Chromatin Accessibility Analysis Reveals Functional Transcription Factors and Regulators in Dental Pulp Stem Cell Differentiation

<p> Purpose: An increase in chromatin accessibility arises from the stretched chromatin structure, which enables the association of transcription complexes with local genomic DNA, and thus induces target mRNA transcription. This is one of the main molecular frameworks for epigenetic regulation of gene expression. In this study, the changes in whole-genomic chromatin accessibility that occur during induced differentiation of human dental pulp stem cells (hDPSC) were analyzed by assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq).</p><p> Methods: hDPSC were cultured in a mineralization induction medium. ATAC-seq samples were prepared before and after the 12-day culture. Bioinformatics analyses were conducted, involving open chromatin peak extraction, sample comparison, consensus DNA binding (CDB) sequence identification, and gene ontology (GO) analysis of neighboring genes for each peak.</p><p> Results: The bioinformatics analyses identified 45,493 and 45,370 open chromatin peaks at day 0 and day 12, respectively. The CDB sequences of transcription factors, including TEADs, bZIPs, and RUNXs, and insulators, including CTCF and BORIS, were commonly enriched in these peaks. Furthermore, GO analysis of neighboring genes of CTCF-CDB revealed an accumulation of the genes associated with the Hippo signaling pathway, in which TEADs act as the essential transcription factors, at day 12 but not at day 0. Additionally, the gene loci of the odonto/osteogenic genes, such as BMP2 and BMPR1B, were identified as accessible chromatin regions controlled by CTCF-CDB at day 12 but not at day 0. From these results, the types of CDB in the stretched chromatin region were not drastically changed before and after hDPSC differentiation. However, because CTCF participates in the dynamic genomic organization to regulate local chromatin accessibility and restrict the territory of distal enhancers and suppressors, CTCF aids TEADs in finding and associating with odonto/osteogenic gene loci, inducing odonto/osteogenic gene expression during differentiation.</p><p> Conclusion: Bioinformatics analysis indicated that local epigenetic alteration mediated by the insulators may play pivotal roles in hDPSC differentiation, and therefore, chromatin accessibility modulation may have therapeutic potential for inducing odontogenic differentiation of hDPSC.</p>