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.
Highlights
Facioscapulohumeral muscular dystrophy (FSHD) is one of the most prevalent genetic myopathies1
To identify the earliest gene expression changes induced by DUX4, we evaluated RNA-seq data from LHCN-M2-iDUX4 cells treated with doxycycline for 6 hours
Similar results were observed when DUX4 was expressed in mouse myoblasts12, the significance of that result was questioned because of the differences between the target genes of DUX4 in the mouse and human systems
Summary
Facioscapulohumeral muscular dystrophy (FSHD) is one of the most prevalent genetic myopathies. DUX4 expression levels are higher in differentiated myotubes than in proliferating myoblasts, DUX4 is detected in undifferentiated human myoblasts, for example by immunostaining in very rare cells mentioned above, and at levels that can activate expression of a 5xDUX4-binding site GFP reporter gene delivered by lentivector. It is reasonable to consider the possibility that many DUX4+ proliferating myoblasts are expressing low levels of DUX4 protein below the threshold for detection by immunostaining, levels that do not lead to cell death, but that may impair regeneration. Whether human cells expressing low levels of DUX4 would show impaired differentiation potential like their mouse counterparts is not self-evident This is because DUX4 induces overlapping but distinct sets of target genes in mouse and human cells. We directly test this proposition, and find that DUX4 impairs differentiation of human myogenic progenitors; we discover the unusual mechanism by which DUX4 perturbs expression of the myogenic regulator, MYF5; and probe regions of the DUX4 protein necessary for inhibition of myogenesis in the human system
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