Figure 2

Fine resolution mapping of histone modifications across the DES LCR and DES in myoblasts and myotubes compared to PBMCs. Chromatin was prepared from myoblasts, myotubes and PBMCs for nChIP (as Figure 1) and amplified using Q-PCR to gain a comprehensive picture of histone modifications status across the DES LCR and DES. (A) Histone H3 acetylation in unfused myoblasts (UF, black bars), in fused myotubes, which express DES 2-fold higher than myoblasts (F, white bars) and in non-expressing PBMCs (grey bars) across DES and the DES LCR. (B) As panel (A) but showing the distribution of histone H4 acetylation. (C) Histone H3K4 di-methylation (H3K4me2) in unfused myoblasts (UF, black bars), in fused myotubes (F) and in non-expressing PBMCs (grey bars) across DES and the DES LCR. (D) As panel (C) but showing histone H3K4me3 enrichment across DES and the DES LCR. Positive controls include the housekeeping genes DNPEP and HTATIP2 and as a non-expressing negative control, HBB. (E) Illustration of DES and DES LCR genomic region. The black rectangles and horizontal arrow denote the exons and direction of transcription of DES respectively. Vertical arrows indicate the relative positions of the DES LCR HS and evolutionarily conserved regions. Vertical bars designated A-J denote the positions of the PCR amplicons following nChIP. Results are presented as fold-enrichment of IP samples over input DNA. Error bars represent the standard deviation about the mean fold-enrichment from at least 2 separate PCRs performed in duplicate from 2 independent nChIP experiments. Enrichment across the muscle-specific DES locus was above non-specific binding background controls (no antibody and IgG) in the myoblast/myotube cultures but not PBMCs. P value < 0.05 denoted by an asterisk (*).