Unveiling the Role of N⁶-Methyladenosine on L1PA in Pluripotency and Totipotency

Transposable elements constitute nearly half of the mammalian genome, exhibit remarkable diversity across species, and have acquired new regulatory functions during evolution and development. In both human and mouse embryos, the robust silencing of transposons—particularly LTR elements—is essential for nor mal development, yet the underlying molecular mechanisms remain incompletely understood. Previous work in mouse embryonic stem cells revealed that caRNAs (chromatin-associated RNAs) are highly enriched for METTL3 installed m⁶A modifications. Loss of METTL3 impairs caRNA degradation and leads to enhanced chromatin acces sibility, demonstrating that m⁶A can directly regulate chromatin states via RNA. Further investigations further iden tified FTO as a key demethylase controlling m⁶A on L1 RNA; FTO depletion results in m⁶A accumulation, reduced L1 RNA stability, repression of L1-containing genes, and aberrant activation of 2C-specific transcripts, underscor ing the central role of the m⁶A-LINE1 axis in maintaining chromatin homeostasis. To investigate whether a similar mechanism operates in human cells, METTL3 was inhibited in METTL3 in naïve hESCs (human embryonic stem cells). METTL3 inhibition triggered widespread activation of totipotency-associated transcripts, including 8C-spe cific ERV1 and ERVL-MaLR LTR families, accompanied by markedly increased chromatin accessibility and a shift toward an 8C-like state. Strikingly, targeted reduction of m⁶A on the primate-specific L1PA family recapitulated the transcriptomic reprogramming induced by METTL3 inhibition, indicating that L1PA functions as a key upstream regulator of the human 8C program. Mechanistically, m⁶A on L1PA RNA dictates the selective recruitment of chro matin regulators: m⁶A restricts EP300 binding at ERV1 regions while promoting KAP1 enrichment at ERVL-MaLRloci, thereby suppressing aberrant LTR activation and maintaining differentiation-associated chromatin compac tion. Loss of m⁶A disrupts this balance, enhancing EP300-mediated H3K27ac deposition and weakening the KAP1 H3K9me3 silencing pathway, ultimately re-activating 8C gene networks and LTR elements. Together, this findings uncover an evolutionarily conserved yet species-adapted m⁶A-L1-LTR regulatory axis that governs LTR silencing in naïve hESCs. This work highlights the critical and conserved role of m⁶A modifications on repetitive RNA in shaping chromatin structure and directing cell-fate transitions.

CSTR: 32200.14.cjcb.2026.02.0001