TET Proteins Control Early-Stage Stem Cell Differentiation via H3K27me3, Not DNA Demethylation

TET proteins reportedly have dual functions as mediators of DNA demethylation and H3K27me3 deposition in embryonic stem cells (ESCs) and during differentiation. To further explore the role of TET proteins in early ESC differentiation, we conducted mapping experiments on wild-type and cells lacking all three TET proteins (TET-TKO) at three stages: in ESCs, immediately after exit from pluripotency, and at the early differentiation stage. We observed minimal differences in transcriptional and DNA methylation levels between wild-type and TET-TKO cells during ESCs and after exit from pluripotency. However, H3K27ac sequential chromatin immunoprecipitation followed by sequencing (ChIP-seq) and bisulfite-seq and single-cell RNA-seq maps demonstrated that during differentiation TET-TKO cells lost their ability to activate the differentiation to extraembryonic endoderm (XEN). This inability was not due solely to higher levels of DNA methylation, as enhancers that were unaffected by TET also showed higher levels of DNA methylation. Loss of XEN differentiation was associated with reduced levels of H3K27me3 during the pluripotency stage. We were able to rescue the effect of TET deficiency by co-culturing TET-TKO ESCs with wild-type ESCs. The co-culturing approach restored XEN differentiation, even though TET proteins were absent during differentiation. We propose that DNA demethylation catalyzed by TET proteins is of minimal importance during the initial stages of differentiation. Instead, the key regulatory function of TETs lies in establishing the ESC ground state through H3K27me3 chromatin deposition.