TET-family dioxygenases catalyze conversion of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) and oxidized methylcytosines in DNA. Here, we show that mouse embryonic stem cells (mESCs), either lacking Tet3 alone or with triple deficiency of Tet1/2/3, displayed impaired adoption of neural cell fate and concomitantly skewed toward cardiac mesodermal fate. Conversely, ectopic expression of Tet3 enhanced neural differentiation and limited cardiac mesoderm specification. Genome-wide analyses showed that Tet3 mediates cell-fate decisions by inhibiting Wnt signaling, partly through promoter demethylation and transcriptional activation of the Wnt inhibitor secreted frizzled-related protein 4 (Sfrp4). Tet1/2/3-deficient embryos (embryonic day 8.0-8.5) showed hyperactivated Wnt signaling, as well as aberrant differentiation of bipotent neuromesodermal progenitors (NMPs) into mesoderm at the expense of neuroectoderm. Our data demonstrate a key role for TET proteins in modulating Wnt signaling and establishing the proper balance between neural and mesodermal cell fate determination in mouse embryos and ESCs.

The discovery of Ten Eleven Translocation proteins, enzymes that oxidize 5-methylcytosine (5mC) in DNA, has revealed novel mechanisms for the regulation of DNA methylation. We have mapped 5-hydroxymethylcytosine (5hmC) at different stages of T-cell development in the thymus and T-cell differentiation in the periphery. We show that 5hmC is enriched in the gene body of highly expressed genes at all developmental stages and that its presence correlates positively with gene expression. Further emphasizing the connection with gene expression, we find that 5hmC is enriched in active thymus-specific enhancers and that genes encoding key transcriptional regulators display high intragenic 5hmC levels in precursor cells at those developmental stages where they exert a positive effect. Our data constitute a valuable resource that will facilitate detailed analysis of the role of 5hmC in T-cell development and differentiation.