Relationship of DUX4 and target gene expression in FSHD myocytes.
Relationship of DUX4 and target gene expression in FSHD myocytes.
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DOI：
10.1002/humu.24171
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发表时间：
2021-04
期刊：
影响因子：
3.9
通讯作者：
Yokomori K
中科院分区：
文献类型：
--
作者：
Chau J;Kong X;Viet Nguyen N;Williams K;Ball M;Tawil R;Kiyono T;Mortazavi A;Yokomori K
面肩肱营养不良 (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 DUX4 上调很难检测，并且检查 DUX4 和靶基因表达的关系和动态一直具有挑战性。使用 RNAScope 原位杂交和高度特异性探针，我们在 13 天的体外分化过程中原位检测患者骨骼肌管中的内源性 DUX4 和靶基因转录本。我们发现，与重组 DUX4 转录物在细胞质中的积累相比，内源性 DUX4 转录物主要定位在一或两个细胞核中。我们还发现 DUX4 和靶基因阳性肌管在第 3 天后持续增加，这与预期的立即细胞毒性相悖。有趣的是，随着 DUX4 阳性/靶基因阴性以及 DUX4 阴性/靶基因阳性肌管的增加，DUX4 和靶基因表达在分化后期变得不一致。 DUX4激活的转录因子DUXA和LEUTX的耗尽，在分化后期特异性抑制了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，这项研究为 FSHD 患者肌细胞中 DUX4 转录网络的动态提供了重要的新见解。
Facioscapulohumeral dystrophy (FSHD) is associated with 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 compared to 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 becomes 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 following the initial activation by DUX4, target genes themselves contribute to the maintenance of downstream gene expression. Together, the study provides important new insights into dynamics of DUX4 transcriptional network in FSHD patient myocytes.
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