Abstract
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.
Highlights
Stem cells are characterized by two features: the ability to differentiate into multiple cell types and the ability to selfrenew.[1,2] Mesenchymal stem cells (MSCs) are one type of postnatal stem cell with a pluripotent differentiation potential that is broader than originally envisioned or perhaps as broad as that of embryonic stem cells.[2]
This observation was confirmed by western blot analysis (Figure 1b). These results suggest that ALKBH1 may have a role in the osteogenic differentiation of MSCs
Knockdown of ALKBH1 restricted this binding (Figure 5c) and increased the abundance of N6mA on the promoter (Figure 5d), which led to transcription silencing. These findings indicated that ALKBH1 may regulate the osteoblastic differentiation of MSCs by removing the N6-mA modifications on activating transcription factor 4 (ATF4)
Summary
Stem cells are characterized by two features: the ability to differentiate into multiple cell types and the ability to selfrenew.[1,2] Mesenchymal stem cells (MSCs) are one type of postnatal stem cell with a pluripotent differentiation potential that is broader than originally envisioned or perhaps as broad as that of embryonic stem cells.[2]. The epigenetic activation of bone-specific genes mediated by promoter demethylation typically occurs when MSCs differentiate into osteoblasts,[22] and the inhibition of stem-cell-specific genes by promoter methylation is a crucial epigenetic mechanism during stem cell differentiation.[23]
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