Abstract
BackgroundFacioscapulohumeral 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 FindingsWe 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/SignificanceThese results constitute a proof of concept for the development of therapeutic approaches for FSHD targeting DUX4 expression.
Highlights
Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal dominant disorder affecting 1/17,000 births
We identify FSHD markers associated with muscle atrophy that are induced by double homeobox 4 (DUX4) expression and inhibited by its suppression either with short interfering RNAs or antisense oligonucleotides (AOs)
In order to investigate whether DUX4 might interfere with the differentiation to myotubes, we transfected immortalized human control myoblasts with pCIneo vectors expressing DUX4 (Fig. S1A) or the shorter DUX1 protein, a non-4q35 homologue limited to the homeodomains [8]
Summary
Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal dominant disorder affecting 1/17,000 births. We could detect stable mRNAs comprising the full DUX4 ORF in FSHD but not control muscle cells. These DUX4 mRNAs derived from the most distal unit, and unexpectedly extended within the flanking pLAM region that provided an intron and a polyadenylation signal (Fig. 1A, [8]). Investigations of genetic polymorphisms in a large cohort of patients and non-affected individuals confirmed this polyadenylation signal is needed to develop FSHD resulting in the production of stable DUX4 mRNAs [9]. 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. The DUX4 protein initiates a large transcription deregulation cascade leading to muscle atrophy and oxidative stress, which are FSHD key features
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