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bioRxiv · 10.1101/2024.01.17.575536

Visualization of DNA Methylation in Cell Cycle Genes during iPSC Cardiac Differentiation

Li, N.·Nguyen, B. T.·Zhang, Z.·Zhang, Y.
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Abstract

BackgroundEpigenetic DNA methylation is an essential mechanism controlling gene expression and cellular function. Existing analyses with conventional assays have generated significant insights into static states of DNA methylation, but were unable to visualize the dynamics of epigenetic regulation. AimWe utilized a genomic DNA methylation reporter (GMR) system to track changes in DNA methylation during cardiac differentiation. Methods and ResultsThe promoter region of Cdk1 (Cyclin-dependent kinase 1) or Sox2 (SRY-Box Transcription Factor 2) gene was cloned upstream of the small nuclear ribonucleoprotein polypeptide N (Snrpn) minimal promoter followed by a fluorescent reporter gene. Mouse induced pluripotent stem cells (iPSCs) carrying Sox2 GMR rapidly lost fluorescent reporter signal upon the induction of differentiation. Cdk1 GMR reporter signal was strong in undifferentiated iPSCs, and gradually decreased during directed cardiomyocyte (CM) differentiation. RT-qPCR and pyrosequencing demonstrated that the reduction of Sox2 and Cdk1 was regulated by hypermethylation of their CpG regions during cardiac differentiation. The present study demonstrated the dynamic DNA methylation along the course of cell cycle withdrawal during CM differentiation. ConclusionThe GMR reporter system can be a useful tool to monitor real-time epigenetic DNA modification at single-cell resolution.

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Li, N., Nguyen, B. T., Zhang, Z., Zhang, Y.. 2024-01-20. Visualization of DNA Methylation in Cell Cycle Genes during iPSC Cardiac Differentiation. https://doi.org/10.1101/2024.01.17.575536

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