Department of Biological Chemistry, School of Medicine, University of California, Irvine, California, USA.
Department of Developmental and Cell Biology, School of Biological Sciences, University of California, Irvine, California, USA.
Hum Mutat. 2021 Apr;42(4):421-433. doi: 10.1002/humu.24171. Epub 2021 Feb 4.
Facioscapulohumeral dystrophy (FSHD) is associated with the upregulation of the DUX4 transcription factor and its target genes. However, low-frequency DUX4 upregulation in patient myocytes is difficult to detect and examining the relationship and dynamics of DUX4 and target gene expression has been challenging. Using RNAScope in situ hybridization with highly specific probes, we detect the endogenous DUX4 and target gene transcripts in situ in patient skeletal myotubes during 13-day differentiation in vitro. We found that the endogenous DUX4 transcripts primarily localize as foci in one or two nuclei as compared with the accumulation of the recombinant DUX4 transcripts in the cytoplasm. We also found the continuous increase of DUX4 and target gene-positive myotubes after Day 3, arguing against its expected immediate cytotoxicity. Interestingly, DUX4 and target gene expression become discordant later in differentiation with the increase of DUX4-positive/target gene-negative as well as DUX4-negative/target gene-positive myotubes. Depletion of DUX4-activated transcription factors, DUXA and LEUTX, specifically repressed a DUX4-target gene, KDM4E, later in differentiation, suggesting that after the initial activation by DUX4, target genes themselves contribute to the maintenance of downstream gene expression. Together, the study provides important new insights into the dynamics of the DUX4 transcriptional network in FSHD patient myocytes.
面肩肱型肌营养不良症（FSHD）与 DUX4 转录因子及其靶基因的上调有关。然而，患者肌细胞中低频的 DUX4 上调很难检测到，而且研究 DUX4 和靶基因表达的关系和动态一直具有挑战性。我们使用高度特异性探针的 RNAScope 原位杂交技术，在体外分化的 13 天内，在患者骨骼肌肌管中检测到内源性 DUX4 和靶基因转录本的原位。我们发现，与重组 DUX4 转录本在细胞质中的积累相比，内源性 DUX4 转录本主要定位于一个或两个核中的焦点。我们还发现，在第 3 天之后，持续增加的 DUX4 和靶基因阳性肌管数量，这与 DUX4 预期的即刻细胞毒性相矛盾。有趣的是，在分化过程中，随着 DUX4 阳性/靶基因阴性以及 DUX4 阴性/靶基因阳性肌管数量的增加，DUX4 和靶基因表达变得不一致。DUX4 激活转录因子 DUXA 和 LEUTX 的缺失特异性抑制了分化后期的一个 DUX4 靶基因 KDM4E，这表明在 DUX4 最初激活后，靶基因本身有助于维持下游基因表达。总之，该研究为 FSHD 患者肌细胞中 DUX4 转录网络的动态提供了重要的新见解。
