Anisms predominate later in skeletal differentiation. Alternatively, there are other demethylase(s) that can compensate for the lack of JHDM3A function in differentiating myocytes. JHDM3A along with JMJD2B, C and D belong to the JmjC domain-family of histone demethylase. JHDM3A, JMJD2C and JMJD2D are all capable of demethylating tri-methylated H3K9 [17]. 25033180 Although we did not detect JMJD2D in C2C12 cells, JMJD2C is expressed in C2C12 cells and knockdown of JHDM3A with siRNA did not affect JMJD2C 15755315 expression. Methylation of H3K9 has been strongly implicated in HP1 recruitment and formation of heterochromatin [28]. Thus, the interaction of HP1 with histone deacetylases and methytransferases has been well studied [12,37]. However, there is little data related to the interaction of HP1 with demethylases in mammalian cells. It has been reported that Swi6, a homolog of HP1 in yeast, recruits Epe1, a JmjC domain protein, to heterochromatin loci to facilitate transcription [38]. Recently Lin et al [39] reported that HP1a specifically interact with the Drosophila KDM4A demethylase and stimulates histone H3 lysine 36 demethylation. Our study is the first to suggest that similar interaction between HP1a and the demethylase JHDM3A occur in mammalian cells, suggesting a new paradigm for the regulation of tissue-specific gene expression.HP1 Alpha Facilitates Myogenic Gene ExpressionFigure 5. H3K9me3 levels on myogenic genes increased in C2C12 myoblasts after depleting HP1a. A. Schematic diagram of the genomic structure 
of the mouse Lbx1 gene and locations of primers used for subsequent ChIP experiments. B. Protein-DNA complexes from cross-linked chromatin extracted from C2C12 myoblasts cultured in GM were immunoprecipitated with HP1a or mouse IgG. Bound DNA was amplified using the indicated PCR primers. C, D, C2C12 myoblasts were transfected with indicated siRNA, 48 hours after transfection, cross-linked chromatin was extracted and immunoprecipitated with indicted antibodies. Lbx1 exon 2 (C) or Lbx1 genomic sequences including exon 1, intron and exon 2 (D) were amplified. E. C2C12 myoblasts were transfected with the indicated siRNA, 48 hours after transfection total cell lysates were subjected to Western blotting with the indicated antibodies. F. C2C12 myoblasts were transfected with indicated siRNA, 48 hours after transfection cross-linked chromatin was extracted and immunoprecipitated with anti-H3K9me3 antibody. Precipitated DNA was used for PCR with primers spanning the MEF2-binding site on the myogenin gene promoter. doi:10.1371/journal.pone.0058319.gOur study proposes a novel function for HP1a in maintenance of myogenic gene expression in myoblasts by inhibiting H3K9me3 via interacting with JHDM3A, which is consistent with previous findings that HP1 can activate gene expression in Drosophila [9,40]. HP1a has also been reported to inhibit MEF2-dependent transcription by interacting with MITR and HDAC9 to form a potent transcriptional repressor complex in undifferentiatedmyoblasts [12]. Thus the roles of HP1 family MedChemExpress Mirin members in differentiation are likely complex. HP1 may play multiple, developmentally dependent JSI-124 site functions in differentiation, and it’s 
positive versus negative transcriptional effects might be determined by interacting partners. The basis for specificity in recruitment of these partners is unknown at this time; however, all three HP1 isoforms can be heavily modified and these posttranslationalHP1 Alpha Facilitates Myogenic Gene ExpressionH.Anisms predominate later in skeletal differentiation. Alternatively, there are other demethylase(s) that can compensate for the lack of JHDM3A function in differentiating myocytes. JHDM3A along with JMJD2B, C and D belong to the JmjC domain-family of histone demethylase. JHDM3A, JMJD2C and JMJD2D are all capable of demethylating tri-methylated H3K9 [17]. 25033180 Although we did not detect JMJD2D in C2C12 cells, JMJD2C is expressed in C2C12 cells and knockdown of JHDM3A with siRNA did not affect JMJD2C 15755315 expression. Methylation of H3K9 has been strongly implicated in HP1 recruitment and formation of heterochromatin [28]. Thus, the interaction of HP1 with histone deacetylases and methytransferases has been well studied [12,37]. However, there is little data related to the interaction of HP1 with demethylases in mammalian cells. It has been reported that Swi6, a homolog of HP1 in yeast, recruits Epe1, a JmjC domain protein, to heterochromatin loci to facilitate transcription [38]. Recently Lin et al [39] reported that HP1a specifically interact with the Drosophila KDM4A demethylase and stimulates histone H3 lysine 36 demethylation. Our study is the first to suggest that similar interaction between HP1a and the demethylase JHDM3A occur in mammalian cells, suggesting a new paradigm for the regulation of tissue-specific gene expression.HP1 Alpha Facilitates Myogenic Gene ExpressionFigure 5. H3K9me3 levels on myogenic genes increased in C2C12 myoblasts after depleting HP1a. A. Schematic diagram of the genomic structure of the mouse Lbx1 gene and locations of primers used for subsequent ChIP experiments. B. Protein-DNA complexes from cross-linked chromatin extracted from C2C12 myoblasts cultured in GM were immunoprecipitated with HP1a or mouse IgG. Bound DNA was amplified using the indicated PCR primers. C, D, C2C12 myoblasts were transfected with indicated siRNA, 48 hours after transfection, cross-linked chromatin was extracted and immunoprecipitated with indicted antibodies. Lbx1 exon 2 (C) or Lbx1 genomic sequences including exon 1, intron and exon 2 (D) were amplified. E. C2C12 myoblasts were transfected with the indicated siRNA, 48 hours after transfection total cell lysates were subjected to Western blotting with the indicated antibodies. F. C2C12 myoblasts were transfected with indicated siRNA, 48 hours after transfection cross-linked chromatin was extracted and immunoprecipitated with anti-H3K9me3 antibody. Precipitated DNA was used for PCR with primers spanning the MEF2-binding site on the myogenin gene promoter. doi:10.1371/journal.pone.0058319.gOur study proposes a novel function for HP1a in maintenance of myogenic gene expression in myoblasts by inhibiting H3K9me3 via interacting with JHDM3A, which is consistent with previous findings that HP1 can activate gene expression in Drosophila [9,40]. HP1a has also been reported to inhibit MEF2-dependent transcription by interacting with MITR and HDAC9 to form a potent transcriptional repressor complex in undifferentiatedmyoblasts [12]. Thus the roles of HP1 family members in differentiation are likely complex. HP1 may play multiple, developmentally dependent functions in differentiation, and it’s positive versus negative transcriptional effects might be determined by interacting partners. The basis for specificity in recruitment of these partners is unknown at this time; however, all three HP1 isoforms can be heavily modified and these posttranslationalHP1 Alpha Facilitates Myogenic Gene ExpressionH.
