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Genome-wide screens that have viability as a readout have been instrumental to identify essential genes. The development of gene knockout screens with the use of CRISPR-Cas has provided a more sensitive method to identify these genes. Here, we performed an exhaustive genome-wide CRISPR/Cas9 phenotypic rescue screen to identify modulators of cytotoxicity induced by the pioneer transcription factor, DUX4. Misexpression of DUX4 due to a failure in epigenetic repressive mechanisms underlies facioscapulohumeral muscular dystrophy (FHSD), a complex muscle disorder that thus far remains untreatable. As the name implies, FSHD generally starts in the muscles of the face and shoulder girdle. Our CRISPR/Cas9 screen revealed no key effectors other than DUX4 itself that could modulate DUX4 cytotoxicity, suggesting that treatment efforts in FSHD should be directed towards direct modulation of DUX4 itself. Our screen did however reveal some rare and unexpected genomic events, that had an important impact on the interpretation of our data. Our findings may provide important considerations for planning future CRISPR/Cas9 phenotypic survival screens., Competing Interests: The authors have declared that no competing interests exist.

FSHD is characterized by the misexpression of DUX4 in skeletal muscle. Although DUX4 upregulation is thought to be the pathogenic cause of FSHD, DUX4 is lowly expressed in patient samples, and analysis of the consequences of DUX4 expression has largely relied on artificial overexpression. To better understand the native expression profile of DUX4 and its targets, we performed bulk RNA-seq on a 6-day differentiation time-course in primary FSHD2 patient myoblasts. We identify a set of 54 genes upregulated in FSHD2 cells, termed FSHD-induced genes. Using single-cell and single-nucleus RNA-seq on myoblasts and differentiated myotubes, respectively, we captured, for the first time, DUX4 expressed at the single-nucleus level in a native state. We identified two populations of FSHD myotube nuclei based on low or high enrichment of DUX4 and FSHD-induced genes ("FSHD-Lo" and "FSHD Hi", respectively). FSHD-Hi myotube nuclei coexpress multiple DUX4 target genes including DUXA, LEUTX and ZSCAN4, and also upregulate cell cycle-related genes with significant enrichment of E2F target genes and p53 signaling activation. We found more FSHD-Hi nuclei than DUX4-positive nuclei, and confirmed with in situ RNA/protein detection that DUX4 transcribed in only one or two nuclei is sufficient for DUX4 protein to activate target genes across multiple nuclei within the same myotube. DUXA (the DUX4 paralog) is more widely expressed than DUX4, and depletion of DUXA suppressed the expression of LEUTX and ZSCAN4 in late, but not early, differentiation. The results suggest that the DUXA can take over the role of DUX4 to maintain target gene expression. These results provide a possible explanation as to why it is easier to detect DUX4 target genes than DUX4 itself in patient cells and raise the possibility of a self-sustaining network of gene dysregulation triggered by the limited DUX4 expression., Competing Interests: The authors have declared that no competing interests exist.

Facioscapulohumeral dystrophy (FSHD) is caused by the mis-expression of DUX4 in skeletal muscle cells. DUX4 is a transcription factor that activates genes normally associated with stem cell biology and its mis-expression in FSHD cells results in apoptosis. To identify genes and pathways necessary for DUX4-mediated apoptosis, we performed an siRNA screen in an RD rhabdomyosarcoma cell line with an inducible DUX4 transgene. Our screen identified components of the MYC-mediated apoptotic pathway and the double-stranded RNA (dsRNA) innate immune response pathway as mediators of DUX4-induced apoptosis. Further investigation revealed that DUX4 expression led to increased MYC mRNA, accumulation of nuclear dsRNA foci, and activation of the dsRNA response pathway in both RD cells and human myoblasts. Nuclear dsRNA foci were associated with aggregation of the exon junction complex component EIF4A3. The elevation of MYC mRNA, dsRNA accumulation, and EIF4A3 nuclear aggregates in FSHD muscle cells suggest that these processes might contribute to FSHD pathophysiology.

Background: Facioscapulohumeral muscular dystrophy type 1 (FSHD1) is the third most common inherited muscular dystrophy. Considering the highly variable clinical expression and the slow disease progression, sensitive outcome measures would be of interest., Methods and Findings: Using muscle MRI, we assessed muscular fatty infiltration in the lower limbs of 35 FSHD1 patients and 22 healthy volunteers by two methods: a quantitative imaging (qMRI) combined with a dedicated automated segmentation method performed on both thighs and a standard T1-weighted four-point visual scale (visual score) on thighs and legs. Each patient had a clinical evaluation including manual muscular testing, Clinical Severity Score (CSS) scale and MFM scale. The intramuscular fat fraction measured using qMRI in the thighs was significantly higher in patients (21.9 ± 20.4%) than in volunteers (3.6 ± 2.8%) (p<0.001). In patients, the intramuscular fat fraction was significantly correlated with the muscular fatty infiltration in the thighs evaluated by the mean visual score (p<0.001). However, we observed a ceiling effect of the visual score for patients with a severe fatty infiltration clearly indicating the larger accuracy of the qMRI approach. Mean intramuscular fat fraction was significantly correlated with CSS scale (p ≤ 0.01) and was inversely correlated with MMT score, MFM subscore D1 (p ≤ 0.01) further illustrating the sensitivity of the qMRI approach. Overall, a clustering analysis disclosed three different imaging patterns of muscle involvement for the thighs and the legs which could be related to different stages of the disease and put forth muscles which could be of interest for a subtle investigation of the disease progression and/or the efficiency of any therapeutic strategy., Conclusion: The qMRI provides a sensitive measurement of fat fraction which should also be of high interest to assess disease progression and any therapeutic strategy in FSHD1 patients.

Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal-dominant disease with no effective treatment. The genetic cause of FSHD is complex and the primary pathogenic insult underlying the muscle disease is unknown. Several disease candidate genes have been proposed including DUX4 and FRG1. Expression analysis studies of FSHD report the deregulation of genes which mediate myoblast differentiation and fusion. Transgenic mice overexpressing FRG1 recapitulate the FSHD muscular dystrophy phenotype. Our current study selectively examines how increased expression of FRG1 may contribute to myoblast differentiation defects. We generated stable C2C12 cell lines overexpressing FRG1, which exhibited a myoblast fusion defect upon differentiation. To determine if myoblast fusion defects contribute to the FRG1 mouse dystrophic phenotype, this strain was crossed with skeletal muscle specific FHL1-transgenic mice. We previously reported that FHL1 promotes myoblast fusion in vitro and FHL1-transgenic mice develop skeletal muscle hypertrophy. In the current study, FRG1 mice overexpressing FHL1 showed an improvement in the dystrophic phenotype, including a reduced spinal kyphosis, increased muscle mass and myofiber size, and decreased muscle fibrosis. FHL1 expression in FRG1 mice, did not alter satellite cell number or activation, but enhanced myoblast fusion. Primary myoblasts isolated from FRG1 mice showed a myoblast fusion defect that was rescued by FHL1 expression. Therefore, increased FRG1 expression may contribute to a muscular dystrophy phenotype resembling FSHD by impairing myoblast fusion, a defect that can be rescued by enhanced myoblast fusion via expression of FHL1.

Background: Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal-dominant disorder and is one of the most common forms of muscular dystrophy. We have recently shown that some hallmarks of FSHD are already expressed in fetal FSHD biopsies, thus opening a new field of investigation for mechanisms leading to FSHD. As microRNAs (miRNAs) play an important role in myogenesis and muscle disorders, in this study we compared miRNAs expression levels during normal and FSHD muscle development., Methods: Muscle biopsies were obtained from quadriceps of both healthy control and FSHD1 fetuses with ages ranging from 14 to 33 weeks of development. miRNA expression profiles were analyzed using TaqMan Human MicroRNA Arrays., Results: During human skeletal muscle development, in control muscle biopsies we observed changes for 4 miRNAs potentially involved in secondary muscle fiber formation and 5 miRNAs potentially involved in fiber maturation. When we compared the miRNA profiles obtained from control and FSHD biopsies, we did not observe any differences in the muscle specific miRNAs. However, we identified 8 miRNAs exclusively expressed in FSHD1 samples (miR-330, miR-331-5p, miR-34a, miR-380-3p, miR-516b, miR-582-5p, miR-517* and miR-625) which could represent new biomarkers for this disease. Their putative targets are mainly involved in muscle development and morphogenesis. Interestingly, these FSHD1 specific miRNAs do not target the genes previously described to be involved in FSHD., Conclusions: This work provides new candidate mechanisms potentially involved in the onset of FSHD pathology. Whether these FSHD specific miRNAs cause deregulations during fetal development, or protect against the appearance of the FSHD phenotype until the second decade of life still needs to be investigated.

Emerging evidence has demonstrated that miRNA sequences can regulate skeletal myogenesis by controlling the process of myoblast proliferation and differentiation. However, at present a deep analysis of miRNA expression in control and FSHD myoblasts during differentiation has not yet been derived. To close this gap, we used a next-generation sequencing (NGS) approach applied to in vitro myogenesis. Furthermore, to minimize sample genetic heterogeneity and muscle-type specific patterns of gene expression, miRNA profiling from NGS data was filtered with FC ≥ 4 (log(2)FC ≥ 2) and p-value<0.05, and its validation was derived by qRT-PCR on myoblasts from seven muscle districts. In particular, control myogenesis showed the modulation of 38 miRNAs, the majority of which (34 out 38) were up-regulated, including myomiRs (miR-1, -133a, -133b and -206). Approximately one third of the modulated miRNAs were not previously reported to be involved in muscle differentiation, and interestingly some of these (i.e. miR-874, -1290, -95 and -146a) were previously shown to regulate cell proliferation and differentiation. FSHD myogenesis evidenced a reduced number of modulated miRNAs than healthy muscle cells. The two processes shared nine miRNAs, including myomiRs, although with FC values lower in FSHD than in control cells. In addition, FSHD cells showed the modulation of six miRNAs (miR-1268, -1268b, -1908, 4258, -4508- and -4516) not evidenced in control cells and that therefore could be considered FSHD-specific, likewise three novel miRNAs that seem to be specifically expressed in FSHD myotubes. These data further clarify the impact of miRNA regulation during control myogenesis and strongly suggest that a complex dysregulation of miRNA expression characterizes FSHD, impairing two important features of myogenesis: cell cycle and muscle development. The derived miRNA profiling could represent a novel molecular signature for FSHD that includes diagnostic biomarkers and possibly therapeutic targets.

Background: In Facioscapulohumeral muscular dystrophy (FSHD), the upper girdle is early involved and often difficult to assess only relying on physical examination. Our aim was to evaluate the pattern and degree of involvement of upper girdle muscles in FSHD compared with other muscle diseases with scapular girdle impairment., Methods: We propose an MRI protocol evaluating neck and upper girdle muscles. One hundred-eight consecutive symptomatic FSHD patients and 45 patients affected by muscular dystrophies and myopathies with prominent upper girdle involvement underwent this protocol. Acquired scans were retrospectively analyzed., Results: The trapezius (100% of the patients) and serratus anterior (85% of the patients) were the most and earliest affected muscles in FSHD, followed by the latissimus dorsi and pectoralis major, whilst spinati and subscapularis (involved in less than 4% of the patients) were consistently spared even in late disease stages. Asymmetry and hyperintensities on short-tau inversion recovery (STIR) sequences were common features, and STIR hyperintensities could also be found in muscles not showing signs of fatty replacement. The overall involvement appears to be disease-specific in FSHD as it significantly differed from that encountered in the other myopathies., Conclusions: The detailed knowledge of single muscle involvement provides useful information for correctly evaluating patients' motor function and to set a baseline for natural history studies. Upper girdle imaging can also be used as an additional tool helpful in supporting the diagnosis of FSHD in unclear situations, and may contribute with hints on the currently largely unknown molecular pathogenesis of this disease.

Facioscapulohumeral muscular dystrophy (FSHD) is an untreatable disease, characterized by asymmetric progressive weakness of skeletal muscle with fatty infiltration. Although the main genetic defect has been uncovered, the downstream mechanisms causing FSHD are not understood. The objective of this study was to determine natural disease state and progression in muscles of FSHD patients and to establish diagnostic biomarkers by quantitative MRI of fat infiltration and phosphorylated metabolites. MRI was performed at 3T with dedicated coils on legs of 41 patients (28 men/13 women, age 34-76 years), of which eleven were re-examined after four months of usual care. Muscular fat fraction was determined with multi spin-echo and T1 weighted MRI, edema by TIRM and phosphorylated metabolites by 3D (31)P MR spectroscopic imaging. Fat fractions were compared to clinical severity, muscle force, age, edema and phosphocreatine (PCr)/ATP. Longitudinal intramuscular fat fraction variation was analyzed by linear regression. Increased intramuscular fat correlated with age (p<0.05), FSHD severity score (p<0.0001), inversely with muscle strength (p<0.0001), and also occurred sub-clinically. Muscles were nearly dichotomously divided in those with high and with low fat fraction, with only 13% having an intermediate fat fraction. The intramuscular fat fraction along the muscle's length, increased from proximal to distal. This fat gradient was the steepest for intermediate fat infiltrated muscles (0.07±0.01/cm, p<0.001). Leg muscles in this intermediate phase showed a decreased PCr/ATP (p<0.05) and the fastest increase in fatty infiltration over time (0.18±0.15/year, p<0.001), which correlated with initial edema (p<0.01), if present. Thus, in the MR assessment of fat infiltration as biomarker for diseased muscles, the intramuscular fat distribution needs to be taken into account. Our results indicate that healthy individual leg muscles become diseased by entering a progressive phase with distal fat infiltration and altered energy metabolite levels. Fat replacement then relatively rapidly spreads over the whole muscle.

The human double-homeodomain retrogene DUX4 is expressed in the testis and epigenetically repressed in somatic tissues. Facioscapulohumeral muscular dystrophy (FSHD) is caused by mutations that decrease the epigenetic repression of DUX4 in somatic tissues and result in mis-expression of this transcription factor in skeletal muscle. DUX4 binds sites in the human genome that contain a double-homeobox sequence motif, including sites in unique regions of the genome as well as many sites in repetitive elements. Using ChIP-seq and RNA-seq on myoblasts transduced with DUX4 we show that DUX4 binds and activates transcription of mammalian apparent LTR-retrotransposons (MaLRs), endogenous retrovirus (ERVL and ERVK) elements, and pericentromeric satellite HSATII sequences. Some DUX4-activated MaLR and ERV elements create novel promoters for genes, long non-coding RNAs, and antisense transcripts. Many of these novel transcripts are expressed in FSHD muscle cells but not control cells, and thus might contribute to FSHD pathology. For example, HEY1, a repressor of myogenesis, is activated by DUX4 through a MaLR promoter. DUX4-bound motifs, including those in repetitive elements, show evolutionary conservation and some repeat-initiated transcripts are expressed in healthy testis, the normal expression site of DUX4, but more rarely in other somatic tissues. Testis expression patterns are known to have evolved rapidly in mammals, but the mechanisms behind this rapid change have not yet been identified: our results suggest that mobilization of MaLR and ERV elements during mammalian evolution altered germline gene expression patterns through transcriptional activation by DUX4. Our findings demonstrate a role for DUX4 and repetitive elements in mammalian germline evolution and in FSHD muscular dystrophy., Competing Interests: The authors have declared that no competing interests exist.

DUX4 (Double Homeobox Protein 4) is a nuclear transcription factor encoded at each D4Z4 unit of a tandem-repeat array at human chromosome 4q35. DUX4 constitutes a major candidate pathogenic protein for facioscapulohumeral muscular dystrophy (FSHD), the third most common form of inherited myopathy. A low-level expression of DUX4 compromises cell differentiation in myoblasts and its overexpression induces apoptosis in cultured cells and living organisms. In this work we explore potential molecular determinants of DUX4 mediating nuclear import and cell toxicity. Deletion of the hypothetical monopartite nuclear localization sequences RRRR(23), RRKR(98) and RRAR(148) (i.e. NLS1, NLS2 and NLS3, respectively) only partially delocalizes DUX4 from the cell nuclei. Nuclear entrance guided by NLS1, NLS2 and NLS3 does not follow the classical nuclear import pathway mediated by α/β importins. NLS and homeodomain mutants from DUX4 are dramatically less cell-toxic than the wild type molecule, independently of their subcellular localization. A triple ΔNLS1-2-3 deletion mutant is still partially localized in the nuclei, indicating that additional sequences in DUX4 contribute to nuclear import. Deletion of ≥111 amino acids from the C-terminal of DUX4, on a ΔNLS1-2-3 background, almost completely re-localizes DUX4 to the cytoplasm, indicating that the C-ter tail contributes to subcellular trafficking of DUX4. Also, C-terminal deletion mutants of DUX4 on a NLS wild type background are less toxic than wild type DUX4. Results reported here indicate that DUX4 possesses redundant mechanisms to assure nuclear entrance and that its various transcription-factor associated domains play an essential role in cell toxicity.

Generation of skeletal muscles with forms adapted to their function is essential for normal movement. Muscle shape is patterned by the coordinated polarity of collectively migrating myoblasts. Constitutive inactivation of the protocadherin gene Fat1 uncoupled individual myoblast polarity within chains, altering the shape of selective groups of muscles in the shoulder and face. These shape abnormalities were followed by early onset regionalised muscle defects in adult Fat1-deficient mice. Tissue-specific ablation of Fat1 driven by Pax3-cre reproduced muscle shape defects in limb but not face muscles, indicating a cell-autonomous contribution of Fat1 in migrating muscle precursors. Strikingly, the topography of muscle abnormalities caused by Fat1 loss-of-function resembles that of human patients with facioscapulohumeral dystrophy (FSHD). FAT1 lies near the critical locus involved in causing FSHD, and Fat1 mutant mice also show retinal vasculopathy, mimicking another symptom of FSHD, and showed abnormal inner ear patterning, predictive of deafness, reminiscent of another burden of FSHD. Muscle-specific reduction of FAT1 expression and promoter silencing was observed in foetal FSHD1 cases. CGH array-based studies identified deletion polymorphisms within a putative regulatory enhancer of FAT1, predictive of tissue-specific depletion of FAT1 expression, which preferentially segregate with FSHD. Our study identifies FAT1 as a critical determinant of muscle form, misregulation of which associates with FSHD., Competing Interests: The authors have declared that no competing interests exist.

Facioscapulohumeral muscular dystrophy (FSHD) is a common muscle disease whose molecular pathogenesis remains largely unknown. Over-expression of FSHD region gene 1 (FRG1) in mice, frogs, and worms perturbs muscle development and causes FSHD-like phenotypes. FRG1 has been implicated in splicing, and we asked how splicing might be involved in FSHD by conducting a genome-wide analysis in FRG1 mice. We find that splicing perturbations parallel the responses of different muscles to FRG1 over-expression and disease progression. Interestingly, binding sites for the Rbfox family of splicing factors are over-represented in a subset of FRG1-affected splicing events. Rbfox1 knockdown, over-expression, and RNA-IP confirm that these are direct Rbfox1 targets. We find that FRG1 is associated to the Rbfox1 RNA and decreases its stability. Consistent with this, Rbfox1 expression is down-regulated in mice and cells over-expressing FRG1 as well as in FSHD patients. Among the genes affected is Calpain 3, which is mutated in limb girdle muscular dystrophy, a disease phenotypically similar to FSHD. In FRG1 mice and FSHD patients, the Calpain 3 isoform lacking exon 6 (Capn3 E6-) is increased. Finally, Rbfox1 knockdown and over-expression of Capn3 E6- inhibit muscle differentiation. Collectively, our results suggest that a component of FSHD pathogenesis may arise by over-expression of FRG1, reducing Rbfox1 levels and leading to aberrant expression of an altered Calpain 3 protein through dysregulated splicing., Competing Interests: The authors have declared that no competing interests exist.

Background: Facioscapulohumeral muscular dystrophy (FSHD) is one of the most common muscular dystrophies and is characterized by a non-conventional genetic mechanism activated by pathogenic D4Z4 repeat contractions. By muscle Magnetic Resonance Imaging (MRI) we observed that T2-short tau inversion recovery (T2-STIR) sequences identify two different conditions in which each muscle can be found before the irreversible dystrophic alteration, marked as T1-weighted sequence hyperintensity, takes place. We studied these conditions in order to obtain further information on the molecular mechanisms involved in the selective wasting of single muscles or muscle groups in this disease., Methods: Histopathology, gene expression profiling and real time PCR were performed on biopsies from FSHD muscles with different MRI pattern (T1-weighted normal/T2-STIR normal and T1-weighted normal/T2-STIR hyperintense). Data were compared with those from inflammatory myopathies, dysferlinopathies and normal controls. In order to validate obtained results, two additional FSHD samples with different MRI pattern were analyzed., Results: Myopathic and inflammatory changes characterized T2-STIR hyperintense FSHD muscles, at variance with T2-STIR normal muscles. These two states could be easily distinguished from each other by their transcriptional profile. The comparison between T2-STIR hyperintense FSHD muscles and inflammatory myopathy muscles showed peculiar changes, although many alterations were shared among these conditions., Conclusions: At the single muscle level, different stages of the disease correspond to the two MRI patterns. T2-STIR hyperintense FSHD muscles are more similar to inflammatory myopathies than to T2-STIR normal FSHD muscles or other muscular dystrophies, and share with them upregulation of genes involved in innate and adaptive immunity. Our data suggest that selective inflammation, together with perturbation in biological processes such as neoangiogenesis, lipid metabolism and adipokine production, may contribute to the sequential bursts of muscle degeneration that involve individual muscles in an asynchronous manner in this disease.

Background: Determine global gene dysregulation affecting 4q-linked (FSHD-1) and non 4q-linked (FSHD-2) cells during early stages of myogenic differentiation. This approach has been never applied to FSHD pathogenesis., Methodology/principal Findings: By in vitro differentiation of FSHD-1 and FSHD-2 myoblasts and gene chip analysis we derived that gene expression profile is altered only in FSHD-1 myoblasts and FSHD-2 myotubes. The changes seen in FSHD-1 regarded a general defect in cell cycle progression, probably due to the upregulation of myogenic markers PAX3 and MYOD1, and a deficit of factors (SUV39H1 and HMGB2) involved in D4Z4 chromatin conformation. On the other hand, FSHD-2 mytubes were characterized by a general defect in RNA metabolism, protein synthesis and degradation and, to a lesser extent, in cell cycle. Common dysregulations regarded genes involved in response to oxidative stress and in sterol biosynthetic process. Interestingly, our results also suggest that miRNAs might be implied in both FSHD-1 and FSHD-2 gene dysregulation. Finally, in both cell differentiation systems, we did not observe a gradient of altered gene expression throughout the 4q35 chromosome., Conclusions/significance: FSHD-1 and FSHD-2 cells showed, in different steps of myogenic differentiation, a global deregulation of gene expression rather than an alteration of expression of 4q35 specific genes. In general, FSHD-1 and FSHD-2 global gene deregulation interested common and distinctive biological processes. In this regard, defects of cell cycle progression (FSHD-1 and to a lesser extent FSHD-2), protein synthesis and degradation (FSHD-2), response to oxidative stress (FSHD-1 and FSHD-2), and cholesterol homeostasis (FSHD-1 and FSHD-2) may in general impair a correct myogenesis. Taken together our results recapitulate previously reported defects of FSHD-1, and add new insights into the gene deregulation characterizing both FSHD-1 and FSHD-2, in which miRNAs may play a role.

Background: Facioscapulohumeral muscular dystrophy (FSHD) is linked to deletions in 4q35 within the D4Z4 repeat array in which we identified the double homeobox 4 (DUX4) gene. We found stable DUX4 mRNAs only derived from the most distal D4Z4 unit and unexpectedly extended to the flanking pLAM region that provided an intron and a polyadenylation signal. DUX4 encodes a transcription factor expressed in FSHD but not control primary myoblasts or muscle biopsies. The DUX4 protein initiates a large transcription deregulation cascade leading to muscle atrophy and oxidative stress, which are FSHD key features., Methodology/principal Findings: We now show that transfection of myoblasts with a DUX4 expression vector leads to atrophic myotube formation associated with the induction of E3 ubiquitin ligases (MuRF1 and Atrogin1/MAFbx) typical of muscle atrophy. DUX4 induces expression of downstream targets deregulated in FSHD such as mu-crystallin and TP53. We developed specific siRNAs and antisense oligonucleotides (AOs) targeting the DUX4 mRNA. Addition of these antisense agents to primary FSHD myoblast cultures suppressed DUX4 protein expression and affected expression of the above-mentioned markers., Conclusions/significance: These results constitute a proof of concept for the development of therapeutic approaches for FSHD targeting DUX4 expression.

Each unit of the D4Z4 macrosatellite repeat contains a retrotransposed gene encoding the DUX4 double-homeobox transcription factor. Facioscapulohumeral dystrophy (FSHD) is caused by deletion of a subset of the D4Z4 units in the subtelomeric region of chromosome 4. Although it has been reported that the deletion of D4Z4 units induces the pathological expression of DUX4 mRNA, the association of DUX4 mRNA expression with FSHD has not been rigorously investigated, nor has any human tissue been identified that normally expresses DUX4 mRNA or protein. We show that FSHD muscle expresses a different splice form of DUX4 mRNA compared to control muscle. Control muscle produces low amounts of a splice form of DUX4 encoding only the amino-terminal portion of DUX4. FSHD muscle produces low amounts of a DUX4 mRNA that encodes the full-length DUX4 protein. The low abundance of full-length DUX4 mRNA in FSHD muscle cells represents a small subset of nuclei producing a relatively high abundance of DUX4 mRNA and protein. In contrast to control skeletal muscle and most other somatic tissues, full-length DUX4 transcript and protein is expressed at relatively abundant levels in human testis, most likely in the germ-line cells. Induced pluripotent (iPS) cells also express full-length DUX4 and differentiation of control iPS cells to embryoid bodies suppresses expression of full-length DUX4, whereas expression of full-length DUX4 persists in differentiated FSHD iPS cells. Together, these findings indicate that full-length DUX4 is normally expressed at specific developmental stages and is suppressed in most somatic tissues. The contraction of the D4Z4 repeat in FSHD results in a less efficient suppression of the full-length DUX4 mRNA in skeletal muscle cells. Therefore, FSHD represents the first human disease to be associated with the incomplete developmental silencing of a retrogene array normally expressed early in development., Competing Interests: The authors have declared that no competing interests exist.

Facioscapulohumeral muscular dystrophy (FSHD) is a dominant disease linked to contractions of the D4Z4 repeat array in 4q35. We have previously identified a double homeobox gene (DUX4) within each D4Z4 unit that encodes a transcription factor expressed in FSHD but not control myoblasts. DUX4 and its target genes contribute to the global dysregulation of gene expression observed in FSHD. We have now characterized the homologous DUX4c gene mapped 42 kb centromeric of the D4Z4 repeat array. It encodes a 47-kDa protein with a double homeodomain identical to DUX4 but divergent in the carboxyl-terminal region. DUX4c was detected in primary myoblast extracts by Western blot with a specific antiserum, and was induced upon differentiation. The protein was increased about 2-fold in FSHD versus control myotubes but reached 2-10-fold induction in FSHD muscle biopsies. We have shown by Western blot and by a DNA-binding assay that DUX4c over-expression induced the MYF5 myogenic regulator and its DNA-binding activity. DUX4c might stabilize the MYF5 protein as we detected their interaction by co-immunoprecipitation. In keeping with the known role of Myf5 in myoblast accumulation during mouse muscle regeneration DUX4c over-expression activated proliferation of human primary myoblasts and inhibited their differentiation. Altogether, these results suggested that DUX4c could be involved in muscle regeneration and that changes in its expression could contribute to the FSHD pathology.