Double Homeobox Research Articles

Are Antioxidants a Potential Therapy for FSHD? A Review of the Literature.

Facioscapulohumeral muscular dystrophy (FSHD) is an inherited myopathy affecting approximately 1 in 7500 individuals worldwide. It is a progressive disease characterised by skeletal muscle weakness and wasting. A genetic mutation on the 4q35 chromosome results in the expression of the double homeobox 4 gene (DUX4) which drives oxidative stress, inflammation, toxicity, and atrophy within the skeletal muscle. FSHD is characterised by oxidative stress, and there is currently no cure and a lack of therapies for the disease. Antioxidants have been researched for many years, with investigators aiming to use antioxidants therapeutically for oxidative stress-associated diseases. This has included both natural and synthetic antioxidants. The use of antioxidants in preclinical or clinical models has been largely successful with a plethora of research reporting positive results. However, when translated to clinical trials, the use of antioxidants as a therapeutic intervention for a variety of disease has been largely unsuccessful. Moreover, specifically focusing on FSHD, limited research has been conducted on the use of antioxidants as a therapy in either preclinical or clinical models. This review summarises the current state of antioxidant use in the treatment of FSHD and discusses their potential avenue for therapeutic use for FSHD patients.

Integrating clinical and genetic observations in facioscapulohumeral muscular dystrophy

This review gives an overview of the currently known key clinical and (epi)genetic aspects of facioscapulohumeral muscular dystrophy (FSHD) and provides perspectives to facilitate future research. Clinically, imaging studies have contributed to a detailed characterization of the FSHD phenotype, and a model is proposed with five stages of disease progression. A number of clinical trials have been conducted regarding exercise and diet aiming to reduce symptoms. Genetically, at least two different mechanisms (FSHD1 and FSHD2) lead to double homeobox 4 (DUX4) expression in skeletal myocytes, which is expected to be necessary for the disease. Disease severity is most likely determined by a combination of the D4Z4 repeat size and its epigenetic state. FSHD is one of the most common muscular dystrophies and is characterized by a typical distribution of muscle weakness. Progress has been made on clinical as well as on (epi)genetic aspects of the disease. Currently, there is no cure available for FSHD. For successful development of new treatments targeting the disease process, integration of clinical and pathogenetic knowledge is essential. A clinical trial toolbox that consists of patient registries, biomarkers and clinical outcome measures will be required to effectively conduct future clinical trials.

Dux4 controls migration of mesenchymal stem cells through the Cxcr4-Sdf1 axis.

We performed transcriptome profiling of human immortalized myoblasts (MB) transiently expressing double homeobox transcription factor 4 (DUX4) and double homeobox transcription factor 4 centromeric (DUX4c) and identified 114 and 70 genes differentially expressed in DUX4- and DUX4c-transfected myoblasts, respectively. A significant number of differentially expressed genes were involved in inflammation, cellular migration and chemotaxis suggesting a role for DUX4 and DUX4c in these processes. DUX4 but not DUX4c overexpression resulted in upregulation of the CXCR4 (C-X-C motif Receptor 4) and CXCL12 (C-X-C motif ligand 12 also known as SDF1) expression in human immortalized myoblasts. In a Transwell cell migration assay, human bone marrow-derived mesenchymal stem cells (BMSCs) were migrating more efficiently towards human immortalized myoblasts overexpressing DUX4 as compared to controls; the migration efficiency of DUX4-transfected BMSCs was also increased. DUX4c overexpression in myoblasts or in BMSCs had no impact on the rate of BMSC migration. Antibodies against SDF1 and CXCR4 blocked the positive effect of DUX4 overexpression on BMSC migration. We propose that DUX4 controls the cellular migration of mesenchymal stem cells through the CXCR4 receptor.

DUX4-induced constitutive DNA damage and oxidative stress contribute to aberrant differentiation of myoblasts from FSHD patients

Facioscapulohumeral dystrophy (FSHD) is one of the three most common muscular dystrophies in the Western world, however, its etiology remains only partially understood. Here, we provide evidence of constitutive DNA damage in in vitro cultured myoblasts isolated from FSHD patients and demonstrate oxidative DNA damage implication in the differentiation of these cells into phenotypically-aberrant myotubes. Double homeobox 4 (DUX4), the major actor in FSHD pathology induced DNA damage accumulation when overexpressed in normal human myoblasts, and RNAi-mediated DUX4 inhibition reduced the level of DNA damage in FSHD myoblasts. Addition of tempol, a powerful antioxidant, to the culture medium of proliferating DUX4-transfected myoblasts and FSHD myoblasts reduced the level of DNA damage, suggesting that DNA alterations are mainly due to oxidative stress. Antioxidant treatment during the myogenic differentiation of FSHD myoblasts significantly reduced morphological defects in myotube formation. We propose that the induction of DNA damage is a novel function of the DUX4 protein affecting myogenic differentiation of FSHD myoblasts.

Influence of Repressive Histone and DNA Methylation upon D4Z4 Transcription in Non-Myogenic Cells.

We looked at a disease-associated macrosatellite array D4Z4 and focused on epigenetic factors influencing its chromatin state outside of the disease-context. We used the HCT116 cell line that contains the non-canonical polyadenylation (poly-A) signal required to stabilize somatic transcripts of the human double homeobox gene DUX4, encoded from D4Z4. In HCT116, D4Z4 is packaged into constitutive heterochromatin, characterized by DNA methylation and histone H3 tri-methylation at lysine 9 (H3K9me3), resulting in low basal levels of D4Z4-derived transcripts. However, a double knockout (DKO) of DNA methyltransferase genes, DNMT1 and DNMT3B, but not either alone, results in significant loss of DNA and H3K9 methylation. This is coupled with upregulation of transcript levels from the array, including DUX4 isoforms (DUX4-fl) that are abnormally expressed in somatic muscle in the disease Facioscapulohumeral muscular dystrophy (FSHD) along with DUX4 protein, as indicated indirectly by upregulation of bondafide targets of DUX4 in DKO but not HCT116 cells. Results from treatment with a chemical inhibitor of histone methylation in HCT116 suggest that in the absence of DNA hypomethylation, H3K9me3 loss alone is sufficient to facilitate DUX4-fl transcription. Additionally, characterization of a cell line from a patient with Immunodeficiency, Centromeric instability and Facial anomalies syndrome 1 (ICF1) possessing a non-canonical poly-A signal and DNA hypomethylation at D4Z4 showed DUX4 target gene upregulation in the patient when compared to controls in spite of retention of H3K9me3. Taken together, these data suggest that both DNA methylation and H3K9me3 are determinants of D4Z4 silencing. Moreover, we show that in addition to testis, there is appreciable expression of spliced and polyadenylated D4Z4 derived transcripts that contain the complete DUX4 open reading frame (ORF) along with DUX4 target gene expression in the thymus, suggesting that DUX4 may provide normal function in this somatic tissue.

Exploring the long noncoding RNAs-based biomarkers and pathogenesis of malignant transformation from dysplasia to oral squamous cell carcinoma by bioinformatics method.

Long noncoding RNAs (lncRNAs) play an important role in many biological processes and carcinogenesis. We aimed to explore lncRNA-based pathogenesis, diagnostic biomarkers, and predictive factors of malignant transformation from dysplasia to oral squamous cell carcinoma (OSCC). Microarray data of GSE30784 consisting of 167 OSCC, 17 dysplasia, and 45 normal oral tissues were downloaded from the GEO database. The differentially expressed genes (DEGs) and lncRNAs between the three samples were identified using R, followed by lncRNA-mRNA coexpression and coregulation network analysis for the prediction of lncRNA target genes. Gene Ontology and Kyoto encydopedia of gene and genomes pathway analysis were performed to further characterize potential interactions. A total of 4462 DEGs and 76 differentially expressed lncRNAs were screened between the three groups, and 200 DEGs and only double homeobox A pseudogene 10 (DUXAP10) were screened among the three groups. A total of 1662 interactions of 46 lncRNAs and their coexpressed target genes were predicted, and 38 pairs of lncRNA-lncRNA coregulated 843 target genes. The coregulated target genes significantly enriched in antigen adaptive immune response, activation of phagocytosis receptor signaling, mast granule NF-κB inflammation, etc. Overall, lncRNAs were differentially expressed in OSCC and dysplasia. The target genes might play an important role in the carcinogenesis and development of OSCC. These results improve our understanding regarding the lncRNA-based pathogenesis and identify some potential targets for early diagnosis of malignant transformation from dysplasia to OSCC.

Deciphering “B-others”: Novel fusion genes driving B-cell acute lymphoblastic leukemia

Deciphering “B-others”: Novel fusion genes driving B-cell acute lymphoblastic leukemia

A Human Pluripotent Stem Cell Model of Facioscapulohumeral Muscular Dystrophy-Affected Skeletal Muscles.

: Facioscapulohumeral muscular dystrophy (FSHD) represents a major unmet clinical need arising from the progressive weakness and atrophy of skeletal muscles. The dearth of adequate experimental models has severely hampered our understanding of the disease. To date, no treatment is available for FSHD. Human embryonic stem cells (hESCs) potentially represent a renewable source of skeletal muscle cells (SkMCs) and provide an alternative to invasive patient biopsies. We developed a scalable monolayer system to differentiate hESCs into mature SkMCs within 26 days, without cell sorting or genetic manipulation. Here we show that SkMCs derived from FSHD1-affected hESC lines exclusively express the FSHD pathogenic marker double homeobox 4 and exhibit some of the defects reported in FSHD. FSHD1 myotubes are thinner when compared with unaffected and Becker muscular dystrophy myotubes, and differentially regulate genes involved in cell cycle control, oxidative stress response, and cell adhesion. This cellular model will be a powerful tool for studying FSHD and will ultimately assist in the development of effective treatments for muscular dystrophies. This work describes an efficient and highly scalable monolayer system to differentiate human pluripotent stem cells (hPSCs) into skeletal muscle cells (SkMCs) and demonstrates disease-specific phenotypes in SkMCs derived from both embryonic and induced hPSCs affected with facioscapulohumeral muscular dystrophy. This study represents the first human stem cell-based cellular model for a muscular dystrophy that is suitable for high-throughput screening and drug development.

153. Third Generation Antisense Targeted to Double Homeobox Protein 4 (DUX4) Reduced DUX4 Expression and Improved Differentiation of FSHD Myoblasts

Facioscapulohumeral muscular dystrophy (FSHD) is caused by aberrant expression of double homeobox protein 4 (DUX4) gene at chromosome 4q35. To date there is no effective treatment for the disease. In the current study, we have evaluated selected Third Generation Antisense compounds (3GAs) targeted to DUX4 for their effectiveness in knocking-down DUX4 in FSHD myoblasts. In addition, we examined the effects of the treatments on myogenic differentiation of FSHD myoblasts. Multiple 3GA compounds (3GA1-5) showed significant knock-down of DUX4 in the FSHD myoblasts, as examined by quantitative RT-PCR. We evaluated two 3GA candidates (3GA3 and 3GA5) at 4 different concentrations, 2.5 nM, 5nM, 25nM, and 50nM, and observed dose-response effects. One of the 3GA compounds completely knocked-down DUX4 expression at 50nM. The effects of 3GAs at25nM on cell differentiation were determined by fusion index, myotube size and number of atrophic myotubes after 7 days of muscle differentiation in culture. Our results showed that the treatments increased fusion index and reduced the number of atrophic myotubes. The studies showed that 3GAs successfully knocked-down DUX4, which improved the myogenic capacity of FSHD myoblasts. The findings demonstrated that the 3GAs are promising therapeutic molecules to be further developed for FSHD treatment.

Facioscapulohumeral dystrophy myoblasts efficiently repair moderate levels of oxidative DNA damage.

Facioscapulohumeral dystrophy (FSHD) is a progressive muscular dystrophy linked to a deletion of a subset of D4Z4 macrosatellite repeats accompanied by a chromatin relaxation of the D4Z4 array on chromosome 4q. In vitro, FSHD primary myoblasts show altered expression of oxidative-related genes and are more susceptible to oxidative stress. Double homeobox 4 (DUX4) gene, encoded within each D4Z4 unit, is normally transcriptionally silenced but is found aberrantly expressed in skeletal muscles of FSHD patients. Its expression leads to a deregulation of DUX4 target genes including those implicated in redox balance. Here, we assessed DNA repair efficiency of oxidative DNA damage in FSHD myoblasts and DUX4-transfected myoblasts. We have shown that the DNA repair activity is altered neither in FSHD myoblasts nor in immortalized human myoblasts transiently expressing DUX4. DNA damage caused by moderate doses of an oxidant is efficiently repaired while FSHD myoblasts exposed for 24h to high levels of oxidative stress accumulated more DNA damage than normal myoblasts, suggesting that FSHD myoblasts remain more vulnerable to oxidative stress at high doses of oxidants.

Homologous Transcription Factors DUX4 and DUX4c Associate with Cytoplasmic Proteins during Muscle Differentiation

Hundreds of double homeobox (DUX) genes map within 3.3-kb repeated elements dispersed in the human genome and encode DNA-binding proteins. Among these, we identified DUX4, a potent transcription factor that causes facioscapulohumeral muscular dystrophy (FSHD). In the present study, we performed yeast two-hybrid screens and protein co-purifications with HaloTag-DUX fusions or GST-DUX4 pull-down to identify protein partners of DUX4, DUX4c (which is identical to DUX4 except for the end of the carboxyl terminal domain) and DUX1 (which is limited to the double homeodomain). Unexpectedly, we identified and validated (by co-immunoprecipitation, GST pull-down, co-immunofluorescence and in situ Proximal Ligation Assay) the interaction of DUX4, DUX4c and DUX1 with type III intermediate filament protein desmin in the cytoplasm and at the nuclear periphery. Desmin filaments link adjacent sarcomere at the Z-discs, connect them to sarcolemma proteins and interact with mitochondria. These intermediate filament also contact the nuclear lamina and contribute to positioning of the nuclei. Another Z-disc protein, LMCD1 that contains a LIM domain was also validated as a DUX4 partner. The functionality of DUX4 or DUX4c interactions with cytoplasmic proteins is underscored by the cytoplasmic detection of DUX4/DUX4c upon myoblast fusion. In addition, we identified and validated (by co-immunoprecipitation, co-immunofluorescence and in situ Proximal Ligation Assay) as DUX4/4c partners several RNA-binding proteins such as C1QBP, SRSF9, RBM3, FUS/TLS and SFPQ that are involved in mRNA splicing and translation. FUS and SFPQ are nuclear proteins, however their cytoplasmic translocation was reported in neuronal cells where they associated with ribonucleoparticles (RNPs). Several other validated or identified DUX4/DUX4c partners are also contained in mRNP granules, and the co-localizations with cytoplasmic DAPI-positive spots is in keeping with such an association. Large muscle RNPs were recently shown to exit the nucleus via a novel mechanism of nuclear envelope budding. Following DUX4 or DUX4c overexpression in muscle cell cultures, we observed their association with similar nuclear buds. In conclusion, our study demonstrated unexpected interactions of DUX4/4c with cytoplasmic proteins playing major roles during muscle differentiation. Further investigations are on-going to evaluate whether these interactions play roles during muscle regeneration as previously suggested for DUX4c.

Antisense targeting of 3' end elements involved in DUX4 mRNA processing is an efficient therapeutic strategy for facioscapulohumeral dystrophy: a new gene-silencing approach.

Defects in mRNA 3'end formation have been described to alter transcription termination, transport of the mRNA from the nucleus to the cytoplasm, stability of the mRNA and translation efficiency. Therefore, inhibition of polyadenylation may lead to gene silencing. Here, we choose facioscapulohumeral dystrophy (FSHD) as a model to determine whether or not targeting key 3' end elements involved in mRNA processing using antisense oligonucleotide drugs can be used as a strategy for gene silencing within a potentially therapeutic context. FSHD is a gain-of-function disease characterized by the aberrant expression of the Double homeobox 4 (DUX4) transcription factor leading to altered pathogenic deregulation of multiple genes in muscles. Here, we demonstrate that targeting either the mRNA polyadenylation signal and/or cleavage site is an efficient strategy to down-regulate DUX4 expression and to decrease the abnormally high-pathological expression of genes downstream of DUX4. We conclude that targeting key functional 3' end elements involved in pre-mRNA to mRNA maturation with antisense drugs can lead to efficient gene silencing and is thus a potentially effective therapeutic strategy for at least FSHD. Moreover, polyadenylation is a crucial step in the maturation of almost all eukaryotic mRNAs, and thus all mRNAs are virtually eligible for this antisense-mediated knockdown strategy.

PARP1 Differentially Interacts with Promoter region of DUX4 Gene in FSHD Myoblasts.

ObjectiveThe goal of the study is to identity proteins, which interact with the promoter region of double homeobox protein 4 (DUX4) gene known to be causative for the autosomal dominant disorder Facioscapulohumeral Muscular Dystrophy (FSHD).MethodsWe performed a DNA pull down assay coupled with mass spectrometry analysis to identify proteins that interact with a DUX4 promoter probe in Rhabdomyosarcomca (RD) cells. We selected the top ranked protein poly (ADP-ribose) polymerase 1 (PARP1) from our mass spectrometry data for further ChIP-qPCR validation using patients' myoblasts. We then treated FSHD myoblasts with PARP1 inhibitors to investigate the role of PARP1 in the FSHD myoblasts.ResultsIn our mass spectrometry analysis, PARP1 was found to be the top ranked protein interacting preferentially with the DUX4 promoter probe in RD cells. We further validated this interaction by immunoblotting in RD cells (2-fold enrichment compared to proteins pulled down by a control probe, p<0.05) and ChIP-qPCR in patients' myoblasts (65-fold enrichment, p<0.01). Interestingly, the interaction was only observed in FSHD myoblasts but not in the control myoblasts. Upon further treatment of FSHD myoblasts with PARP1 inhibitors, we showed that treatment with a PARP1 inhibitor, 3-aminobenzamide (0.5 mM), for 24 h had a suppression of DUX4 (2.6 fold, p<0.05) and ZSCAN4, a gene previously shown to be upregulated by DUX4, (1.6 fold, p<0.01) in FSHD myoblasts. Treatment with fisetin (0.5 mM), a polyphenol compound with PARP1 inhibitory property, for 24 h also suppressed the expression of DUX4 (44.8 fold, p<0.01) and ZSCAN4 (2.2 fold, p<0.05) in the FSHD myoblasts. We further showed that DNA methyltransferase 1 (DNMT1), a gene regulated by PARP1 was also enriched at the DUX4 promoter in RD cells through immunoblotting (2-fold, p<0.01) and immortalized FSHD myoblasts (42-fold, p<0.01) but not control myoblasts through ChIP qPCR.ConclusionOur results showed that PARP1 and DNMT1 interacted with DUX4 promoter and may be involved in modulating DUX4 expression in FSHD.

Targeting mRNA for the treatment of facioscapulohumeral muscular dystrophy.

Facioscapulohumeral muscular dystrophy (FSHD) is an inherited autosomal dominant disorder characterized clinically by progressive muscle degeneration. Currently, no curative treatment for this disorder exists. FSHD patients are managed through physiotherapy to improve function and quality of life. Over the last two decades, FSHD has been better understood as a disease genetically characterized by a pathogenic contraction of a subset of macrosatellite repeats on chromosome 4. Specifically, several studies support an FSHD pathogenesis model involving the aberrant expression of the double homeobox protein 4 (DUX4) gene. Hence, potential therapies revolving around inhibition of DUX4 have been explored. One of the potential treatment options is the use of effective antisense oligonucleotides (AOs) to knockdown expression of the myopathic DUX4 gene and its downstream molecules including paired-like homeodomain transcription factor 1 (PITX1). Success in the suppression of PITX1 expression has already been demonstrated systemically in vivo in recent studies. In this article, we will review the pathogenesis of FSHD and the latest research involving the use of antisense knockdown therapy.

The Role of D4Z4-Encoded Proteins in the Osteogenic Differentiation of Mesenchymal Stromal Cells Isolated from Bone Marrow.

Facioscapulohumeral muscular dystrophy (FSHD) is associated with an activation of the double homeobox 4 (DUX4) gene, which we previously identified within the D4Z4 repeated elements in the 4q35 subtelomeric region. The pathological DUX4 mRNA is derived from the most distal D4Z4 unit and extends unexpectedly within the flanking pLAM region, which provides an intron and polyadenylation signal. The conditions that are required to develop FSHD are a permissive allele providing the polyadenylation signal and hypomethylation of the D4Z4 repeat array compared with the healthy muscle. The DUX4 protein is a 52-kDa transcription factor that initiates a large gene deregulation cascade leading to muscle atrophy, inflammation, differentiation defects, and oxidative stress, which are the key features of FSHD. DUX4 is a retrogene that is normally expressed in germline cells and is submitted to repeat-induced silencing in adult tissues. Since DUX4 mRNAs have been detected in human embryonic and induced pluripotent stem cells, we investigated whether they could also be expressed in human mesenchymal stromal cells (hMSCs). We found that DUX4 mRNAs were induced during the differentiation of hMSCs into osteoblasts and that this process involved DUX4 and new longer protein forms (58 and 70 kDa). A DUX4 mRNA with a more distant 5' start site was characterized that presented a 60-codon reading frame extension and encoded the 58-kDa protein. Transfections of hMSCs with an antisense oligonucleotide targeting DUX4 mRNAs decreased both the 52- and 58-kDa protein levels and confirmed their identity. Gain- and loss-of-function experiments in hMSCs suggested these DUX4 proteins had opposite roles in osteogenic differentiation as evidenced by the alkaline phosphatase activity and calcium deposition. Differentiation was delayed by the 58-kDa DUX4 expression and it was increased by 52-kDa DUX4. These data indicate a role for DUX4 protein forms in the osteogenic differentiation of hMSCs.

Endogenous DUX4 expression in FSHD myotubes is sufficient to cause cell death and disrupts RNA splicing and cell migration pathways

Facioscapulohumeral muscular dystrophy (FSHD) is caused by chromatin relaxation that results in aberrant expression of the transcription factor Double Homeobox 4 (DUX4). DUX4 protein is present in a small subset of FSHD muscle cells, making its detection and analysis of its effects historically difficult. Using a DUX4-activated reporter, we demonstrate the burst expression pattern of endogenous DUX4, its method of signal amplification in the unique shared cytoplasm of the myotube, and FSHD cell death that depends on its activation. Transcriptome analysis of DUX4-expressing cells revealed that DUX4 activation disrupts RNA metabolism including RNA splicing, surveillance and transport pathways. Cell signaling, polarity and migration pathways were also disrupted. Thus, DUX4 expression is sufficient for myocyte death, and these findings suggest mechanistic links between DUX4 expression and cell migration, supporting recent descriptions of phenotypic similarities between FSHD and an FSHD-like condition caused by FAT1 mutations.

Culture Conditions Affect Expression of DUX4 in FSHD Myoblasts.

Facioscapulohumeral muscular dystrophy (FSHD) is believed to be caused by aberrant expression of double homeobox 4 (DUX4) due to epigenetic changes of the D4Z4 region at chromosome 4q35. Detecting DUX4 is challenging due to its stochastic expression pattern and low transcription level. In this study, we examined different cDNA synthesis strategies and the sensitivity for DUX4 detection. In addition, we investigated the effects of dexamethasone and knockout serum replacement (KOSR) on DUX4 expression in culture. Our data showed that DUX4 was consistently detected in cDNA samples synthesized using Superscript III. The sensitivity of DUX4 detection was higher in the samples synthesized using oligo(dT) primers compared to random hexamers. Adding dexamethasone to the culture media significantly suppressed DUX4 expression in immortalized (1.3 fold, p < 0.01) and primary (4.7 fold, p < 0.01) FSHD myoblasts, respectively. Culture medium with KOSR increased DUX4 expression and the response is concentration dependent. The findings suggest that detection strategies and culture conditions should be carefully considered when studying DUX4 in cultured cells.

Individual epigenetic status of the pathogenic D4Z4 macrosatellite correlates with disease in facioscapulohumeral muscular dystrophy.

BackgroundBoth forms of facioscapulohumeral muscular dystrophy (FSHD) are associated with aberrant epigenetic regulation of the chromosome 4q35 D4Z4 macrosatellite. Chromatin changes due to large deletions of heterochromatin (FSHD1) or mutations in chromatin regulatory proteins (FSHD2) lead to relaxation of epigenetic repression and increased expression of the deleterious double homeobox 4 (DUX4) gene encoded within the distal D4Z4 repeat. However, many individuals with the genetic requirements for FSHD remain asymptomatic throughout their lives. Here we investigated family cohorts of FSHD1 individuals who were either affected (manifesting) or without any discernible weakness (nonmanifesting/asymptomatic) and their unaffected family members to determine if individual epigenetic status and stability of repression at the contracted 4q35 D4Z4 array in myocytes correlates with FSHD disease.ResultsFamily cohorts were analyzed for DNA methylation on the distal pathogenic 4q35 D4Z4 repeat on permissive A-type subtelomeres. We found DNA hypomethylation in FSHD1-affected subjects, hypermethylation in healthy controls, and distinctly intermediate levels of methylation in nonmanifesting subjects. We next tested if these differences in DNA methylation had functional relevance by assaying DUX4-fl expression and the stability of epigenetic repression of DUX4-fl in myogenic cells. Treatment with drugs that alter epigenetic status revealed that healthy cells were refractory to treatment, maintaining stable repression of DUX4, while FSHD1-affected cells were highly responsive to treatment and thus epigenetically poised to express DUX4. Myocytes from nonmanifesting subjects had significantly higher levels of DNA methylation and were more resistant to DUX4 activation in response to epigenetic drug treatment than cells from FSHD1-affected first-degree relatives containing the same contraction, indicating that the epigenetic status of the contracted D4Z4 array is reflective of disease.ConclusionsThe epigenetic status of the distal 4qA D4Z4 repeat correlates with FSHD disease; FSHD-affected subjects have hypomethylation, healthy unaffected subjects have hypermethylation, and nonmanifesting subjects have characteristically intermediate methylation. Thus, analysis of DNA methylation at the distal D4Z4 repeat could be used as a diagnostic indicator of developing clinical FSHD. In addition, the stability of epigenetic repression upstream of DUX4 expression is a key regulator of disease and a viable therapeutic target.Electronic supplementary materialThe online version of this article (doi:10.1186/s13148-015-0072-6) contains supplementary material, which is available to authorized users.

A feedback loop between nonsense-mediated decay and the retrogene DUX4 in facioscapulohumeral muscular dystrophy

Facioscapulohumeral muscular dystrophy (FSHD) is a muscular dystrophy caused by inefficient epigenetic repression of the D4Z4 macrosatellite array and somatic expression of the DUX4 retrogene. DUX4 is a double homeobox transcription factor that is normally expressed in the testis and causes apoptosis and FSHD when misexpressed in skeletal muscle. The mechanism(s) of DUX4 toxicity in muscle is incompletely understood. We report that DUX4-triggered proteolytic degradation of UPF1, a central component of the nonsense-mediated decay (NMD) machinery, is associated with profound NMD inhibition, resulting in global accumulation of RNAs normally degraded as NMD substrates. DUX4 mRNA is itself degraded by NMD, such that inhibition of NMD by DUX4 protein stabilizes DUX4 mRNA through a double-negative feedback loop in FSHD muscle cells. This feedback loop illustrates an unexpected mode of autoregulatory behavior of a transcription factor, is consistent with 'bursts' of DUX4 expression in FSHD muscle, and has implications for FSHD pathogenesis.

DUX4 promotes transcription of FRG2 by directly activating its promoter in facioscapulohumeral muscular dystrophy.

BackgroundThe most common form of facioscapulohumeral muscular dystrophy (FSHD) is caused by a genetic contraction of the polymorphic D4Z4 macrosatellite repeat array in the subtelomeric region of chromosome 4q. In some studies, genes centromeric to the D4Z4 repeat array have been reported to be over-expressed in FSHD, including FRG1 and FRG2, presumably due to decreased long-distance repression by the shorter array through a mechanism similar to position-effect variegation. Differential regulation of FRG1 in FSHD has never been unequivocally proven, however, FRG2 has been reproducibly shown to be induced in primary FSHD-derived muscle cells when differentiated in vitro. The molecular function of FRG2 and a possible contribution to FSHD pathology remain unclear. Recent evidence has identified the mis-expression of DUX4, located within the D4Z4 repeat unit, in skeletal muscle as the cause of FSHD. DUX4 is a double homeobox transcription factor that has been shown to be toxic when expressed in muscle cells.MethodsWe used a combination of expression analysis by qRT/PCR and RNA sequencing to determine the transcriptional activation of FRG2 and DUX4. We examined this in both differentiating control and FSHD derived muscle cell cultures or DUX4 transduced control cell lines. Next, we used ChIP-seq analysis and luciferase reporter assays to determine the potential DUX4 transactivation effect on the FRG2 promoter.ResultsWe show that DUX4 directly activates the expression of FRG2. Increased expression of FRG2 was observed following expression of DUX4 in myoblasts and fibroblasts derived from control individuals. Moreover, we identified DUX4 binding sites at the FRG2 promoter by chromatin immunoprecipitation followed by deep sequencing and confirmed the direct regulation of DUX4 on the FRG2 promoter by luciferase reporter assays. Activation of luciferase was dependent on both DUX4 expression and the presence of the DUX4 DNA binding motifs in the FRG2 promoter.ConclusionWe show that the FSHD-specific upregulation of FRG2 is a direct consequence of the activity of DUX4 protein rather than representing a regional de-repression secondary to fewer D4Z4 repeats.