ALKBH1 was recently discovered as a demethylase for DNA N6-methyladenine (N6-mA), a new epigenetic modification, and interacts with the core transcriptional pluripotency network of embryonic stem cells. However, the role of ALKBH1 and DNA N6-mA in regulating osteogenic differentiation is largely unknown. In this study, we demonstrated that the expression of ALKBH1 in human mesenchymal stem cells (MSCs) was upregulated during osteogenic induction. Knockdown of ALKBH1 increased the genomic DNA N6-mA levels and significantly reduced the expression of osteogenic-related genes, alkaline phosphatase activity, and mineralization. ALKBH1-depleted MSCs also exhibited a restricted capacity for bone formation in vivo. By contrast, the ectopic overexpression of ALKBH1 enhanced osteoblastic differentiation. Mechanically, we found that the depletion of ALKBH1 resulted in the accumulation of N6-mA on the promoter region of ATF4, which subsequently silenced ATF4 transcription. In addition, restoring the expression of ATP by adenovirus-mediated transduction successfully rescued osteogenic differentiation. Taken together, our results demonstrate that ALKBH1 is indispensable for the osteogenic differentiation of MSCs and indicate that DNA N6-mA modifications area new mechanism for the epigenetic regulation of stem cell differentiation.

中文翻译：

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DNA N6-甲基腺嘌呤脱甲基酶ALKBH1增强人MSC的成骨分化。

最近发现ALKBH1是DNA N6-甲基腺嘌呤（N6-mA）的脱甲基酶，它是一种新的表观遗传修饰，并与胚胎干细胞的核心转录多潜能网络相互作用。但是，ALKBH1和DNA N6-mA在调节成骨分化中的作用尚不清楚。在这项研究中，我们证明了成骨诱导过程中人间充质干细胞（MSCs）中ALKBH1的表达上调。剔除ALKBH1可增加基因组DNA N6-mA水平，并显着降低成骨相关基因的表达，碱性磷酸酶活性和矿化作用。贫ALKBH1的MSC在体内也显示出有限的骨形成能力。相反，ALKBH1的异位表达增强了成骨细胞的分化。机械上，我们发现，ALKBH1的耗竭导致N6-mA在ATF4的启动子区域积累，随后使ATF4转录沉默。此外，通过腺病毒介导的转导恢复ATP的表达成功地挽救了成骨细胞的分化。综上所述，我们的结果表明，ALKBH1对于MSC的成骨分化是必不可少的，并且表明DNA N6-mA修饰是干细胞分化的表观遗传调控的新机制。