Double homeobox 4 (DUX4), the causative gene of facioscapulohumeral muscular dystrophy (FSHD), is ectopically expressed in the skeletal muscle cells of FSHD patients because of chromatin relaxation at 4q35. The diminished heterochromatic state at 4q35 is caused by either large genome contractions [FSHD type 1 (FSHD1)] or mutations in genes encoding chromatin regulators, such as SMCHD1 [FSHD type 2 (FSHD2)]. However, the mechanism by which DUX4 expression is regulated remains largely unknown. Here, using a myocyte model developed from patient-derived induced pluripotent stem cells, we determined that DUX4 expression was increased by oxidative stress (OS), a common environmental stress in skeletal muscle, in both FSHD1 and FSHD2 myocytes. We generated FSHD2-derived isogenic control clones with SMCHD1 mutation corrected by clustered regularly interspaced short palindromic repeats (CRISPR)/ CRISPR associated 9 (Cas9) and homologous recombination and found in the myocytes obtained from these clones that DUX4 basal expression and the OS-induced upregulation were markedly suppressed due to an increase in the heterochromatic state at 4q35. We further found that DNA damage response (DDR) was involved in OS-induced DUX4 increase and identified ataxia-telangiectasia mutated, a DDR regulator, as a mediator of this effect. Our results suggest that the relaxed chromatin state in FSHD muscle cells permits aberrant access of OS-induced DDR signaling, thus increasing DUX4 expression. These results suggest OS could represent an environmental risk factor that promotes FSHD progression.
Facioscapulohumeral muscular dystrophy (FSHD), a progressive skeletal muscle disorder, is epigenetically characterized by DNA hypomethylation of the D4Z4 repeats in the 4q35 region, which enables aberrant DUX4 expression. Sustainable DUX4 suppression is thus a promising therapeutic strategy by which to prevent disease progression, but most of the supposed methods to achieve this depend on the expression of a mediator biochemical entity that would potentially narrow the quality of life of individuals with FSHD in the clinical context. In this study, we report that by applying hit-and-run silencing with dCas9-mediated epigenetic editing targeting DNA hypomethylation on D4Z4 repeats, we could achieve the suppression of endogenous DUX4 in our FSHD patient-derived iPSC model. Notably, DNA methylation was significantly upregulated in FSHD cells and suppression effects were observed for at least two weeks after intervention, which was not the case with transient treatments of typical dCas9-KRAB alone. Off-target analysis showed that despite the potential genome-wide risk for DNA methylation, the impact on the transcriptome was limited. We propose that hit-and-run silencing could be a promising option to prevent disease progression with minimum intervention for individuals with FSHD, motivating further study for clinical development.
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