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

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 first performed pooled RNA-seq on a 6-day differentiation time-course in FSHD2 patient-derived primary myoblasts. We identify a set of 54 FSHD-induced genes upregulated in FSHD2 cells starting at day 2 of differentiation through the end of the time-course. Using single-cell and single-nucleus RNA-seq on FSHD2 myoblasts and day 3 and day 5 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 nuclei upregulate many cell cycle related genes with significant enrichment of E2F target genes and p53 signaling activation. In FSHD-Hi myotube nuclei, multiple DUX4 target genes are co-expressed including a set of transcription factors, such as DUXA, ZSCAN4 and LEUTX. 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 indicate that the DUXA can take over the role of DUX4 and maintain target gene expression. These results may provide explanation as to why it is easier to detect and monitor DUX4 target genes than DUX4 itself in patient cells and suggest a self-sustaining network of gene dysregulation that perpetuates this disease after DUX4 is no longer expressed. Full length single-cell and single-nucleus RNA-seq using SmartSeq on the Fluidigm C1 platform for control and FSHD2 myoblast cells and myotube nuclei.

面肩肱型肌营养不良症（FSHD）以骨骼肌中双同源框蛋白4（DUX4）的异常表达为特征。尽管学界普遍认为DUX4的上调是FSHD的致病诱因，但患者样本中DUX4的表达水平极低，因此对DUX4表达后果的分析大多依赖于人工过表达体系。为深入解析DUX4的天然表达谱及其靶基因，我们首先对源自FSHD2型患者的原代成肌细胞开展了为期6天的分化时间序列实验，并进行了混合RNA测序（pooled RNA-seq）分析。我们鉴定出一组共54个FSHD诱导基因，这些基因在分化第2天起直至该时间序列结束的FSHD2型细胞中均呈上调表达。我们分别针对FSHD2型成肌细胞、分化第3天及第5天的肌管，采用单细胞RNA测序与单细胞核RNA测序技术，首次在天然状态下于单细胞核层面检测到DUX4的表达。基于DUX4与FSHD诱导基因的富集程度差异，我们将FSHD肌管细胞核划分为两个群体：分别为DUX4及FSHD诱导基因低富集的FSHD-Lo群体，与高富集的FSHD-Hi群体。FSHD-Hi细胞核上调了大量细胞周期相关基因，其中E2F靶基因与p53信号通路激活呈现显著富集。在FSHD-Hi肌管细胞核中，多个DUX4靶基因共表达，包括双同源框A（DUXA）、ZSCAN4与LEUTX等转录因子。其中DUXA作为DUX4的旁系同源基因，其表达范围较DUX4更为广泛；在分化后期（而非早期）敲低DUXA的表达，会抑制LEUTX与ZSCAN4的转录水平。研究结果表明，DUXA可替代DUX4的功能，维持靶基因的表达。上述结果或可解释为何在患者细胞中，相较于DUX4本身，其靶基因更易于被检测与监测，并提示了一个自我维持的基因失调网络：该网络可在DUX4不再表达后，持续推动疾病进程。本研究还借助Fluidigm C1平台的SmartSeq技术，针对对照组、FSHD2型成肌细胞及肌管细胞核开展了全长单细胞与单细胞核RNA测序。