The METTL3-YTHDC1 axis mediates architectural RNA m(6)A modification to modulate the integrity of chromatin TADs in MLLr + AML genome.

BACKGROUND: The RNA methyltransferase METTL3 as a key regulator of acute myeloid leukemia (AML) contributes to malignant transformation. Chromatin topologically associating domains (TADs) are critical for maintaining AML genome integrity, but the mechanism by which METTL3 facilitates TADs integrity in AML progression remains unclear. METHODS: To determine whether METTL3 is transcriptionally activated by MLL in MLL-rearranged (MLLr+) AML cells, we analyzed MLL ChIP-seq data. Additionally, we performed a multi-omics approach—including RNA-seq, immunoprecipitation-mass spectrometry (IP-MS) for METTL3 and YTHDC1, DNA: RNA hybrid immunoprecipitation sequencing (DRIP-seq), METTL3 ChIP-seq, CTCF ChIP-seq, H3K4me3 and H3K27ac ChIP-seq—to delineate the functional interplay among METTL3-YTHDC1 axis, R-loops, and CTCF in the MLLr + AML genome. Furthermore, METTL3-RIPseq, YTHDC1 RIPseq, CTCF RIPseq, m(6)A-seq, and Hi-C-seq assays were conducted to elucidate the function of the METTL3-YTHDC1 axis-mediated m(6)A modification of architectural RNAs (arcRNAs) in regulating CTCF-dependent TAD boundary activity. RESULTS: METTL3 is transcriptionally activated by MLL and forms a complex with YTHDC1 and CTCF, colocalizing at promoters and enhancers in MLLr + AML cells. METTL3 depletion disrupts CTCF binding sites (CBSs) and reduces chromatin accessibility at key leukemic genes (e.g. MYB and RUNX1). Hi-C analysis further reveals that YTHDC1 loss compromises CTCF-dependent 3D genome organization. METTL3-mediated m(6)A modification stabilizes arcRNAs and R-loops, which are crucial for maintaining TAD integrity at leukemic loci. Mechanistically, YTHDC1 recognizes m(6)A-modified arcRNAs (e.g. MALAT1) to enhance R-loop formation, thereby sustaining CTCF-mediated TAD activity in the MLLr + AML genome. CONCLUSIONS: Our study identifies the METTL3-YTHDC1-CTCF axis as a critical regulator of AML signature gene expression by orchestrating 3D genome organization. These findings provide novel insights into AML pathogenesis and reveal new therapeutic targets for this kind of aggressive disease. GRAPHICAL ABSTRACT: [Image: see text] SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s12943-025-02545-x.