Dual gene activating and repressive functions of TET1 in germ layer lineage bifurcation distinguished by genomic context and dependence on 5-methylcytosine oxidation [CUT&RUN]
Gastrulation initiates when the epiblast differentiates into either definitive ectoderm or primitive streak. During the lineage bifurcation, the DNA dioxygenase TET1 plays dual roles in both transcriptional activation and repression, but how it exerts this bipartite control via 5-methylcytosine (5mC) oxidation-dependent and independent activities remains unclear. Here, we perform a monolayer differentiation of mouse embryonic stem cells (ESCs) into neuronal precursors to define at single-cell resolution how Tet1-/- cells undergo a lineage switch to primitive streak and subsequently form mesoderm and endoderm. We identify the Wnt repressor Tcf7l1 as a direct target of TET1 that controls a signaling cascade of Wnt/ß-catenin upstream of Nodal. Disrupting the endogenous catalytic site of Tet1 in ESCs contributes to activation of Nodal and subsequently Wnt/ß-catenin signaling, promoting tri-lineage differentiation into ectoderm, mesoderm and endoderm. Catalytically dead TET1 is sufficient in sustaining open neuroectoderm enhancers, by which gene expression is uncoupled from enhancer DNA demethylation, and indirectly keeping primitive streak enhancers inaccessible to Wnt regulators and effectors. DNA hypermethylation caused by TET1 catalytic dysfunction instead promotes precocious primitive streak gene activation when associated with CpG islands overlapping bivalent gene promotors. Moreover, hypermethylated regions amplify in numbers in the absence of TET1 through differentiation to affect genes associated with neurological functions. Our results reveal two-way safeguarding activities of TET1 separable by genomic features, where at CpG-poor distal enhancers TET1 maintains accessible chromatin permissive for neural fate independently of 5mC oxidation; at CpG-rich bivalent promoters it prevents premature gene activation inducing alternative fates by harnessing 5mC oxidation in Polycomb gene repression. Overall design: WT, Tet1 MUT and Tet1 KO mESCs were differentiated to antNPCs.