Urs. Cell cycle reentry was determined by immunostaining for BrdU incorporation (Blue: DAPI, Red: sarcomeric myosin heavy chain; Green; BrdU). Scale bar equals 50 mm. doi:10.1371/journal.pone.0058319.gexpressed normally in HP1a deficient myotubes, compensate for HP1a’s function will need to be determined. Although overexpression of a siRNA resistant 
HP1a 25033180 was able to rescue the defect in myogenic gene expression in transfected cells there was a persistent defect in myotube formation (Fig. 2). Although we achieved very high 
transfection efficiency (.90 ) for siRNA in C2C12 cells, the transfection efficiency of pLPC-EHGF-rHP1 alpha in C2C12 is low but consistent with published transient transfection rates in these cells [34]. Thus, we can not determine if the defect in myotube formation is related to the expected low rate of rescue due to low transient transfection efficiency, high levels of HP1a expression by transfected construct versus a true physiologic difference. Although HP1’s ability to silence gene transcription is well documented, few examples exist of its ability to activate gene expression in mammalian cells and little is known regarding the possible mechanisms. However, it was known that when activating gene expression HP1 can associate anywhere within genomic sequences [9]. It has been recently demonstrated that Pax7 SMER-28 custom synthesis activates Myf5 expression by recruitment of a histone methytransferase complex to Myf5 coding regions [35]. However HP1a’s function on transcription activation appeared independent of histone acetyltransferases and methyltransferase since there was no discernable change in the level of H3K9Ac and H3K4me2 after knockdown of HP1a expression. Instead, we found total H3K9me3 levels increased after knockdown of HP1a either in promoter or transcribed regions generally is associated with gene silencing [36]. Depleting HP1a decreased Lbx1 expression in C2C12 myoblasts correlating with increased H3K9me3 on Lbx1 exon 2. This appears to be a direct effect since HP1a binds directly to this same region of Lbx1. Interestingly, despite previous reports that HP1a interacted with MEF2C to inhibit ML 281 site myogenin expression, depleting HP1a resulted in decreased endogenous myogenin expression, which was associated with a higher level of H3K9me3 at the myogenin promoter. These results suggest that HP1a might be promoting demethylation of H3K9me3 at myogenic promoters. Recent studies have identified a family of JmjC domaincontaining proteins that possess histone demethylation activity andfacilitate transcription activation [26,27,28]. We were able to demonstrate an association of JHDM3A with genomic sequence of Lbx1 and myogenin and this interaction was HP1a-dependent. JHDM3A, also known as JMJD2A, is a JmjC domain-containing protein that possesses histone demethylation activity, which specifically demethylates tri- and di- methylation of H3K9 [26,27]. We demonstrated that JHDM3A interacts with HP1a and the effects of JHDM3A knockdown on myogenic gene transcription were similar to HP1a deficiency. These results suggest that HP1a recruits JHDM3A to transcriptional regions of Lbx1, demethylating H3K9me3, and facilitating transcription to promote myogenic differentiation. However, knockdown of JHDM3A delayed myogenic gene expression but did not block C2C12 differentiation completely (Fig. S5 and data not shown). Several possible explanations exist for this observation. JHDM3A may be critical early for myogenic gene transcription but other mech.Urs. Cell cycle reentry was determined by immunostaining for BrdU incorporation (Blue: DAPI, Red: sarcomeric myosin heavy chain; Green; BrdU). Scale bar equals 50 mm. doi:10.1371/journal.pone.0058319.gexpressed normally in HP1a deficient myotubes, compensate for HP1a’s function will need to be determined. Although overexpression of a siRNA resistant HP1a 25033180 was able to rescue the defect in myogenic gene expression in transfected cells there was a persistent defect in myotube formation (Fig. 2). Although we achieved very high transfection efficiency (.90 ) for siRNA in C2C12 cells, the transfection efficiency of pLPC-EHGF-rHP1 alpha in C2C12 is low but consistent with published transient transfection rates in these cells [34]. Thus, we can not determine if the defect in myotube formation is related to the expected low rate of rescue due to low transient transfection efficiency, high levels of HP1a expression by transfected construct versus a true physiologic difference. Although HP1’s ability to silence gene transcription is well documented, few examples exist of its ability to activate gene expression in mammalian cells and little is known regarding the possible mechanisms. However, it was known that when activating gene expression HP1 can associate anywhere within genomic sequences [9]. It has been recently demonstrated that Pax7 activates Myf5 expression by recruitment of a histone methytransferase complex to Myf5 coding regions [35]. However HP1a’s function on transcription activation appeared independent of histone acetyltransferases and methyltransferase since there was no discernable change in the level of H3K9Ac and H3K4me2 after knockdown of HP1a expression. Instead, we found total H3K9me3 levels increased after knockdown of HP1a either in promoter or transcribed regions generally is associated with gene silencing [36]. Depleting HP1a decreased Lbx1 expression in C2C12 myoblasts correlating with increased H3K9me3 on Lbx1 exon 2. This appears to be a direct effect since HP1a binds directly to this same region of Lbx1. Interestingly, despite previous reports that HP1a interacted with MEF2C to inhibit myogenin expression, depleting HP1a resulted in decreased endogenous myogenin expression, which was associated with a higher level of H3K9me3 at the myogenin promoter. These results suggest that HP1a might be promoting demethylation of H3K9me3 at myogenic promoters. Recent studies have identified a family of JmjC domaincontaining proteins that possess histone demethylation activity andfacilitate transcription activation [26,27,28]. We were able to demonstrate an association of JHDM3A with genomic sequence of Lbx1 and myogenin and this interaction was HP1a-dependent. JHDM3A, also known as JMJD2A, is a JmjC domain-containing protein that possesses histone demethylation activity, which specifically demethylates tri- and di- methylation of H3K9 [26,27]. We demonstrated that JHDM3A interacts with HP1a and the effects of JHDM3A knockdown on myogenic gene transcription were similar to HP1a deficiency. These results suggest that HP1a recruits JHDM3A to transcriptional regions of Lbx1, demethylating H3K9me3, and facilitating transcription to promote myogenic differentiation. However, knockdown of JHDM3A delayed myogenic gene expression but did not block C2C12 differentiation completely (Fig. S5 and data not shown). Several possible explanations exist for this observation. JHDM3A may be critical early for myogenic gene transcription but other mech.