Higher order structure of the facioscapulohumeral muscular dystrophy (FSHD) locus. (a) Diagram of the genomic region analyzed in the chromosome conformation capture (3C) experiments, indicating the PvuII restriction sites (thin vertical lines); the arrowheads indicate the primer positions, the F series in FSHD-related gene 1(FRG1), the C series in DUX4c, the D series in the D4Z4 repeats; the numbers from 1 to 5 indicate the restriction sites near the D1 bait used as positive controls. The red rectangles show the locations of CarG boxes. (b) The Y axis represents the crosslinking frequency expressed as the ratio of polymerase chain reaction (PCR) performed on 3C samples relative to bacterial artificial chromosome (BAC) controls between the fixed PvuII fragment D1 (D4Z4 repeats) and the rest of the FSHD locus after the correction for digestion and ligation. The calculation of the relative crosslinking frequency between two given fragments, performed as described previously , allows a direct comparison between the different cell types used in the 3C assay by correcting for possible variants. All data points were generated from an average of three independent experiments. The standard error of the mean is indicated. One-way analysis of variance (ANOVA) was applied for statistical analysis; control myoblasts: α = 0.05, P = 1.81*10-12; control myotubes: α = 0.05, P = 3.14*10-6. To compare myoblasts and myotubes, two-way ANOVA was applied for statistical analysis: α = 0.05, P = 0.0073. (c) Frequency of interaction between the FRG1 promoter and D4Z4 sequences in FSHD and control myoblasts, indicating the standard error of the mean. Quantitative (q)PCR was performed on 3C templates using a TaqMan probe complementary to the FRG1 promoter portion of the PCR product obtained using F7 and D1 primers. The results are normalized to digestion, ligation and crosslinking efficiency, as described in Additional file 5. A two-tailed t test was used for statistical analysis; the asterisks indicate the statistically significant differences at α = 0.05. Controls, n = 3; FSHD, n = 4; P = 0.021. The primers are shown in the detailed 3C protocol section of Additional file 5.