An isogenetic myoblast expression screen identifies DUX4-mediated FSHD-associated molecular pathologies

Abstract

Facioscapulohumeral muscular dystrophy (FSHD) is caused by an unusual deletion with neomorphic activity. This deletion derepresses genes in cis; however which candidate gene causes the FSHD phenotype, and through what mechanism, is unknown. We describe a novel genetic tool, inducible cassette exchange, enabling rapid generation of isogenetically modified cells with conditional and variable transgene expression. We compare the effects of expressing variable levels of each FSHD candidate gene on myoblasts. This screen identified only one gene with overt toxicity: DUX4 (double homeobox, chromosome 4), a protein with two homeodomains, each similar in sequence to Pax3 and Pax7. DUX4 expression recapitulates key features of the FSHD molecular phenotype, including repression of MyoD and its target genes, diminished myogenic differentiation, repression of glutathione redox pathway components, and sensitivity to oxidative stress. We further demonstrate competition between DUX4 and Pax3/Pax7: when either Pax3 or Pax7 is expressed at high levels, DUX4 is no longer toxic. We propose a hypothesis for FSHD in which DUX4 expression interferes with Pax7 in satellite cells, and inappropriately regulates Pax targets, including myogenic regulatory factors, during regeneration.

Figure 1

Generation of ICE-recipient cell lines. (A) Schematic representation of the lentiviral constructs carrying the components of the ICE system, and the proviral ICE locus before and after recombination. (B) Flow cytometry of iC2C12 cells, which carry an inducible cre–ires–GFP proviral locus before recombination, and derivative cell lines carrying DsRed2 or luciferase, after recombination. (C) Southern blot analyses with GFP-specific probe DNA to detect copy number and recombination status of the ICE locus in iC2C12, iC2C12-DUX4 and i3T3 cell lines. Note that a single band hybridized with the GFP probe in iC2C12 and i3T3 cells but was missing following recombination (in iC2C12-DUX4 cells, in which GFP was replaced with DUX4). DNA from a spermatogonial cell line carrying GFP was used as a positive control. (D) Dose–response and time course (500 ng/ml) of luciferase gene expression in response to doxycycline.

Figure 2

Identification of DUX4-specific cell pathological phenotypes. (A) Cell viability (ATP content), after 24 h in 100 or 500 ng/ml doxycycline-treated cells. DUX4 has a unique dose-dependent effect on viability. (B) Morphology of iC2C12 myoblasts 24 h after DUX4 induction with 500 ng/ml dox. (C) Western blotting analysis of FLAG tag fusion protein expression in FLAG–DUX4, FLAG–ANT1, FLAG–TUBB4q and FRG1-3xFLAG cell lines. Cells were induced with 125 and 500 ng/ml doxycycline for 14 h. (D) Immunofluorescence showing DUX4 (red) accumulation in the nucleus and cell morphological changes (lamin, green) in iC2C12-DUX4 cells over a 16 h time course (500 ng/ml). (E) Dose–response at 20 h (upper panels) and time course (in 500 ng/ml doxycycline, lower panels) of DUX4 expression in iC2C12-DUX4 cells. (F) Western blot analyses of DUX4 expression in differentiated and proliferating cells. iC2C12-DUX4 cells were differentiated into myotubes for 4 days and DUX4 was induced for 14 h for comparison to proliferating cells. (G) Western blots for p21 and cyclin E during a time course of DUX4 induction at 500 ng/ml dox. (H) FACS analyses of DUX4-induced apoptosis and cell death using annexin V (x axis) and 7AAD (y axis) staining. Early apoptotic cells are annexin V⁺, dead cells are annexin V⁺/7AAD⁺. (I) Western blots for activated caspases 6, 8 and 9 in DUX4-expressing myoblasts over a time course. (J) Morphology of cells expressing DUX4 at low levels while proliferating, and viability (K) of these cells by ATP assay. (L) Morphology of DUX4-expressing myotubes and their viability (M).

Figure 3

DUX4 target genes. (A) DUX4 target genes (left chart, 4 h post-induction, right chart 12 h) represented by gene ontology. (B) Hierarchical clustering. Signal values for genes differentially expressed at 4 or 12 h were normalized by Z score and clustered using Spotfire DecisionSite software. Bright green represents low signal values, bright red indicates very high signal values, and black represents median signal values. Experiments are clustered by columns, and individual genes by rows. C1 through C3 represent 0 h controls, and 4 and 12 h represent samples treated with doxycycline for the indicated times. As shown, all controls clustered together as did the 4 and 12 h replicates. The majority of changes in gene expression increased over time (i.e. became brighter red by 12 h, the latest time point test. (C–F) Real-time PCR confirmation of expression changes for key FSHD-related genes: (C) glutathione redox pathway components, (D) heat-shock proteins, (E) Lamin A and p21, and (F) MyoD, which made the cutoff only in one replicate due to sensitivity of the microarray.

Figure 4

Effect of antioxidants on DUX4-expressing myoblasts. (A) ATP assay for cell survival of DUX4-expressing myoblasts exposed to different stress-inducing agents. Oxidative stressors (tBHP and Paraquat) show a synergistic interaction with DUX4 at low levels. (B) Cell rescue with various concentrations of antioxidants demonstrated by ATP assay after 24 h of DUX4 induction (500 ng/ml dox). (C) Cell morphology of iC1C12-DUX4 myoblasts induced with 500 ng/ml dox for 24 h and treated with various antioxidants. (D) Western blot demonstrating that DUX4 was still expressed in the rescued cells (24 h post-induction). (E) qRT–PCR analyses of MyoD and Myf5 in antioxidant-rescued, DUX4-expressing cells. Data represent the fold difference compared with the level of GAPDH, error bars are STDEV (n=3). Antioxidants have no effect on the expression of these downstream genes.

Figure 5

DUX4 interferes with myogenic regulators and diminishes myotube formation. (A) qRT–PCR analyses of myogenic-specific genes in iC1C12-DUX4. DUX4 was induced with various concentrations of dox for 12 h. (B) Western blot analyses for the expression of MyoD and Myf5 in iC2C12-DUX4 over 16 h. (C) Immunofluorescence for MyoD (red) in 16 h DUX4-induced cells. Nuclei were contrastained with DAPI (blue). (D) Morphology of iC2C12-DUX4 after 4 days of differentiation in 2% horse serum. Note that DUX4-expressing cells, depending on the dox concentration, showed impaired to diminished differentiation. (E) Immunofluorescence for MyHC (red) on day 4 of differentiation of iC2C12-DUX4 cells. (F) Morphology of control (iC2C12 target) cells after 4 days of differentiation induced with 50 ng/ml doxycycline and evaluated by immunofluorescence for MyHC (G). Note that doxycycline alone does not have any significant effect on iC2C12 differentiation.

Figure 6

Pax3 and Pax7 compete with DUX4 and rescue toxicity. (A) Unrooted tree representing the sequence relationship of different homeodomains. The length of arms indicates the distance between a given sequence and the nearest hypothetical sequence in common with the rest of the tree. (B) Cell viability demonstrated by ATP assay at 24 and 48 h post-DUX4 induction iC2C12-DUX4, iC2C12-DUX4 & GFP, iC2C12-DUX4 & Pax3 and iC2C12-DUX4 & Pax7. Note complete cell rescue in Pax3- and Pax7-expressing cells at 100 ng/ml dox. (C) Cell morphology of iC2C12-DUX4, iC2C12-DUX4 & Pax3 and iC2C12-DUX4 & Pax7 24 h after DUX4 induction with 125 and 500 ng/ml dox. Note the elongated cells at 125 ng/ml dox in the control (left) panel versus stellate cells in the Pax3- or Pax7-overexpressing groups (middle and right panels). (D) Western blot analyses for DUX4, Pax3 and Pax7 at 24 h of induction with different concentrations of dox, demonstrating that DUX4 is still expressed in these myoblasts. (E) qRT–PCR for MyoD and Myf5 in rescued cells at 12 and 24 h post-induction with varying levels of dox. Data represent the fold difference compared with the level of GAPDH, error bars are STDEV (n=3).