Histone H3 trimethylation at lysine 36 guides m⁶A RNA modification co-transcriptionally

DNA and histone modifications have notable effects on gene expression¹. Being the most prevalent internal modification in mRNA, the N⁶-methyladenosine (m⁶A) mRNA modification is as an important post-transcriptional mechanism of gene regulation^2–4 and has crucial roles in various normal and pathological processes^5–12. However, it is unclear how m⁶A is specifically and dynamically deposited in the transcriptome. Here we report that histone H3 trimethylation at Lys36 (H3K36me3), a marker for transcription elongation, guides m⁶A deposition globally. We show that m⁶A modifications are enriched in the vicinity of H3K36me3 peaks, and are reduced globally when cellular H3K36me3 is depleted. Mechanistically, H3K36me3 is recognized and bound directly by METTL14, a crucial component of the m⁶A methyltransferase complex (MTC), which in turn facilitates the binding of the m⁶A MTC to adjacent RNA polymerase II, thereby delivering the m⁶A MTC to actively transcribed nascent RNAs to deposit m⁶A co-transcriptionally. In mouse embryonic stem cells, phenocopying METTL14 knockdown, H3K36me3 depletion also markedly reduces m⁶A abundance transcriptome-wide and in pluripotency transcripts, resulting in increased cell stemness. Collectively, our studies reveal the important roles of H3K36me3 and METTL14 in determining specific and dynamic deposition of m⁶A in mRNA, and uncover another layer of gene expression regulation that involves crosstalk between histone modification and RNA methylation.