Mus musculus musculus Genome sequencing. Mus musculus musculus

During cellular reprogramming to induced pluripotent stem cells (iPSCs), somatic cells rebuild their epigenetic architecture to acquire a steady self-renewing state1-7. Here we systematically characterize the epigenomic roadmap to pluripotency by performing whole genome bisulfite sequencing of the “Project Grandiose” (PG) samples8-10 (Extended Data Fig.1a-b), 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.