Abstract
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.
Highlights
Cell fate specification is achieved through spatiotemporal gene expression during embryonic development, tissue regeneration, or cell reprogramming (Spitz and Furlong, 2012)
Because nucleosome-free regions (NFRs) enriched in mouse dental papilla cells (mDPCs)-D9 groups are associated with odontoblast terminal differentiation, we explored whether these regions were regulated by any specific transcription factors (TFs)
RNA-seq along with ChIP-seq against histone modifications in postnatal dental pulp perivascular-derived mesenchymal stem cells illustrated that odontoblast-specific genes, such as Dspp and Dmp1, were in a transcriptionally permissive state inhibiting by RING1B (Yianni and Sharpe, 2018)
Summary
Cell fate specification is achieved through spatiotemporal gene expression during embryonic development, tissue regeneration, or cell reprogramming (Spitz and Furlong, 2012). A set of tissuespecific transcription factors (TFs) regulate these genes at the transcriptional level They potentially recognize and interact with their specific DNA-binding motifs in the genome to drive lineagespecific gene expression at different developmental stages (Lee and Young, 2013). A majority of potential DNA-binding sites are inaccessible because the genomic DNA in eukaryotic cells is occluded by higher-order chromatin structures (Luger et al, 1997). Within this context, gene regulation occurs at gene regulatory regions in the opened chromatin, which allows for the binding of TFs and functioning of RNA polymerase (Calo and Wysocka, 2013). The mechanism by which TFs recognize their binding motifs or influence the chromatin accessibility to initiate different biological processes remains largely unknown
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