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DUX4 expression activates JNK and p38 MAP kinases in myoblasts

ABSTRACT: Facioscapulohumoral muscular dystrophy (FSHD) is a progressive muscle wasting disease caused by misexpression of the DUX4 transcription factor. DUX4 expression in skeletal muscle cells results in a dramatic alteration in their transcriptional profile and cellular phenotype and subsequently, cell death. To gain insight into the kinetics of DUX4-induced stresses, we activated DUX4 expression in myoblasts and performed longitudinal RNA sequencing paired with proteomics and phosphoproteomics. This analysis revealed changes in cellular physiology such as DNA damage and altered mRNA splicing before detectable changes in protein levels. Implementation of phosphoproteomics uncovered that widespread changes in protein phosphorylation preceded changes in protein levels indicating that alterations in kinase signaling may play a role in DUX4-mediated stress and cell death. Here we demonstrate that two stress-responsive MAP kinase pathways, JNK and p38, are activated in response to DUX4 expression. Pharmacological inhibition of either of these pathways ameliorated DUX4-mediated cell death in myoblasts. These findings uncover JNK as a novel pathway involved in DUX4-mediated cell death, as well as provide additional insights into the role of the p38 pathway, a clinical target for the treatment of FSHD.

ORGANISM(S): Homo sapiens

PROVIDER: GSE205421 | GEO | 2022/10/09

REPOSITORIES: GEO

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DUX4 expression activates JNK and p38 MAP kinases in myoblasts

Project description:Facioscapulohumoral muscular dystrophy (FSHD) is caused by misexpression of the DUX4 transcription factor in skeletal muscle that results in transcriptional alterations, abnormal phenotypes, and cell death. To gain insight into the kinetics of DUX4-induced stresses, we activated DUX4 expression in myoblasts and performed longitudinal RNA sequencing paired with proteomics and phosphoproteomics. This analysis revealed changes in cellular physiology including DNA damage and altered mRNA splicing. Phosphoproteomic analysis uncovered widespread changes in protein phosphorylation rapidly following DUX4 induction indicating that alterations in kinase signaling may play a role in DUX4-mediated stress and cell death. Indeed, we demonstrate that two stress-responsive MAP kinase pathways, JNK and p38, are activated in response to DUX4 expression. Inhibition of each of these pathways ameliorated DUX4-mediated cell death in myoblasts. These findings uncover JNK as a novel pathway involved in DUX4-mediated cell death as well as provide additional insights into the role of the p38 pathway, a clinical target for the treatment of FSHD.

2022-07-05 | MSV000089812 | MassIVE

DUX4 activates germline genes, retroelements and immune-mediators: Implications for facioscapulohumeral dystrophy

Project description:Facioscapulohumeral dystrophy (FSHD) is one of the most common inherited muscular dystrophies. The causative gene remains controversial and the mechanism of pathophysiology unknown. Here we identify genes associated with germline and early stem cell development as targets of the DUX4 transcription factor, a leading candidate gene for FSHD. The genes regulated by DUX4 are reliably detected in FSHD muscle but not in controls, providing direct support for the model that misexpression of DUX4 is a causal factor for FSHD. Additionally, we show that DUX4 binds and activates LTR elements from a class of MaLR endogenous primate retrotransposons and suppresses the innate immune response to viral infection, at least in part through the activation of DEFB103, a human defensin that can inhibit muscle differentiation. These findings suggest specific mechanisms of FSHD pathology and identify candidate biomarkers for disease diagnosis and progression. [Overexpression experiment] Quadruplicate total RNA samples were collected from control human primary myoblasts transduced with lentivirus carrying DUX4-fl, DUX4-s or GFP (MOI = 15) for 24 h and from untransduced myoblasts. [Defensin experiment] Quadruplicate samples were also collected from myoblasts and myotubes grown in media containing human beta-defensin 3 peptide or in control media.

2012-01-03 | E-GEOD-33799 | biostudies-arrayexpress

SMCHD1 activates the expression of genes required for the expansion of human myoblasts (ChIP-seq)

Project description:In human muscle, SMCHD1 mutations are associated with the onset of FSHD2, but the mechanism driving the disease onset remains unclear. A commonly accepted explanation is the loss of SMCHD1 binding to the D4Z4 locus activates the expression of DUX4 in FSHD2 muscle. In this study, we used human myoblasts having DUX4 non-permissive 4qB alleles as a model to study DUX4-independent functions of SMCHD1 on myoblast cell growth. Surprisingly, depletion of SMCHD1 in these cells resulted in a cell proliferation defect. Despite the absence of DUX4 target genes’ activation, these cells showed a repression of PAX7 target genes (a hallmark of FSHD) and similar changes in expression profile compared to FSHD myoblasts. Interestingly, downregulation of cell proliferation-related genes and dysregulation of fibroblasts-specific genes were observed in SMCHD1 knockdown myoblasts and FSHD2 myoblasts but not FSHD1 myoblasts. Additionally, we identified LAP2 as direct targets of SMCHD1. Depletion of LAP2 leads to cell proliferation defect similar to the effect after SMCHD1 knockdown. These data imply that DUX4 is not the only driver for the onset of FSHD, and SMCHD1 has DUX4-independent functions in muscle growth and development.

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SMCHD1 activates the expression of genes required for the expansion of human myoblasts (RNA-seq)

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Analysis of genes regulated by DUX4 via oxidative stress reveals potential therapeutic targets for treatment of facioscapulohumeral dystrophy

Project description:Muscles of patients with facioscapulohumeral dystrophy (FSHD) are characterized by sporadic DUX4 expression and oxidative stress which is at least partially induced by DUX4 protein. Nevertheless, targeting oxidative stress with antioxidants has a limited impact on FSHD patients, and the exact role of oxidative stress in the pathology of FSHD, as well as its interplay with the DUX4 expression, remain unclear. Here we set up a screen for genes that are upregulated by DUX4 via oxidative stress with the aim to target these genes rather than the oxidative stress itself. Immortalized human myoblasts expressing DUX4 (MB135-DUX4) have an increased level of reactive oxygen species (ROS) and exhibit differentiation defects which can be reduced by treating the cells with classic (Tempol) or mitochondria-targeted antioxidants (SkQ1). The transcriptome analysis of antioxidant-treated MB135 and MB135-DUX4 myoblasts allowed us to identify 200 genes with expression deregulated by DUX4 but normalized upon antioxidant treatment. Several of these genes, including PITX1, have been already associated with FSHD and/or muscle differentiation. We confirmed that PITX1 was indeed deregulated in MB135-DUX4 cells and primary FSHD myoblasts and revealed a redox component in PITX1 regulation. PITX1 silencing partially reversed the differentiation defects of MB135-DUX4 myoblasts. Our approach can be used to identify and target redox-dependent genes involved in human diseases.

2021-05-04 | GSE173762 | GEO

Model systems of DUX4 expression recapitulate the transcriptional profile of FSHD cells

Project description:Facioscapulohumeral dystrophy (FSHD) is caused by the mis-expression of the double-homeodomain transcription factor DUX4 in skeletal muscle cells. Many different cell culture models have been developed to study the pathophysiology of FSHD, frequently based on endogenous expression of DUX4 in FSHD cells or by mis-expression of DUX4 in control human muscle cells. Although results generated using each model are generally consistent, differences have also been reported, making it unclear which model(s) faithfully recapitulate DUX4 and FSHD biology. In this study, we systematically compared RNA-seq data generated from three different models of FSHD—lentiviral-based DUX4 expression in myoblasts, doxycycline-inducible DUX4 in myoblasts, and differentiated human FSHD myocytes expressing endogenous DUX4—and show that the DUX4-associated gene expression signatures of each dataset are highly correlated (Pearson’s correlation coefficient, r ~ 0.75-0.85). The few robust differences were attributable to different states of cell differentiation and other differences in experimental design. Our study describes a model system for inducible DUX4 expression that enables reproducible and synchronized experiments and validates the fidelity and FSHD relevance of multiple distinct models of DUX4 expression.

2016-09-21 | GSE85461 | GEO

DUX4-induced dsRNA and MYC mRNA Stabilization Activate Apoptotic Pathways in Human Cell Models of Facioscapulohumeral Dystrophy

Project description:Facioscapulohumeral muscular dystophy (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.

2016-12-31 | GSE87495 | GEO

DUX4 expression activates JNK and p38 MAP kinases in myoblasts

Project description:DUX4 expression activates JNK and p38 MAP kinases in myoblasts

| PRJNA844998 | ENA

DUX4-induced gene expression is the major molecular signature in FSHD skeletal muscle

Project description:Facioscapulohumeral dystrophy (FSHD) is caused by decreased epigenetic repression of the D4Z4 macrosatellite array and recent studies have shown that this results in the expression of low levels of the DUX4 mRNA in skeletal muscle. Several other mechanisms have been suggested for FSHD pathophysiology and it remains unknown whether DUX4 expression can account for most of the molecular changes seen in FSHD. Since DUX4 is a transcription factor, we used RNA-seq to measure gene expression in muscle cells transduced with DUX4, and in muscle cells and biopsies from control and FSHD individuals. We show that DUX4 target gene expression is the major molecular signature in FSHD muscle together with a gene expression signature consistent with an immune cell infiltration. In addition, one unaffected individual without a known FSHD-causing mutation showed expression of DUX4 target genes. This individual has a sibling with FSHD and also without a known FSHD-causing mutation, suggesting the presence of yet unidentified modifier locus for DUX4 expression and FSHD. These findings demonstrate that expression of DUX4 accounts for the majority of the gene expression changes in FSHD skeletal muscle together with an immune cell infiltration. RNA-seq for muscle cells and biopsies from control and FSHD individuals.

2014-07-03 | E-GEOD-56787 | biostudies-arrayexpress

Single-nucleus RNA-seq identifies divergent populations of FSHD2 myotube nuclei [Fluidigm]

Project description:FSHD is characterized by the misexpression of DUX4 in skeletal muscle. Although DUX4 upregulation is thought to be the pathogenic cause of FSHD, DUX4 is lowly expressed in patient samples, and analysis of the consequences of DUX4 expression has largely relied on artificial overexpression. To better understand the native expression profile of DUX4 and its targets, we first performed pooled RNA-seq on a 6-day differentiation time-course in FSHD2 patient-derived primary myoblasts. We identify a set of 54 FSHD-induced genes upregulated in FSHD2 cells starting at day 2 of differentiation through the end of the time-course. Using single-cell and single-nucleus RNA-seq on FSHD2 myoblasts and day 3 and day 5 differentiated myotubes respectively, we captured, for the first time, DUX4 expressed at the single-nucleus level in a native state. We identified two populations of FSHD myotube nuclei based on low or high enrichment of DUX4 and FSHD-induced genes (FSHD-Lo and “FSHD Hi”, respectively). FSHD-Hi nuclei upregulate many cell cycle related genes with significant enrichment of E2F target genes and p53 signaling activation. In FSHD-Hi myotube nuclei, multiple DUX4 target genes are co-expressed including a set of transcription factors, such as DUXA, ZSCAN4 and LEUTX. DUXA (the DUX4 paralog) is more widely expressed than DUX4, and depletion of DUXA suppressed the expression of LEUTX and ZSCAN4 in late, but not early, differentiation. The results indicate that the DUXA can take over the role of DUX4 and maintain target gene expression. These results may provide explanation as to why it is easier to detect and monitor DUX4 target genes than DUX4 itself in patient cells and suggest a self-sustaining network of gene dysregulation that perpetuates this disease after DUX4 is no longer expressed.

2020-04-07 | GSE143492 | GEO

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