Nywhere within genomic sequences when activating gene expression. To clarify the mechanism underlying the altered expression of myogenicHP1 Alpha Facilitates Myogenic Gene ExpressionHP1 Alpha 
Facilitates Myogenic Gene inhibitor ExpressionFigure 2. HP1a is required for skeletal muscle differentiation. C2C12 cells were transfected with the indicated siRNA in GM and 24 hours after transfection, DM was added and cultured for 72 hours. A. Phase contrast images were obtained (A a ). Confocal fluorescence microscopy was performed after immunostaining for skeletal sarcomeric myosin heavy chain (red) and DAPI (blue) (A e ). Scale bar equals 50 mm (A a ) and 6 mm 
(A e ). B, Total protein lysates were subjected to Western blotting. C. C2C12 cells were transfected with NSsiRNA or HP1asiRNA (#1: ID# 60593; #2: ID# 60497) and cultured in DM for 72 hours. Total protein lysates were subjected to Western blotting. D. Reintroduction of siRNA resistant version of HP1a rescued myogenic differentiation in HP1asiRNA transfected cells. C2C12 myoblasts were co-transfected with the indicated siRNA and pLPCEHGF or pLPC-EHGF-rHP1a plasmids. 24 hours after transfection, DM was added and cultured for another 72 hours. Cells were stained with anti-GFP (green) and anti-Troponin C (red), nuclei were counterstained with DAPI (blue). Scale bar equals 50 mm. E. The total number of GFP positive cells and the cells positive for both GFP and Troponin C were counted and percentage of Troponin C positive cells in GFP positive cells were calculated. *P,0.01 for siHP1a/GFP versus siHP1a/GFP-rHP1a cells. Expression of GFP and GFP fusion HP1 were detected by Western blotting using anti-GFP antibody. doi:10.1371/journal.pone.0058319.gregulatory genes in HP1a depleted myocytes, we examined if HP1a directly associates with the genomic sequences of Lbx1. Lbx1 plays a critical in hypaxial muscle development [21,22]. It has been reported that Lbx1 functions upstream of MyoD [23] and is thus the earliest myogenic gene whose expression is altered by HP1a-deficiency. We performed ChIP assays using primers that span the entire Lbx1 genomic sequence including putative promoter, exons and intronic regions (Fig. 5A). Primers within the promoter of the Col11a2 gene, a known HP1a target, were used as a positive control [24]. As shown in Figure 5B, endogenous HP1a binds preferably to exon 2 of Lbx1 in myoblasts while the binding of HP1a to the other genomic sequences of Lbx1 was undetectable. HP1 has been shown to stimulate gene expression in Drosophila and these HP1-induced target genes were marked by the presence of dimethylated histone 3 lysine 4 (H3K4me2), which is found, along with histone acetylation (H3K9Ac), on active chromatin [25]. To determine the effects of HP1a depletion on histone modifications present on Lbx1 exon 2, we examined the inhibitor status of H3K9Ac and H3K4me2 in C2C12 myoblasts transfected with NSsiRNA and HP1asiRNA. Surprisingly, there were no significant changes in levels of H3K4me2 or H3K9Ac, however, levels of H3K9me3, typically associated with gene silencing were increased by 59 in HP1a depleted myoblasts as compared to control cells (Fig. 5C and Fig. S4A P,0.01). This effect was specific to exon 2 as H3K9me3 in exon 1 or the intronic region was unchanged (Fig. 5D). These data suggest that HP1a paradoxically might be mediating demethylation of H3K9me3 within exon 2 of Lbx1 in myoblasts. To investigate if the effect of HP1a on H3K9me3 is specific to the Lbx1 gene or possibly a more g.Nywhere within genomic sequences when activating gene expression. To clarify the mechanism underlying the altered expression of myogenicHP1 Alpha Facilitates Myogenic Gene ExpressionHP1 Alpha Facilitates Myogenic Gene ExpressionFigure 2. HP1a is required for skeletal muscle differentiation. C2C12 cells were transfected with the indicated siRNA in GM and 24 hours after transfection, DM was added and cultured for 72 hours. A. Phase contrast images were obtained (A a ). Confocal fluorescence microscopy was performed after immunostaining for skeletal sarcomeric myosin heavy chain (red) and DAPI (blue) (A e ). Scale bar equals 50 mm (A a ) and 6 mm (A e ). B, Total protein lysates were subjected to Western blotting. C. C2C12 cells were transfected with NSsiRNA or HP1asiRNA (#1: ID# 60593; #2: ID# 60497) and cultured in DM for 72 hours. Total protein lysates were subjected to Western blotting. D. Reintroduction of siRNA resistant version of HP1a rescued myogenic differentiation in HP1asiRNA transfected cells. C2C12 myoblasts were co-transfected with the indicated siRNA and pLPCEHGF or pLPC-EHGF-rHP1a plasmids. 24 hours after transfection, DM was added and cultured for another 72 hours. Cells were stained with anti-GFP (green) and anti-Troponin C (red), nuclei were counterstained with DAPI (blue). Scale bar equals 50 mm. E. The total number of GFP positive cells and the cells positive for both GFP and Troponin C were counted and percentage of Troponin C positive cells in GFP positive cells were calculated. *P,0.01 for siHP1a/GFP versus siHP1a/GFP-rHP1a cells. Expression of GFP and GFP fusion HP1 were detected by Western blotting using anti-GFP antibody. doi:10.1371/journal.pone.0058319.gregulatory genes in HP1a depleted myocytes, we examined if HP1a directly associates with the genomic sequences of Lbx1. Lbx1 plays a critical in hypaxial muscle development [21,22]. It has been reported that Lbx1 functions upstream of MyoD [23] and is thus the earliest myogenic gene whose expression is altered by HP1a-deficiency. We performed ChIP assays using primers that span the entire Lbx1 genomic sequence including putative promoter, exons and intronic regions (Fig. 5A). Primers within the promoter of the Col11a2 gene, a known HP1a target, were used as a positive control [24]. As shown in Figure 5B, endogenous HP1a binds preferably to exon 2 of Lbx1 in myoblasts while the binding of HP1a to the other genomic sequences of Lbx1 was undetectable. HP1 has been shown to stimulate gene expression in Drosophila and these HP1-induced target genes were marked by the presence of dimethylated histone 3 lysine 4 (H3K4me2), which is found, along with histone acetylation (H3K9Ac), on active chromatin [25]. To determine the effects of HP1a depletion on histone modifications present on Lbx1 exon 2, we examined the status of H3K9Ac and H3K4me2 in C2C12 myoblasts transfected with NSsiRNA and HP1asiRNA. Surprisingly, there were no significant changes in levels of H3K4me2 or H3K9Ac, however, levels of H3K9me3, typically associated with gene silencing were increased by 59 in HP1a depleted myoblasts as compared to control cells (Fig. 5C and Fig. S4A P,0.01). This effect was specific to exon 2 as H3K9me3 in exon 1 or the intronic region was unchanged (Fig. 5D). These data suggest that HP1a paradoxically might be mediating demethylation of H3K9me3 within exon 2 of Lbx1 in myoblasts. To investigate if the effect of HP1a on H3K9me3 is specific to the Lbx1 gene or possibly a more g.
