Mll4 regulates tooth enamel development

Amelogenesis, or enamel development, is a highly regulated process that leads to the formation of tooth enamel, which is critical for protecting teeth from decay and wear. Disruptions in the amelogenesis process can result in amelogenesis imperfecta, a group of genetic conditions characterized by inadequately formed enamel. This condition can include enamel hypoplasia, marked by thinning or underdevelopment of the enamel layer. Mutations in the MLL4 (KMT2D) gene, which encodes a histone H3-lysine 4-methyltransferase, are associated with Kabuki syndrome, a developmental disorder that can involve dental anomalies such as enamel hypoplasia. However, the specific role of MLL4 in amelogenesis and its underlying mechanisms remain poorly understood. To investigate the role of Mll4 in amelogenesis, we generated a conditional knockout mouse line with an ectoderm-specific deletion of Mll4 (Krt14-Cre;Mll4 ^fl/fl , or Mll4-cKO) and examined the gross, radiographic, histological, cellular, and molecular features in these mice. Micro-computed tomography and scanning electron microscopy analyses revealed that adult Mll4-cKO mice exhibited 100% penetrant amelogenesis imperfecta, characterized by hypoplastic and hypomineralized enamel, partially phenocopying human Kabuki syndrome. Additionally, Mll4-cKO neonates developed molar tooth germs with minor cusp shape alterations and mild delays in ameloblast differentiation at birth. RNA-seq analysis of the first molar tooth germ at birth revealed that approximately 33.7% of known amelogenesis-related genes were significantly downregulated in the Mll4-cKO teeth. Intersection with Mll4 CUT&RUN-seq results identified 8 overlapping genes directly targeted by Mll4. Re-analysis of a single-cell RNA-seq dataset in the developing mouse incisor teeth revealed distinct roles for these genes in Mll4-regulated differentiation across various cell subtypes within the dental epithelium. Among these genes, Satb1 and Sp6 are likely directly targeted by Mll4 during the differentiation of pre-ameloblasts into ameloblasts. Taken together, we propose that Mll4 plays a crucial role in amelogenesis by directly activating key genes involved in ameloblast differentiation.