Highlights d Allele-specific reporters revealed dynamic DNA methylation of Sox2 and miR290 SEs d DNMTs and transcription factor binding regulate methylation dynamics d SE DNA methylation directly regulates transcription in cis d Dynamic DNA methylation is co-regulated with MED1 recruitment and H3K27ac level

Summary Parent-specific differentially methylated regions (DMRs) are established during gametogenesis and regulate parent-specific expression of imprinted genes. Monoallelic expression of imprinted genes is essential for development suggesting that imprints are faithfully maintained in embryo and adult. To test this hypothesis, we targeted a reporter for genomic methylation to the imprinted Dlk1-Dio3 intergenic DMR (IG-DMR) to assess the methylation of both parental alleles at single cell resolution. Biallelic gain or loss of IGDMR methylation occurred in a small fraction of mouse embryonic stem cells significantly affecting developmental potency. Mice carrying the reporter in either parental allele showed striking parent-specific changes in IG-DMR methylation causing substantial and consistent tissue- and cell type-dependent signatures in embryos and postnatal animals. Furthermore, dynamics in DNA methylation persisted during adult neurogenesis resulting in inter-individual diversity. This substantial cell-cell DNA methylation heterogeneity implies that dynamic DNA methylation variations in the adult may be of functional importance.

Reversible lysine methylation is essential for regulating histones and emerges to critically regulate non-histone proteins as well. Here we show that the master transcription factor OCT4 in pluripotent stem cells (PSCs) was methylated at multiple lysine residues. LSD1 that is highly expressed in PSCs can directly interact with and demethylate OCT4 at lysine 222 (K222) in the flexible linker region. Reduced LSD1 activity led to the methylation of OCT4-K222 that diminished the differentiation potential of PSCs while facilitating proteasome-independent degradation of OCT4 proteins. Furthermore, site-specifically replacing K222 with phenylalanine to mimic the constitutively methylated lysine promoted the ‘locked-in’ mode engagement of the OCT4 PORE-homodimers that tightly bind to and block the transcription of multiple PORE-motif-containing target genes regulating cell fate determination and cell junction organization, and thereby reducing the pluripotency of PSCs. Thus, LSD1-mediated demethylation of OCT4 plays a crucial role in restricting the ‘locked-in’ mode binding of OCT4 PORE-homodimers to the PORE-motif-containing genes and thereby maintaining their transcription to safeguard the pluripotency of PSCs.