H3K27me3 is dispensable for early differentiation but required to maintain differentiated cell identity

Polycomb repressive complex 2 (PRC2) catalyzes trimethylation of histone H3 on lysine 27 and is required for normal development of complex eukaryotes. The requirement for H3K27me3 in various aspects of mammalian differentiation is not clear. Though associated with repressed genes, the modification is not sufficient to induce gene repression, and in some instances is not required. To examine the role of the modification in mammalian differentiation, we blocked trimethylation of H3K27 with both a small molecule inhibitor, GSK343, and by introducing a point mutation into EZH2, the catalytic subunit of PRC2. We found that cells with substantively decreased H3K27 tri-methylation were able to differentiate, which contrasts with EZH2 null cells. Different PRC2 targets had varied requirements for K27me3 in repressive regulation with a subset that maintained normal levels of repression in the absence of methylation. The primary cellular phenotype where H3K27 tri-methylation was blocked was the inability of the altered cells to maintain a differentiated state when challenged. This phenotype depended upon H3K27me3 deposition both in embryonic stem cells and in the first four days of differentiation. H3K27 tri-methylation therefore was not necessary for formation of differentiated cell states but was required to maintain a stable differentiated state. RNA-seq and Cut&Run analysis of GSK343-treated, EZH2-mutant or WT ESC or differentiated embryoid bodies.