METTL3 suppresses cardiac differentiation via the PRC2/SMARCA2 Axis

Abstract Background Identifying the regulatory factors and uncovering the mechanisms of human cardiac differentiation are useful for studying cardiac development and therapeutic application. METTL3, the core methyltransferase of m 6 A modification, is essential for tissue development and various pathological processes, however, its role in human cardiac differentiation remains elusive. Methods Cardiac differentiation of human embryonic stem cells (hESCs) was induced using a monolayer differentiation approach. An inducible METTL3 overexpression cell line was generated using a doxycycline (DOX)-inducible system. A METTL3-degraded cell line was generated by introducing an SMASh tag into the METTL3 locus. Cardiac differentiation efficiency was assessed by quantitative real-time PCR (qRT-PCR) and immunofluorescence staining. Genome-wide m 6 A enrichment was analyzed by MeRIP-seq. METTL3-mediated m 6 A methylation activity was assessed via dot blot analysis. Genome-wide H3K27me3 distribution was profiled by H3K27me3 ChIP-seq, and locus-specific enrichment of H3K27me3 was validated by ChIP-qPCR. The effect of METTL3 on EED and EZH2 expression and stability was evaluated by qRT-PCR, polysome profiling, western blot, and ubiquitination analysis, respectively. The function of ESC-to-mesoderm (MES) METTL3-degraded cardiomyocytes (CMs) was investigated after transplantation into a myocardial infarction (MI) mouse model. Results We found that METTL3 expression decreased during cardiac differentiation. Overexpression of METTL3 inhibited cardiac differentiation, whereas its degradation promoted this process. Mechanistically, METTL3 regulated ESC-to-MES transition independently of its m 6 A methyltransferase activity. METTL3 degradation reduced H3K27me3 levels at cardiac lineage-associated loci. Specifically, METTL3 physically interacted with EED and EZH2 and maintained their protein stability by suppressing ubiquitin-mediated proteasomal degradation. SMARCA2 degradation abolished the pro-differentiation effect of METTL3 degradation. Furthermore, early-stage METTL3-degraded CMs derived from hESC retained therapeutic potential in the MI model. Conclusions METTL3 acts as a negative regulator of cardiac differentiation by modulating the ESC-to-MES transition through stabilization of EED and EZH2 against ubiquitin-dependent proteasomal degradation. Early-stage degradation of METTL3 enhanced CMs yield while maintaining the therapeutic potential of hESC-derived CMs for cardiac repair. These findings reveal a previously unrecognized role of METTL3 in human cardiac lineage commitment and suggest that stage-specific METTL3 degradation represents a promising strategy to improve the yield of CMs for regenerative therapy.