-CT images, length of tibia, femur, radius humerus and vertabra in PHF2-t/g mice and their littermates on P1 was analyzed using the software Amira version 5.4.1. (PDF 57 kb)
Supplementary information, Figure S2
(A) Representative gross appearances of 3 months old PHF2-t/g and wild-type mice. (PDF 325 kb)
Supplementary information, Figure S3
C2C12 cells, which had been transfected with Flag vector or Flag-PHF2, were treated with BMP2 for indicated time. (PDF 185 kb)
Supplementary information, Figure S4
C2C12 stable cells expressing Sh-Con or Sh-PHF2 shRNA were further transfected with F/S-R2. (PDF 192 kb)
Supplementary information, Figure S5
C2C12 cells were co-transfected with Myc-Runx2 (or its K245A mutant), 6xOSE or OG2 promoter luciferase, Flag-PHF2, and β-gal plasmids. (PDF 136 kb)
Supplementary information, Figure S6
C2C12 cells were transfected with Myc-Runx2 (or its K245R mutant) and Flag-PHF2 (A) or Flag-SUV39H1 (B) plasmids. (PDF 195 kb)
Supplementary information, Figure S7
C2C12 cells were transfected with SUV39H1 plamsid or siRNA, and treated with BMP2 for 2 days. (PDF 140 kb)
Supplementary information, Table S1
Nucleotide sequences of RNAs and primers used in experiments (PDF 136 kb)
Rights and permissions
About this article
Cite this article
Kim, HJ., Park, JW., Lee, KH. et al. Plant homeodomain finger protein 2 promotes bone formation by demethylating and activating Runx2 for osteoblast differentiation. Cell Res 24, 1231–1249 (2014). https://doi.org/10.1038/cr.2014.127
Received:
Revised:
Accepted:
Published:
Issue date:
DOI: https://doi.org/10.1038/cr.2014.127
Keywords
This article is cited by
-
Palmitoylation-driven PHF2 ubiquitination remodels lipid metabolism through the SREBP1c axis in hepatocellular carcinoma
Nature Communications (2023)
-
The osteoprotective role of USP26 in coordinating bone formation and resorption
Cell Death & Differentiation (2022)
-
Epigenetic Regulators of Mesenchymal Stem/Stromal Cell Lineage Determination
Current Osteoporosis Reports (2020)
-
Histone demethylase KDM4A regulates adipogenic and osteogenic differentiation via epigenetic regulation of C/EBPα and canonical Wnt signaling
Cellular and Molecular Life Sciences (2020)
-
Histone demethylase UTX counteracts glucocorticoid deregulation of osteogenesis by modulating histone-dependent and -independent pathways
Journal of Molecular Medicine (2017)
