Expression of Dux4 targets in FSHD biopsy samples. (A) Heatmap of the expression of the most robust 114 DUX4 targets across all control and FSHD skeletal muscle biopsy samples. The 114 genes were selected as the subset of 213 robust DUX4-regulated genes that met a threshold of average expression across all of the samples (transformed expression level >0.5, see Materials and Methods). The expression levels were transformed and centered by subtracting the median for each gene and the color coding indicates the log-fold-change relative to the median. The columns are colored coded as: red, control; light blue, FSHD1; dark blue, FSHD2. (B–D) Pairwise comparisons of gene expression levels in (B) FSHD and control biopsy samples, (C) FSHD and control cultured myotubes and (D) MB541 transduced with DUX4 or GFP as a control. Colors indicate: green, DUX4 targets called up-regulated in FSHD biopsy samples; purple, DUX4 targets not called up-regulated in FSHD biopsy samples; orange, up-regulated in FSHD biopsies but not DUX4 targets; red, up-regulated in control biopsy samples. (E) DUX4-binding sites within ± 4 kb of TSSs of a set of candidate biomarker genes. The red vertical bar corresponds to the presence of a consensus motif for the DUX4 binding site, and height of the bar indicate the strength of the motif based on the PWM score. The black line indicates the DUX4 ChIP-Seq coverage in DUX4-transduced muscle cells. The blue gradient along the x-axis indicates the sequence uniqueness based on the ENCODE CRG Align 40 track. Gene names colored in red have annotated starting exons, those colored in green have internal exons activated by DUX4 as starting exons, and those colored in blue have novel starting exons activated by DUX4 in intergenic or intronic regions. The zero position on the y-axis represents the predicted TSS based on the RNA-seq. (F) The PRAMEF locus. The gene names in bold type correspond to members of the 67 candidate biomarker genes expressed in FSHD biopsies. The uniqueness tracks and RNA-seq tracks for one FSHD and one control myotube sample are also shown.