Facioscapulohumeral muscular dystrophy (FSHD) is a skeletal muscle disorder that is caused by derepression of the transcription factor DUX4 in skeletal muscle cells. Apart from SMCHD1, DNMT3B was recently identified as a disease gene and disease modifier in FSHD. However, the exact role of DNMT3B at the D4Z4 repeat array remains unknown. To determine the role of Dnmt3b on DUX4 repression, hemizygous mice with a FSHD-sized D4Z4 repeat array (D4Z4-2.5 mice) were cross-bred with mice carrying an in-frame exon skipping mutation in Dnmt3b (Dnmt3bMommeD14 mice). Additionally, siRNA knockdowns of Dnmt3b were performed in mouse embryonic stem cells (mESCs) derived from the D4Z4-2.5 mouse model. In mESCs derived from D4Z4-2.5 mice, Dnmt3b was enriched at the D4Z4 repeat array and DUX4 transcript levels were upregulated after a knockdown of Dnmt3b. In D4Z4-2.5/Dnmt3bMommeD14 mice, Dnmt3b protein levels were reduced; however, DUX4 RNA levels in skeletal muscles were not enhanced and no pathology was observed. Interestingly, D4Z4-2.5/Dnmt3bMommeD14 mice showed a loss of DNA methylation at the D4Z4 repeat array and significantly higher DUX4 transcript levels in secondary lymphoid organs. As these lymphoid organs seem to be more sensitive to epigenetic modifiers of the D4Z4 repeat array, different immune cell populations were quantified in the spleen and inguinal lymph nodes of D4Z4-2.5 mice crossed with Dnmt3bMommeD14 mice or Smchd1MommeD1 mice. Only in D4Z4-2.5/Smchd1MommeD1 mice the immune cell populations were disturbed. Our data demonstrates that loss of Dnmt3b results in derepression of DUX4 in lymphoid tissues and mESCs but not in myogenic cells of D4Z4-2.5/Dnmt3bMommeD14 mice. In addition, the Smchd1MommeD1 variant seems to have a more potent role in DUX4 derepression. Our studies suggest that the immune system is particularly but differentially sensitive to D4Z4 chromatin modifiers which may provide a molecular basis for the yet underexplored immune involvement in FSHD.

中文翻译：

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Dnmt3b 在转基因 D4Z4 小鼠中以组织依赖性方式调节 DUX4 表达


面肩肱型肌营养不良症 (FSHD) 是一种骨骼肌疾病，由骨骼肌细胞中转录因子 DUX4 的去抑制引起。除了 SMCHD1 之外，DNMT3B 最近被确定为 FSHD 的疾病基因和疾病修饰因子。然而，DNMT3B 在 D4Z4 重复阵列中的确切作用仍然未知。为了确定 Dnmt3b 对 DUX4 抑制的作用，将具有 FSHD 大小的 D4Z4 重复阵列的半合子小鼠（D4Z4-2.5 小鼠）与 Dnmt3b 中携带框内外显子跳跃突变的小鼠（Dnmt3bMommeD14 小鼠）进行杂交。此外，在源自 D4Z4-2.5 小鼠模型的小鼠胚胎干细胞 (mESC) 中进行了 Dnmt3b 的 siRNA 敲低。在源自 D4Z4-2.5 小鼠的 mESC 中，Dnmt3b 在 D4Z4 重复阵列中富集，并且在 Dnmt3b 敲低后 DUX4 转录物水平上调。在D4Z4-2.5/Dnmt3bMommeD14小鼠中，Dnmt3b蛋白水平降低；然而，骨骼肌中的 DUX4 RNA 水平没有增强，也没有观察到病理学。有趣的是，D4Z4-2.5/Dnmt3bMommeD14 小鼠在 D4Z4 重复阵列中表现出 DNA 甲基化缺失，并且次级淋巴器官中的 DUX4 转录物水平显着升高。由于这些淋巴器官似乎对 D4Z4 重复阵列的表观遗传修饰剂更敏感，因此在与 Dnmt3bMommeD14 小鼠或 Smchd1MommeD1 小鼠杂交的 D4Z4-2.5 小鼠的脾脏和腹股沟淋巴结中对不同的免疫细胞群进行了定量。仅在 D4Z4-2.5/Smchd1MommeD1 小鼠中，免疫细胞群受到干扰。我们的数据表明，Dnmt3b 的缺失会导致淋巴组织和 mESC 中 DUX4 的去抑制，但不会导致 D4Z4-2.5/Dnmt3bMommeD14 小鼠的肌原细胞中 DUX4 的去抑制。此外，Smchd1MommeD1 变体似乎在 DUX4 去抑制中具有更有效的作用。 我们的研究表明，免疫系统对 D4Z4 染色质修饰剂特别敏感，但存在差异，这可能为 FSHD 中尚未充分探索的免疫参与提供分子基础。