Engineering CpG island DNA methylation in pluripotent cells through synthetic CpG-free ssDNA insertion.

Cellular differentiation requires global changes to DNA methylation (DNAme), where it functions to regulate transcription factor, chromatin remodeling activity, and genome interpretation. Here, we describe a simple DNAme engineering approach in pluripotent stem cells (PSCs) that stably extends DNAme across target CpG islands (CGIs). Integration of synthetic CpG-free single-stranded DNA (ssDNA) induces a target CpG island methylation response (CIMR) in multiple PSC lines, Nt2d1 embryonal carcinoma cells, and mouse PSCs but not in highly methylated CpG island hypermethylator phenotype (CIMP)+ cancer lines. MLH1 CIMR DNAme spanned the CGI, was precisely maintained through cellular differentiation, suppressed MLH1 expression, and sensitized derived cardiomyocytes and thymic epithelial cells to cisplatin. Guidelines for CIMR editing are provided, and initial CIMR DNAme is characterized at TP53 and ONECUT1 CGIs. Collectively, this resource facilitates CpG island DNAme engineering in pluripotency and the genesis of novel epigenetic models of development and disease.

Mapping Human Pluripotent-to-Cardiomyocyte Differentiation: Methylomes, Transcriptomes, and Exon DNA Methylation "Memories".

The directed differentiation of human cardiomyocytes (CMs) from pluripotent cells provides an invaluable model for understanding mechanisms of cell fate determination and offers considerable promise in cardiac regenerative medicine. Here, we utilize a human embryonic stem cell suspension bank, produced according to a good manufacturing practice, to generate CMs using a fully defined and small molecule-based differentiation strategy. Primitive and cardiac mesoderm purification was used to remove non-committing and multi-lineage populations and this significantly aided the identification of key transcription factors, lncRNAs, and essential signaling pathways that define cardiomyogenesis. Global methylation profiles reflect CM development and we report on CM exon DNA methylation "memories" persisting beyond transcription repression and marking the expression history of numerous developmentally regulated genes, especially transcription factors.