Development and differentiation in eukaryotic systems are regulated by master transcription factors (TFs) that govern cell-specific transcriptional programs (Li et al., 2007). During cell fate transitions, master TFs target specific DNA sites by their DNA-binding domains, modify chromatin structure and reset genetic networks (Voss and Hager, 2014). Besides, TFs have transcription activating domains that are functionally phosphorylated and involved in post-translational modification by phosphoswitches (Lynch et al., 2011). Phosphorylation can rapidly alter the activity and control function of TFs (Whitmarsh and Davis, 2000). Histone modifications regulate chromatin accessibility and nucleosome unfolding, allowing cells to adjust their transcriptional programs (Li et al., 2007). However, mechanisms in which TFs are phosphorylated to modify chromatin and activate new genetic networks need further investigation.

Cranial neural crest-derived dental mesenchymal cells differentiate into dental papilla cells, odontoblasts and form dentin after the bell stage during odontogenesis (Ruch, 1998). During odontoblastic differentiation, several TFs have diverse functions by regulating gene expression programs, such as RUNX2, DLX3, SOX2, and KLF6 (Li et al., 2011; Yang et al., 2017; Chen et al., 2021). Our previous studies demonstrateed that KLF4, SALL1, and ZEB1 promote odontoblastic differentiation through different mechanisms (Tao et al., 2019; Lin et al., 2021; Xiao et al., 2021). While the roles of numerous TFs have been elucidated during odontoblastic differentiation, the initial changes in chromatin accessibility and related genetic networks are still not definite.

Our recent study on the global mapping of open chromatin regulatory elements during dentinogenesis demonstrated that the differentiation stage of the in vitro differentiation of mouse dental papilla cells (mDPCs) represented the secretary and mature odontoblasts in vivo. During this stage, the transcription network shifted during the odontogenesis to mineralization transition, which was accompanied by the occupation of the basic leucine zipper (bZIP) TF family in a specific cluster of open chromatin regions (Zhang et al., 2021). Given that those regions are functionally enriched in biomineralization, we ask whether members of the bZIP TF family could alter the chromatin conformation in preparation for the differentiation process.

Among all members of the bZIP TF family, activating transcription factor 2 (ATF2) was characterized as a mediator response to various stimuli (Shaulian and Karin, 2002). ATF2 binds to different response elements on target genes and elicits distinct transcriptional programs (Gong et al., 2002; Lopez-Bergami et al., 2010). Phosphorylation of ATF2 (p-ATF2) at threonine-69 and 71 (T69, T71) was reported to allow p-ATF2 to bind to gene promoters (Hayakawa et al., 2004; Kirsch et al., 2020), controlling intrinsic histone acetyltransferase (HAT) activity with specificity for histones H2B and H4 (Kawasaki et al., 2000). Another study found that when bound to amino acid response elements, p-ATF2 acetylates H2B (Bruhat et al., 2007). It was demonstrated that p-ATF2 binds to the TGF-β2 promoter and increases histone H2B acetylation (Namachivayam et al., 2015). Thus, ATF2 not only controls the transcription of target genes through direct binding to promoter, but also has a regulatory role in chromatin restructuring. Deletion or mutation of ATF2 phosphorylation sites resulted in epithelial-mesenchymal transition (EMT)-related developmental defects (Breitwieser et al., 2007). Atf2 itself is required for early development, and complete knockout of Atf2 leads to postnatal lethality due to meconium aspiration syndrome (Maekawa et al., 1999), whereas ATF2 deficiency leads to defects in endochondral ossification and neurological abnormalities (Reimold et al., 1996; Ackermann et al., 2011). In addition, few studies have focused on the expression pattern of ATF2 or p-ATF2 in teeth (Nishikawa, 2004; Bolat and Keklikoglu, 2010; Keklikoglu and Akinci, 2015). ATF2 immunoreactivity is observed in human dental follicles and terminally differentiated odontoblasts (Bolat and Keklikoglu, 2010; Keklikoglu and Akinci, 2015). A transient increase in p-ATF2 is also observed in the nuclei of late secretion ameloblast of rat incisors (Nishikawa, 2004). These investigations suggest that Atf2 participates in tooth development, but whether ATF2 or p-ATF2 is essential in regulating odontoblastic differentiation remains unclear.

In this study, we identified the p-ATF2 was crucial for promoting odontoblastic differentiation at initiation stage, accelerating mineralization. When mDPCs were treated with the differentiation medium or the extracellular signaling molecule BMP2, ATF2 was rapidly phosphorylated by p-p38 within several hours. Then, p-ATF2 was able to promote differentiation by targeting the chromatin regions near mineralization-related genes, such as Alpl, encouraging H2BK12 acetylation and increasing the chromatin accessibility of adjacent regions. Collectively, our findings indicated that p-ATF2 promoted odontoblastic differentiation by phosphorylation and provided insights into the transcriptional regulation of odontoblastic differentiation.