Chromatin Accessibility Predetermines Odontoblast Terminal Differentiation

Embryonic development and stem cell differentiation are orchestrated by changes in sequential binding of regulatory transcriptional factors to their motifs. These processes are invariably accompanied by the alternations in chromatin accessibility, conformation, and histone modification. Odontoblast lineage originates from cranial neural crest cells and is crucial in dentinogenesis. Our previous work revealed several transcription factors (TFs) that promote odontoblast differentiation. However, it remains elusive as to whether chromatin accessibility affects odontoblast terminal differentiation. Herein, integration of single-cell RNA-seq and bulk RNA-seq revealed that in vitro odontoblast differentiation using dental papilla cells at E18.5 was comparable to the crown odontoblast differentiation trajectory of OC (osteocalcin)-positive odontogenic lineage. Before in vitro odontoblast differentiation, ATAC-seq and H3K27Ac CUT and Tag experiments demonstrated high accessibility of chromatin regions adjacent to genes associated with odontogenic potential. However, following odontoblastic induction, regions near mineralization-related genes became accessible. Integration of RNA-seq and ATAC-seq results further revealed that the expression levels of these genes were correlated with the accessibility of nearby chromatin. Time-course ATAC-seq experiments further demonstrated that odontoblast terminal differentiation was correlated with the occupation of the basic region/leucine zipper motif (bZIP) TF family, whereby we validated the positive role of ATF5 in vitro. Collectively, this study reports a global mapping of open chromatin regulatory elements during dentinogenesis and illustrates how these regions are regulated via dynamic binding of different TF families, resulting in odontoblast terminal differentiation. The findings also shed light on understanding the genetic regulation of dentin regeneration using dental mesenchymal stem cells.