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Low level DUX4 expression disrupts myogenesis through deregulation of myogenic gene expression.

ABSTRACT: Loss of silencing of the DUX4 gene on chromosome 4 causes facioscapulohumeral muscular dystrophy. While high level DUX4 expression induces apoptosis, the effects of low level DUX4 expression on human myogenic cells are not well understood. Low levels and sporadic expression of DUX4 have been reported in FSHD biopsy samples and myoblast cultures. Here, we show that a large set of human myogenic genes is rapidly deregulated by DUX4, including MYOD1 and MYF5, which are efficiently repressed even by low, non-toxic levels of DUX4. Human myoblasts modified to express low nontoxic levels of DUX4 were significantly impaired from differentiating into myotubes in vitro. Surprisingly, inhibition of differentiation does not require the transcriptional activation domain, thus is likely a feature of all mammalian DUX genes. DUX4 does not bind near the MYF5 gene, but has a prominent ChIP-seq peak within the MYF5 -118 kb enhancer. We find that when DUX4 binds at this site, it directs enhancer activity towards a nearby transcriptional start site for a noncoding nonfunctional RNA we name DIME (DUX4-induced MYF5 enhancer) transcript. These data highlight the anti-myogenic properties of DUX4 in human myogenic progenitor cells, and provide an example of enhancer disruption in the downregulation of MYF5.

SUBMITTER: Bosnakovski D

PROVIDER: S-EPMC6240038 | biostudies-literature | 2018 Nov

REPOSITORIES: biostudies-literature

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Low level DUX4 expression disrupts myogenesis through deregulation of myogenic gene expression.

Bosnakovski Darko D Gearhart Micah D MD Toso Erik A EA Ener Elizabeth T ET Choi Si Ho SH Kyba Michael M

Scientific reports 20181116 1

Loss of silencing of the DUX4 gene on chromosome 4 causes facioscapulohumeral muscular dystrophy. While high level DUX4 expression induces apoptosis, the effects of low level DUX4 expression on human myogenic cells are not well understood. Low levels and sporadic expression of DUX4 have been reported in FSHD biopsy samples and myoblast cultures. Here, we show that a large set of human myogenic genes is rapidly deregulated by DUX4, including MYOD1 and MYF5, which are efficiently repressed even by ...[more]

PMID: 30446688

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Project description:BackgroundNuclear bodies, such as nucleoli, PML bodies, and SC35 speckles, are dynamic sub-nuclear structures that regulate multiple genetic and epigenetic processes. Additional regulation is provided by RNA/DNA handling proteins, notably TDP-43 and FUS, which have been linked to ALS pathology. Previous work showed that mouse cell line myotubes have fewer but larger nucleoli than myoblasts, and we had found that nuclear aggregation of TDP-43 in human myotubes was induced by expression of DUX4-FL, a transcription factor that is aberrantly expressed and causes pathology in facioscapulohumeral dystrophy (FSHD). However, questions remained about nuclear bodies in human myogenesis and in muscle disease.MethodsWe examined nucleoli, PML bodies, SC35 speckles, TDP-43, and FUS in myoblasts and myotubes derived from healthy donors and from patients with FSHD, laminin-alpha-2-deficiency (MDC1A), and alpha-sarcoglycan-deficiency (LGMD2D). We further examined how these nuclear bodies and proteins were affected by DUX4-FL expression.ResultsWe found that nucleoli, PML bodies, and SC35 speckles reorganized during differentiation in vitro, with all three becoming less abundant in myotube vs. myoblast nuclei. In addition, though PML bodies did not change in size, both nucleoli and SC35 speckles were larger in myotube than myoblast nuclei. Similar patterns of nuclear body reorganization occurred in healthy control, MDC1A, and LGMD2D cultures, as well as in the large fraction of nuclei that did not show DUX4-FL expression in FSHD cultures. In contrast, nuclei that expressed endogenous or exogenous DUX4-FL, though retaining normal nucleoli, showed disrupted morphology of some PML bodies and most SC35 speckles and also co-aggregation of FUS with TDP-43.ConclusionsNucleoli, PML bodies, and SC35 speckles reorganize during human myotube formation in vitro. These nuclear body reorganizations are likely needed to carry out the distinct gene transcription and splicing patterns that are induced upon myotube formation. DUX4-FL-induced disruption of some PML bodies and most SC35 speckles, along with co-aggregation of TDP-43 and FUS, could contribute to pathogenesis in FSHD, perhaps by locally interfering with genetic and epigenetic regulation of gene expression in the small subset of nuclei that express high levels of DUX4-FL at any one time.

Project description:BACKGROUND:Facioscapulohumeral muscular dystrophy (FSHD) is a dominant disease linked to contraction of an array of tandem 3.3-kb repeats (D4Z4) at 4q35. Within each repeat unit is a gene, DUX4, that can encode a protein containing two homeodomains. A DUX4 transcript derived from the last repeat unit in a contracted array is associated with pathogenesis but it is unclear how. METHODS:Using exon-based microarrays, the expression profiles of myogenic precursor cells were determined. Both undifferentiated myoblasts and myoblasts differentiated to myotubes derived from FSHD patients and controls were studied after immunocytochemical verification of the quality of the cultures. To further our understanding of FSHD and normal myogenesis, the expression profiles obtained were compared to those of 19 non-muscle cell types analyzed by identical methods. RESULTS:Many of the ~17,000 examined genes were differentially expressed (>2-fold, p<0.01) in control myoblasts or myotubes vs. non-muscle cells (2185 and 3006, respectively) or in FSHD vs. control myoblasts or myotubes (295 and 797, respectively). Surprisingly, despite the morphologically normal differentiation of FSHD myoblasts to myotubes, most of the disease-related dysregulation was seen as dampening of normal myogenesis-specific expression changes, including in genes for muscle structure, mitochondrial function, stress responses, and signal transduction. Other classes of genes, including those encoding extracellular matrix or pro-inflammatory proteins, were upregulated in FSHD myogenic cells independent of an inverse myogenesis association. Importantly, the disease-linked DUX4 RNA isoform was detected by RT-PCR in FSHD myoblast and myotube preparations only at extremely low levels. Unique insights into myogenesis-specific gene expression were also obtained. For example, all four Argonaute genes involved in RNA-silencing were significantly upregulated during normal (but not FSHD) myogenesis relative to non-muscle cell types. CONCLUSIONS:DUX4's pathogenic effect in FSHD may occur transiently at or before the stage of myoblast formation to establish a cascade of gene dysregulation. This contrasts with the current emphasis on toxic effects of experimentally upregulated DUX4 expression at the myoblast or myotube stages. Our model could explain why DUX4's inappropriate expression was barely detectable in myoblasts and myotubes but nonetheless linked to FSHD.

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Low level DUX4 expression disrupts myogenesis through deregulation of myogenic gene expression.

Publications

Low level DUX4 expression disrupts myogenesis through deregulation of myogenic gene expression.

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