DUX4 induces a transcriptome more characteristic of a less-differentiated cell state and inhibits myogenesis

Abstract

Skeletal muscle wasting in facioscapulohumeral muscular dystrophy (FSHD) results in substantial morbidity. On a disease-permissive chromosome 4qA haplotype, genomic and/or epigenetic changes at the D4Z4 macrosatellite repeat allows transcription of the DUX4 retrogene. Analysing transgenic mice carrying a human D4Z4 genomic locus from an FSHD-affected individual showed that DUX4 was transiently induced in myoblasts during skeletal muscle regeneration. Centromeric to the D4Z4 repeats is an inverted D4Z4 unit encoding DUX4c. Expression of DUX4, DUX4c and DUX4 constructs, including constitutively active, dominant-negative and truncated versions, revealed that DUX4 activates target genes to inhibit proliferation and differentiation of satellite cells, but that it also downregulates target genes to suppress myogenic differentiation. These transcriptional changes elicited by DUX4 in mouse have significant overlap with genes regulated by DUX4 in man. Comparison of DUX4 and DUX4c transcriptional perturbations revealed that DUX4 regulates genes involved in cell proliferation, whereas DUX4c regulates genes engaged in angiogenesis and muscle development, with both DUX4 and DUX4c modifing genes involved in urogenital development. Transcriptomic analysis showed that DUX4 operates through both target gene activation and repression to orchestrate a transcriptome characteristic of a less-differentiated cell state.

The double homeodomain protein DUX4c is associated with regenerating muscle fibers and RNA-binding proteins

“…Lastly, in agreement with GTEx data (Table S3), we demonstrated that the DUX4c protein was expressed in testis, as previously shown for the homologous DUX4 protein [35]. Knopp et al [14] proposed that both DUX4 and DUX4c target genes were involved in urogenital development. However, in contrast to DUX4, which was detected in spermatogonia and spermatocytes I [35], we found DUX4c staining generally located Fig.…”

“…S3A) and appeared like comets with a DUX4c cytoplasmic staining at one side (as reported in immortalized cells, [12]). We also observed DUX4c in larger myotubes, specifically in TnT intense areas found either next to clusters of nuclei (circle), at a tip of the cell, next to the membrane, at cell-cell contacts or next to a very thin extension (characteristic of DUX4-expressing muscle cells, [14]). At day 6, we unexpectedly observed in only two myotubes close to each other (of all the analyzed fields) a DUX4c nuclear labeling with the rat antiserum (Fig.…”

“…We also demonstrated that only an siRNA targeting DUX4c, not DUX4, could reverse the disorganized FSHD myotube phenotype [16]. In a transcriptomic study on primary mouse myoblasts transduced with retroviruses expressing DUX4 or DUX4c, Knopp et al [14] showed that in contrast to DUX4 targets, genes deregulated by DUX4c were associated with muscle development. Previous studies had already suggested that DUX4c might be involved in muscle regeneration [2,3,12,13].…”

“…DUX4c loss-or gain-of-function studies in human muscle cells showed that excess DUX4c affected proliferation of human TE671 rhabdomyosarcoma or primary muscle cells and inhibited their differentiation [2,3,12]. DUX4c excess also interfered with mouse myoblast fusion in cell cultures [13,14]. We further showed that DUX4c excess at a later stage of primary myoblast differentiation altered the organization of myofibrils and led to the formation of large clusters of nuclei [3].…”

“…The membrane defects described in FSHD muscle cells and in myofibers of DUX4 mouse models [38] might be linked to fusion anomalies. Furthermore, both DUX4 and DUX4c overexpression negatively impacted myoblast fusion [14]. Future studies need to explore proteins involved in MYOD regulation in FSHD, such as (i) the MyoD family inhibitor (MDFIC) that maintains MyoD in the cytoplasm by masking its nuclear localization signal [42], (ii) the Id proteins that inhibit MYOD transcriptional activity [43], (iii) MYC that is reported to inhibit MyoD and muscle differentiation [44] and found stabilized by DUX4 ectopic expression [45], as well as other factors involved in muscle cell fusion.…”