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The lysine methyltransferase Ehmt2/G9a is dispensable for skeletal muscle development and regeneration Zhang, Regan-Heng; Judson, Robert N; Liu, David Y; Kast, Jürgen; Rossi, Fabio M V May 27, 2016

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RESEARCH Open AccessThe lysine methyltransferase Ehmt2/G9a isdispensable for skeletal muscledevelopment and regenerationRegan-Heng Zhang, Robert N. Judson, David Y. Liu, Jürgen Kast and Fabio M. V. Rossi*AbstractBackground: Euchromatic histone-lysine N-methyltransferase 2 (G9a/Ehmt2) is the main enzyme responsible for theapposition of H3K9 di-methylation on histones. Due to its dual role as an epigenetic regulator and in the regulationof non-histone proteins through direct methylation, G9a has been implicated in a number of biological processesrelevant to cell fate control. Recent reports employing in vitro cell lines indicate that Ehmt2 methylates MyoD torepress its transcriptional activity and therefore its ability to induce differentiation of activated myogenic cells.Methods: To further investigate the importance of G9a in modulating myogenic regeneration in vivo, we crossedEhmt2floxed mice to animals expressing Cre recombinase from the Myod locus, resulting in efficient knockout in theentire skeletal muscle lineage (Ehmt2ΔmyoD).Results: Surprisingly, despite a dramatic drop in the global levels of H3K9me2, knockout animals did not show anydevelopmental phenotype in muscle size and appearance. Consistent with this finding, purified Ehmt2ΔmyoD satellitecells had rates of activation and proliferation similar to wild-type controls. When induced to differentiate in vitro,Ehmt2 knockout cells differentiated with kinetics similar to those of control cells and demonstrated normal capacityto form myotubes. After acute muscle injury, knockout mice regenerated as efficiently as wildtype. To exclude possiblecompensatory mechanisms elicited by the loss of G9a during development, we restricted the knockout within adultsatellite cells by crossing Ehmt2floxed mice to Pax7CreERT2 and also found normal muscle regeneration capacity.Conclusions: Thus, Ehmt2 and H3K9me2 do not play significant roles in skeletal muscle development and regenerationin vivo.Keywords: Euchromatic methyltransferase, Ehmt2, Ehmt1, G9a, GLP, Myogenesis, Skeletal muscle, Development,Regeneration, MyodBackgroundThe formation of skeletal muscle begins in the embry-onic somites [1], which generate the primary myotomeand the first primitive myogenic structure containingmuscle progenitors. Morphogen gradients includingsonic hedgehog (Shh) [2, 3] and Wingless (Wnt) [4]ensure initial myogenic specification by controlling theexpression of myogenic regulatory factors (MRFs—Myf5,Myod, Myog, and Mrf4)—a conserved family of muscle-specific basic helix-loop-helix (bHLH) transcription fac-tors responsible for myogenic lineage commitment anddifferentiation. Embryonic muscle progenitors migrate,expand, and undergo subsequent waves of myogenesispersisting through fetal and early neonatal developmentresulting in the formation of the different skeletal mus-cles of the adult.A proportion of fetal myoblasts also become localizedunderneath the basal lamina of newly formed myofibers.These cells become specified as satellite cells—the quies-cent, tissue resident stem cell of the skeletal muscle,identifiable by the expression of paired box transcriptionfactor, paired box 7 (Pax7). Satellite cells are responsiblefor the regenerative potential of the muscle and, uponacute injury, break quiescence and mimic their develop-mental programs by expanding rapidly, upregulating* Correspondence: fabio@brc.ubc.caThe Biomedical Research Centre, The University of British Columbia,Vancouver, Canada© 2016 Zhang et al. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, andreproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link tothe Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.Zhang et al. Skeletal Muscle  (2016) 6:22 DOI 10.1186/s13395-016-0093-7MRFs and differentiating to form new myofibers. Inmice, this process leads to near-complete tissue regener-ation and restoration of muscle function.Despite much knowledge about the key transcriptionfactors regulating myogenesis, the epigenetic landscapesrequired for the control of gene expression in skeletalmuscle differentiation remain less well understood.Epigenetic mechanisms including different modificationson the unstructured C-terminals (tails) of histone pro-teins have an increasingly appreciated role in controllinggene expression during myogenesis [5]. Some studies[6–8] have found that specific histone acetyltransferasescan interact with Myod and acetylate histones associatedwith muscle-specific genes, thereby activating their tran-scription. Recent genome-wide analyses have uncovereddynamic epigenetic changes during myogenesis [9], in-cluding the loss of histone 2B (H2B) ubiquitination [10].Chromatin immunoprecipitation (ChIP)-seq analyseshave also revealed the importance of bivalent domainscontaining both H3K4me1 and H3K27ac in regulatingmuscle enhancers during myogenesis [9]. These dataalso point to Myod as playing a key role in the recruit-ment of chromatin-modifying enzymes and transcriptionfactors to activate such enhancers [11, 12].A less well-characterized histone mark in myogenesis isH3K9me2, produced by euchromatic histone-lysine N-methyltransferase 2 (Ehmt2) (MGI: 2148922), also knownas G9a. This Su (var)3-9 and enhancer of zeste (SET) do-main containing methyltransferase dimerizes with its closehomologue Ehmt1 (aka Glp) to induce H3K9me2 [13].Knockout of Ehmt2 leads to a global reduction ofH3K9me2 levels and early embryonic lethality in mice,underscoring its importance and the fact that Ehmt1 can-not fully compensate for its loss [14]. Conditional knock-outs of Ehmt2 also demonstrated its importance in germcell development [15], heart development [16], lympho-cyte development [17, 18], leukemia [19], drug addiction[20], cognition, and adaptive behaviors [21, 22]. H3K9me2appears to occur on repressed genes in euchromatin,whereas H3K9me3 is also a repressive mark, butassociated with pericentromeric heterochromatin [23].The repressive function is led by recruitment of proteinssuch as HP1, which bind preferentially to H3K9me2 andestablish a repressive chromatin conformation [15]. TheSET domain in Ehmt2 and other histone methyltransfer-ases also have the potential to methylate non-histonepolypeptides [24, 25], allowing possible regulation of thelocalization and activity of a variety on non-histone pro-teins [26]. Ehmt2 contains an ankyrin domain, enablingprotein–protein interactions [27] with a variety of partnersincluding DNA methyltransferases (DNMT) [28], whichprovide an alternative mechanism for gene repression[29]. Finally, Ehmt2 can act as a gene activator in additionto its repressive roles, by interacting with coactivatorssuch as in nuclear receptor-mediated transcription regula-tion [23, 30].Despite Ehmt2 being capable of regulating a diverserange of cellular and biological functions, little is knownabout its role in skeletal muscle. Working with thewidely used murine cell line C2C12, Ling et al. [31]recently highlighted Ehmt2 as a possible regulator ofmyogenic differentiation. Using in vitro overexpressionand knockdown strategies, Ehmt2 was shown to act asan inhibitor of myotube formation. Biochemical analysessuggested that EHMT2 also has the capability to directlymethylate MYOD at K104 [31], revealing a novelBhlhe41/Sharp1-dependent mechanism inhibiting myo-genesis that is controlled by Ehmt2 through both directmodulation of MYOD and the repressive H3K9me2modification of Myod target genes [32, 33]. In spite ofthese initial findings, whether Ehmt2 plays an equivalentrole in a more complex biological system such as in vivoskeletal muscle development or regeneration is yet to beevaluated. In this study, we generated transgenic mousestrains to genetically delete Ehmt2 during muscle devel-opment as well as in adult satellite cells. We found thatproliferation and differentiation of satellite cells was notinfluenced by the absence of Ehmt2. Knocking outEhmt2 also failed to result in significant consequencesfor skeletal muscle development and in adult muscleregeneration in vivo. Thus, Ehmt2 is completely dispens-able for the normal functioning, maintenance, and dam-age response of murine skeletal muscle.MethodsMice and animal careC57BL/6 mice harboring the Ehmt2floxed allele were pre-viously generated by Lehnertz et al. [17]. In this strain,Cre-mediated targeting of the Ehmt2floxed allele resultsin a genomic deletion from exon 4 to exon 20 and aframeshift mutation that places the downstream codingsequence out of frame. All other transgenic strains usedherein were generated by other groups [34–36] and ob-tained from The Jackson Laboratory. Inducible Cre re-combinase was activated by intraperitoneal injection oftamoxifen dissolved in corn oil (250 mg/kg of bodyweight per day) for 5 consecutive days, followed by7 days without any treatment to allow sufficientactivation. The mice were housed in a pathogen-freefacility, and all experiments were performed accordingto the Canadian Council on Animal Care (CCAC)regulations.Acute muscle injuryThe tibialis anterior (TA) muscle of 8–12-week-old con-trol or experimental mice was injected with the myo-toxin notexin (7 μl), as previously described [37].Zhang et al. Skeletal Muscle  (2016) 6:22 Page 2 of 10Flow cytometry/FACSSkeletal muscle tissue was prepared as described previ-ously [37]. Cell preparations were then incubated withprimary antibodies for 30 min at 4 °C in supplementedPBS containing 2 mM EDTA and 2 % fetal bovine serumat ~3 × 10 [7] cells per milliliter. We used the followingmonoclonal-conjugated primary antibodies: anti-plateletendothelial cell adhesion molecule (PECAM)-1 (CD31)(clones MEC13.3, Becton Dickinson, and 390, CedarlaneLaboratories); anti-protein tyrosine phosphatase receptortype C (PTPRC) (CD45) (clone 30-F11, Becton Dickinson);anti-lymphocyte antigen 6A/E (LY6A/E) (Sca-1) (clone D7,eBioscience); anti-vascular cell adhesion molecule (VCAM)(produced in-house); and anti-integrin alpha-7 (producedin-house). Satellite cells were identified as PECAM−,PTPRC−, LY6A/E−, VCAM+, and integrin alpha-7+.Antibody dilution and staining volume were determinedexperimentally. Where necessary, biotinylated primaryantibodies were detected using streptavidin coupled tophycoerythrin (PE), allophycocyanin (APC), phycoerythrin-cyanine 7 tandem complex (PE-Cy7), or fluorescein iso-thiocyanate (FITC) (Caltag). To assess viability, cells werestained with propidium iodide (1 μg/ml) and Hoechst33342 (2.5 μg/ml) and resuspended at ~1 × 10 cells/ml [7]immediately before flow cytometry analysis or sorting.Analysis was performed on LSRII (Becton Dickinson)equipped with three lasers. Data were collected usingFACSDiva software. Sorts were performed on a FACSInflux (Becton Dickinson) or FACSAria (Becton Dickinson),both equipped with three lasers, using a 100-μm nozzle at18 psi to minimize the effects of pressure on the cells.Sorting gates were strictly defined based on “fluorescenceminus one” stains.Cell culture and immunocytochemistryViable single myofibers were isolated from the extensordigitorum longus (EDL) muscle of 6–8-week-old micefollowing dissociation with collagenase I as previouslydescribed [38]. Myofibers and their associated satellitecells were maintained ex vivo for up to 72 h in high-glucose Dulbecco’s modified Eagle’s medium (DMEM)supplemented with 20 % v/v FBS, 0.5 % v/v chick em-bryo extract, and pen-strep. Following culture, singlemyofibers were fixed in 4 % PFA and then stained over-night with the following primary antibodies: mouse anti-Pax7 (Developmental Studies Hybridoma Bank (DSHB)),mouse anti-MyoD (Dako, clone 5.8A), mouse anti-Myogenin (DSHB, clone FD5).Fluorescence-activated cell sorting (FACS)-sorted cellswere grown in high-glucose DMEM, supplemented with2.5 ng/ml bFGF (Invitrogen) 20 % v/v FBS, and 10 % v/vhorse serum. This medium is hereafter referred to as“growth medium.” Cells were seeded in tissue culture-treated plastics coated with Matrigel (BD Biosciences).The media were changed every 24–48 h. To inducemyogenic differentiation, confluent myoblasts were cul-tured in DMEM supplemented with 5 % horse serum forup to 96 h, before being fixed in 4 % PFA and stainedovernight with mouse anti-Myosin (DSHB, clone MF20).HistologyTA muscles were dissected from mice, fixed in 4 % para-formaldehyde overnight followed by 70 % ethanol over-night, and then embedded in paraffin following standardprotocols. Tissues were cut with a microtome in a cross-sectional orientation through the entire length of themuscle. Cross sections of 5 mm thickness were thenmounted onto glass slides (Thermo Fisher Scientific,USA) and stained with Masson’s trichrome or Picrosiriusred following standard protocols. The cross-sectionalarea was used as a measure of myofiber size, which wasproduced by semi-automated measurements on stitchedwhole-section images (Nikon).Gene deletion efficiency measured by allele countAt least 10,000 FACS-purified satellite cells per samplewere used for measuring the efficiency of Ehmt2 condi-tional knockouts. Cells were lysed and purified for genomicDNA, of which 50 ng per sample was mixed with digitaldroplet PCR supermix (Bio-Rad) and two TaqMan CopyNumber Assays for a duplex (fluorescein amidite (FAM)and VIC) digital droplet PCR assay. The “functional assay”is a TaqMan Copy Number Assay with FAM dye (ThermoFisher Scientific #4400291 Mm00466045_cn) that detectsEhmt2 in a region from intron 14 to exon 15, which isfound only in the wildtype or floxed alleles (functionalalleles) of the gene. The “reference assay” is a TaqManCopy Number Assay with VIC dye (Thermo FisherScientific #4400291 Mm00466690_cn) that detects Ehmt2in a region from intron 25 to intron 26, which is found inany null, wildtype, or floxed alleles of the gene. Droplets ofthe mixture were generated according to standard digitaldroplet PCR protocol (Bio-Rad) and ran in a thermocyclerfor 40 PCR cycles. PCR products were read droplet-wise induplex (FAM and VIC) following standard protocol (Bio-Rad), and a signal ratio was calculated by dividing theabsolute copy number of the functional assay to the copynumber of the reference assay. The signal ratio was thenused to interpolate the functional allele frequency (%) froma known standard curve (see Additional file 1: Figure S1).The standard curve of signal ratios was produced by per-forming digital droplet polymerase chain reaction (ddPCR)using genomic DNA that were mixed at known propor-tions from Ehmt2wt/wt and Ehmt2null/null mouse embryonicfibroblasts. The standard curve has a Pearson coefficient of0.993, and a statistical test of linearity yielded a p value of0.0001. The results of this measurement are presentedeither as the frequency of functional alleles in eachZhang et al. Skeletal Muscle  (2016) 6:22 Page 3 of 10experimental sample or as the efficiency of gene deletion,which is calculated as the reciprocal of the functional allelefrequency.StatisticsMouse weight measurements plotted against age weresubjected to linear regression analysis. A sum-of-squaresF test was performed on a shared model to test the nullhypothesis that one curve fits all groups. The result-ing p value was used to conclude the differences ingrowth pattern between the groups. Error for mean ofmeans is propagated by weighted pooled variance.ResultsEhmt2 (G9a) is dispensable in skeletal muscledevelopmentTo examine the role of Ehmt2 in myogenesis in vivo, wefirst established a transgenic mouse model in which theEhmt2 gene was conditionally knocked out in theskeletal muscle lineage. Mice harboring loxP-flankedEhmt2 alleles (Ehmt2floxed) were crossed to mice with aCre recombinase gene knocked in to the Myod locus(MyodCre) [34]. To verify the efficiency of the conditionalknockout, we performed an exon-specific allele-countingassay using digital droplet PCR to measure functionalallele frequency [19] (see the “Methods” section for de-tails). In FACS-purified satellite cells from control mice(Myodwt/wt Ehmt2floxed/floxed), the Ehmt2 functional allelefrequency was 100 %; whereas in the knockout mice(Myodwt/Cre Ehmt2floxed/floxed), the functional allele fre-quency was reduced to 2.9–7.9 % (95 % CI) (Fig. 1a).These genomic results were consistent with immuno-staining quantification of EHMT2 protein in satellitecells. In wildtype mice, EHMT2 was robustly expressedin activated satellite cells whereas no detectable stainingwas present in the knockout mice (Fig. 1c). Furthermore,western blot analysis of whole skeletal muscle lysatesfrom the conditional knockout mice showed reduction ofH3K9me2 levels compared to those of the wildtype(Fig. 1b), congruent with previous reports that H3K9me2is diminished in Ehmt2null/null models [14].Knockout and control group progenies from MyodCreand Ehmt2floxed breeding were born at expected Mendelianfrequencies (Fig. 1d), and neonatal weights were similarbetween both groups (Fig. 1e). Growth patterns of knock-out and control group progenies were charted by bodyweight, which showed no statistically significant differ-ences (Fig. 1f). Mature skeletal muscles in these mice areof similar size (Fig. 1g) and showed no difference uponhistological examination, which included a comparison ofawt/wt wt/f f/f020406080100Ehmt2 genotypefunctional allelefrequency (%)Cre+ f/f Cre+ f/wt Cre- f/f Cre- f/wt01020304050no. of progenyd expectedobservedmale female0. WTneonatalweight (g)KOns ns10 20 30 400510152025age (days)weight (g)WTHETKON.S.fWTKOcDAPI EHMT2gWTKOWT KO010203040TA weight (mg)nshWT KO05001000150020002500average myo fiber CSA (µm2 )insbH3H3K9me2normalized H3K9me2WT KO01234WT KOFig. 1 Ehmt2 is dispensable for normal muscle development. a Ehmt2 deletion efficiency as measured by functional allele frequency in FACS-purifiedsatellite cells. Analysis by gDNA allele counting using ddPCR, n≥ 3. b Relative abundance of H3K9me2 in whole skeletal muscle tissue lysate of wildtypeand knockout mice, normalized to histone H3. c Immunofluorescence detection of EHMT2 on myofiber. d Number of live births from n≥ 3 matingpairs of MyodCre Ehmt2floxed/floxed mice. e, f Neonatal weight and growth curve of wildtype and knockout mice at D0 −D1. g Masson’s trichrome stain ofhistological sections of the tibialis anterior muscle of adult mice. h Whole muscle weight. i Myofiber size measurement by cross-sectional areaZhang et al. Skeletal Muscle  (2016) 6:22 Page 4 of 10myofiber size by measuring the cross-sectional area ofmyofibers (Fig. 1h, i). These findings indicate that Ehmt2in the skeletal muscle lineage is dispensable in embryonicand fetal development.Ehmt2 (G9a) knockout satellite cells have normalproliferation kinetics and differentiation capacity in vitroAs previous reports have suggested that Ehmt2 is animportant regulator of C2C12 myogenesis, we next assessedwhether it plays a similar role in satellite cell and primarymyoblast cultures ex vivo. To analyze satellite cell prolifera-tion, we performed a 4-h 5-ethynyl-2′-deoxyuridine (EdU)pulse on myofiber-associated satellite cells from the wild-type and conditional knockout mice after 72 h in culture.No differences in EdU incorporation were detectedbetween the control and knockout groups (Fig. 2a, e).Similarly, quantification of immunofluorescent stainingshowed similar numbers of PAX7+ (Fig. 2c) and MYOD+(Fig. 2b) satellite cells after 72 h in culture. These resultsindicate that satellite cells lacking Ehmt2 show compar-able rates of proliferation and myogenic activation towild-type cells ex vivo.Next, we assessed the requirement of Ehmt2 for satel-lite cells to undergo myogenic differentiation. Satellitecell-derived myofibers from wildtype (WT) and knock-out (KO) mice were expanded to confluence, induced todifferentiate, and then analyzed by immunostaining ofMYOG and myosin heavy chain after 4, 24, and 48 h.Under differentiating conditions, MYOG-expressing cellsincreased, but no significant differences in the percentagesof cells expressing MYOG were observed between controland KO myoblasts (Fig. 2e). Similarly, we found no signifi-cant differences in myogenic fusion index (ratio of fusednuclei found in myosin-expressing cells to total nuclei)following 48 h of differentiation (Fig. 2f), providing furthersupport that deletion of Ehmt2 does not have significanteffects on the progress or timing of myogenic differenti-ation in primary myoblasts.Together, these data provide little evidence that Ehmt2plays a major role in the regulation of satellite cell prolif-eration or myogenic differentiation in vitro.Skeletal muscle- and satellite cell-specific deletion ofEhmt2 (G9a) has little effect on muscle regenerationin vivoBefore evaluating the requirement of Ehmt2 in the re-sponse of skeletal muscle to acute injury in vivo, we firstanalyzed the expression of the gene in our injury modelin WT mice, which involved an intramuscular injectionof notexin (a snake venom toxin) in the TA muscle.During the ensuing regenerative process, we performedtranscriptome sequencing of WT primary myoblasts ataDAPIEdUWT KOWT KO020406080100% EdU+ cellsN.S.DAPIMYODWT KObWT KO020406080100% MYOD+ cellsN.S.DAPIPAX7WT KOcWT KO020406080100% PAX7+ cellsN.S.DAPIMYOGWT KOe4H 24H 48H020406080100% MYOG+ cellstime in differentiation mediaf WT KODAPIMYH2WT KO0204060myoblastfusion index (%)N.S.DAPIEHMT2WT KOd4H 24H 48H020406080100% EHMT2+ cellstime in differentiation mediaKOWTWTKOFig. 2 Ehmt2 knockout satellite cells proliferate and differentiate normally. Myofibers were isolated from WT and Myod-Cre Ehmt2 KO mice andcultured under growth conditions for 72 h and satellite cells visualized by confocal microscopy. a Detection of proliferating cells after 4 h of EdUtreatment. b, c Immunofluorescence detection of MYOD and PAX7. Myoblasts were seeded at equal densities and induced with differentiationmedia for 4, 24, and 48 h. d, e Immunofluorescence detection of EHMT2 and MYOG, respectively. f Immunofluorescence detection of myosinheavy chain in myotubes, myoblast fusion index calculated as % nuclei inside myosin-expressing myotubesZhang et al. Skeletal Muscle  (2016) 6:22 Page 5 of 10different timepoints; in our results, neither Ehmt2 norEhmt1 showed any dynamic changes in expression dur-ing the process, in contrast to key myogenic regulators(Additional file 1: Figure S2).Then, we evaluated the aforementioned MyodCreEhmt2floxed/floxed model, which deletes Ehmt2 in all skel-etal muscles and satellite cells during their development[39]. Following injury, we quantified the cross-sectionalarea of centrally nucleated myofibers as a measure of re-generation [40]. Despite a trend toward an increasednumber of the largest fibers in KO samples, no statisticallysignificant difference was found between the control andKO mice in the distribution of myofiber size at 7, 14, or21 days post injury, indicating comparable regenerativecapacities (Fig. 3b, Additional file 1: Figure S3).Myod-driven CRE leads to target deletion early indevelopment and could therefore trigger compensatoryeffects that mask the regulatory role of Ehmt2 in adultregenerative myogenesis. To mitigate this risk, weperformed a satellite cell-specific, inducible Ehmt2 KOusing a strain carrying the tamoxifen-activated CreERT2recombinase knocked in to the Pax7 locus [35]. Thisallowed us to confine the gene deletion within adult sat-ellite cells and to use a Rosa26YFP reporter to monitorthe efficiency of induction. One week after the end oftamoxifen treatment, we found that the YFP reporterwas activated in 70 % of cells in the satellite cell popula-tion (Additional file 1: Figure S4). Since this was not adefinitive measure of gene deletion, we further per-formed Ehmt2 allele count and found that the functionalallele frequency had been reduced to 27.2 ± 4.2 % (SEM)in the satellite cell population purified by FACS.Acute muscle injury by notexin was performed at7 days after the final CreERT2 induction on mice har-boring Pax7CreERT2 and Ehmt2floxed/null alleles. At 7, 14,and 21 days after the injury, no significant differences inmyofiber size distribution were observed between thecontrol and KO groups (Fig. 3f, Additional file 1: FigureS5, data not shown), suggesting that lack of Ehmt2 inadult satellite cells does not significantly affect repairand regeneration in vivo.Together, our data provide little evidence for a role ofEhmt2 in regulating skeletal muscle development,homeostasis, or regeneration in vivo.DiscussionIn this study, we provide a comprehensive assessment ofthe biological consequences of G9a deletion in skeletalmuscle progenitors. Our data strongly suggests thatEhmt2 is dispensable for both developmental and re-generative myogenesis in vivo and is not required fornormal satellite cell proliferation and myogenic differ-entiation in vitro.Although germ line deletion of Ehmt2 is embryonicallylethal, conditional KO models suggest that Ehmt2 playsdistinct roles in different tissues. Studies have shown aninvolvement of Emht2 in the regulation of embryogen-esis [14, 28, 41], cardiac morphogenesis [16], lymphopoi-esis [17, 18], myelopoiesis [19], germ cell development[15], brain and cognitive development [21, 22], and drugaddiction [20], confirming Ehmt2 is capable of control-ling a diverse range of biological processes. In the caseof myogenesis, we showed that conditional loss of Ehmt2in vivo does not induce any significant developmentalimpact or any significant alterations to the regenerativecapacity of myogenic progenitors in response to skeletalmuscle injury. Deletion of Ehmt2 in primary myoblastsalso fails to induce any significant alterations in prolifer-ative kinetics or differentiation capacity in vitro, suggest-ing little role for Ehmt2 in regulating myogenesis.These results are surprising given the previous reportssuggesting an important role for Ehmt2 in negativelyregulating myogenic differentiation of C2C12 cells, animmortalized myogenic line. In particular, it was demon-strated that siRNA knockdown of Ehmt2 led to en-hanced and/or premature differentiation of C2C12myoblasts [31]. It was further reported that Ehmt2 is anintegral component of the mechanism with whichBhlhe41/Sharp1 regulates in myogenesis, in that it is re-cruited by SUMOylated BHLHE41/SHARP1 [33], meth-ylates MYOD at lysine 104 [31], and leads to repressiveH3K9me2 modifications on Myod targets [32]. Theseresults were not consistent with observations in thecurrent study when examining the differentiation cap-acity of primary myoblasts lacking Ehmt2, which showedno premature differentiation, no alterations in myogenicfusion, and normal proliferation. This discrepancy infindings could stem from the fundamental differencesbetween the biological models being analyzed; unlikeC2C12 cells, which were derived from a different strainof mouse (C3H) [42], immortalized [43], and have amuch shorter doubling time [44], the primary myoblastsin our study were not serially passaged and were ana-lyzed in their myofiber niche during proliferation and onMatrigel during differentiation, in addition to in vivoanalyses. These differences may be particularly relevantin the case of Ehmt2, as we have recently reported, inanother tissue system, that its absence has drasticallydifferent effects on transformed compared to naturalhematopoietic cells [19].To date, no genome-wide analysis of the Ehmt2-medi-ated H3K9me2 in myogenic cells exists. Dynamicchanges in H3K9me2 have been reported at specificgene bodies and regulatory regions [45, 46], suggestingthat modulating this epigenetic mark may affect genetranscription. However, in our experiments, lack ofEhmt2 led to a dramatic drop in the global levels ofZhang et al. Skeletal Muscle  (2016) 6:22 Page 6 of 10wt/wt f/null020406080100Ehmt2 genotypefunctional allelefrequency (%)400600800100012001400160018002000220024002600280030003200340036003800400042004400460048000246810relative frequency (%)fi bre size (µm2)cWTKOeWT KO0500100015002000250030003500average myo fibe rCSA (µm2 )fnsb WT KOg WT KOD7a MyodCreEhmt2f/fD14 D21D0notexininjury histologyhistologyhistologyD12D5D0notexininjurytamoxifend Pax7CreERT2Ehmt2f/nullD19 D26 D33histologyhistologyhistologyFig. 3 (See legend on next page.)Zhang et al. Skeletal Muscle  (2016) 6:22 Page 7 of 10detectable H3K9me2 in the absence of any effects onskeletal muscle development. This indicates that Ehmt2activity is mostly non-redundant and questions the im-portance of Ehmt2-mediated histone modifications inmyogenesis in particular and in the control of differenti-ation in general.What remains unclear is the status of EHMT2-mediated methylation of MYOD at lysine 104 [31] in thein vivo model. Ling et al. reported the identification ofthis residue by mass spectrometry of peptides resultingfrom the digestion of MYOD with trypsin [31]. However,the reported MYOD peptides, ACKACKRKTT and itsmethylated forms, do not appear to be obtainable bytrypsin digestion alone or by any commonly used diges-tion method. The reported MYOD peptide is also notfound in the tandem mass spectrometry proteomicsrepository PeptideAtlas (https://db.systemsbiology.net/sbeams/cgi/shortURL?key=1mk36ybs). More intriguingly,the proposed mechanism [31] was based on liquid chro-matography–mass spectrometry (LC-MS) results, showingthat the different methylation states of the MYOD peptideare separated by only 1 m/z unit each. Methylation adds14 Da to the peptide mass; thus, each peptide would haveto carry 14 charges on 10 residues. The LC-MS resultscould not possibly correspond to methylation of the re-ported MYOD peptide. The uncertainty of MYOD methy-lation, together with the dispensability of Ehmt2-mediatedH3K9me2 in vivo, casts doubts on the proposed role ofEhmt2 in Bhlhe41/Sharp1-mediated regulation of myo-genesis [32]. Nevertheless, SUMOylated Bhlhe41/Sharp1[33] may still regulate Myod and downstream targetsthrough an alternative mechanism.Although we have shown here that the loss of EHMT2’shistone methylation function in our model was not com-pensated, the possibility exists that its potential interactionwith myogenic regulators could be compensated by an-other gene, such as its close homologue Ehmt1 (GLP).These two genes are highly similar in structure, as theirprotein products contain highly similar catalytic domainsfor lysine methylation (SET domain) and a set of ankyrinrepeats for protein–protein interaction. These two en-zymes are known to form heterodimers [14] but also playunique roles depending on the cell type and developmen-tal stage [47]. Using domain-specific mutations, theEHMT1 but not the EHMT2 ankyrin repeats were foundto be required for mouse viability [48], suggesting that thisdomain function in EHMT2 could be compensated byEHMT1. On the other hand, the SET domain in EHMT1is dispensable for mouse viability [16], suggesting EHMT2could compensate for EHMT1’s methyltransferase activity.In our Ehmt2 KO cells, even though the histone methyl-transferase activity, a function shared by both pro-teins, is not compensated by EHMT1, its ankyrin repeat-dependent protein–protein interactions may be compen-sated. Both proteins have been reported to methylate anumber of non-histone targets beyond MYOD [49, 50],and it is unknown if EHMT1 could replace EHMT2 inbinding and methylating these targets. Interestingly, suchcompensation by Ehmt1 was not observed in C2C12studies [31], and in our transcriptome data, neither of thegenes showed any dynamic changes during regeneration.Nevertheless, to fully address this concern, a conditionaldouble KO of Ehmt2 and Ehmt1 in myogenesis would berequired.ConclusionsIn this study, we analyzed tissue-specific KO models ofEhmt2 both during development and in adult satellitecells, and unlike as previously reported, we found no evi-dence for a significant role of this methyltransferase inthe development or regeneration of skeletal muscle. Thedrop in H3K9me2 levels observed in cells lacking Ehmt2strongly suggests that this histone modification isdispensable for the regulation of myogenesis. Primarycultures revealed that Ehmt2 does not significantly alterthe proliferation and differentiation processes of satellitecells. Thus, the proposed regulatory role of Ehmt2 inmyogenesis cannot be validated in vivo.Additional fileAdditional file 1: Main figures are referred to as Fig. # in the article.Figure legends are supplied on the next page. Supplementary figureswith figure legends are referred to as Supplementary Fig. # in the article.AbbreviationsAPC: allophycocyanin; bHLH: basic helix-loop-helix; Bhlhe41: basic helix-loop-helix family member e41, aka Sharp1; CCAC: Canadian Council on AnimalCare; ChIP: chromatin immunoprecipitation; CI: confidence interval;Cre: Causes Recombination; ddPCR: digital droplet polymerase chainreaction; DMEM: Dulbecco’s modified Eagle’s medium; DNMT: DNAmethyltransferase; DSHB: Developmental Studies Hybridoma Bank;EDL: extensor digitorum longus; EdU: 5-ethynyl-2′-deoxyuridine;Ehmt1: euchromatic histone-lysine N-methyltransferase 1, aka GLP (G9a-like(See figure on previous page.)Fig. 3 Ehmt2 is dispensable for muscle regeneration. a Schematic diagram of leg injury timeline for MyodCre Ehmt2floxed/floxed mice. b, c Masson’strichrome stain of histological sections of the tibialis anterior muscle of adult MyodCre Ehmt2floxed/floxed mice at 21 days after injury and myofibersize measurement by cross-sectional area, respectively. d Schematic diagram of leg injury timeline for Pax7CreERT2 Ehmt2floxed/null mice. e Ehmt2deletion efficiency by Pax7CreERT2 as measured by functional allele frequency in FACS-purified satellite cells. Analysis by gDNA allele counting usingddPCR, n≥ 3. f, g Myofiber size measurement by cross-sectional area and Masson’s trichrome stain of histological sections of the tibialis anteriormuscle of induced adult Pax7CreERT2 Ehmt2floxed/floxed mice at 21 days after injury, respectivelyZhang et al. Skeletal Muscle  (2016) 6:22 Page 8 of 10protein); Ehmt2: euchromatic histone-lysine N-methyltransferase 2, aka G9a;FACS: fluorescence-activated cell sorting; FAM: fluorescein amidite;FITC: fluorescein isothiocyanate; H2B: histone 2B; H3K27ac: acetylation oflysine 27 on histone H3 protein subunit; H3K4me1: unimethylation of lysine4 on histone H3 protein subunit; H3K9me2: trimethylation of lysine 9 onhistone H3 protein subunit; HP1: heterochromatin protein 1; KO: knockout;LC-MS: liquid chromatography–mass spectrometry; LY6A/E: lymphocyteantigen 6A/E, aka SCA-1 (stem cell antigen 1); MRF: myogenic regulatoryfactor; Mrf4: myogenic regulatory factor 4, aka myogenic factor 6 (herculin);Myf5: myogenic factor 5; Myog: myogenin; Pax7: paired box 7;PE: phycoerythrin; PECAM: platelet endothelial cell adhesion molecule, akaCD31 (cluster of differentiation 31); PE-Cy7: phycoerythrin-cyanine 7 tandemcomplex; PTPRC: protein tyrosine phosphatase receptor type C, aka CD45(cluster of differentiation 45); SEM: standard error of mean; SET: Su (var)3-9and enhancer of zeste; Shh: sonic hedgehog; SUMO: small ubiquitin-likemodifier; TA: tibialis anterior; VCAM: vascular cell adhesion molecule, akaCD106 (cluster of differentiation 106); WT: wildtype.AcknowledgementsWe thank all the lab members and alumni for the technical and scientificsupport, especially Ms Lin Yi, Mr Chihkai Chang, Ms Vittoria Canale,Ms Claudia Hopkins, Dr Bernhard Lehnertz, Dr Dario Lemos, Dr PretheebanThavaneetharajah, Dr Anuradha Natarajan, Dr Farshad Babaei, Ms ChristineEisner, Dr Coral-Ann Lewis, Dr Marcela Low, Ms Joey Nguyen, Dr HeshamSoliman, Ms Gloria Loi, Mr Alan Wong, Mr Ryan Cheng, Mr Alvin Tsuei, andespecially Dr Elena Groppa for the transcriptome-sequencing database ofskeletal muscle regeneration. The Animal Unit staff at The BiomedicalResearch Centre were instrumental in supporting the in vivo research,especially Ms Helen Merkens, Ms Krista Ranta, Mr Wei Yuan, and Mr JerryChen. We also owe tremendous help from Mr Andy Johnson and Mr JustinWong of UBC Flow Cytometry, Ms Ingrid Barta of the UBC Histology Lab, andMr John Schipilow and Dr Nancy Ford of the Centre for High-ThroughputPhenogenomics.FundingThis research is supported by the Canadian Institutes of Health Researchgrant number MOP-97856.Availability of supporting dataNot applicable.Authors’ contributionsRHZ contributed to the conception of the strategies, project management,design of the experiments, carrying out the in vivo and in vitro experiments,data analysis, statistical analysis, writing all the sections of the article, andfigure designs. RNJ contributed to the design of the in vitro experiments,carrying out the myofiber culture experiments, writing the “Methods” sectionfor cell culture and immunocytochemistry, and editing of the article. DYLcontributed to carrying out the in vivo experiments, collecting the data frommicroscopy, and data analysis. JK contributed to writing the discussion. FMRcontributed to the conception of the strategies, supervision of the project,design of the experiments, and writing the background, results, discussion,abstract, and all the correspondences. All authors read and approved thefinal manuscript.Competing interestsThe authors declare that they have no competing interests.Consent for publicationNot applicable.Ethics approval and consent to participateWe obtained ethical approval from the UBC Animal Care and Use Program.Received: 8 April 2016 Accepted: 17 May 2016References1. Parker MH, Seale P, Rudnicki MA. Looking back to the embryo: definingtranscriptional networks in adult myogenesis. 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Mol Syst Biol. 2014;10:724.•  We accept pre-submission inquiries •  Our selector tool helps you to find the most relevant journal•  We provide round the clock customer support •  Convenient online submission•  Thorough peer review•  Inclusion in PubMed and all major indexing services •  Maximum visibility for your researchSubmit your manuscript atwww.biomedcentral.com/submitSubmit your next manuscript to BioMed Central and we will help you at every step:Zhang et al. Skeletal Muscle  (2016) 6:22 Page 10 of 10


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