METTL3-dependent m6A RNA methylation represses transposable elements derived enhancers to suppress naïve pluripotency in human pluripotent stem cells [4C]

Using a METTL3 inducible knockout (iKO) system in human expanded potential stem cells (hEPSCs), we uncovered that unlike in mESCs, METTL3 was indispensable for hPSCs’ maintenance, and that loss of METTL3 caused significant upregulation of naïve pluripotency genes accompanied with impaired hEPSCs differentiation particularly towards to both trophoblastic and amnionic lineage. Mechanistically, METTL3 iKO in hPSCs substantially increased expression of two primate-specific transposable elements (TEs), SVA_D and HERVK/LTR5_Hs. To investigate the function of RNA m6A methylation in hPSCs, we attempted to knock out the main methyltransferase METTL3 in hEPSCs. We used a knockout and inducible transgenic expression (iKO) system to transgenically express a METTL3 cDNA and rescue the KO phenotype. We firstly introduced a doxycycline (DOX)-inducible METTL3 cDNA stably integrated into hEPSCs by piggyBac transposition followed by knocking out the endogenous METTL3 by the CRISPR/Cas9. In the presence of DOX, two independent iKO cell lines with different METTL3 mutations were selected for subsequent investigation. To ensure comparable METTL3 expression levels in METTL3 iKO hEPSCs as in normal cells, we titrated DOX concentration and selected 5.0 ng/ml. Upon DOX withdrawal, exogenous METTL3 protein became undetectable after 5 days of culturing over 2 passages