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Relationship of DUX4 and target gene expression in FSHD myocytes

Facioscapulohumeral dystrophy (FSHD) is linked to misexpression of the transcription factor, DUX4. Although DUX4 target gene expression is often readily detectable, analysis of DUX4 expression has been limited due to its low expression in patient samples. Recently, single cell/nucleus RNA-sequencing was used to detect the native expression of DUX4 for the first time, but important spatial relationships with its target gene expression was missing. Furthermore, dynamics of DUX4 expression during myoblast differentiation has not been fully explored. In order to study the spatiotemporal relationship of DUX4 and key target genes, we performed RNA FISH on immortalized FSHD2 patient skeletal muscle cells. Using two probe sets, DUX4 transcripts were detected in 1-4% of myotubes after 3-day differentiation in vitro. We found that DUX4 transcripts mainly localize as foci in one or two nuclei in a myotube compared to abundant accumulation of the target gene transcripts in the cytoplasm. Over a 13-day differentiation timecourse, DUX4 expression without target gene expression significantly increased and peaked at day 7. Target gene expression correlates better with DUX4 expression early in differentiation while the expression of target genes without detectable DUX4 transcripts increases later. Consistently, shRNA depletion of DUX4-activated transcription factors, DUXA and LEUTX, specifically repressed a DUX4-target gene, KDM4E, later in differentiation, suggesting that following the initial activation by DUX4, target genes themselves contribute to the maintenance of downstream gene expression. Together, in situ detection of the DUX4 and target gene transcripts provided new insight into dynamics of DUX4 transcriptional network in FSHD patient myocytes. Significance Statement FSHD is the third most common muscular dystrophy and is associated with upregulation of DUX4, a transcription factor, and its target genes. Although target genes are easily detectable in FSHD, low frequency DUX4 upregulation in patient myocytes is difficult to detect, and examining the relationship and dynamics of DUX4 and target gene expression without artificial overexpression of DUX4 has been challenging. Using RNAScope with highly specific probes, we detect the endogenous DUX4 and target gene transcripts in situ in patient skeletal myotubes during differentiation in vitro. Our study reveals a unique DUX4 expression pattern and its relationship to the expression of target genes, and evidence for self-sustainability of the target gene network. The study provides important new insights into the FSHD disease mechanism.