DNA methylation as a reprogramming modulator: An epigenomic roadmap to induced pluripotency. mus musculus

During cellular reprogramming to induced pluripotent stem cells (iPSCs), somatic cells rebuild their epigenetic architecture to acquire a steady self-renewing state. Here we systematically characterize the epigenomic roadmap to pluripotency by performing whole genome bisulfite sequencing of the “Project Grandiose” (PG) samples, along with analysis of histone modification and the transcriptome. The differentially methylated regions (DMRs) of the DNA samples revealed that hypermethylated-DMRs (Hyper-DMRs) gradually accumulated throughout reprogramming whereas there was a distinct gain in hypomethylated-DMRs (Hypo-DMRs) once cells reached the ESC-like state. ESC-associated transcription factor binding sites (ESC-TFBSs) were highly enriched in the Hypo-DMR group. During high transgene expression, leading to the pluripotent F-class state10, cells exhibited focal DNA demethylation at the binding sites of the activated reprogramming factors, whereas ESC-like pluripotent cells exhibited widespread demethylation surrounding these sites. Genes associated with CpG rich promoters demonstrated a stable low DNA methylation state with strong engagement of H3K4me3 and H3K27me3 histone marks (histone modification-driven control). CpG poor promoter-related genes were repressed by DNA methylation, with transcriptional activity occurring upon promoter demethylation (DNA methylation-driven control). We further report that a proportion of genes proposed to be ESC-pluripotency predictors, including Dppa2, Eras, and Esrrb, are associated with CpG poor promoters and become transcriptionally activated upon DNA demethylation in ESC-like cells. Gene expression leading to ESC-like pluripotency is impacted and specifically orchestrated by dynamic changes of DNA methylation and histone modification. These results suggest that DNA methylation plays a crucial role within the epigenetic switch that drives somatic cells to pluripotency.