Fig. 4

SMCHD1 binding at the tRNA cluster and transcriptional expression of the chromosome tRNA, HOXB, and 5S rRNA clusters. a qPCR analysis on SMCHD1 ChIPed DNA for the identified SMCHD1 binding site on chromosome 1 within the tRNA cluster using n = 4 for control, n = 2 for FSHD1, and n = 5 for FSHD2 myoblast cell lines. Results represent the log10 input normalized binding enrichment relative to IgG, which has been arbitrarily set to 1. Asterisks directly over bars represent a significant increase in binding relative to IgG *Bonferroni adjusted P < 0.05, t test; error bars = SEM. RNA expression analysis on isoforms within the chromosome 1 tRNA cluster and tRNA Val_CAC directly proximal to the cluster repeat in primary (b) myoblast and (c) myotube cells, the HOXB cluster isoform members in primary (d) myoblast and (e) myotube cells, and the 5S rRNA cluster on chromosome 1 in (f) myoblast and (g) myotube cells. RNA expression analysis for the 5S rRNA cluster on chromosome 1 for (h) myoblasts in SMCHD1 shRNA KD in comparison with control KD shRNAs in non-DUX4 permissive 4qB cells. Results represent qRT-PCR analysis of the indicated gene after normalization to the internal control gene GUS1. For each gene, the value of expression in control individuals was then arbitrarily set to 1. *Bonferroni adjusted P < 0.05, t test; n = 4 for controls, n = 10 for FSHD1, n = 7 for FSHD2, n = 6 for non-DUX4 permissive 4qB cells (two shRNA constructs for each target on three independent cell lines); error bars = SEM