A comprehensive study of odontoblastic differentiation is essential to understand the process of tooth development and to achieve the ability of tooth regeneration in the future. Zinc finger E-box-binding homeobox 1 (Zeb1) is a transcription factor expressed in various neural crest-derived tissues, including the mesenchyme of the tooth germ. However, its role in odontoblastic differentiation remains unknown. In this study, we found the expression of Zeb1 gradually increased during odontoblast differentiation in vivo, as well as during induced differentiation of cultured primary murine dental papilla cells (mDPCs) in vitro. In addition, the differentiation of mDPCs was repressed in Zeb1-silenced cells. We used RNA sequencing (RNA-seq) to identify the transcriptome-wide targets of Zeb1 and used assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) to explore the direct targets of Zeb1 in both the early stage (embryonic day 16.5; E16.5) and the late stage (postnatal day 0; PN0) of tooth development. We identified the motifs of transcription factors enriched in Zeb1-dependent accessible chromatin regions and observed that only in the early stage of mDPCs could Zeb1 significantly change the accessibility of chromatin regions. In vivo and in vitro experiments confirmed that silencing of Zeb1 at E16.5 inhibited dentinogenesis. Analysis of RNA-seq and ATAC-seq resulted in the identification of Runx2, a gene directly regulated by Zeb1 during early odontoblast differentiation. Zeb1 enhances the expression of Runx2 by binding to its cis-elements, and ZEB1 interacts with RUNX2. In the late stage of tooth development, we found that ZEB1 could directly bind to and increase the enhancer activity of an element upstream of Dspp and promote dentinogenesis. In this study, for the first time, we revealed that ZEB1 promoted odontoblast differentiation in the early stage by altering chromatin accessibility of cis-elements near genes such as Runx2, while in the late stage, it directly enhanced Dspp transcription, thereby performing a dual role.

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