The FSHD2 Gene SMCHD1 Is a Modifier of Disease Severity in Families Affected by FSHD1

Facioscapulohumeral muscular dystrophy type 1 (FSHD1) is caused by contraction of the D4Z4 repeat array on chromosome 4 to a size of 1–10 units. The residual number of D4Z4 units inversely correlates with clinical severity, but significant clinical variability exists. Each unit contains a copy of the DUX4 retrogene. Repeat contractions are associated with changes in D4Z4 chromatin structure that increase the likelihood of DUX4 expression in skeletal muscle, but only when the repeat resides in a genetic background that contains a DUX4 polyadenylation signal. Mutations in the structural maintenance of chromosomes flexible hinge domain containing 1 (SMCHD1) gene, encoding a chromatin modifier of D4Z4, also result in the increased likelihood of DUX4 expression in individuals with a rare form of FSHD (FSHD2). Because SMCHD1 directly binds to D4Z4 and suppresses somatic expression of DUX4, we hypothesized that SMCHD1 may act as a genetic modifier in FSHD1. We describe three unrelated individuals with FSHD1 presenting an unusual high clinical severity based on their upper-sized FSHD1 repeat array of nine units. Each of these individuals also carries a mutation in the SMCHD1 gene. Familial carriers of the FSHD1 allele without the SMCHD1 mutation were only mildly affected, suggesting a modifier effect of the SMCHD1 mutation. Knocking down SMCHD1 in FSHD1 myotubes increased DUX4 expression, lending molecular support to a modifier role for SMCHD1 in FSHD1. We conclude that FSHD1 and FSHD2 share a common pathophysiological pathway in which the FSHD2 gene can act as modifier for disease severity in families affected by FSHD1.     

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Background

Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal dominant neuromuscular disorder associated with the partial deletion of integral numbers of 3.3 kb D4Z4 DNA repeats within the subtelomere of chromosome 4q. A number of candidate FSHD genes, adenine nucleotide translocator 1 gene (ANT1), FSHD-related gene 1 (FRG1), FRG2 and DUX4c, upstream of the D4Z4 array (FSHD locus), and double homeobox chromosome 4 (DUX4) within the repeat itself, are upregulated in some patients, thus suggesting an underlying perturbation of the chromatin structure. Furthermore, a mouse model overexpressing FRG1 has been generated, displaying skeletal muscle defects.

Results

In the context of myogenic differentiation, we compared the chromatin structure and tridimensional interaction of the D4Z4 array and FRG1 gene promoter, and FRG1 expression, in control and FSHD cells. The FRG1 gene was prematurely expressed during FSHD myoblast differentiation, thus suggesting that the number of D4Z4 repeats in the array may affect the correct timing of FRG1 expression. Using chromosome conformation capture (3C) technology, we revealed that the FRG1 promoter and D4Z4 array physically interacted. Furthermore, this chromatin structure underwent dynamic changes during myogenic differentiation that led to the loosening of the FRG1/4q-D4Z4 array loop in myotubes. The FRG1 promoter in both normal and FSHD myoblasts was characterized by H3K27 trimethylation and Polycomb repressor complex binding, but these repression signs were replaced by H3K4 trimethylation during differentiation. The D4Z4 sequences behaved similarly, with H3K27 trimethylation and Polycomb binding being lost upon myogenic differentiation.

Conclusion

We propose a model in which the D4Z4 array may play a critical chromatin function as an orchestrator of in cis chromatin loops, thus suggesting that this repeat may play a role in coordinating gene expression.     

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

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

Aberrant Splicing in Transgenes Containing Introns,Exons, and V5 Epitopes: Lessons from Developing an FSHD Mouse Model Expressing a D4Z4 Repeat with Flanking Genomic Sequences

The DUX4 gene, encoded within D4Z4 repeats on human chromosome 4q35, has recently emerged as a key factor in the pathogenic mechanisms underlying Facioscapulohumeral muscular dystrophy (FSHD). This recognition prompted development of animal models expressing the DUX4 open reading frame (ORF) alone or embedded within D4Z4 repeats. In the first published model, we used adeno-associated viral vectors (AAV) and strong viral control elements (CMV promoter, SV40 poly A) to demonstrate that the DUX4 cDNA caused dose-dependent toxicity in mouse muscles. As a follow-up, we designed a second generation of DUX4-expressing AAV vectors to more faithfully genocopy the FSHD-permissive D4Z4 repeat region located at 4q35. This new vector (called AAV.D4Z4.V5.pLAM) contained the D4Z4/DUX4 promoter region, a V5 epitope-tagged DUX4 ORF, and the natural 3’ untranslated region (pLAM) harboring two small introns, DUX4 exons 2 and 3, and the non-canonical poly A signal required for stabilizing DUX4 mRNA in FSHD. AAV.D4Z4.V5.pLAM failed to recapitulate the robust pathology of our first generation vectors following delivery to mouse muscle. We found that the DUX4.V5 junction sequence created an unexpected splice donor in the pre-mRNA that was preferentially utilized to remove the V5 coding sequence and DUX4 stop codon, yielding non-functional DUX4 protein with 55 additional residues on its carboxyl-terminus. Importantly, we further found that aberrant splicing could occur in any expression construct containing a functional splice acceptor and sequences resembling minimal splice donors. Our findings represent an interesting case study with respect to AAV.D4Z4.V5.pLAM, but more broadly serve as a note of caution for designing constructs containing V5 epitope tags and/or transgenes with downstream introns and exons.     

Biphasic Myopathic Phenotype of Mouse DUX,an ORF within Conserved FSHD-Related Repeats

Facioscapulohumeral muscular dystrophy (FSHD) is caused by contractions of D4Z4 repeats at 4q35.2 thought to induce misregulation of nearby genes, one of which, DUX4, is actually localized within each repeat. A conserved ORF (mDUX), embedded within D4Z4-like repeats, encoding a double-homeodomain protein, was recently identified on mouse chromosome 10. We show here that high level mDUX expression induces myoblast death, while low non-toxic levels block myogenic differentiation by down-regulating MyoD and Myf5. Toxicity and MyoD/Myf5 expression changes were competitively reversed by overexpression of Pax3 or Pax7, implying mechanistic similarities with the anti-myogenic activity of human DUX4. We tested the effect of mDUX expression on Xenopus development, and found that global overexpression led to abnormalities in gastrulation. When targeted unilaterally into blastomeres fated to become tail muscle in 16-cell embryos, mDUX caused markedly reduced tail myogenesis on the injected side. These novel cell and animal models highlight the myopathic nature of sequences within the FSHD-related repeat array.