Expression patterns of FSHD-causing DUX4 and myogenic transcription factors PAX3 and PAX7 are spatially distinct in differentiating human stem cell cultures
Expression patterns of FSHD-causing DUX4 and myogenic transcription factors PAX3 and PAX7 are spatially distinct in differentiating human stem cell cultures
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DOI：
10.1186/s13395-017-0130-1
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发表时间：
2017-06-21
期刊：
影响因子：
4.9
通讯作者：
Miller, Daniel G.
中科院分区：
文献类型：
--
作者：
Haynes, Premi;Kernan, Kelly;Miller, Daniel G.
背景：面肩肱型肌营养不良症 (FSHD) 最常见的是常染色体显性遗传，由骨骼肌中 DUX4 的异常表达引起。 DUX4 转录因子具有与几种配对类同源异型转录因子相似的 DNA 结合域，但只有生肌因子 PAX3 和 PAX7 在小鼠成肌细胞中与 DUX4 共表达时才能挽救细胞活力。这一观察结果表明卫星细胞中 DNA 结合位点的竞争可能会限制肌肉修复，并且可能是 DUX4 相关肌肉毒性的一个方面。竞争假说要求 DUX4 和 PAX3/7 在发育过程中的某个时刻或在成体组织中在相同细胞中表达。我们使用人同基因 iPS 和 ES 细胞模拟肌发生，并检查了 DUX4、PAX3 和 PAX7 的表达模式，以确定在细胞培养物中促进 PAX3 和 PAX7 表达的条件是否也会促进相同细胞中 DUX4 的表达。 方法：从两个患有 FSHD 的体细胞嵌合个体的人成纤维细胞中产生同基因 iPSC。从同一个体中分离出含有缩短的引起 FSHD 的 D4Z4 阵列或长的非致病性阵列的克隆。我们还检查了来自受 FSHD 影响和未受影响胚胎的市售 hES 细胞系的肌生成。在 40 天分化方案中对 DUX4、PAX3 和 PAX7 信使 RNA (mRNA) 进行定量，并使用抗体来识别不同分化阶段的细胞类型，以确定 DUX4 和 PAX3 或 PAX7 是否存在于同一细胞中。 结果：人 iPS 和 ES 细胞分化为骨骼肌细胞，如在 40 天分化方案结束时出现的 Titin 阳性多核纤维所证明。 PAX3 和 PAX7 在分化过程中在相似的时间表达，并且在包含短 D4Z4 阵列的细胞中在分化末期观察到 DUX4 阳性细胞核。在多能细胞生肌分化过程中的五个不同时间点检查免疫染色细胞核后，未观察到同时表达 DUX4 和 PAX3 或 DUX4 和 PAX7 的细胞核。结论：我们得出结论，DUX4、PAX3 和 PAX7 在干细胞生肌分化过程中具有不同的表达模式。我们的研究结果与以下假设一致：FSHD 中的肌肉损伤是由于 DUX4 介导的毒性导致终末分化肌纤维破坏所致。虽然这些研究检查了干细胞成肌过程中 DUX4、PAX3 和 PAX7 的表达模式，但它们不应推广到成体肌肉组织中的组织修复。
Background: Facioscapulohumeral muscular dystrophy (FSHD) is most commonly inherited in an autosomal dominant pattern and caused by the abnormal expression of DUX4 in skeletal muscle. The DUX4 transcription factor has DNA binding domains similar to several paired class homeotic transcription factors, but only myogenic factors PAX3 and PAX7 rescue cell viability when co-expressed with DUX4 in mouse myoblasts. This observation suggests competition for DNA binding sites in satellite cells might limit muscle repair and may be one aspect of DUX4-associated myotoxicity. The competition hypothesis requires that DUX4 and PAX3/7 be expressed in the same cells at some point during development or in adult tissues. We modeled myogenesis using human isogenic iPS and ES cells and examined expression patterns of DUX4, PAX3, and PAX7 to determine if conditions that promote PAX3 and PAX7 expression in cell culture also promote DUX4 expression in the same cells.Methods: Isogenic iPSCs were generated from human fibroblasts of two FSHD-affected individuals with somatic mosaicism. Clones containing the shortened FSHD-causing D4Z4 array or the long non-pathogenic array were isolated from the same individuals. We also examined myogenesis in commercially available hES cell lines derived from FSHD-affected and non-affected embryos. DUX4, PAX3, and PAX7 messenger RNAs (mRNAs) were quantified during a 40-day differentiation protocol, and antibodies were used to identify cell types in different stages of differentiation to determine if DUX4 and PAX3 or PAX7 are present in the same cells.Results: Human iPS and ES cells differentiated into skeletal myocytes as evidenced by Titin positive multinucleated fibers appearing toward the end of a 40-day differentiation protocol. PAX3 and PAX7 were expressed at similar times during differentiation, and DUX4 positive nuclei were seen at terminal stages of differentiation in cells containing the short D4Z4 arrays. Nuclei that expressed both DUX4 and PAX3, or DUX4 and PAX7 were not observed after examining immunostained nuclei at five different time points during myogenic differentiation of pluripotent cells.Conclusions: We conclude that DUX4, PAX3, and PAX7 have distinct expression patterns during myogenic differentiation of stem cells. Our findings are consistent with the hypothesis that muscle damage in FSHD is due to DUX4-mediated toxicity causing destruction of terminally differentiated myofibers. While these studies examine DUX4, PAX3, and PAX7 expression patterns during stem cell myogenesis, they should not be generalized to tissue repair in adult muscle tissue.