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

Abstract

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 performed bulk RNA-seq on a 6-day differentiation time-course in primary FSHD2 patient myoblasts. We identify a set of 54 genes upregulated in FSHD2 cells, termed FSHD-induced genes. Using single-cell and single-nucleus RNA-seq on myoblasts and 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 myotube nuclei coexpress multiple DUX4 target genes including DUXA, LEUTX and ZSCAN4, and also upregulate cell cycle-related genes with significant enrichment of E2F target genes and p53 signaling activation. We found more FSHD-Hi nuclei than DUX4-positive nuclei, and confirmed with in situ RNA/protein detection that DUX4 transcribed in only one or two nuclei is sufficient for DUX4 protein to activate target genes across multiple nuclei within the same myotube. 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 suggest that the DUXA can take over the role of DUX4 to maintain target gene expression. These results provide a possible explanation as to why it is easier to detect DUX4 target genes than DUX4 itself in patient cells and raise the possibility of a self-sustaining network of gene dysregulation triggered by the limited DUX4 expression.

Relationship of DUX4 and target gene expression in FSHD myocytes

“…We further confirmed the presence of DUX4 protein in the same myotube that contains the endogenous DUX4 transcript‐positive nuclei (Figure 1d). Consistent with previous observations (Jiang et al, 2020; Rickard et al, 2015; Tassin et al, 2013), the expression of DUX4 RNA transcripts in even one nucleus appears to be sufficient for DUX4 protein localization in most of the nuclei in the same myotube. Taken together, these results strongly support the specificity of the 6ZZ probe set and indicate that the majority of DUX4 transcripts are retained in the nucleus forming foci in FSHD myotubes.…”

“…Thus, to minimize crossreactivity and maximize the preferential detection of the full‐length DUX4 (DUX4fl) shown to be most relevant to FSHD, we custom‐designed an RNAScope probe set with the lowest possible number of ZZ probe pairs (“ZZ” represents a pair of RNAScope target probes) for the fluorescent detection system (Figure 1a; 6ZZ). Interestingly, we observed the major DUX4 transcript signal as foci in the nucleus using this set in primary FSHD myotubes (Figure 1b) (Jiang et al, 2020).…”

“…Primary and immortalized control and FSHD1 and FSHD2 skeletal myoblast cells were grown in high‐glucose Dulbecco's modified Eagle's medium (DMEM) (Gibco) supplemented with 20% fetal bovine serum (FBS) (Omega Scientific, Inc.), 1% pen–strep (Gibco), and 2% Ultrasor G (Crescent Chemical Co.). Primary control and FSHD2 (4qA161, SMCHD1 mutation: g.2697999_2698003del) myoblasts (Jiang et al, 2020), as well as FSHD1 (4qA161, two D4Z4 units) myoblasts, were immortalized using hTERT with p16INK4a‐resistant R24C mutant CDK4 (mtCDK4) and cyclin D1, as previously described (Shiomi et al, 2011). After immortalization, CD56‐positive cells were selected by magnetic‐activated cell sorting conjugated with anti‐CD56 antibody (130‐050‐401; MiltenyiBiotec).…”

“…However, assessment of the endogenous DUX4 and target gene expression in FSHD patient myocytes has been limited. Recently, we detected DUX4 and target gene transcripts using single‐nucleus RNA sequencing (snRNA‐seq) (Jiang et al, 2020). Unlike the previous single‐cell RNA‐seq of fusion‐blocked myotubes (van den Heuvel et al, 2019), our isolation and analyses of nuclei from naturally fused multinucleated myotubes provided the first evidence that DUX4 target gene expression is much more widespread than DUX4 transcription itself, which explains easier detection of the target gene transcripts rather than DUX4 itself (Broucqsault et al, 2013; Ferreboeuf et al, 2014; Geng et al, 2012; Jones et al, 2012; Rahimov et al, 2012; Rickard et al, 2015).…”

Membrane Repair Deficit in Facioscapulohumeral Muscular Dystrophy

“…Indeed, previous studies have estimated that the expression of DUX4 occurs in as few as 1/1000 nuclei in cultured FSHD myoblasts [72]. Single-cell RNAseq studies have reported DUX4 expression in a small percentage of FSHD1 and FSHD2 myoblasts with as few as 1/217 cells demonstrating an FSHD transcriptional signature (expressing >5 DUX4 biomarkers per cell) [73,74]. However, we identified deficits in membrane repair in the majority of myoblasts we tested from all three FSHD donors, with~60-80% failing to repair based on the kinetics of FM1-43 dye entry after injury.…”