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Relationship of DUX4 and target gene expression in FSHD myocytes

By Jonathan Chau, Xiangduo Kong, Nam Nguyen, Katherine Williams, Rabi Tawil, Tohru Kiyono, Ali Mortazavi, Kyoko Yokomori

Posted 25 May 2020
bioRxiv DOI: 10.1101/2020.05.24.109710

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. ### Competing Interest Statement The authors have declared no competing interest.

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