The Role of Active DNA Demethylation in Mammalian Epigenetic Reprogramming

DNA methylation in the form of 5-methylcytosine (5mC) is an essential epigenetic regulator of gene expression and cellular identity in mammals. As a dynamic epigenetic feature, DNA methylation can be reversed through several distinct pathways. Whereas global DNA demethylation is achieved through the suppression of DNA methyltransferase (DNMT) enzymes, targeted demethylation at individual CpG sites is regulated through the activity of Ten-eleven Translocation (TET) family enzymes. TET enzymes mediate the sequential oxidation of 5mC to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), or 5-carboxycytosine (5caC). These oxidized residues are poorly recognized by maintenance methyltransferase DNMT1, allowing their steady dilution over several rounds of cellular division. Alternatively, 5fC and 5caC may be targeted for excision by thymine DNA glycosylase (TDG), restoring the unmodified cytosine through the base excision repair pathway. While these two mechanisms are collectively referred to as “active DNA demethylation”, little is known about their relative contributions to DNA demethylation in vivo. Here, using newly discovered “5hmC-stalling” TET mutants with impaired 5fC and 5caC oxidation, we tested the requirement of the fC/caC branch of active DNA demethylation for epigenetic reprogramming in two model systems. First, using an induced pluripotent stem cell (iPSC) model, we demonstrate that the fC/caC activity of TET2 is essential for DNA demethylation at reprogramming enhancers during iPSC induction, and that loss of this activity reduces the reprogramming potential of mouse embryonic fibroblasts. Next, we used CRISPR/Cas9 engineering to generate two new mouse models of TET1 activity: a 5hmC-stalling Tet1T1642V mutant, and a catalytically inactive Tet1HxD mutant. Our preliminary results indicate that the fC/caC activity of TET1 is critical for epigenetic reprogramming in the developing mouse germline, and that loss of TET1 catalytic activity in the adult mouse cortex leads to hypermethylation in promoters of genes involved in neurodevelopment and maintenance. In total, our studies represent the first in vivo characterization of the multiple activities of TET enzymes and the roles they play in regulating genomic 5mC levels and shaping cellular identity