Methyltransferase 1-mediated tRNA <i>N</i>7-methylguanosine modification regulates dentin formation during tooth root development via p53 signaling

tRNA N7-methylguanosine (m7G) is one of the most abundant epigenetic modifications in mammals, which is catalyzed by the methyltransferase 1-WD repeat-containing protein 4 (METTL1-WDR4) complex. Missense mutations in WDR4 have been linked to primordial dwarfism, which shows severe craniofacial developmental deformities including small teeth, but the underlying molecular mechanisms remain elusive. In this study, we explore the effect of m7G modification on dentin formation during tooth root development. METTL1 was actively expressed in mice developing tooth roots, and its expression became undetectable after tooth root formation. Next, we generated Prrx1-Cre driven Mettl1 (Prrx1Cre;Mettl1fl/fl) conditional KO mice to delete Mettl1 in dental mesenchyme and explored its regulation during tooth development. Micro-computed tomography revealed that the roots of the mandibular first molar in Prrx1Cre;Mettl1fl/fl mice were shorter and smaller compared to littermate control, with a reduction in the width of dentin and pre-dentin in both the root area and the crown area. Wdr4R215L/R215L mice also exhibited tooth root shortening and dentin thinning, phenocopying the Prrx1Cre;Mettl1fl/fl mice. Moreover, METTL1-depleted human dental pulp cells (hDPCs) showed decreased ability of proliferation, migration, and odontogenic differentiation. RNA-seq revealed upregulation of the p53 signaling pathway and cell cycle arrest after deletion of Mettl1. The proliferation and odontogenic differentiation of METTL1-depleted hDPCs is partially rescued with pifithrin-α (PFT-α), a p53 signaling inhibitor. Taken together, our results demonstrate that loss of METTL1-mediated tRNA m7G modification impairs the proliferation and odontogenic differentiation of hDPCs via the p53 signaling pathway and affects the dentin formation during tooth root development, providing a novel epigenetic mechanism underlying small teeth.