RUNX1 N6-methyladenosine methylation enhances cytoskeleton remodelling and boosts cardiac fibrosis

The RUNX family of transcription factors is critical for heart development, physiology, and cardiovascular disease. However, current models of transcription factor binding seldom incorporate RNA modifications, and the latest methods that include them remain limited. This gap impedes accurate profiling of transcription factor affinities. In particular, the role of N6-methyladenosine (m⁶A)-mediated mechanisms in regulating RUNX factors during cardiac fibrosis is still poorly understood.

RNA sequencing of human atrial fibrillation tissues identified transcription factors with enriched expression associated with cardiac gene expression. Cardiac fibroblast-specific Ythdf1 conditional knockout mice (Postn-Cre × Ythdf1^flox/flox), along with Cre and wild-type controls, were subjected to ISO/TAC treatment to induce cardiac fibrosis. AAV9 vectors carrying Postn promoter-driven shRNA targeting Runx1 were administered to ISO-treated mice to evaluate its role in cardiac fibrosis. Multi-omics approaches, including MeRIP-seq, single-cell RNA-seq, RNA-seq, and ChIP-seq, combined with histological and biochemical analyses, were employed to elucidate the mechanism by which YTHDF1 regulates Runx1 expression. Runx1 was reconstituted in Ythdf1-deficient cardiac fibroblasts and mouse hearts to assess its effects on fibroblast proliferation and fibrosis.

Runx1 expression was elevated in human atrial fibrillation samples, experimental cardiac fibrosis models, and TGF-β1-stimulated cardiac fibroblasts. Fibroblast-specific Runx1 knockdown attenuated cytoskeletal remodelling, suppressed fibroblast proliferation, and inhibited cardiac fibrosis. Mechanistically, Runx1 upregulation was associated with increased m⁶A methylation on its mRNA. Site-specific m⁶A modification at peak_21317 was essential for promoting YTHDF1 binding to Runx1 mRNA and enhancing its translation. This led to increased transcriptional activation of connective tissue growth factor (Ctgf), promoting cytoskeletal reorganization and collagen deposition. Importantly, epitranscriptomic inhibition of Runx1 ameliorated experimental cardiac fibrosis.

Our study reveals a novel epitranscriptomic pathway wherein YTHDF1 recognizes m⁶A-modified Runx1 mRNA, enhancing its translation and thereby stimulating RUNX1-mediated Ctgf transcription. This process drives cytoskeletal remodelling, cardiac fibroblast proliferation, and fibrosis in an m⁶A-dependent manner. These findings offer new perspectives for developing preventive strategies against cardiac fibrosis.