The interplay between 3D chromatin architecture and gene silencing is incompletely understood. Here, we report a novel point mutation in the non-canonical SMC protein SMCHD1 that enhances its... Show moreThe interplay between 3D chromatin architecture and gene silencing is incompletely understood. Here, we report a novel point mutation in the non-canonical SMC protein SMCHD1 that enhances its silencing capacity at endogenous developmental targets. Moreover, it also results in enhanced silencing at the facioscapulohumeral muscular dystrophy associated macrosatellite-array, D4Z4, resulting in enhanced repression of DUX4 encoded by this repeat. Heightened SMCHD1 silencing perturbs developmental Hox gene activation, causing a homeotic transformation in mice. Paradoxically, the mutant SMCHD1 appears to enhance insulation against other epigenetic regulators, including PRC2 and CTCF, while depleting long range chromatin interactions akin to what is observed in the absence of SMCHD1. These data suggest that SMCHD1's role in long range chromatin interactions is not directly linked to gene silencing or insulating the chromatin, refining the model for how the different levels of SMCHD1-mediated chromatin regulation interact to bring about gene silencing in normal development and disease.Here the authors reveal that a neomorphic mutation in chromatin protein SMCHD1 enhances SMCHD1-mediated gene silencing, including at the FSHD disease-relevant locus, while depleting SMCHD1-mediated chromatin interactions, suggesting these SMCHD1 functions are unlinked. Show less
Facioscapulohumeral muscular dystrophy (FSHD) is a progressive skeletal muscle disorder that mainly affects the muscles of the face, shoulders and upper arms. Skeletal muscle wasting in FSHD is... Show moreFacioscapulohumeral muscular dystrophy (FSHD) is a progressive skeletal muscle disorder that mainly affects the muscles of the face, shoulders and upper arms. Skeletal muscle wasting in FSHD is caused by the failure to epigenetically repress the transcription factor DUX4 that is typically expressed during early development. DUX4 expression in skeletal muscle induces several myotoxic cascades that ultimately lead to the death of skeletal muscles cells. At the moment there is no molecular therapy that can delay or stop disease progression. The work described in this thesis mainly aims to gain more insight in the different FSHD mouse models and the in vivo testing of new therapies in FSHD mice. We describe the generation of one new mouse model and the characterization of two other FSHD mouse models. In addition, we tested a RNA therapy that blocks the DUX4 transcript in vivo. We show that this therapy could reduce DUX4 and DUX4 target genes in FSHD mice. In addition, the therapy alleviated the severity of skeletal muscle pathology. With the data described in this thesis we hope to accelerate the development and testing of new therapies for a disease that cannot be treated until this day. Show less
Bouwman, L.F.; Hamer, B. den; Heuvel, A. van den; Franken, M.; Jackson, M.; Dwyer, C.A.; ... ; Greef, J.C. de 2021
Facioscapulohumeral muscular dystrophy (FSHD) is one of the most prevalent skeletal muscle dystrophies. Skeletal muscle pathology in individuals with FSHD is caused by inappropriate expression of... Show moreFacioscapulohumeral muscular dystrophy (FSHD) is one of the most prevalent skeletal muscle dystrophies. Skeletal muscle pathology in individuals with FSHD is caused by inappropriate expression of the transcription factor DUX4, which activates different myotoxic pathways. At the moment there is no molecular therapy that can delay or prevent skeletal muscle wasting in FSHD. In this study, a systemically delivered antisense oligonucleotide (ASO) targeting the DUX4 transcript was tested in vivo in ACTA1-MCM;FLExDUX4 mice that express DUX4 in skeletal muscles. We show that the DUX4 ASO was well tolerated and repressed the DUX4 transcript, DUX4 protein, and mouse DUX4 target gene expression in skeletal muscles. In addition, the DUX4 ASO alleviated the severity of skeletal muscle pathology and partially prevented the dysregulation of inflammatory and extracellular matrix genes. DUX4 ASOtreated ACTA1-MCM;FLExDUX4 mice performed better on a treadmill; however, the hanging grid and four-limb grip strength tests were not improved compared to control ASOtreated ACTA1-MCM;FLExDUX4 mice. This study shows that systemic delivery of ASOs targeting DUX4 is a promising therapeutic strategy for FSHD and strategies that further improve the ASO efficacy in skeletal muscle are warranted. Show less
Bouwman, L.F.; Maarel, S.M. van der; Greef, J.C. de 2020
Purpose of review Facioscapulohumeral muscular dystrophy (FSHD) is a neuromuscular disorder, which is caused by incomplete repression of the transcription factor double homeobox 4 (DUX4) in... Show morePurpose of review Facioscapulohumeral muscular dystrophy (FSHD) is a neuromuscular disorder, which is caused by incomplete repression of the transcription factor double homeobox 4 (DUX4) in skeletal muscle. To date, there is no DUX4-targeting treatment to prevent or delay disease progression. In the present review, we summarize developments in therapeutic strategies with the focus on inhibiting DUX4 and DUX4 target gene expression. Recent findings Different studies show that DUX4 and its target genes can be repressed with genetic therapies using diverse strategies. Additionally, different small compounds can reduce DUX4 and its target genes in vitro and in vivo. Most studies that show DUX4 repression by genetic therapies have only been tested in vitro. More efforts should be made to test them in vivo for clinical translation. Several compounds have been shown to prevent DUX4 and target gene expression in vitro and in vivo. However, their efficiency and specificity has not yet been shown. With emerging clinical trials, the clinical benefit from DUX4 repression in FSHD will likely soon become apparent. Show less
Bouwman, L.F.; Hamer, B. den; Verveer, E.P.; Lerink, L.J.S.; Krom, Y.D.; Maarel, S.M. van der; Greef, J.C. de 2020
Background Facioscapulohumeral muscular dystrophy (FSHD) is a skeletal muscle disorder that is caused by derepression of the transcription factor DUX4 in skeletal muscle cells. Apart from SMCHD1,... Show moreBackground Facioscapulohumeral muscular dystrophy (FSHD) is a skeletal muscle disorder that is caused by derepression of the transcription factor DUX4 in skeletal muscle cells. Apart from SMCHD1, DNMT3B was recently identified as a disease gene and disease modifier in FSHD. However, the exact role of DNMT3B at the D4Z4 repeat array remains unknown. Methods To determine the role of Dnmt3b on DUX4 repression, hemizygous mice with a FSHD-sized D4Z4 repeat array (D4Z4-2.5 mice) were cross-bred with mice carrying an in-frame exon skipping mutation inDnmt3b(Dnmt3b(MommeD14)mice). Additionally, siRNA knockdowns ofDnmt3bwere performed in mouse embryonic stem cells (mESCs) derived from the D4Z4-2.5 mouse model. Results In mESCs derived from D4Z4-2.5 mice, Dnmt3b was enriched at the D4Z4 repeat array and DUX4 transcript levels were upregulated after a knockdown ofDnmt3b. In D4Z4-2.5/Dnmt3b(MommeD14)mice, Dnmt3b protein levels were reduced; however, DUX4 RNA levels in skeletal muscles were not enhanced and no pathology was observed. Interestingly, D4Z4-2.5/Dnmt3b(MommeD14)mice showed a loss of DNA methylation at the D4Z4 repeat array and significantly higher DUX4 transcript levels in secondary lymphoid organs. As these lymphoid organs seem to be more sensitive to epigenetic modifiers of the D4Z4 repeat array, different immune cell populations were quantified in the spleen and inguinal lymph nodes of D4Z4-2.5 mice crossed with Dnmt3b(MommeD14)mice or Smchd1(MommeD1)mice. Only in D4Z4-2.5/Smchd1(MommeD1)mice the immune cell populations were disturbed. Conclusions Our data demonstrates that loss of Dnmt3b results in derepression of DUX4 in lymphoid tissues and mESCs but not in myogenic cells of D4Z4-2.5/Dnmt3b(MommeD14)mice. In addition, the Smchd1(MommeD1)variant seems to have a more potent role in DUX4 derepression. Our studies suggest that the immune system is particularly but differentially sensitive to D4Z4 chromatin modifiers which may provide a molecular basis for the yet underexplored immune involvement in FSHD. Show less
