Chromatin structure of D4Z4 units in human myoblasts. (a)(i) A simplified schema of the D4Z4 unit showing the position of two CarG box responsive elements (sequences in red). The arrowheads indicate the primer positions for the Lsau, D4Z4 binding element (DBE)1 and DBE2 subregions; the PvuII restriction site positions are indicated. (ii) Chromatin immunoprecipitation (ChIP) and methylated DNA immunoprecipitation (MeDIP) experiments on myoblasts and myotubes using anti-H3K27me3 (K27me3), Ezh2, YY1, and 5-methyl cytidine (5meCy) antibodies (iii) Examples of ChIP experiments on the Lsau and DBE1 subregions in myoblasts and myotubes. (b) H3K27 trimethylation of D4Z4 sequences before (day 0) and after (day 8) myogenic differentiation in healthy control (CN) and facioscapulohumeral muscular dystrophy (FSHD) cell lines, as revealed by ChIP experiments on the DBE1 subregion using anti-H3K27me3 antibody (red), indicating the standard error of the mean. A two-tailed t test was used for statistical analysis; the asterisks indicate the statistically significant differences at α = 0.05. CN-day 0/CN-day 8: P = 0.0172, n = 3; FSHD-day 0/FSHD-day 8: P = 0.0003, n = 4. All of the polymerase chain reaction (PCR) experiments were performed in a linear range of amplification, and band intensities were measured using a Typhoon 9200 phosphoscanner and Image Quant analysis software; after subtracting the signals derived from immunoprecipitation with IgG antibody, the results were expressed as percentages of input DNA. The primer pairs are shown in Additional file 3.