DNA methylation cooperates with H3K9me2 at HCN4 promoter to regulate the differentiation of bone marrow mesenchymal stem cells into pacemaker-like cells

Sick sinus syndrome (SSS) is a a life-threatening disease, and biological pacemakers derived from bone marrow mesenchymal stem cells (BMSCs) have practical clinical applications. Previous studies demonstrated that epigenetics plays an important role in the differentiation of BMSCs into pacemaker-like cells. However, the underlying mechanisms remain unclear. In the present study, we investigated the role of DNA methylation and histone methylation in pacemaker cells formation and found that changes in DNA and H3K9 methylation occur in the promoter region of the pacemaker cell-specific gene HCN4. In addition, the combined addition of methylation inhibitors was able to improve the efficiency of transduction of Tbx18 in inducing the differentiation of BMSCs into pacemaker-like cells. In vitro experiments have shown that inhibition of DNA methylation and H3K9 methylation can enhance the activity of the HCN4 promoter activity, and both can affect the binding of the transcription factor NKx2.5to the HCN4 promoter region. Further research on the interaction mechanism between DNA methylation and H3K9me2 in the HCN4 promoter region revealed that the two may be coupled, and that the methylesterase G9a and DNMT1 may directly interact to bind as a complex that affects DNA methylation and H3K9me2 regulation of HCN4 transcription. In conclusion, our studies suggest that the mutual coupling of DNA and H3K9 methylation plays a critical role in regulating the differentiation of BMSCs into pacemaker-like cells from the perspective of interactions between epigenetic modifications, and combined methylation is a promising strategy to optimise pacemaker-like cells for in vitro applications.