Ession via Interacting with the Histone Demethylase JHDM3AIt has been recently reported that the mammalian demethylase, JHDM3A, is capable of removing the me3 group from modified H3K9 and H3K36 [26,27,28]. To determine if an interaction between HP1a and JHDM3A might account for HP1a’s paradoxical effect on transcription and methylation of CI-1011 price 256373-96-3 site Myogenic genes, 25033180 we examined whether JHDM3A could also associate with these genes in myoblasts. ChIP assays performed on myoblasts transfected with either NSsiRNA or HP1asiRNA demonstrated that endogenous JHDM3A is also present at the Lbx1 exon 2 and myogenin promoter, and that this association is impaired in HP1a deficient cells (Fig. 6A and Fig. S4B). The impairment ofHP1 Alpha Facilitates Myogenic Gene ExpressionFigure 3. HP1a-deficient myocytes demonstrate a defect at the committed myoblast stage with reduced Lbx and MyoD expression. A, B, C. C2C12 myoblasts were transfected with indicated siRNA. 24 hours after transfection, transfection medium were changed to GM or DM, cells were incubated for an additional 24 hours. Total RNA was isolated and semiquantitative PCR analysis of gene expression was performed (A, C). mRNA levels of Lbx1 and MyoD (B) in cells cultured in GM quantified by real-time PCR normalized to GAPDH. *P,0.05 for Lbx1 levels siNS vs. siHP1a; **P,0.05 for MyoD levels siNS vs. siHP1a. D. EDU assay were performed 24 hours after indicated siRNA transfection in C2C12 myoblasts. Percentage of EDU positive cells was calculated. *P,0.05 for sins versus siHP1a. E. C2C12 cells were transfected with indicated siRNA. Total RNA was isolated 36 hours after transfection. Semiquantitative PCR analysis of gene expression was performed. F. C2C12 myoblasts were co-transfected with indicated siRNA and GFP-expressing plasmid. Total RNA was extracted from GFP-positive cells isolated by fluorescence-activated cell sorting (FACS). Semiquantitative PCR analysis of gene expression was performed. doi:10.1371/journal.pone.0058319.gmyoblasts was accompanied by reduced Lbx1, MyoD, MEF2C and myogenin expression without significantly effecting Pax7 or Myf5. Reduction 1081537 in expression of these key regulators of myogenesis is likely sufficient to account for the subsequent defect in myogenic differentiation we observed. This dependence of skeletal muscle differentiation was specific to myoblasts since no defect was seen when HP1a was knockdown in myotubes. Thisdata is reminiscent of recent studies exploring Rb’s role in skeletal muscle differentiation, where Rb is critical for progression through myogenic differentiation [29,30,31] but is dispensable for the maintenance of the terminally differentiated state [32,33]. Although our study demonstrates that HP1a is dispensable for maintenance of terminal differentiation, whether this implies HP1a has no role in myotubes 
or whether HP1b and c, which areHP1 Alpha Facilitates Myogenic Gene ExpressionFigure 4. HP1a 
is dispensable for maintenance of terminal differentiation. C2C12 cells were cultured in DM for 72 hours and then transfected with indicated siRNA. A, 48 hours after transfection, total RNA was isolated and semiquantitative PCR analysis of gene expression was performed. B. 24 hours after siRNA transfection, DMEM contain 20 FBS were added and cells were cultured for another 24 hours. Total protein were extracted and probed for indicated antibodies. C. 24 hours after siRNA transfection, cells were stimulated with DMEM contain 20 FBS in the presence of BrdU for 24 ho.Ession via Interacting with the Histone Demethylase JHDM3AIt has been recently reported that the mammalian demethylase, JHDM3A, is capable of removing the me3 group from modified H3K9 and H3K36 [26,27,28]. To determine if an interaction between HP1a and JHDM3A might account for HP1a’s paradoxical effect on transcription and methylation of myogenic genes, 25033180 we examined whether JHDM3A could also associate with these genes in myoblasts. ChIP assays performed on myoblasts transfected with either NSsiRNA or HP1asiRNA demonstrated that endogenous JHDM3A is also present at the Lbx1 exon 2 and myogenin promoter, and that this association is impaired in HP1a deficient cells (Fig. 6A and Fig. S4B). The impairment ofHP1 Alpha Facilitates Myogenic Gene ExpressionFigure 3. HP1a-deficient myocytes demonstrate a defect at the committed myoblast stage with reduced Lbx and MyoD expression. A, B, C. C2C12 myoblasts were transfected with indicated siRNA. 24 hours after transfection, transfection medium were changed to GM or DM, cells were incubated for an additional 24 hours. Total RNA was isolated and semiquantitative PCR analysis of gene expression was performed (A, C). mRNA levels of Lbx1 and MyoD (B) in cells cultured in GM quantified by real-time PCR normalized to GAPDH. *P,0.05 for Lbx1 levels siNS vs. siHP1a; **P,0.05 for MyoD levels siNS vs. siHP1a. D. EDU assay were performed 24 hours after indicated siRNA transfection in C2C12 myoblasts. Percentage of EDU positive cells was calculated. *P,0.05 for sins versus siHP1a. E. C2C12 cells were transfected with indicated siRNA. Total RNA was isolated 36 hours after transfection. Semiquantitative PCR analysis of gene expression was performed. F. C2C12 myoblasts were co-transfected with indicated siRNA and GFP-expressing plasmid. Total RNA was extracted from GFP-positive cells isolated by fluorescence-activated cell sorting (FACS). Semiquantitative PCR analysis of gene expression was performed. doi:10.1371/journal.pone.0058319.gmyoblasts was accompanied by reduced Lbx1, MyoD, MEF2C and myogenin expression without significantly effecting Pax7 or Myf5. Reduction 1081537 in expression of these key regulators of myogenesis is likely sufficient to account for the subsequent defect in myogenic differentiation we observed. This dependence of skeletal muscle differentiation was specific to myoblasts since no defect was seen when HP1a was knockdown in myotubes. Thisdata is reminiscent of recent studies exploring Rb’s role in skeletal muscle differentiation, where Rb is critical for progression through myogenic differentiation [29,30,31] but is dispensable for the maintenance of the terminally differentiated state [32,33]. Although our study demonstrates that HP1a is dispensable for maintenance of terminal differentiation, whether this implies HP1a has no role in myotubes or whether HP1b and c, which areHP1 Alpha Facilitates Myogenic Gene ExpressionFigure 4. HP1a is dispensable for maintenance of terminal differentiation. C2C12 cells were cultured in DM for 72 hours and then transfected with indicated siRNA. A, 48 hours after transfection, total RNA was isolated and semiquantitative PCR analysis of gene expression was performed. B. 24 hours after siRNA transfection, DMEM contain 20 FBS were added and cells were cultured for another 24 hours. Total protein were extracted and probed for indicated antibodies. C. 24 hours after siRNA transfection, cells were stimulated with DMEM contain 20 FBS in the presence of BrdU for 24 ho.