ASH2L, Core Subunit of H3K4 Methylation Complex, Regulates Amelogenesis

Abstract

Histone methylation assumes a crucial role in the intricate process of enamel development. Our study has illuminated the substantial prevalence of H3K4me3 distribution, spanning from the cap stage to the late bell stage of dental germs. In order to delve into the role of H3K4me3 modification in amelogenesis and unravel the underlying mechanisms, we performed a conditional knockout of Ash2l, a core subunit essential for the establishment of H3K4me3 within the dental epithelium of mice. The absence of Ash2l resulted in reduced H3K4me3 modification, subsequently leading to abnormal morphology of dental germ at the late bell stage. Notably, knockout of Ash2l resulted in a loss of polarity in ameloblasts and odontoblasts. The proliferation and apoptosis of the inner enamel epithelium cells underwent dysregulation. Moreover, there was a notable reduction in the expression of matrix-related genes, Amelx and Dspp, accompanied with impaired enamel and dentin formation. Cut&Tag-seq (cleavage under targets and tagmentation sequencing) analysis substantiated a reduction of H3K4me3 modification on Shh, Trp63, Sp6, and others in the dental epithelium of Ash2l knockout mice. Validation through real-time polymerase chain reaction, immunohistochemistry, and immunofluorescence consistently affirmed the observed downregulation of Shh and Sp6 in the dental epithelium following Ash2l knockout. Intriguingly, the expression of Trp63 isomers, DNp63 and TAp63, was perturbed in Ash2l defect dental epithelium. Furthermore, the downstream target of TAp63, P21, exhibited aberrant expression within the cervical loop of mandibular first molars and incisors. Collectively, our findings suggest that ASH2L orchestrates the regulation of crucial amelogenesis-associated genes, such as Shh, Trp63, and others, by modulating H3K4me3 modification. Loss of ASH2L and H3K4me3 can lead to aberrant differentiation, proliferation, and apoptosis of the dental epithelium by affecting the expression of Shh, Trp63, and others genes, thereby contributing to the defects of amelogenesis.