A conserved function of EZH2-PRC2 in the repression of primitive endoderm cell fate [CUT&Tag]

In early blastocyst, inner cell mass (ICM) initiates the second wave of cell fate decisions to generate epiblast and primitive endoderm (PrE), crucial for proper embryo development. However, the epigenetic mechanisms underlying this segregation remain incompletely understood. Here, we reveal that treatment of embryonic stem cells (ESCs) with the EZH2 inhibitor EPZ-6438 significantly enhances transdifferentiation efficiency from ESCs to primitive endoderm stem cells (PrESCs). EZH2 knockout in ESCs markedly reduces H3K27me3 modification near the promoter regions of PrE-specific genes, upregulating their expression and promoting transition to PrESCs. Overexpression of wildtype EZH2, but not catalytic site or EED-binding motif mutants, prevents this transition. Notably, the ICM of mouse blastocysts treated with EPZ6438 at 8-cell stage contain almost no epiblast but only GATA6 positive cells. Finally, EPZ6438 treatment in human naïve ESCs upregulates hypoblast-associated genes. These findings demonstrated the conserved function of EZH2-PRC2 in protecting the pluripotency from primitive endoderm cell fate. Overall design: We aim to investigate the conserved function of EZH2-PRC2 in protecting the pluripotency from primitive endoderm cell fate. We established two EPZ-6438-induced PrESCs (EPZ-iPrESCs-1 and EPZ-iPrESCs-2) from wild-type (WT) mouse embryonic stem cells (mESCs). Duplicate RNA-seq samples of WT mESCs, EPZ-iPrESCs-1, EPZ-iPrESCs-2, and C57-PrESCs were analyzed to characterize the transcriptomic profiles of iPrESCs. Subsequently, we generated two Ezh2 knockout lines (Ezh2KO-10 and Ezh2KO-12) and their rescued counterparts (Ezh2KO-10-OE and Ezh2KO-12-OE). These cell lines were cultured in either serum/LIF (SL) or serum/2i/LIF (S2iL) medium. Duplicate RNA-seq samples were collected from WT mESCs (SL and S2iL), Ezh2KO-10 (SL and S2iL), and Ezh2KO-10-OE. For histone modification analysis, CUT&Tag profiling of H3K27me3 was performed on WT mESCs, C57-PrESCs, Ezh2KO-10, Ezh2KO-12, Ezh2KO-10-OE, and Ezh2KO-12-OE. Additionally, triplicate samples of PXGL-H1 human ESCs maintained in N2B27 basal medium (PXGL-H1 N2B27) were treated with 5 µM EPZ-6438 (PXGL-H1 N2B27+EPZ) for 3 days, followed by duplicate RNA-seq analyses.