GSE210315
Active DNA demethylation promotes cell fate specification and the DNA damage response [SEAL]
Neurons harbor high levels of endogenous single strand DNA breaks (SSBs) that are targeted to neuronal enhancers and correlate with marks of DNA demethylation. To determine the source of SSBs at neuronal enhancers, we depleted the thymidine DNA glycosylase TDG, which excises TET-mediated oxidized methylcytidines 5fC and 5caC, to produce unmodified C. In differentiating neurons, induced degradation of TDG led to the disappearance of SSBs, demonstrating the existence of ongoing TET-mediated oxidation. Using an independent model of macrophage differentiation from reprogrammed pre-B cells, we demonstrate that TET/TDG-mediated active demethylation may be a general mechanism underlying post-mitotic lineage specification. We find that macrophage differentiation prefers short patch base excision repair (SP-BER) to fill-in single nucleotide gaps, whereas neurons also frequently utilize the long-patch (LP-BER) sub-pathway. By measuring the distribution of SSBs relative to sites of oxidized cytosine, we observed that stretches of 2-30 bases are synthesized distal from the methylated CpG site during repair. Disrupting gap-filling using anti-neoplastic nucleoside analogs resulted in continuous DNA damage/repair events at enhancers each resolving within 1-2 hours, but ultimately triggering neuronal cell death. This DNA damage response and toxicity was dependent on TDG activity. Thus, TET-mediated active DNA demethylation promotes endogenous DNA damage at regulatory elements, a process which normally contributes to cell identity but can also provoke neurotoxicity following anti-cancer treatments. Overall design: We developed the oxditative end sequencing (oxEND-seq) to detect 5fC/5caC sites in postmotitic neurons and macrophages. Combined with ChIP-seqs, SEAL-seq, END-seq,  S1-END-seq, and RNA-seq, we were able to study the active DNA demethylation and its roles in cell fate determination. Meanwhile, we used anti-neoplastic nucleoside analogs to measure the DNA damage/repair events at enhancers and the relationship with TET/TDG activity.