METTL3 safeguards cell identity and epigenome of human trophoblast stem cells

N6-methyladenosine (m6A) is the most prevalent mRNA modification with diverse regulatory functions in mammalian cells. However, its significance in the human trophoblast lineage and human placenta development remains largely unexplored. Here, we found that in human pregnancy, METTL3 and METTL14, the primary enzymes responsible for m6A deposition, specifically mark cytotrophoblast cells in the human placenta. By utilizing a METTL3 inducible knockout (iKO) system, we demonstrated the essential role of METTL3 in generating human trophoblast stem cells (hTSCs) from human expanded potential stem cells (hESPCs). Furthermore, METTL3 is critical for the self-renewal ability and cellular identity in the established hTSCs. Mechanistically, METTL3-dependent m6A modification regulates histone modifiers such as EP300 and EZH2 to maintain the epigenetic homeostasis of hTSCs. The depletion of METTL3 in hTSCs results in epigenetic dysregulation, leading to activation of inflammation and premature senescence, which are associated with the preeclampsia phenotype. Notably, our findings from in vitro model show that inhibiting EP300 could efficiently rescue the preeclampsia signature at the transcriptomic level. These results underscore the critical roles of METTL3-dependent m6A RNA methylation in human placentation and the development of placental diseases. Overall design: we performed RNA sequencing (RNA-seq) to analyze global gene expression profiles in P0, P2 and P3 hTSCs using two independent cell lines. We performed methylated RNA immunoprecipitation sequencing (MeRIP-seq) to analyze m6A modification profiles in P0 and P2 hTSCs. We performed RNA stability analysis, in which we introduced Actinomycin D to transiently suppress global transcription then performed RNA-seq experiments at different time points to evaluate the transcription dynamics in P0 and P2 hTSCs. we conducted CUT&Tag for H3K27ac, H3K27me3, H3K9me3, H3K4me1 and JUND, as well as ATAC-seq for chromatin accessibility in P0 and P3 hTSCs.