Integrated methylome and hydroxymethylome analysis identifies CAMK2G, NFATC4, and SFRP2 as TET1-regulated drivers of odontoblastic differentiation in human dental pulp cells.

Abstract

DNA 5-methylcytosine (5mC) dynamics are pivotal for tooth differentiation. However, the 5mC landscape during human dental pulp cell (hDPCs) odontoblastic differentiation and the role of Ten-eleven translocation 1 (TET1)-mediated 5mC regulation remain unclear. This study aimed to characterize methylation dynamics and identify TET1-regulated drivers of odontoblastic differentiation. Genome-wide DNA methylation was profiled by microarray, and hydroxymethylation changes after TET1 knockdown were assessed by hMeDIP-seq. Methylation and demethylation maintained a dynamic equilibrium during odontoblastic differentiation. A total of 9752 differentially methylated genes were identified and were enriched in pathways related to mesenchymal stem cell and ameloblast differentiation and neurotrophin signaling. hMeDIP-seq demonstrated that TET1 knockdown in hDPCs resulted in 2237 peaks with decreased 5-hydroxymethylcytosine (5hmC) levels and 3285 peaks with increased 5hmC levels compared to controls, corresponding to 1477 hypo-hydroxymethylated genes and 1905 hyper-hydroxymethylated genes. Integrated analysis of hypo-methylated genes and hypo-hydroxymethylated genes identified 88 overlapping candidates. Functional enrichment analyses highlighted tooth mineralization, osteoblast differentiation, TGF-β, and Wnt pathways. Among these, CAMK2G, NFATC4, and SFRP2 were specifically enriched in Wnt signaling and exhibited reduced 5hmC levels after TET1 knockdown. scRNA-seq data further confirmed increased expression of these three genes during odontoblast differentiation. This study delineated the genome-wide DNA methylation landscape during odontoblastic differentiation and identifies CAMK2G, NFATC4, and SFRP2 as novel TET1-regulated epigenetic drivers via 5hmC modification. These findings highlight potential therapeutic targets for epigenetic intervention in dentin regeneration and dental tissue engineering.