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ORIGINAL ARTICLEnpgCell Research(2014):1-19.2014 IBCB,SIBS,CAS All rights reserved 1001-0602/14$ homeodomain finger protein 2 promotes bone formation by demethylating and activating Runx2 for osteoblast differentiationHye-Jin Kim1,Jong-Wan Park1,2,3,Kyoung-Hwa Lee1,2,Haejin Yoon1,Dong Hoon Shin1,2,Uk-Il Ju1,Seung Hyeok Seok4,Seung Hyeon Lim5,Zang Hee Lee6,Hong-Hee Kim6,Yang-Sook Chun1,21Department of Biomedical Sciences,Seoul National University College of Medicine,Seoul 110-799,Republic of Korea;2Ischemic/Hypoxic Disease Institute,Seoul National University College of Medicine,Seoul 110-799,Republic of Korea;3Cancer Research Institute,Seoul National University College of Medicine,Seoul 110-799,Republic of Korea;4Department of Microbiology and Im-munology,Seoul National University College of Medicine,Seoul 110-799,Republic of Korea;5Institute for Experimental Animals,Seoul National University College of Medicine,Seoul 110-799,Republic of Korea;6Department of Cell and Developmental Biolo-gy,Seoul National University School of Dentistry,Seoul 110-749,Republic of KoreaPlant 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 tran-scription 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 differ-entiating 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 cal-varial 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 for-mation 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 sta-tus 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-transla-tional 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.Keywords:PHF2;SUV39H1;Runx2;lysine methylation;osteoblast differentiationCell Research advance online publication 26 September 2014;doi:10.1038/cr.2014.127Correspondence:Jong-Wan Parka,Yang-Sook Chunb aE-mail:parkjwsnu.ac.krbE-mail:chunyssnu.ac.krReceived 5 January 2014;revised 9 July 2014;accepted 5 August 2014IntroductionPlant homeodomain finger protein 2(PHF2)belongs to the lysine demethylase 7 family in the Jumonji-C su-perfamily because it contains both a plant homeodomain(PHD)and a Jumonji-C(JmjC)domain at its N-terminus.The PHD recognizes methylated lysine residues,and the JmjC domain catalyzes the removal of methyl groups from lysine residues by using Fe2+and 2-oxoglutarate as cofactors 1.Functionally,PHF2 has been identified as a histone demethylase that reverses dimethylation of histone H3 at lysine 9(H3K9me2).H3K9 methylation is regarded as transcription-repressive because of its association with heterochromatin assembly 2.However,H3K9 methyla-tion also occurs at active genes in mammalian chromatin and is associated with transcription elongation 3.Thus,the precise consequences of PHF2-mediated H3K9 de-methylation on gene expression remain unclear.Nonethe-less,PHF2 is generally believed to act as a transcriptional coactivator by reversing histone methylation.For example,2PHF2 promotes osteoblastogenesis by demethylating Runx2npgCell Research| has been found to participate in energy homeostasis by activating HNF4 and FXR and to induce adipocyte dif-ferentiation by activating CEBP 4,5.In addition,PHF2 has been shown to promote inflammatory responses by in-ducing the expression of NF-B,which then enhances ex-pression of proinflammatory genes 6.Given that H3K9 modification is widely involved in epigenetic regulation,PHF2 likely participates in diverse biological processes beyond those mentioned above.Runt-related transcription factor 2(Runx2)plays es-sential roles in osteoblast differentiation during bone de-velopment and remodeling.Runx2 binds to a conserved promoter sequence(R/TACCRCA)and transactivates genes encoding osteogenic proteins such as collagen 1,osteopontin,bone sialoprotein(BSP),and osteocalcin(OCN)7,8.Runx2 is post-translationally activated during the early stage of osteoblast differentiation.Runx2 is activated by phosphorylation at serine or threonine resi-dues through the MAPK-ERK pathway but inactivated by phosphorylation at other residues by GSK3 and CDK4 9,10.In addition,acetylation is involved in Runx2 signal-ing.For instance,p300 and p300/CBP-associated factor have been reported to enhance the transcriptional activity of Runx2 by acetylating Runx2 at lysine residues as well as to stabilize Runx2 by inhibiting Runx2 ubiquitination by Smurf1 11,12.In contrast,the deacetylase HDAC3 is known to interact with Runx2 to repress the expression of Runx2 target genes 13.Similarly,several members of the class II HDAC family have been shown to act as repressors in regulating the expression of Runx2-driven genes 14.In addition,Runx2 gene expression is posi-tively regulated by the JmjC-domain-containing histone demethylase Jmjd3,which demethylates H3K27me3 at the promoter region of Runx2 15.The current study was motivated by two reports link-ing PHF2 to bone development and Runx2 signaling,respectively.Hansen et al.16 observed that mutation of the PHF2 gene causes dwarfism,following random mu-tagenesis by retroviral transduction of gene-trap vectors into mouse embryonic stem cells.Zhao et al.17 found that the expression of PHF2 is strongly related to that of Runx2 across all stages of thymocyte differentiation.Giv-en these two studies,we hypothesized that PHF2 might interact with Runx2 to regulate bone formation.Indeed,the results of our in vitro and in vivo experiments indicate that PHF2 plays an essential role in bone formation as a positive regulator of Runx2 activity,suggesting that PHF2 could be a novel target for enhancing bone formation.ResultsBone development is facilitated in PHF2 transgenic miceTo examine the role of PHF2 in bone development,PHF2 transgenic(t/g)heterozygous mice were generated,and their whole skeletons on postnatal day 1 were com-pared with those of their wild-type littermates.Bone and cartilage were co-stained using Alizarin red and Alcian blue,respectively.Alizarin red staining was denser in PHF2-t/g mice than in wild-type mice,whereas Alcian blue staining intensity did not differ significantly(Fig-ure 1A,left panel).A close examination of three skele-tal regions revealed that they were better developed in PHF2-t/g mice than in wild-type mice(Figure 1A,right panel).However,the limb and spine lengths were similar between the two groups(Supplementary information,Figure S1),suggesting that bone growth in length due to endochondral ossification is not affected by PHF2 over-expression.Following confirmation of PHF2 overexpres-sion in the calvarial bones of PHF2-t/g mice by western blotting and immunostaining(Figure 1B),calvarial bone area and density of PHF2-t/g mice were compared with those of wild-type mice by micro-CT.PHF2-t/g mice displayed larger and denser bones than wild-type mice(Figure 1C).To analyze the extent of new bone formation in fetal mice,we first stained fetal bones with Calcein green for 2 days and further stained them with Alizalin red for another 2 days,through intraperitoneal injection of these dyes,into pregnant mice(see details in Materi-als and Methods).The area of new bone formed over 2 days(revealed by red staining in the absence of green)was significantly larger in PHF2-t/g mice(Figure 1D).Moreover,bone formation was verified using von Kossa staining(dark purple color generated by silver deposition to calcium phosphate)and OCN immunofluorescence(Figure 1E).We next examined osteoblast differentiation in calvarial bones by measuring the mRNA levels of os-teogenic genes,which revealed higher expression of these genes in PHF2-t/g calvarias(Figure 1F).In contrast to newborn mice,the gross appearance and skeletal structure of 3-month-old mice did not differ significantly between PHF2-t/g and wild-type groups(Supplementary informa-tion,Figure S2).These results suggest that PHF2 overex-pression facilitates bone development during the neonatal stage,but does not affect bone growth afterwards.PHF2 promotes osteoblast differentiationGiven the earlier onset of bone development observed in PHF2-t/g mice,the involvement of PHF2 in osteoblast differentiation,a critical step in bone development,was examined.Primary osteoblasts were isolated from cal-varias of PHF2-t/g and wild-type mice on postnatal day 1 and treated with BMP2 to induce differentiation.In-deed,western blotting revealed that PHF2-t/g osteoblasts showed a higher expression level of PHF2 than wild-type ones(Figure 2A).In addition,PHF2 seems to be ex-pressed in parallel with Runx2 because the expression of Hye-Jin Kim et al.3npgwww.cell-|Cell Researchboth proteins was induced during the early stage of dif-ferentiation but decreased after day 5 of differentiation in wild-type osteoblasts(Figure 2A).Immunofluorescence analysis revealed that PHF2 and Runx2 were co-local-4PHF2 promotes osteoblastogenesis by demethylating Runx2npgCell Research| in nuclei of wild-type osteoblasts(Figure 2B).To examine whether PHF2 affects the rate of osteoblast dif-ferentiation,alkaline phosphatase(ALP)staining(Figure 2C)and its enzymatic activity assay(Figure 2D)were carried out.Both assays revealed an earlier and more evident increase in ALP activity in PHF2-t/g osteoblasts than in wild-type cells(Figure 2C and 2D).The effects of PHF2 on late osteoblast differentiation(maturation)were examined by using Alizarin red staining to assess matrix mineralization and RT-PCR to measure the expression of differentiation markers.PHF2-t/g osteoblasts displayed greater mineralization(Figure 2E)and enhanced expres-sion of ALP,OCN,and BSP(Figure 2F).Consistently,ectopic expression of PHF2 in C2C12 precursor cells facilitated BMP2-induced ALP activation(Supplemen-tary information,Figure S3).Collectively,these results suggest that PHF2 is a positive regulator of osteoblast differentiation.PHF2 is essential for osteoblast differentiationThe involvement of PHF2 in osteoblast differentiation was also examined by knocking down PHF2 in C2C12 cells using shRNA lentivirus.Western blotting confirmed the stable knockdown of PHF2(Figure 3A).As in prima-ry osteoblasts,BMP2 treatment upregulated the expres-sion of PHF2 and Runx2 in control C2C12 cells(Figure 3A).PHF2 knockdown significantly attenuated ALP ac-tivity and matrix mineralization(Figure 3B-3D),as well as mRNA expression levels of osteoblast differentiation markers(ALP,OCN,and BSP;Figure 3E).These results further demonstrate that PHF2 supports osteoblast differ-entiation.Bone regeneration in rat calvaria is blocked by PHF2 knockdownIn addition to bone development,fracture healing also depends on osteoblast differentiation.Therefore,the role of PHF2 in bone regeneration was examined in rats with surgery-generated calvarial defects.Two shRNAs were designed to target rat PHF2,and the efficiency of PHF2 knockdown was verified in rat fibroblasts by west-ern blotting(Figure 4A).Collagen matrices containing BMP2 and shRNA lentiviruses were placed over the cal-varial defects.After 8 weeks of recovery,newly formed bones were analyzed by reconstructing 3D micro-CT images.PHF2 knockdown significantly decreased the re-generated bone volume(Figure 4B and 4C).Histological examination confirmed that tissue formed at defect mar-gins was bone(Figure 4D).These in vivo data suggest that PHF2 is also required for bone regeneration.PHF2 binds to Runx2 and facilitates its transcriptional activityIt was recently reported that PHF2 and Runx2 share a similar expression pattern in thymocytes of different developmental stages 17,similar to what we have ob-served in osteoblasts(Figures 2A and 3A).Therefore,the involvement of PHF2 in Runx2 signaling was exam-ined using osteocalcin promoter(OG2)-luciferase and 6OSE-luciferase(a construct containing 6 Runx2-bind-ing sites)reporters.BMP2 treatment activated luciferase expression driven by the OG2 promoter in C2C12 cells,and PHF2 knockdown negated this effect(Figure 5A).PHF2 knockdown also attenuated the 6OSE and OG2 reporter activation induced by ectopic expression of Runx2(Figure 5B).Conversely,ectopic expression of PHF2 augmented Runx2-stimulated activation of both reporters and a mutant version of PHF2 with lower ac-tivity(H249A)4 did so to a considerably lesser extent(Figure 5C).We next examined the interaction between Figure 1 PHF2 facilitated bone development in mice.(A)Bones and cartilages of Flag-PHF2-t/g mice and their littermates on postnatal day 1(P1)were stained with Alizarin Red(red color)and Alcian Blue(blue color),respectively.The left panel shows the representative results for whole skeletons,and the right panel shows calvaria(a),forelimb(b),and hindlimb(c).(B)The expression of transgenic Flag-PHF2 in mouse calvarias on P1 was checked by immunoblotting with anti-PHF2 or anti-Flag antibody(left panel),by immunohistochemistry with an anti-Flag antibody(middle panel),and by immunofluorescence with an anti-PHF2 antibody(right panel).The densities of PHF2 and-tubulin blots were quantified using the ImageJ program and the PHF2/tubulin ratios are presented below the PHF2 blot.An arrow indicates a Flag-PHF2-expressing osteoblast in the periosteum.(C)The left panel shows the micro-CT images of bodies(top)and calvarias(bottom)of PHF2-t/g mice and their wild-type littermates on P1.Based on micro-CT images,calvarial bone areas and densities were analyzed using the ImageJ1.36b software(NIH:Maryland,MD)and plotted in the right panels.Horizontal bars represent mean values,and*de-notes P 0.05 for the indicated groups.(D)Bone formation in PHF2-t/g mice and their littermates(WT)on P1 was detected by double labeling with Calcein(green)and Alizarin(red).The area of Alizarin-labeled tissue,which indicates bone formed newly for two days before birth,was analyzed using ImageJ and plotted in the bottom panel.Data shown represent the mean SD(n=3)and*denotes P 0.05.(E)In calvarias of mice on P1,mineralization,OCN expression and histology were ana-lyzed by Von Kossa staining,immunofluorescence with anti-OCN antibody and H&E staining,respectively.(F)The mRNA lev-els of the osteogenic genes(ALP,BSP,and OCN)and PHF2 in calvarias were analyzed by RT-qPCR