Dux4 induced genes mis-expressed in FSHD muscle cells. (A) log-fold change of DUX4 up-regulated genes in MB135 (y-axis) and MB541 (x-axis) cultured myoblast cells transduced with lentiviral DUX4 compared with a control lenti-GFP (purple, genes upregulated by DUX4 > 5 log-fold change in both MB135 and MB541; orange, up-regulated by DUX4 in both cell types but <5 log-fold; red, DUX4 up-regulated only in MB135 cells; green, DUX4 up-regulated only in MB541cells). (B and C) Pairwise comparison of gene expression in DUX4-transduced (x-axes) and the control GFP-transduced (y-axes) MB541 (B) and MB135(C) cells. Gene counts were transformed as described in Materials and Methods. Genes are colored as in (A). (D) Comparison of the log-fold change of genes up-regulated by DUX4 in MB541 cells (y-axis) to the log-fold change of genes up-regulated in FSHD myotubes compared to control myotubes (x-axis) (dark purple, genes increased in FSHD compared with control cells that were up-regulated by DUX4 >5 log-fold in MB541; blue, increased in FSHD cells and up-regulated by DUX4 in MB51 with log-FC between one and five; olive, increased in FSHD cells and not called increased by DUX4 in MB541 (log-FC <1); light purple, not increased in FSHD cells compared with controls but up-regulated by DUX4 in MB541 cells).

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.

(A and B) Association of DUX4-target gene expression with clinical severity. (A) The y-axis is the total number of reads that mapped to any of the 67 candidate biomarker genes in each sample (scaled and square-root transformed). The biopsy samples are color coded as: red, control; light blue, FSHD1; dark blue, FSHD2. The size of the circle corresponds to the corrected clinical severity score (CSS) and the numbers within the circles correspond to the pathology score, with larger size indicating a higher severity. The samples with missing numbers did not have pathology scores. (B) Similar to A but using the total reads mapped to the selected four biomarkers: LEUTX, PRAMEF2, TRIM43 and KHDC1L. (C and D) Genes expressed at higher levels in FSHD samples that are not DUX4 targets. (C) Heatmap of genes expressed more highly in FSHD samples but that are not induced (logFC <1) by DUX4-transduction of human muscle cells with presentation and coding as described in Figure A. (D) Boxplot of the expression distribution for the genes in (C) based on RNA-seq reads from the FSHD samples that were DUX4-target-positive, stratified as immune and non-immune genes. The red dots correspond to the expression of each gene in control sample C5. Note that some loci have copy number variation in the human population, such as GSTT1 that is not present in some individuals. Boxplot: vertical bounds of rectangle represent the 25th and 75th percentiles, the whisker extends to the extreme value (minimum or maximum) bounded by 1.5 times the IQR (25th and 75th inter-quartile range), and the filled circles are the median and the open circles are the outliers.