Klf4 Promotes Dentinogenesis and Odontoblastic Differentiation via Modulation of TGF-β Signaling Pathway and Interaction With Histone Acetylation

Abstract

Transcription factors bind to cell‐specific cis‐regulatory elements, such as enhancers and promoters, to initiate much of the gene expression program of different biological process. Odontoblast differentiation is a necessary step for tooth formation and is also governed by a complex gene regulatory network. Our previous in vitro experiments showed that Krüppel‐like factor 4 (KLF4) can promote odontoblastic differentiation of both mouse dental papillary cells (mDPCs) and human dental pulp cells; however, its mechanism remains unclear. We first used Wnt1‐Cre; KLF4fx/fx (Klf4 cKO) mice to examine the role of KLF4 during odontoblast differentiation in vivo and demonstrated significantly impaired dentin mineralization and enlarged pulp/root canals. Additionally, combinatory analysis using RNA‐seq and ATAC‐seq revealed genomewide direct regulatory targets of KLF4 in mouse odontoblasts. We found that KLF4 can directly activate the TGF‐β signaling pathway at the beginning of odontoblast differentiation with Runx2 as a cofactor. Furthermore, we found that KLF4 can directly upregulate the expression levels of Dmp1 and Sp7, which are markers of odontoblastic differentiation, through binding to their promoters. Interestingly, as a transcription factor, KLF4 can also recruit histone acetylase as a regulatory companion to the downstream target genes to positively or negatively regulate transcription. To further investigate other regulatory companions of KLF4, we chose histone acetylase HDAC3 and P300. Immunoprecipitation demonstrated that KLF4 interacted with P300 and HDAC3. Next, ChIP analysis detected P300 and HDAC3 enrichment on the promoter region of KLF4 target genes Dmp1 and Sp7. HDAC3 mainly interacted with KLF4 on day 0 of odontoblastic induction, whereas P300 interacted on day 7 of induction. These temporal‐specific interactions regulated Dmp1 and Sp7 transcription, thus regulating dentinogenesis. Taken together, these results demonstrated that KLF4 regulates Dmp1 and Sp7 transcription via the modulation of histone acetylation and is vital to dentinogenesis. © 2019 American Society for Bone and Mineral Research.

Epigenetic Regulation of Dental Pulp Stem Cell Fate

“…The data of a CHIP analysis revealed that when dental pulp cells are induced into odontoblasts, HDAC3 mainly interacts with KLF4 on the promoter of Dmp1 and Sp7 on day 0 of induction, while p300 interacts on day 7 of induction. These results reveal that KLF4 can regulate the odontoblast differentiation by affecting the histone acetylation on the promoter regions of DMP1 and Sp7 and by interacting with p300 and HDAC3 [ 134 ].…”

HDAC6 Regulates the Fusion of Autophagosome and Lysosome to Involve in Odontoblast Differentiation

“…The deacetylation role of HDACs has been explored in cell differentiation. Our previous study showed that acetylation was involved in dentinogenesis, and HDAC3 regulated histone acetylation during odontoblast differentiation (Tao et al, 2019). HDAC6 could also deacetylate histones related to Runx2 (Ozaki et al, 2013) and acted as a co-repressor for Runx2 transcription to suppress the osteoblastic differentiation of MC3T3 cells (Zhu et al, 2011).…”

“…For functional knock-down of HDAC6, an HDAC6-targeting small interfering RNA (siRNA; siHDAC6, Sigma) and the universal negative control siRNA were transfected into hDPCs. Cells were seeded into 6-well plates and then transfected with siRNA using Lipo2000 (Invitrogen, Thermo Fisher Scientific, Waltham, MA, United States) according to the manufacturer's instructions (Tao et al, 2019). Knockdown efficacy was confirmed by western blot.…”

“…HDAC1 and HDAC3, which are type I HDACs, are thought to inhibit Runx2 expression and affect osteoblast differentiation (Schroeder et al, 2004;Liu et al, 2015). HDAC3 has been shown to interact with KLF4 during the initiation of odontoblastic induction (Tao et al, 2019). HDAC6 is also involved in human osteoblast mechanosensation (Ehnert et al, 2017).…”

circRNA Expression Profile in Dental Pulp Stem Cells during Odontogenic Differentiation

“…A previous study reported the differentiation of pulp-like tissues in a subcutaneous immunodeficient mouse model, after the implantation of human tooth root segments with 3D-DPSCs [ 2 ]. Although many groups have been studying the odontogenic differentiation [ 13 – 15 ], the exact molecular mechanisms are still poorly understood, and thus, the regeneration of dentin-pulp complex in the clinical context is still not implemented [ 16 ]. Therefore, the search for new biomarkers linked to the molecular mechanisms of odontogenic differentiation is extremely important for studying pulp regeneration.…”