TET-mediated DNA Demethylation Mechanisms and Epigenome Engineering Applications - PROJECT SUMMARY This proposal aims to elucidate the mechanisms by which TET family enzymes act on 5-methylcytosine in genomic DNA and to exploit these insights to devise controllable epigenome editors. Modifications to cytosine bases play an important role in diverse processes, including development, pluripotency, and oncogenesis. The best-studied cytosine modification is methylation at the 5-position (5mC), typically occurring at cytosine-guanine dinucleotides (CpGs) which are most often found in clusters, including CpG islands (CGIs). The methylation status of CpGs over regions of DNA plays a key role in dictating whether associated genes are actively transcribed or silenced. While methylation can be stably maintained as part of the epigenetic code governing cell identity, changes in methylation are equally important, allowing the genome to be dynamically responsive to the cellular environment or during development. This critical process of active DNA demethylation is mediated by TET family enzymes, Fe(II)/α-ketoglutarate-dependent dioxygenases that can oxidize 5mC to generate 5- hydroxymethylcytosine (5hmC). Importantly, 5hmC is itself a substrate for further oxidation, generating 5- formylcytosine (5fC) and 5-carboxylcytosine (5caC). The different oxidized 5mC bases (ox-mCs) open up distinct pathways for DNA demethylation, which differ in their obligate dependence on DNA replication versus DNA repair, resulting in major different implications for the timing and consequences of gene activation. Despite the critical importance of 5mC oxidation in regulating gene expression, the mechanisms by which TET enzymes reactivate silenced genes has remained enigmatic. This gap is due in part to limitations in the tools used to sequence and track dynamics of 5mC and ox-mCs, and the fact that most studies employ all-or-none approaches with TET deletion or inactivation. The urgency for better understanding demethylation dynamics is fueled by the discovery that TET enzymes can also be harnessed as powerful epigenome editing tools when targeted to specific genomic loci by CRISPR-Cas proteins. This proposal aims to apply a series of innovative enzymatic and sequencing tools to reveal and exploit the mechanisms of TET enzymes. Specifically, the mode of TET action on CpG clusters will be revealed by the combined application of novel sequencing methods that can localize multiple different ox-mCs in single DNA molecules and engineered TET variants that can either stall or accelerate through multiple oxidation states of 5mC. These tools will be applied to decipher the footprint of TET on model substrates in vitro and in cells, and these observations will be applied to dissect T cell differentiation as biological model. Building on these mechanistic insights into targeted DNA demethylation, we will devise novel controllable epigenome editors that can allow for efficient and targeted DNA demethylation and mimic physiological gene activation. Together, our aims will thus newly reveal the mechanisms by which TET enzymes act on CpG clusters and advance novel biotechnological tools that leverage TET enzymes to reshape the epigenome.
