Background: As cells divide, they must both replicate their DNA and generate a new set of histone proteins. The newly synthesized daughter strands and histones are unmodified, and must therefore be covalently modified to allow for transmission of important epigenetic marks to daughter cells. Human pluripotent stem cells (hPSCs) display a unique cell cycle profile, and control of the cell cycle is known to be critical for their proper differentiation and survival. A major unresolved question is how hPSCs regulate their DNA methylation status through the cell cycle, namely how passive and active demethylation work to maintain a stable genome. Thymine-DNA glycosylase (TDG), an embryonic essential gene, has been recently implicated as a major enzyme involved in demethylation. Methods: We use human pluripotent stem cells and their derivatives to investigate the role of TDG in differentiation and proliferation. To perform loss of function of TDG, RNA Interference was used. To study the cell cyle, we engineered human pluripotent stem cells to express the FUCCI tool which marks cells at various stages of the cell cycle with distinct patterns of fluorescent proteins. We also used cell cycle profiling by FACS, and DNA methylation analysis to probe a connection between DNA demethylation and cell cycle. Results: Here we present data showing that TDG regulates cell cycle dynamics in human neural progenitors (NPCs) derived from hPSCs, leading to changes in cell cycle related gene expression and neural differentiation capacity. These data show that loss of TDG function can block differentiation by driving proliferation of neural progenitors. We also identify specific cell cycle related genes whose expression changes upon loss of TDG expression. Conclusions: These observations suggest that TDG and active demethylation play an important role in hPSC cell cycle regulation and differentiation.
Gene delivery methods and genome editing of human pluripotent stem cells
