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

Abstract

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.

β - catenin is central to DUX4 -driven network rewiring in facioscapulohumeral muscular dystrophy

“…Additionally, levels of TNF-a are negatively correlated with muscle endurance [42]. FSHD myoblasts undergo cell death in response to non-pathological levels of hydrogen peroxide [41] and other oxidative stress-inducing factors; while antioxidants inhibit DUX4-induced toxicity in FSHD myoblasts [45]. Thus, overactivation of ROS-mediated TNF-a-induced cell death pathways are potentially important pathomechanism in FSHD.…”

“…The role of ROS in FSHD is well reported, and our FSHD network contains many genes in the ROS-mediated pathway, including TNF-a. DUX4 represses genes of the glutathione redox pathway, likely causing ROS accumulation in FSHD muscle [45], which may lead to increased TNF-a as part of a pro-inflammatory response [46]. Additionally, levels of TNF-a are negatively correlated with muscle endurance [42].…”

Facioscapulohumeral muscular dystrophy (FSHD): an enigma unravelled?

“…DUX4 appears to be unstable as treatment of myoblast cultures with a proteasome inhibitor (MG-132) strongly improved its detection (Dixit et al 2007; A Tassin, Unpublished data). Moreover, DUX4 mediated cell death appears more frequent in myoblasts than in myotubes, thus restricting DUX4 detection to few surviving myoblasts while more DUX4 positive nuclei can be found after differentiation to myotubes (Bosnakovski et al 2008b). No specific antibody allowing immunohistochemical detection of DUX4 in muscle sections has been published and so investigating whether DUX4 expression is restricted to satellite cells or regenerating fibres is not yet possible.…”

Double SMCHD1 variants in FSHD2: the synergistic effect of two SMCHD1 variants on D4Z4 hypomethylation and disease penetrance in FSHD2

“…7 Its expression in skeletal muscle activates genes involved in germ line and early stem cell development, as well as specific classes of repeat elements, and overexpression of DUX4 in somatic cells causes cell death. [10][11][12] To cause FSHD, D4Z4 chromatin relaxation must occur on a specific genetic background of chromosome 4 (most often 4A161) that facilitates the production of stable DUX4 mRNA due to the presence of a polymorphic DUX4 polyadenylation signal distal to the D4Z4 repeat array. 8,13 D4Z4 chromatin relaxation on non-permissive chromosomes lacking a DUX4 polyadenylation signal do not cause FSHD in the absence of detectable levels of DUX4 mRNA.…”