DNMT3B supports meso-endoderm differentiation from mouse embryonic stem cells [BSAS]

The correct establishment of DNA methylation patterns during mouse early development is essential for cell fate specification. However, the molecular targets as well as the mechanisms that determine the specificity of the de novo methylation machinery during differentiation are not completely elucidated. Here we show that the DNMT3B-dependent DNA methylation in epiblasts of key developmental regulatory regions provides an epigenetic priming that ensures flawless commitment at later stages. Using in vitro stem cell differentiation and loss of function experiments combined with high-throughput genome-wide bisulfite-, bulk-, and single cell RNA-sequencing, we dissected the specific role of DNMT3B in cell specification, we identified the DNMT3B-dependent regulatory elements on the genome which, in Dnmt3b knockout (3BKO), impair the differentiation into meso-endodermal (ME) progenitors and redirect epiblast-like cells (EpiLCs) towards the neuro-ectodermal lineages. Moreover, ectopic expression of DNMT3B in 3BKO re-establishes the DNA methylation of the master regulator Sox2 super-enhancer, down-modulates its expression, and restores the expression of ME markers. Taken together, our data reveal that DNMT3B-dependent methylation at the epiblast stage is essential for the priming of the meso-endodermal lineages and provide functional characterization of the de novo DNMTs during EpiLCs lineage determination. 1) Targeted bisulfite amplicon sequencing (BSAS) analysis of DNA methylation in Sox2 locus of WT, 3BKO and 3BKO ectopically expressing Dnmt3b (Rescued) mouse ES-derived EpiLCs. 8 samples, 2 independent 3BKO clones (B77,B126). 2) Targeted bisulfite amplicon sequencing (BSAS) analysis of DNA methylation in Sox2 locus of 3BKO and 3BKO ESC transfected with CRISPRoff targeting 4 enhancers (E1-E4). 6 samples, 2 independent 3BKO clones (B77,B126).