Padi2/3 Deficiency Alters the Epigenomic Landscape and Causes Premature Differentiation of Mouse Trophoblast Stem Cells

Histone citrullination is a relatively poorly studied epigenetic modification that involves the irreversible conversion of arginine residues into citrulline. It is conferred by a small family of enzymes known as protein arginine deiminases (PADIs). PADI function supports the pluripotent state of embryonic stem cells, but in other contexts, also promotes efficient cellular differentiation. In the current study, we sought to gain deeper insights into the possible roles of PADIs in mouse trophoblast stem cells (TSCs). We show that Padi2 and Padi3 are the most highly expressed PADI family members in TSCs and are rapidly down-regulated upon differentiation. Padi2/3 double knockout (DKO) TSCs express lower levels of stem cell transcription factors CDX2 and SOX2 and are prone to differentiate into extremely large trophoblast giant cells, an effect that may be mediated by centrosome duplication defects. Interestingly, Padi2/3 DKO TSCs display alterations to their epigenomic landscape, with fewer H3K9me3-marked chromocentric foci and globally reduced 5-methylcytosine levels. DNA methylation profiling identifies that this effect is specifically evident at CpG islands of critical trophoblast genes, such as Gata3, Peg3, Socs3 and Hand1. As a consequence of the hypomethylated state, these factors are up-regulated in Padi2/3 DKO TSCs, driving their premature differentiation. Our data uncover a critical epigenetic role for PADI2/3 in safeguarding the stem cell state of TSCs by modulating the DNA methylation landscape to restrict precocious trophoblast differentiation.