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.

中文翻译：

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FSHD心肌细胞中DUX4与靶基因表达的关系

面肩肱型营养不良（FSHD）与转录因子DUX4的错误表达有关。尽管DUX4靶基因的表达通常很容易被检测到，但由于DUX4在患者样品中的表达较低，因此对DUX4表达的分析受到了限制。最近，单细胞/核RNA测序首次用于检测DUX4的天然表达，但缺少与其靶基因表达的重要空间关系。此外，尚未完全探讨成肌细胞分化过程中DUX4表达的动力学。为了研究DUX4和关键靶基因的时空关系，我们对永生化的FSHD2患者骨骼肌细胞进行了RNA FISH分析。使用两个探针组，在体外分化3天后，在1-4％的肌管中检测到DUX4转录本。我们发现，与靶基因转录本在细胞质中的大量积累相比，DUX4转录本主要定位为肌管中一两个核中的病灶。在13天的分化过程中，没有靶基因表达的DUX4表达显着增加并在第7天达到峰值。在分化早期，靶基因表达与DUX4表达更好地相关，而没有可检测DUX4转录本的靶基因表达则随后增加。一致地，DUX4激活的转录因子DUXA和LEUTX的shRNA耗竭在分化后期特别抑制了DUX4靶基因KDM4E，这表明在DUX4最初激活后，靶基因本身就有助于维持下游基因的表达。一起，