DUX4-induced dsRNA and MYC mRNA stabilization activate apoptotic pathways in human cell models of facioscapulohumeral dystrophy

Abstract

Facioscapulohumeral dystrophy (FSHD) is caused by the mis-expression of DUX4 in skeletal muscle cells. DUX4 is a transcription factor that activates genes normally associated with stem cell biology and its mis-expression in FSHD cells results in apoptosis. To identify genes and pathways necessary for DUX4-mediated apoptosis, we performed an siRNA screen in an RD rhabdomyosarcoma cell line with an inducible DUX4 transgene. Our screen identified components of the MYC-mediated apoptotic pathway and the double-stranded RNA (dsRNA) innate immune response pathway as mediators of DUX4-induced apoptosis. Further investigation revealed that DUX4 expression led to increased MYC mRNA, accumulation of nuclear dsRNA foci, and activation of the dsRNA response pathway in both RD cells and human myoblasts. Nuclear dsRNA foci were associated with aggregation of the exon junction complex component EIF4A3. The elevation of MYC mRNA, dsRNA accumulation, and EIF4A3 nuclear aggregates in FSHD muscle cells suggest that these processes might contribute to FSHD pathophysiology.

Quantitative proteomics reveals key roles for post-transcriptional gene regulation in the molecular pathology of FSHD

“…Facioscapulohumeral muscular dystrophy is caused by the inappropriate expression of an early embryonic transcriptional activator, DUX4, in adult muscle, leading to cell death (1, 2). Decades of work have generated a detailed parts list of the genes and pathways affected by DUX4 that may underlie FSHD pathophysiology (3–10). However, an integrated model for how those DUX4-induced changes lead to disease has remained elusive (11–13).…”

“…DUX4 induces changes in the expression of hundreds of genes that impact dozens of highly interconnected pathways (3–10), making a cause-and-effect relationship between dysregulated gene expression and FSHD pathology difficult to discern. Because DUX4 is a strong transcriptional activator, most studies of DUX4 activity have focused on measuring gene expression at the transcript level (3, 14, 15), thereby implicitly assuming that the transcriptome accurately represents the cellular proteome in DUX4-expressing cells.…”

Transgenic mice expressing tunable levels of DUX4 develop characteristic facioscapulohumeral muscular dystrophy-like pathophysiology ranging in severity

“…The GO analysis of differentially induced genes showed that muscles from the moderate and severe models are enriched for genes in apoptotic pathways (Figure 7, Table 2). As mentioned, DUX4-FL is a pro-apoptotic protein, its expression is highly toxic to muscle cells in culture [19,36,77,92], and apoptosis is a feature of FSHD muscle [93]. To assess apoptosis in the FSHD-like models, TUNEL assays were performed on TA muscles across a DUX4-FL induction time-course in both the moderate and severe models (Figures 12 and 13, respectively).…”

DUX4-induced bidirectional HSATII satellite repeat transcripts form intranuclear double stranded RNA foci in human cell models of FSHD

“…DUX4 also activates the expression of normally silenced repetitive elements that are transcribed during EGA such as endogenous retroviruses (ERVs) and the pericentric human satellite II (HSATII) repeats (3, 4, 8, 9). The expression of DUX4 in skeletal muscle cells is toxic and causes apoptosis (10–13), however, the specific molecular pathways leading to cell toxicity are not fully understood. Previous studies have suggested multiple, non-mutually exclusive mechanisms for DUX4 toxicity including an increased sensitivity to oxidative stress (12, 14), interference with PAX3/PAX7 (12, 15) and the formation of insoluble TDP-43 nuclear aggregates due to impaired protein turnover (16).…”

“…More recently, we reported that the expression of DUX4 in skeletal muscle cells resulted in the accumulation of intranuclear foci of dsRNAs (13). The accumulation of DUX4-induced dsRNAs correlated with PKR and eIF2α phosphorylation, both proapoptotic characteristics of the cellular innate immune response typically triggered by viral dsRNAs (17).…”