Title: An Essential Role of TET-meditated DNA Modification in Enamel Formation
Jun Wang (Presenter)
Texas A&M College of Dentistry
Wei Han, Institute of Biosciences and Technology, Texas A&M University
Yun Huang, Institute of Biosciences and Technology, Texas A&M University
Jian Feng, Texas A&M College of Dentistry
Objectives: DNA methylation and demethylation homeostasis is an essential epigenetic regulatory mechanism during normal development and pathogenesis of various diseases. TET (ten-eleven translocation)-family dioxygenases (TET-1, -2 and -3) are one of the key enzymes in mediating DNA demethylation. Defects in TETs function are directly linked to diseases, but their roles in enamel formation are largely unknown. The goal of this study is to determine whether and how TETs control amelogenesis.
Methods: The K14-Cre mice were crossed with Tet1 flox/flox, Tet2 flox/flox and Tet3 flox/flox (single or the combination of Tet1-Tet2 or Tet1-Tet3 or Tet2-Tet3) to specifically inactivate Tet genes in the epithelial cells, including the dental epithelial cells that are responsible for enamel formation. The combined approaches of μCT, histology, immunohistochemistry and RNA-seq were used to determine the pathological changes of ameloblasts after TETs abrogation qualitatively and quantitatively.
Results: None of single Tet conditional knockout (cKO) or Tet1-Tet2 or Tet1-Tet3 double knockout (dKO) mice developed apparent enamel defects. In contrast, the Tet2-Tet3 dKO mice displayed severe hypoplastic amelogenesis imperfecta similar to human cases. μCT analysis revealed >93% losses in first molar enamel volume by post-natal day 18. Among the remaining enamel, the enamel density reduced by nearly 30% (n=4, P＜0.001). Histological analyses revealed a lack of enamel matrices, a loss in cell polarity, and sharp reductions in cytoplasm volume but great increase in nuclear volume in dKO ameloblasts. Molecular mechanism studies showed an essential absence of ameloblastin expression in dKO ameloblasts. Importantly, there was a great reduction in 5-hydroxymethylcytosine (a key oxidative product in the active demethylation process) in dKO ameloblast nuclei, which is partially responsible for the ameloblasts differentiation defects.
Conclusions: TETs control amelogenesis via balancing DNA methylation and demethylation process, which maintains ameloblast nuclear size, the ratio of nucleus/cytoplasm, cell polarity, enamel matrix production and mineralization.