Ufl1 ‐Mediated UFMylation Sustains Amelogenesis by Stabilizing RUNX2

UFMylation is a conserved ubiquitin-like post-translational modification that controls protein stability and tissue homeostasis, while its role in amelogenesis remains largely uncharacterized. Here, we investigated the function of UFL1, the sole E3 ligase of the UFMylation pathway, in mammalian enamel development using K14-Cre-mediated epithelial-specific Ufl1 knockout mice. Ufl1 ablation caused severe amelogenesis imperfecta, with impaired enamel deposition, hypomineralization, and progressive tooth damage, accompanied by abnormal cervical loop development. Transcriptomic profiling revealed elevated endoplasmic reticulum stress and dysregulated expression of enamel mineralization genes, including significant downregulation of downstream targets of RUNX2, a master regulator of amelogenesis. We further demonstrated that UFL1 and DDRGK1 directly interacted with RUNX2, and UFL1-mediated UFMylation stabilized RUNX2 protein at the post-translational level. Taken together, this study identifies a novel UFMylation-RUNX2 regulatory axis essential for amelogenesis, providing new mechanistic insights into amelogenesis imperfecta and potential therapeutic targets for dental enamel defects.