Meta-analysis towards FSHD reveals misregulation of neuromuscular junction, nuclear envelope, and spliceosome.

Facioscapulohumeral muscular dystrophy (FSHD) is one of the most common autosomal dominant muscle disorders, yet no cure or amelioration exists. The clinical presentation is diverse, making it difficult to identify the actual driving pathomechanism among many downstream events. To unravel this complexity, we performed a meta-analysis of 13 original omics datasets (in total 171 FSHD and 129 control samples). Our approach confirmed previous findings about the disease pathology and specified them further. We confirmed increased expression of former proposed DUX4 biomarkers, and furthermore impairment of the respiratory chain. Notably, the meta-analysis provides insights about so far not reported pathways, including misregulation of neuromuscular junction protein encoding genes, downregulation of the spliceosome, and extensive alterations of nuclear envelope protein expression. Finally, we developed a publicly available shiny app to provide a platform for researchers who want to search our analysis for genes of interest in the future.

DNMT3B splicing dysregulation mediated by SMCHD1 loss contributes to DUX4 overexpression and FSHD pathogenesis.

Structural maintenance of chromosomes flexible hinge domain-containing 1 (SMCHD1) is a noncanonical SMC protein and an epigenetic regulator. Mutations in SMCHD1 cause facioscapulohumeral muscular dystrophy (FSHD), by overexpressing DUX4 in muscle cells. Here, we demonstrate that SMCHD1 is a key regulator of alternative splicing in various cell types. We show how SMCHD1 loss causes splicing alterations of DNMT3B, which can lead to hypomethylation and DUX4 overexpression. Analyzing RNA sequencing data from muscle biopsies of patients with FSHD and Smchd1 knocked out cells, we found mis-splicing of hundreds of genes upon SMCHD1 loss. We conducted a high-throughput screen of splicing factors, revealing the involvement of the splicing factor RBM5 in the mis-splicing of DNMT3B. Subsequent RNA immunoprecipitation experiments confirmed that SMCHD1 is required for RBM5 recruitment. Last, we show that mis-splicing of DNMT3B leads to hypomethylation of the D4Z4 region and to DUX4 overexpression. These results suggest that DNMT3B mis-splicing due to SMCHD1 loss plays a major role in FSHD pathogenesis.

Muscle strength, quantity and quality and muscle fat quantity and their association with oxidative stress in patients with facioscapulohumeral muscular dystrophy: Effect of antioxidant supplementation.

The purpose of this study was to identify causes of quadriceps muscle weakness in facioscapulohumeral muscular dystrophy (FSHD). To this aim, we evaluated quadriceps muscle and fat volumes by magnetic resonance imaging and their relationships with muscle strength and oxidative stress markers in adult patients with FSHD (n = 32) and healthy controls (n = 7), and the effect of antioxidant supplementation in 20 of the 32 patients with FSHD (n = 10 supplementation and n = 10 placebo) (NCT01596803). Compared with healthy controls, the dominant quadriceps strength and quality (muscle strength per unit of muscle volume) were decreased in patients with FSHD. In addition, fat volume was increased, without changes in total muscle volume. Moreover, in patients with FSHD, the lower strength of the non-dominant quadriceps was associated with lower muscle quality compared with the dominant muscle. Antioxidant supplementation significantly changed muscle and fat volumes in the non-dominant quadriceps, and muscle quality in the dominant quadriceps. This was associated with improved muscle strength (both quadriceps) and antioxidant response. These findings suggest that quadriceps muscle strength decline may not be simply explained by atrophy and may be influenced also by the muscle intrinsic characteristics. As FSHD is associated with increased oxidative stress, supplementation might reduce oxidative stress and increase antioxidant defenses, promoting changes in muscle function.

The DUX4-HIF1α Axis in Murine and Human Muscle Cells: A Link More Complex Than Expected.

FacioScapuloHumeral muscular Dystrophy (FSHD) is one of the most prevalent inherited muscle disorders and is linked to the inappropriate expression of the DUX4 transcription factor in skeletal muscles. The deregulated molecular network causing FSHD muscle dysfunction and pathology is not well understood. It has been shown that the hypoxia response factor HIF1α is critically disturbed in FSHD and has a major role in DUX4-induced cell death. In this study, we further explored the relationship between DUX4 and HIF1α. We found that the DUX4 and HIF1α link differed according to the stage of myogenic differentiation and was conserved between human and mouse muscle. Furthermore, we found that HIF1α knockdown in a mouse model of DUX4 local expression exacerbated DUX4-mediated muscle fibrosis. Our data indicate that the suggested role of HIF1α in DUX4 toxicity is complex and that targeting HIF1α might be challenging in the context of FSHD therapeutic approaches.

Muscle eosinophilia is a hallmark of chronic disease in facioscapulohumeral muscular dystrophy.

Facioscapulohumeral muscular dystrophy (FSHD) is a progressive myopathy caused by the aberrant increased expression of the DUX4 retrogene in skeletal muscle cells. The DUX4 gene encodes a transcription factor that functions in zygotic genome activation and then is silenced in most adult somatic tissues. DUX4 expression in FSHD disrupts normal muscle cell function; however, the downstream pathogenic mechanisms are still unclear. Histologically, FSHD affected muscles show a characteristic dystrophic phenotype that is often accompanied by a pronounced immune cell infiltration, but the role of the immune system in FSHD is not understood. Previously, we used ACTA1;FLExDUX4 FSHD-like mouse models varying in severity as discovery tools to identify increased Interleukin 6 and microRNA-206 levels as serum biomarkers for FSHD disease severity. In this study, we use the ACTA1;FLExDUX4 chronic FSHD-like mouse model to provide insight into the immune response to DUX4 expression in skeletal muscles. We demonstrate that these FSHD-like muscles are enriched with the chemoattractant eotaxin and the cytotoxic eosinophil peroxidase, and exhibit muscle eosinophilia. We further identified muscle fibers with positive staining for eosinophil peroxidase in human FSHD muscle. Our data supports that skeletal muscle eosinophilia is a hallmark of FSHD pathology.

Regional and bilateral MRI and gene signatures in facioscapulohumeral dystrophy: implications for clinical trial design and mechanisms of disease progression.

Identifying the aberrant expression of DUX4 in skeletal muscle as the cause of facioscapulohumeral dystrophy (FSHD) has led to rational therapeutic development and clinical trials. Several studies support the use of MRI characteristics and the expression of DUX4-regulated genes in muscle biopsies as biomarkers of FSHD disease activity and progression. We performed lower-extremity MRI and muscle biopsies in the mid-portion of the tibialis anterior (TA) muscles bilaterally in FSHD subjects and validated our prior reports of the strong association between MRI characteristics and expression of genes regulated by DUX4 and other gene categories associated with FSHD disease activity. We further show that measurements of normalized fat content in the entire TA muscle strongly predict molecular signatures in the mid-portion of the TA, indicating that regional biopsies can accurately measure progression in the whole muscle and providing a strong basis for inclusion of MRI and molecular biomarkers in clinical trial design. An unanticipated finding was the strong correlations of molecular signatures in the bilateral comparisons, including markers of B-cells and other immune cell populations, suggesting that a systemic immune cell infiltration of skeletal muscle might have a role in disease progression.

Autosomal dominant in cis D4Z4 repeat array duplication alleles in facioscapulohumeral dystrophy.

Facioscapulohumeral dystrophy (FSHD) has a unique genetic aetiology resulting in partial chromatin relaxation of the D4Z4 macrosatellite repeat array on 4qter. This D4Z4 chromatin relaxation facilitates inappropriate expression of the transcription factor DUX4 in skeletal muscle. DUX4 is encoded by a retrogene that is embedded within the distal region of the D4Z4 repeat array. In the European population, the D4Z4 repeat array is usually organized in a single array that ranges between 8 and 100 units. D4Z4 chromatin relaxation and DUX4 derepression in FSHD is most often caused by repeat array contraction to 1-10 units (FSHD1) or by a digenic mechanism requiring pathogenic variants in a D4Z4 chromatin repressor like SMCHD1, combined with a repeat array between 8 and 20 units (FSHD2). With a prevalence of 1.5% in the European population, in cis duplications of the D4Z4 repeat array, where two adjacent D4Z4 arrays are interrupted by a spacer sequence, are relatively common but their relationship to FSHD is not well understood. In cis duplication alleles were shown to be pathogenic in FSHD2 patients; however, there is inconsistent evidence for the necessity of an SMCHD1 mutation for disease development. To explore the pathogenic nature of these alleles we compared in cis duplication alleles in FSHD patients with or without pathogenic SMCHD1 variant. For both groups we showed duplication-allele-specific DUX4 expression. We studied these alleles in detail using pulsed-field gel electrophoresis-based Southern blotting and molecular combing, emphasizing the challenges in the characterization of these rearrangements. Nanopore sequencing was instrumental to study the composition and methylation of the duplicated D4Z4 repeat arrays and to identify the breakpoints and the spacer sequence between the arrays. By comparing the composition of the D4Z4 repeat array of in cis duplication alleles in both groups, we found that specific combinations of proximal and distal repeat array sizes determine their pathogenicity. Supported by our algorithm to predict pathogenicity, diagnostic laboratories should now be furnished to accurately interpret these in cis D4Z4 repeat array duplications, alleles that can easily be missed in routine settings.

snRNA-seq analysis in multinucleated myogenic FSHD cells identifies heterogeneous FSHD transcriptome signatures associated with embryonic-like program activation and oxidative stress-induced apoptosis.

The sporadic nature of DUX4 expression in FSHD muscle challenges comparative transcriptome analyses between FSHD and control samples. A variety of DUX4 and FSHD-associated transcriptional changes have been identified, but bulk RNA-seq strategies prohibit comprehensive analysis of their spatiotemporal relation, interdependence and role in the disease process. In this study, we used single-nucleus RNA-sequencing of nuclei isolated from patient- and control-derived multinucleated primary myotubes to investigate the cellular heterogeneity in FSHD. Taking advantage of the increased resolution in snRNA-sequencing of fully differentiated myotubes, two distinct populations of DUX4-affected nuclei could be defined by their transcriptional profiles. Our data provides insights into the differences between these two populations and suggests heterogeneity in two well-known FSHD-associated transcriptional aberrations: increased oxidative stress and inhibition of myogenic differentiation. Additionally, we provide evidence that DUX4-affected nuclei share transcriptome features with early embryonic cells beyond the well-described cleavage stage, progressing into the 8-cell and blastocyst stages. Altogether, our data suggests that the FSHD transcriptional profile is defined by a mixture of individual and sometimes mutually exclusive DUX4-induced responses and cellular state-dependent downstream effects.

Hypoxia enhances human myoblast differentiation: involvement of HIF1α and impact of DUX4, the FSHD causal gene.

BACKGROUND: Hypoxia is known to modify skeletal muscle biological functions and muscle regeneration. However, the mechanisms underlying the effects of hypoxia on human myoblast differentiation remain unclear. The hypoxic response pathway is of particular interest in patients with hereditary muscular dystrophies since many present respiratory impairment and muscle regeneration defects. For example, an altered hypoxia response characterizes the muscles of patients with facioscapulohumeral dystrophy (FSHD). METHODS: We examined the impact of hypoxia on the differentiation of human immortalized myoblasts (LHCN-M2) cultured in normoxia (PO2: 21%) or hypoxia (PO2: 1%). Cells were grown in proliferation (myoblasts) or differentiation medium for 2 (myocytes) or 4 days (myotubes). We evaluated proliferation rate by EdU incorporation, used myogenin-positive nuclei as a differentiation marker for myocytes, and determined the fusion index and myosin heavy chain-positive area in myotubes. The contribution of HIF1α was studied by gain (CoCl2) and loss (siRNAs) of function experiments. We further examined hypoxia in LHCN-M2-iDUX4 myoblasts with inducible expression of DUX4, the transcription factor underlying FSHD pathology. RESULTS: We found that the hypoxic response did not impact myoblast proliferation but activated precocious myogenic differentiation and that HIF1α was critical for this process. Hypoxia also enhanced the late differentiation of human myocytes, but in an HIF1α-independent manner. Interestingly, the impact of hypoxia on muscle cell proliferation was influenced by dexamethasone. In the FSHD pathological context, DUX4 suppressed HIF1α-mediated precocious muscle differentiation. CONCLUSION: Hypoxia stimulates myogenic differentiation in healthy myoblasts, with HIF1α-dependent early steps. In FSHD, DUX4-HIF1α interplay indicates a novel mechanism by which DUX4 could interfere with HIF1α function in the myogenic program and therefore with FSHD muscle performance and regeneration.

Flavones provide resistance to DUX4-induced toxicity via an mTor-independent mechanism.

Facioscapulohumeral muscular dystrophy (FSHD) is among the most common of the muscular dystrophies, affecting nearly 1 in 8000 individuals, and is a cause of profound disability. Genetically, FSHD is linked to the contraction and/or epigenetic de-repression of the D4Z4 repeat array on chromosome 4, thereby allowing expression of the DUX4 gene in skeletal muscle. If the DUX4 transcript incorporates a stabilizing polyadenylation site the myotoxic DUX4 protein will be synthesized, resulting in muscle wasting. The mechanism of toxicity remains unclear, as many DUX4-induced cytopathologies have been described, however cell death does primarily occur through caspase 3/7-dependent apoptosis. To date, most FSHD therapeutic development has focused on molecular methods targeting DUX4 expression or the DUX4 transcript, while therapies targeting processes downstream of DUX4 activity have received less attention. Several studies have demonstrated that inhibition of multiple signal transduction pathways can ameliorate DUX4-induced toxicity, and thus compounds targeting these pathways have the potential to be developed into FSHD therapeutics. To this end, we have screened a group of small molecules curated based on their reported activity in relevant pathways and/or structural relationships with known toxicity-modulating molecules. We have identified a panel of five compounds that function downstream of DUX4 activity to inhibit DUX4-induced toxicity. Unexpectedly, this effect was mediated through an mTor-independent mechanism that preserved expression of ULK1 and correlated with an increase in a marker of active cellular autophagy. This identifies these flavones as compounds of interest for therapeutic development, and potentially identifies the autophagy pathway as a target for therapeutics.

MATR3 is an endogenous inhibitor of DUX4 in FSHD muscular dystrophy.

Facioscapulohumeral muscular dystrophy (FSHD) is one of the most common neuromuscular disorders and has no cure. Due to an unknown molecular mechanism, FSHD displays overlapping manifestations with the neurodegenerative disease amyotrophic lateral sclerosis (ALS). FSHD is caused by aberrant gain of expression of the transcription factor double homeobox 4 (DUX4), which triggers a pro-apoptotic transcriptional program resulting in inhibition of myogenic differentiation and muscle wasting. Regulation of DUX4 activity is poorly known. We identify Matrin 3 (MATR3), whose mutation causes ALS and dominant distal myopathy, as a cellular factor controlling DUX4 expression and activity. MATR3 binds to the DUX4 DNA-binding domain and blocks DUX4-mediated gene expression, rescuing cell viability and myogenic differentiation of FSHD muscle cells, without affecting healthy muscle cells. Finally, we characterize a shorter MATR3 fragment that is necessary and sufficient to directly block DUX4-induced toxicity to the same extent as the full-length protein. Collectively, our data suggest MATR3 as a candidate for developing a treatment for FSHD.

The transcription factor DUX4 orchestrates translational reprogramming by broadly suppressing translation efficiency and promoting expression of DUX4-induced mRNAs.

Translational control is critical for cell fate transitions during development, lineage specification, and tumorigenesis. Here, we show that the transcription factor double homeobox protein 4 (DUX4), and its previously characterized transcriptional program, broadly regulates translation to change the cellular proteome. DUX4 is a key regulator of zygotic genome activation in human embryos, whereas misexpression of DUX4 causes facioscapulohumeral muscular dystrophy (FSHD) and is associated with MHC-I suppression and immune evasion in cancer. We report that translation initiation and elongation factors are disrupted downstream of DUX4 expression in human myoblasts. Genome-wide translation profiling identified mRNAs susceptible to DUX4-induced translation inhibition, including those encoding antigen presentation factors and muscle lineage proteins, while DUX4-induced mRNAs were robustly translated. Endogenous expression of DUX4 in human FSHD myotubes and cancer cell lines also correlated with reduced protein synthesis and MHC-I presentation. Our findings reveal that DUX4 orchestrates cell state conversion by suppressing the cellular proteome while maintaining translation of DUX4-induced mRNAs to promote an early developmental program.

Influence of DUX4 Expression in Facioscapulohumeral Muscular Dystrophy and Possible Treatments.

Facioscapulohumeral muscular dystrophy (FSHD) represents the third most common form of muscular dystrophy and is characterized by muscle weakness and atrophy. FSHD is caused by the altered expression of the transcription factor double homeobox 4 (DUX4), which is involved in several significantly altered pathways required for myogenesis and muscle regeneration. While DUX4 is normally silenced in the majority of somatic tissues in healthy individuals, its epigenetic de-repression has been linked to FSHD, resulting in DUX4 aberrant expression and cytotoxicity in skeletal muscle cells. Understanding how DUX4 is regulated and functions could provide useful information not only to further understand FSHD pathogenesis, but also to develop therapeutic approaches for this disorder. Therefore, this review discusses the role of DUX4 in FSHD by examining the possible molecular mechanisms underlying the disease as well as novel pharmacological strategies targeting DUX4 aberrant expression.

Compromised nonsense-mediated RNA decay results in truncated RNA-binding protein production upon DUX4 expression.

Nonsense-mediated RNA decay (NMD) degrades transcripts carrying premature termination codons. NMD is thought to prevent the synthesis of toxic truncated proteins. However, whether loss of NMD results in widespread production of truncated proteins is unclear. A human genetic disease, facioscapulohumeral muscular dystrophy (FSHD), features acute inhibition of NMD upon expression of the disease-causing transcription factor, DUX4. Using a cell-based model of FSHD, we show production of truncated proteins from physiological NMD targets and find that RNA-binding proteins are enriched for aberrant truncations. The NMD isoform of one RNA-binding protein, SRSF3, is translated to produce a stable truncated protein, which is detected in FSHD patient-derived myotubes. Ectopic expression of truncated SRSF3 confers toxicity, and its downregulation is cytoprotective. Our results delineate the genome-scale impact of NMD loss. This widespread production of potentially deleterious truncated proteins has implications for FSHD biology as well as other genetic diseases where NMD is therapeutically modulated.

Facioscapulohumeral Muscular Dystrophy is Associated With Altered Myoblast Proteome Dynamics.

Proteomic studies in facioscapulohumeral muscular dystrophy (FSHD) could offer new insight into disease mechanisms underpinned by post-transcriptional processes. We used stable isotope (deuterium oxide; D2O) labeling and peptide mass spectrometry to investigate the abundance and turnover rates of proteins in cultured muscle cells from two individuals affected by FSHD and their unaffected siblings (UASb). We measured the abundance of 4420 proteins and the turnover rate of 2324 proteins in each (n = 4) myoblast sample. FSHD myoblasts exhibited a greater abundance but slower turnover rate of subunits of mitochondrial respiratory complexes and mitochondrial ribosomal proteins, which may indicate an accumulation of "older" less viable mitochondrial proteins in myoblasts from individuals affected by FSHD. Treatment with a 2'-O-methoxyethyl modified antisense oligonucleotide targeting exon 3 of the double homeobox 4 (DUX4) transcript tended to reverse mitochondrial protein dysregulation in FSHD myoblasts, indicating the effect on mitochondrial proteins may be a DUX4-dependent mechanism. Our results highlight the importance of post-transcriptional processes and protein turnover in FSHD pathology and provide a resource for the FSHD research community to explore this burgeoning aspect of FSHD.

An in silico FSHD muscle fiber for modeling DUX4 dynamics and predicting the impact of therapy.

Facioscapulohumeral muscular dystrophy (FSHD) is an incurable myopathy linked to the over-expression of the myotoxic transcription factor DUX4. Targeting DUX4 is the leading therapeutic approach, however, it is only detectable in 0.1-3.8% of FSHD myonuclei. How rare DUX4 drives FSHD and the optimal anti-DUX4 strategy are unclear. We combine stochastic gene expression with compartment models of cell states, building a simulation of DUX4 expression and consequences in FSHD muscle fibers. Investigating iDUX4 myoblasts, scRNAseq, and snRNAseq of FSHD muscle we estimate parameters including DUX4 mRNA degradation, transcription and translation rates, and DUX4 target gene activation rates. Our model accurately recreates the distribution of DUX4 and targets gene-positive cells seen in scRNAseq of FSHD myocytes. Importantly, we show DUX4 drives significant cell death despite expression in only 0.8% of live cells. Comparing scRNAseq of unfused FSHD myocytes to snRNAseq of fused FSHD myonuclei, we find evidence of DUX4 protein syncytial diffusion and estimate its rate via genetic algorithms. We package our model into freely available tools, to rapidly investigate the consequences of anti-DUX4 therapy.

Nutritional Status of Patients with Facioscapulohumeral Muscular Dystrophy.

In patients with facioscapulohumeral muscular dystrophy (FSHD), a rare genetic neuromuscular disease, reduced physical performance is associated with lower blood levels of vitamin C, zinc, selenium, and increased oxidative stress markers. Supplementation of vitamin C, vitamin E, zinc, and selenium improves the quadriceps' physical performance. Here, we compared the nutritional status of 74 women and 85 men with FSHD. Calorie intake was lower in women with FSHD than in men. Moreover, we assessed vitamin C, vitamin E, zinc, copper, and selenium intakes in diet and their concentrations in the plasma. Vitamin E, copper, and zinc intake were lower in women with FSHD than in men, whereas plasma vitamin C, copper levels, and copper/zinc ratio were higher in women with FSHD than in men. The dietary intake and plasma concentrations of the studied vitamins and minerals were not correlated in both sexes. A well-balanced and varied diet might not be enough in patients with FSHD to correct the observed vitamin/mineral deficiencies. A low energy intake is a risk factor for suboptimal intake of proteins, vitamins, and minerals that are important for protein synthesis and other metabolic pathways and that might contribute to progressive muscle mass loss. Antioxidant supplementation and higher protein intake seem necessary to confer protection against oxidative stress and skeletal muscle mass loss.

WDR5 is required for DUX4 expression and its pathological effects in FSHD muscular dystrophy.

Facioscapulohumeral muscular dystrophy (FSHD) is one of the most prevalent neuromuscular disorders. The disease is linked to copy number reduction and/or epigenetic alterations of the D4Z4 macrosatellite on chromosome 4q35 and associated with aberrant gain of expression of the transcription factor DUX4, which triggers a pro-apoptotic transcriptional program leading to muscle wasting. As today, no cure or therapeutic option is available to FSHD patients. Given its centrality in FSHD, blocking DUX4 expression with small molecule drugs is an attractive option. We previously showed that the long non protein-coding RNA DBE-T is required for aberrant DUX4 expression in FSHD. Using affinity purification followed by proteomics, here we identified the chromatin remodeling protein WDR5 as a novel DBE-T interactor and a key player required for the biological activity of the lncRNA. We found that WDR5 is required for the expression of DUX4 and its targets in primary FSHD muscle cells. Moreover, targeting WDR5 rescues both cell viability and myogenic differentiation of FSHD patient cells. Notably, comparable results were obtained by pharmacological inhibition of WDR5. Importantly, WDR5 targeting was safe to healthy donor muscle cells. Our results support a pivotal role of WDR5 in the activation of DUX4 expression identifying a druggable target for an innovative therapeutic approach for FSHD.

DUX4 double whammy: The transcription factor that causes a rare muscular dystrophy also kills the precursors of the human nose.

SMCHD1 mutations cause congenital arhinia (absent nose) and a muscular dystrophy called FSHD2. In FSHD2, loss of SMCHD1 repressive activity causes expression of double homeobox 4 (DUX4) in muscle tissue, where it is toxic. Studies of arhinia patients suggest a primary defect in nasal placode cells (human nose progenitors). Here, we show that upon SMCHD1 ablation, DUX4 becomes derepressed in H9 human embryonic stem cells (hESCs) as they differentiate toward a placode cell fate, triggering cell death. Arhinia and FSHD2 patient-derived induced pluripotent stem cells (iPSCs) express DUX4 when converted to placode cells and demonstrate variable degrees of cell death, suggesting an environmental disease modifier. HSV-1 may be one such modifier as herpesvirus infection amplifies DUX4 expression in SMCHD1 KO hESC and patient iPSC. These studies suggest that arhinia, like FSHD2, is due to compromised SMCHD1 repressive activity in a cell-specific context and provide evidence for an environmental modifier.

Human DUX4 and porcine DUXC activate similar early embryonic programs in pig muscle cells: implications for preclinical models of FSHD.

Human DUX4 and its mouse ortholog Dux are normally expressed in the early embryo-the 4-cell or 2-cell cleavage stage embryo, respectively-and activate a portion of the first wave of zygotic gene expression. DUX4 is epigenetically suppressed in nearly all somatic tissue, whereas facioscapulohumeral dystrophy (FSHD)-causing mutations result in its aberrant expression in skeletal muscle, transcriptional activation of the early embryonic program and subsequent muscle pathology. Although DUX4 and Dux both activate an early totipotent transcriptional program, divergence of their DNA binding domains limits the use of DUX4 expressed in mice as a preclinical model for FSHD. In this study, we identify the porcine DUXC messenger ribonucleic acid expressed in early development and show that both pig DUXC and human DUX4 robustly activate a highly similar early embryonic program in pig muscle cells. These results support further investigation of pig preclinical models for FSHD.