METTL3-dependent m6A RNA methylation regulates transposable elements and represses human naïve pluripotency through transposable element-derived enhancers.

N 6-methyladenosine (m6A) is the most prevalent messenger RNA modification with diverse regulatory roles in mammalian cells. While its functions are well-documented in mouse embryonic stem cells (mESCs), its role in human pluripotent stem cells (hPSCs) remains to be fully explored. METTL3 is the main enzyme responsible for m6A deposition. Here, using a METTL3 inducible knockout (iKO) system, we uncovered that, unlike in mESCs, METTL3 was indispensable for hPSC maintenance. Importantly, loss of METTL3 caused significant upregulation of pluripotency factors including naïve pluripotency genes and failure to exit pluripotency, thus impairing stem cell differentiation towards both embryonic and extraembryonic cell lineages. Mechanistically, METTL3 iKO in hPSCs promoted expression and enhancer activities of two primate-specific transposable elements (TEs), SVA_D and HERVK/LTR5_Hs. At SVA_D elements, loss of METTL3 leads to reduced H3K9me3 deposition. On the other hand, the activation of LTR5_Hs in the METTL3 iKO cells is accompanied by increased chromatin accessibility and binding pluripotency factors. The activated SVA_D and LTR5_Hs elements can act as enhancers and promote nearby naïve gene expression by directly interacting with their promoters. Together these findings reveal that METTL3-dependent m6A RNA methylation plays critical roles in suppressing TE expression and in regulating the human pluripotency network.