Genome-wide distribution of 5hmC in the dental pulp of mouse molars and incisors

Abstract

The dental pulp is critical for the production of odontoblasts to create reparative dentin. In recent years, dental pulp has become a promising source of mesenchymal stem cells that are capable of differentiating into multiple cell types. To elucidate the transcriptional control mechanisms specifying the early phases of odontoblast differentiation, we analysed the DNA demethylation pattern associated with 5-hydroxymethylcytosine (5hmC) in the primary murine dental pulp. 5hmC plays an important role in chromatin accessibility and transcriptional control by modelling a dynamic equilibrium between DNA methylation and demethylation. Our research revealed 5hmC enrichment along genes and non-coding regulatory regions associated with specific developmental pathways in the genome of mouse incisor and molar dental pulp. Although the overall distribution of 5hmC is similar, the intensity and location of the 5hmC peaks significantly differs between the incisor and molar pulp genome, indicating cell type-specific epigenetic variations. Our study suggests that the differential DNA demethylation pattern could account for the distinct regulatory mechanisms underlying the tooth-specific ontogenetic programs.

Keywords: 5-hydroxymethylcysteine (5hmC); TET enzymes; dental pulp; gene body; promoter.

Graphical Abstract

Fig. 1

Global distribution of 5hmC in the mouse dental pulp genome. (A) High-resolution map of 5hmC enrichment across the mouse chromosome 2. Genome-browser view of hMeDIP-seq in the mouse dental pulp genome. The molar and incisor hMeDIP-seq datasets along with input DNA controls are shown. Each track is represented as normalized density of reads/bp/million uniquely mapped reads. (B) 5hmC is primarily enriched at the intergenic regions and gene bodies of both the incisor and molar genomes. (C) The regulatory regions flanking the gene body were defined as the upstream promoter regions with an interval from −2 kb to 500 bp from the TSS and the downstream regulatory regions with an interval from 500 bp from the TES of the gene until the 2 kb downstream region. The average plot revealed that 5hmC signal is low around the TSS, and the overall intensity of 5hmC increases in promoters and gene bodies.

Fig. 2

KEGG analysis indicates that 5hmC is enriched at promoters and gene bodies of key developmental genes. (A) In the incisor pulp genome, 5hmC is enriched at promoters of genes associated with cell adhesion, cytotoxicity, cytokine receptor interaction and cell signalling pathways. (B) In the molar pulp genome, 5hmC signal is enriched at promoters of genes involved in cell adhesion. In the gene body region in both incisors and molars, 5hmC is enriched at active genes involved in development, differentiation, cell adhesion and cell signalling.

Fig. 3

The relationship between 5hmC enrichment and gene expression. (A) Genes were separated into five groups according to their RNA expression levels (from low to high: 0–20%, 20–40%, 40–60%, 60–80% and 80–100%) and 5hmC enrichment profiles were plotted across the promoter and gene body regions. In the molar pulp genome, 5hmC signal is relatively low at promoters of highly expressed genes (80–100% group) (A, left). A relatively low level of 5hmC is observed in the 60–80% and 40–60% gene groups. In contrast, 5hmC signal is higher in the 20–40% and 0–20% gene groups. Within the gene bodies, 5hmC is more enriched in the 40–60%, 20–40% and 0–20% groups (A, right). 5hmC signal is relatively modest in the 80–100% and 60–80% gene groups. Interestingly, the intensity of the 5hmC signal is higher at the gene bodies of the 20–40% group. (B) We found that 5hmC showed similar distribution patterns in the incisor pulp genome. The distribution of average FPKM values in dental pulp ranged from 0 to 19,388.6 for molars and from 0 to 6,056.69 for incisors from a total of 16,331 genes.

Fig. 4

The differential enrichment of 5hmC in the regulatory regions and gene bodies of mouse dental pulp. Within the incisor pulp genome, 5hmC is strongly associated with genes engaged in lineage commitment and specification. 5hmC signal is significantly higher (A) at the promoter of Sp7, (B) at the end of the gene body region of Twist2, (C) and along the non-coding regulatory regions and gene bodies of Dspp and Dmp1.