Regulation and function of active DNA demethylation in Cellular Differentiation

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Methylation at the 5' position of cytosine (5mC) plays an essential role in cell differentiation and embryonic development. Despite its importance, 5mC also increases the CpG mutation rate by promoting deamination to form thymine. In humans the incidence of 5mC to thymine mutations is 10 to 50-fold higher than other base transitions. The resulting G:T and G:U (G:T/U) mispairs can alter coding and regulatory sequences. Consequently, there is a need to understand the molecular mechanisms underlying the dynamics of genome methylation. Recent studies have identified a novel DNA demethylation pathway whereby 5mC is oxidized to 5 hydroxymethylcytosine (5hmC) catalyzed by the Ten Eleven Translocation (TET 1-3). 5hmC is metabolized further by TETs into 5 formylcytosine (5fC) and 5 carboxylcytosine (5caC). 5fC and 5caC are recognized and excised by the base excision repair enzyme thymine DNA glycosylase (TDG) to restore unmodified cytosine. The discovery of active demethylation involving the TET/TDG pathway represents a transformative discovery that has greatly advanced our understanding of the dynamics of DNA methylation. Studies have shown that 5mC derivatives have important roles in stem cell differentiation, genomic imprinting and neuronal function. Furthermore, specific interacting proteins for each derivative have been identified thus reflecting the unique biological properties of 5hmC/5fC/5caC. Exactly how these oxidized bases exert their function is unclear and will require a better understanding of their mechanism of deposition, as well as the ability to comprehensively profile the distribution of 5mC derivatives in the mammalian genome. This proposal focuses on two complimentary strategies to test the hypothesis that active demethylation is essential for gene transcription in response to differentiation signals. First using mouse embryonic stem cells, which have the capacity to undergo neural lineage differentiation in response to retinoic acid, we will quantitatively map changes in 5fC/5caC using a new technique known as methylation assisted bisulphite sequencing (MABseq). MABseq provides a direct readout of TET/TDG mediated active demethylation activity at base resolution. Second, to study the molecular mechanisms of active demethylation we will use a protein affinity purification approach known as Proximity-dependent biotin identification (BioID) in combination with mass spectrometry, to identify the protein interaction network for TET/TDG during cellular differentiation. Active DNA demethylation represents a new paradigm in gene regulation that is having important ramifications in all aspects of cell physiology. Collectively, our studies will identify genomic sites where active demethylation occurs and in combination with the experiments proposed in specific aim 2 will provide an important framework towards understanding dynamic DNA methylation.