TET3 regulates cellular identity and DNA methylation in neural progenitor cells

TET enzymes oxidize 5-methylcytosine (5mC) into 5-hydroxymethylcytosine (5hmC), a process thought to be intermediary in an active DNA demethylation mechanism. Notably, 5hmC is highly abundant in the brain and in neuronal cells. Here we show that Tet3 is highly upregulated during neuronal differentiation and necessary to maintain silencing of pluripotency-associated genes in neural precursor cells (NPCs). Indeed, Tet3 knockdown (KD) in NPCs led to a significant increase in Oct4 and Nanog gene expression, with OCT4-positive cells appearing as cellular aggregates. Moreover, Tet3 KD led to a genome-scale loss of DNA methylation and hypermethylation of a small number of CpGs that are notably located at neurogenesis-related genes and at imprinting control regions (ICRs) of three imprinted genes (Peg10, Zrsr1 and Mcts2). Our results suggest that TET3 plays a pivotal role in maintaining neural stem cell identity and DNA methylation levels in neural precursor cells, and point to a non-catalytic role for TET3 in neural differentiation. Overall design: Mouse embryonic stem cells containing inducible shRNAs for Tet3 or a non-targetting control were differentiated to neural precursor cells. Knockdown was induced with doxyxycline for 5 days. Genomic DNA was extracted to generate oxRRBS libraries.