Gene expression during normal and FSHD myogenesis

Abstract

BackgroundFacioscapulohumeral 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.MethodsUsing 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.ResultsMany 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.ConclusionsDUX4'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.

β - catenin is central to DUX4 -driven network rewiring in facioscapulohumeral muscular dystrophy

“…Many genes in this intersection have been associated with FSHD, e.g. TP53 [16], JUNB [20,17], HIF1A [20], WNT3 [4], LMO3 [20], ANXA4 [5] and HSPB1 [22]. Gene Set Enrichment Analysis (GSEA) [23] on the intersection also implicated many FSHD associated processes, such as myogenesis [17] and regulation of the actin cytoskeleton [15], and pathways, including, p53 [16], Wnt [4], and VEGF [5] To identify genes implicated as rewiring specifically in FSHD, we also ran InSpiRe on two datasets each describing skeletal muscle gene expression during ageing (GSE5086 [24] and GSE9676 [25]), disuse atrophy (GSE5110 [26] and GSE8872 [27]) and other muscle diseases involving inflammation and wasting (GSE3307 [19], where juvenile dermatomyositis and limb-girdle muscular dystrophy type 2A datasets were independently analysed).…”

“…HIF1-a signalling has recently been implicated in FSHD, due to identification of several downstream components of the pathway being differentially expressed [20].…”

“…GSE26145 [20] and GSE26061 [21] expression studies of FSHD and control myoblasts and myotubes profiled on the Affymetrix Human Exon 1.0 ST Array, were also analysed.…”

“…Both GSE3307 and GSE10760 were profiled on the Affymetrix Human Genome U133A Array platform. GSE26145 [20] and GSE26061 [21] are exon array studies using the Affymetrix Human Exon 1.0 ST Array each profiling three myoblast and three myotube samples from either quadriceps, rhomboid or deltoid muscles of three FSHD patients and three control individuals. As only isolated cells were arrayed in GSE26145 [20] and GSE26061 [21], nonmuscle gene expression was assumed to be low/negligible.…”

“…GSE26145 [20] and GSE26061 [21] are exon array studies using the Affymetrix Human Exon 1.0 ST Array each profiling three myoblast and three myotube samples from either quadriceps, rhomboid or deltoid muscles of three FSHD patients and three control individuals. As only isolated cells were arrayed in GSE26145 [20] and GSE26061 [21], nonmuscle gene expression was assumed to be low/negligible. These studies were employed in our meta-analysis to refine the larger datasets of primary muscle biopsies of GSE3307 [17,19] and GSE10760 [5].…”