Fig. 4: SMCHD1 and LRIF1 have residual repressive action at hypomethylated D4Z4.

a SMCHD1 and LRIF1 ChIP-qPCR in primary control (n = 3, lines: 2524, 2397, 2333) fibroblasts and fibroblasts carrying either a heterozygous SMCHD1 mutation (n = 3, lines: 2440, 2337, 2332), a heterozygous DNMT3B mutation (n = 2, lines: v294, b974) or biallelic DNMT3B mutations (n = 2, lines: Rf699.3, Rf286.3). Schematic of one D4Z4 unit in which the position of three regions within D4Z4 examined by ChIP-qPCR is indicated (DR1, Q, HOX). Bars and whiskers represent mean ± SEM. Isotype specific IgG was used for background control. Statistical significance between WT and mutant groups was calculated by one-way ANOVA with Dunnett’s post hoc test (*p < 0.05, ns not significant). b Western blot confirmation of successful siRNA-mediated knock-down of SMCHD1, LRIF1L or LRIF1L + S in primary ICF1 myoblasts. Tubulin was used as a loading control. c RT-qPCR of DUX4 and four of its target genes (ZSCAN4, KHDC1L, TRIM43 and MBD3L2) after siRNA-mediated KD of SMCHD1, LRIF1L or LRIF1L + S in ICF1 myoblasts. Expression levels detected in KD cells were normalized to cells transfected with non-targeting (NT) siRNA and further log2 transformed. GUSB was used as a housekeeping gene for intra-sample normalization. Bars and whiskers represent mean ± SEM of three independent experiments. Statistical significance was calculated by one sample t-test (**p < 0.01, *p < 0.05, ns not significant).