Dynamic visualization of DNA methylation in cell cycle genes during iPSC cardiac differentiation

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. We utilized a genomic DNA methylation reporter (GMR) system carrying Snrpn minimal promoter and CpG regions of <i>Cdk1</i> (Cyclin-dependent kinase 1) or <i>Sox2</i> (SRY-Box Transcription Factor 2). Mouse Sox2 GMR iPSCs rapidly lost fluorescent reporter signal upon the induction of cardiac differentiation. Cdk1 GMR reporter signal was strong in undifferentiated iPSCs, and gradually decreased during cardiomyocyte differentiation. RT-qPCR and pyrosequencing demonstrated that the reduction of <i>Sox2</i> and <i>Cdk1</i> was regulated by hypermethylation of their promoters’ CpG regions during cardiac differentiation. The GMR reporter system can be useful for monitoring real-time epigenetic DNA modification at single-cell resolution. This research article introduces a genomics DNA methylation reporter (GMR) system that utilizes Snrpn gene’s mini-promoter along with regulatory regions (e.g. promoter or enhancer) of genes of interest, Cdk1, and Sox2. When mouse stem cells carrying the Sox2 or Cdk1 GMR were differentiated into heart muscle cells, the cells gradually lost their fluorescent reporter signal. Further testing with cell imaging, gene expression, and DNA methylation assays confirmed that these changes were due to increased methylation in specific DNA regions as the cells developed into heart cells. This system offers a way to track real-time changes in DNA methylation at the single-cell level.