Model systems of DUX4 expression recapitulate the transcriptional profile of FSHD cells

Abstract

Facioscapulohumeral dystrophy (FSHD) is caused by the mis-expression of the double-homeodomain transcription factor DUX4 in skeletal muscle cells. Many different cell culture models have been developed to study the pathophysiology of FSHD, frequently based on endogenous expression of DUX4 in FSHD cells or by mis-expression of DUX4 in control human muscle cells. Although results generated using each model are generally consistent, differences have also been reported, making it unclear which model(s) faithfully recapitulate DUX4 and FSHD biology. In this study, we systematically compared RNA-seq data generated from three different models of FSHD—lentiviral-based DUX4 expression in myoblasts, doxycycline-inducible DUX4 in myoblasts, and differentiated human FSHD myocytes expressing endogenous DUX4—and show that the DUX4-associated gene expression signatures of each dataset are highly correlated (Pearson’s correlation coefficient, r ∼ 0.75-0.85). The few robust differences were attributable to different states of cell differentiation and other differences in experimental design. Our study describes a model system for inducible DUX4 expression that enables reproducible and synchronized experiments and validates the fidelity and FSHD relevance of multiple distinct models of DUX4 expression.

Figure 1.

Codon altering allows stable, inducible expression of DUX4 in human myoblasts. (A) Graphical depiction of GC percentage and CpG occurrence of the codon-altered (black) and wild-type (red) DUX4 coding regions. GC percentage was calculated over 50 base pair sliding windows. The positions of CpG dinucleotides are indicated by open circles. (B) Phase contrast images of monoclonal cells encoding wild-type or codon-altered DUX4 expression constructs, with or without doxycycline induction. The number of clonal cell lines that exhibited cell death among all that were tested is shown. (Binomial test for equality of proportions, P Value = 0.001). (C) Western blot analysis for DUX4 expression on lysates from 5 clones encoding wild-type or codon-altered DUX4, with or without induction with doxycycline for 8 hours. Histone 3 (H3) serves as a loading control. Black arrowhead indicates full-length DUX4 product. (D) qRT-PCR analysis of a DUX4 transcriptional target, ZSCAN4, shown as fold-change over uninduced cells in the various clones. The clones that exhibited cell death upon doxycycline induction are highlighted.

Figure 2.

Inducible expression of codon-altered DUX4 activates germline antigens, endogenous retrotransposons and repetitive elements and inhibits RNA quality control. (A) Scatter plot of gene expression (in transcripts per million) in control (uninduced) versus iDUX4 (doxycycline-induced) myoblasts. Red/blue, genes exhibiting increases/decreases of > 2.5 fold. (N) Numbers of genes with increased/decreased expression; (percentages) fraction of genes that are affected by DUX4 expression. (B) Relative mRNA levels of known DUX4 transcriptional targets in iDUX4 versus control myoblasts expressed as log₂ fold-change. (C) Scatter plot of repetitive element expression (in transcripts per million) in control (uninduced) versus iDUX4 (doxycycline-induced) myoblasts. Red/blue, repeat elements exhibiting increases/decreases of > 2.5 fold. (N) Numbers of repetitive elements with increased/decreased expression; (percentages) fraction of repetitive elements that are affected by DUX4 expression. (D) Relative levels of known DUX4-activated repetitive elements in iDUX4 versus control myoblasts expressed as log₂ fold-change. (E, F) Isoform ratios of predicted NMD substrates generated by cassette exon alternative splicing (E) or intron retention (F), comparing iDUX4 versus control myoblasts. Red/blue, cassette exons (E) or retained introns (F) exhibiting increases/decreases of ≥10% in isoform ratios for the isoforms that are predicted NMD substrates. Events that do not change significantly are rendered transparent.

Figure 3.

Transcriptional response of endogenous and exogenous DUX4 expression in human myoblasts. (A) Schematic representation of the RNA-seq data analysis pipeline. (B–D) MA plots for inducible, viral and endogenous DUX4-induced transcriptomes. Genes upregulated by more than 4-fold in red; genes downregulated by more than 4-fold in blue; genes with a significant adjusted P-value (< 0.05) that do not meet 4-fold cutoff for differential expression in green and the genes with adjusted P-value > 0.05 in black. For the vDUX4 sample, genes with log₂ fold-change >8 or < -4 are plotted as ‘triangles’ at the top and bottom edges of the plot, respectively.

Figure 4.

Genes unique to endogenous DUX4 expression are most relevant to muscle differentiation. (A) Venn diagram showing the overlap between the detected genes in iDUX4, enDUX4 and vDUX4 samples. (B–D) MA plot for inducible, viral and endogenous DUX4-induced transcriptome, highlighting the genes that were uniquely detected in each of the samples. Dotted line represents log₂ TPM of 3. Color scheme is same as that of Figure 3B–D. (E–F) Gene Ontology (GO) analysis for the genes unique to vDUX4 and enDUX4, respectively, and expressed at a level above 8 TPM (log₂ TPM of 3). (G-H) Scatter plot of log₂ fold-change of genes robustly and uniquely upregulated by enDUX4 versus log₂ fold-change for the corresponding genes by iDUX4 (G) and vDUX4 (H) calculated without applying a filter for low expressing genes. Black dots represent the discordant genes.

Figure 5.

Regulated gene sets show significant overlap between samples. (A, C) Venn diagram of upregulated genes showing the overlap between genes with > 2 log₂ fold-change (A) or < -2 log₂ fold-change (B) and an adjusted P-value < 0.05 in the three datasets. (B) Percent overlap plot shows the overlap of gene sets that are > 2 log₂ fold upregulated in Sample A with a significant adjusted P value over a sliding scale of 0 to 5 log₂ fold upregulation in Sample B. (D) Percent overlap plot shows the overlap of gene sets that are < -2 log₂ fold downregulated in Sample A with a significant adjusted P value over a sliding scale of 0 to -5 log₂ fold downregulation in Sample B. (E–G) Scatter plot of log₂ fold-change of quantifiable genes in inducible versus viral DUX4 expression (E), endogenous versus viral DUX4 expression (F), and endogenous versus inducible DUX4 expression (G). r - Pearson’s correlation coefficient.

Figure 6.

Differentially regulated genes appear most relevant to the gene expression programs underway during DUX4 expression. (A) Scatter plot of scaled and centered log₂ fold-change values of iDUX4 and vDUX4. Line represents a linear model and the genes marked in red are more over-expressed in vDUX4 compared to iDUX4 (residual > 2) and those in blue are more under-expressed in vDUX4 compared to iDUX4 (residual < -2). (B) Scatter plot of scaled and centered log₂ fold-change values of enDUX4 and iDUX4. Line represents a linear model and the genes marked in red are more over-expressed in iDUX4 compared to enDUX4 (residual > 2) and those in blue are more under-expressed in iDUX4 compared to enDUX4 (residual < -2). (C–D) GO category analysis of significantly under-expressed genes in vDUX4 compared to iDUX4 (C) and significantly over-expressed genes in iDUX4 compared to enDUX4 (D). (E) qPCR data for a few candidate discordant genes in control and iDUX4 cells in growth media (GM) versus differentiation media (DM).

Figure 7.

Gene sets common to endogenous and exogenous DUX4 expression highlight the core functions of DUX4. (A) 3D scatter plot for the three datasets (iDUX4, enDUX4 and vDUX4) highlighting the genes upregulated by more than 2 log₂ fold-change in all samples in red and those downregulated by more than 2 log₂ fold-change in all samples in blue. (B) GO analysis of the upregulated genes (marked ‘red' in 6A). (C) GO analysis of downregulated genes (marked ‘blue' in 6A). (D–F) MA plot for 47 biomarkers identified by Yao et al. (17) for the iDUX4 (D), vDUX4 (E) and enDUX4 (F) datasets; The four high-confidence biomarkers (LEUTX, PRAMEF2, TRIM43, KHDC1L) are marked in ‘red'. The horizontal dotted line represents TPM of 8; the vertical dotted line represents fold change of 4.