TDG orchestrates ATF4-dependent gene transcription during retinoic acid-induced cell lineage committment

Cell differentiation during development is associated to large-scale modifications in the methylome that require the engagement of an active DNA demethylation machinery including Ten-Eleven-Translocation enzymes for oxidation of 5-methylcytosine and the T:G mismatch DNA glycosylase (TDG) for removal of the oxidized bases. Despite this well-defined molecular function, the biological output of TDG activity remains elusive. Here we combined transcriptomic and epigenomic approaches in TDG knock-out embryonal carcinoma cells, an epiblast stem-like cell model, to decipher TDG function in pluripotent cells and their retinoic acid-induced differentiated progeny. We determined that TDG activity is balancing differentiation in favor of a neural fate at the expense of a cardiac mesoderm fate. This process is associated with a sustained activity of a large set of ATF4-dependent genes in relation with TDG engagement at the Atf4 gene promoter and in conjunction with a TDG-dependent regulation of the mammalian target of rapamycin complex 1. These observations highlight the central role of TDG in cell differentiation and support a model linking metabolic reprogramming to cell fate acquisition. To investigate the role of TDG during cell differentiation, we invalidated the Tdg gene by CRISPR-Cas9 in P19 embryonal carcinoma cells. wt and Tdg-null cells were then grown as monolayers or aggregates in the presence or not of retinoic acid. RNA-seq was then applied to the different samples in order to establish the transcriptome associated with these different cell culture conditions.