The three TET enzymes are key epigenetic regulators of DNA methylation dynamics in the genome. Their dioxygenase activity alters DNA methylation status by sequentially oxidizing 5-methylcytosine (5mC) to 5- hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC). Although these oxidized species of 5mC (oxi-mC) represent transient intermediates of DNA demethylation, they are also epigenetic marks that may influence chromatin organization and gene expression. Thus, TET proteins and DNA methyltransferases (DNMTs) together modulate genomic 5mC/5hmC status in cells, thereby influencing many biological processes, such as gene expression, cell fate decisions, oncogenesis, and reprogramming of somatic cells to induced pluripotent stem cells (iPSCs). The involvement of TET enzymes in the regulation of pluripotency was suggested by Tet1 and Tet2 elevated expression in embryonic stem cells (ESCs), in which 5hmC levels are preeminent compared to most cell types other than neurons. Interestingly, Tet1 and Tet2 expression as well as 5hmC levels increase progressively during the reprogramming of mouse embryonic fibroblasts (MEFs) into iPSCs, suggesting that TET enzymes contribute as a minimum to the epigenomic remodeling during this process. This hypothesis was supported by the putative role of Tet1 in modulating the balance between lineage commitment and the maintenance of pluripotency. To address this question we analyzed the reprogramming efficiency of Tet1- and Tet2-deficient MEFs into iPSCs. Our results revealed that these two TETs play opposite roles, Tet1 functioning as a positive regulator of reprogramming, whereas Tet2 has a mild negative effect. In order to identify distinct reprogramming intermediates that are affected by the individual ablation of Tet1 or Tet2, we are presently taking advantage of the CyTOF(r) mass cytometry platform to perform time-course experiments so as to chart continuous molecular roadmaps of WT and TET-deficient reprogramming MEFs. The resulting data will help us identify all the transient reprogramming populations and their molecular signatures, which will be crucial to isolate the reprogramming-prone cells by conventional fluorescence-activated cell sorting (FACS), and further characterize them by RNA-Seq and genome-wide CMS-IP to decipher the link between TET enzymatic activity, deposition of 5hmC epigenetic marks and activation of gene regulatory networks favoring reprogramming. Our main objective is to uncover how the TET enzymes alleviate some of the reprogramming roadblocks by enabling DNA demethylation and modulating the targeted deposition of 5hmC epigenetic marks in order to reset the epigenome of reprogrammable-prone intermediate subpopulations. Overall, this proposal will allow deciphering how the fine-tuning of the 5hmC dynamics during somatic reprogramming plays a crucial role in the activation and stabilization of pluripotency- related regulatory networks that are essential for generating high-quality iPSCs.
During somatic cell reprogramming, epigenetic mechanisms are crucial for orchestrating the induction of pluripotency-related regulatory networks, which in turn are essential for supporting complete reprogramming of somatic cells. The proposed research project will decipher the roles of the TET enzymes during somatic cell reprogramming in both mouse and human systems. The long-term biomedical significance of this work is to elucidate whether TET enzymes contribute to resetting the epigenome by alleviating some reprogramming barriers, in order to bring us closer to generating clinical-grade iPSCs for regenerative medicine.
