by Lawrence J. Hayward, MD PhD, University of Massachusetts Medical School
To develop robust therapies for FSHD, we need a better understanding of the sequence of events occurring at the single-cell level upon expression of DUX4, the causative gene. A recent paper by Chau et al. from the Yokomori lab at the University of California at Irvine provides novel insights into the dynamics of DUX4-triggered gene expression and associated regulatory networks in individual FSHD muscle cells. The paper also shows that both the amount of DUX4 expression and its location inside muscle cells might strongly influence its downstream consequences.
DUX4 expression is known to be triggered in subsets of FSHD muscle cells, but this has been notoriously difficult to visualize directly. Yokomori’s team overcame this challenge by using a highly sensitive and specific method, RNAscope, to detect the location of DUX4 expression and that of its target genes within individual muscle cells. RNAscope amplifies very low signals while nearly eliminating background noise and allowed the researchers to follow snapshots of expression over nearly a 2-week culture period. A key report by Chermahini et al. in 2019 first showed that DUX4 expression could be detected in FSHD1 muscle cultures using custom RNAscope probes and that the signal decreased following treatment with a therapeutic microRNA under development.
The present study compared the original probe set from the Chermahini study and a new one designed to detect more specifically the full-length DUX4 transcript shown to be injurious in FSHD. Experimentally forced high-level expression of an artificial DUX4 RNA known to be rapidly toxic produced a signal mostly in the cell cytoplasm, similar to previous observations. In contrast, when the Yokomori lab revealed a very low signal emanating only from the native DUX4 gene, it localized to small clumps within cell nuclei. Furthermore, this nuclear-retained DUX4 signal was detected in subsets of FSHD myotubes over nearly two weeks in culture, suggesting that if the low-level DUX4 transcript remains sequestered in nuclei, it might not immediately kill muscle cells. The researchers also used a fluorescent readout that allowed co-detection of multiple transcripts in single cells so that the expression of other genes triggered by DUX4 activation could be monitored. Two of the known DUX4 target genes, LEUTX and KDM4E, were visualized together in cells in a highly linked pattern even with DUX4 no longer detectable, and evidence was shown that LEUTX, once activated by DUX4, could help to sustain KDM4E expression.
What do these results mean, and why is this paper important?
Chau et al. show the inter-related and dynamic expression patterns of DUX4 and its target genes directly in muscle cells from FSHD patients. Their observations provide a framework for more comprehensive studies to dissect the mechanisms by which DUX4 may initially be triggered, or perhaps kept in check in the nucleus, and to understand how downstream DUX4 target gene expression might become self-perpetuating. Such insights could help to identify novel points of therapeutic intervention in FSHD. Furthermore, RNAscope technology might be applied to investigate DUX4 and its biomarkers at different stages of disease progression in FSHD muscle biopsies. As envisioned by Chermahini et al., robust measurement of DUX4 expression or its targets using RNAscope could be developed as an outcome measure to assess DUX4 inhibition strategies directly in future FSHD clinical trials.
Chau J, Kong X, Nguyen N, Williams K, Tawil R, Kiyono T, Mortazavi A, Yokomori K. Relationship of DUX4 and target gene expression in FSHD myocytes. Hum Mutat. 2021 Jan 27. doi: 10.1002/humu.24171. PMID: 33502067
Chermahini, G. A., Rashnonejad, A., & Harper, S. Q. (2019). RNAscope in situ hybridization-based method for detecting DUX4 RNA expression in vitro. RNA, 25, 1211-1217.