Epigenetic regulation of embryonic stem cell biology by Tet enzymes

ABSTRACT The central goal of my research program is to establish the epigenetic mechanisms of gene regulation that govern the biology of embryonic stem cells (ESCs). We are interested in defining how ESC gene expression programs and biology are regulated by DNA methylation and the Tet family of DNA demethylases. Tet enzymes oxidize 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5- carboxylcytosine (5caC) in a step-wise manner. 5fC and 5caC are removed from the genome by the DNA repair machinery leading to active DNA demethylation. The conventional understanding in the field has been that Tet- mediated stepwise oxidation of 5mC mainly promotes DNA demethylation. However, the stability and abundance of 5hmC in ESCs and other cell types suggests that not all DNA hydroxylation leads to DNA demethylation. This poses a major question in the field that DNA hydroxylation and the base 5hmC may have gene regulatory functions besides being an intermediate in DNA demethylation. We hypothesize that the early DNA hydroxylation and the later formylation/carboxylation activities of Tet enzymes uniquely regulate gene expression programs in ESCs, and have distinct contributions to the developmental potential of ESCs. We also hypothesize that 5hmC may serve as an independent epigenetic mark having its own readers and impact on gene regulation. We will pursue these hypotheses in the context of two projects. The first project will dissect the molecular and biological requirements of Tet-driven DNA hydroxylation vs. formylation/carboxylation in ESCs biology. The second project will identify readers of 5hmC and the potential of 5hmC as an independent epigenetic mark in regulation of ESC biology. This work builds logically on our prior work and expertise in defining Tet-mediated epigenetic regulation of gene expression in stem cell biology and development. Our findings will establish new layers of epigenetic regulation by Tet enzymes, which is otherwise canonically thought to be only DNA demethylation. Knowing which Tet enzymatic activity or which of these modified bases is the most important for pluripotency network and differentiation will help us better target Tets to enhance ESC applications in regenerative medicine. Moreover, identifying novel readers of 5hmC can help us better understand gene regulatory mechanisms in ESCs. Our findings will also have implications for the etiology of human diseases where Tet enzymes are mutated/ dysregulated or 5hmC levels are perturbed, and identify the relevant Tet activities for therapeutic targeting.