Odontoblasts are primarily responsible for synthesizing and secreting extracellular matrix proteins,which are crucial for dentinogenesis.Our previous single-cell profile and RNAscope for odontoblast lineage revealed that cyclic adenosine monophosphate responsive element-binding protein 3 like 1(Creb3l1)was specifically enriched in the terminal differentiated odontoblasts.In this study,deletion of Creb3l1 in the Wnt1+lineage led to insufficient root elongation and dentin deposition.Assay for transposase-accessible chromatin with high-throughput sequencing(ATAC-seq)and RNA sequencing were performed to revealed that in CREB3L1-deficient mouse dental papilla cells(mDPCs),the genes near the closed chromatin regions were mainly associated with mesenchymal development and the downregulated genes were primarily related to biological processes including cell differentiation,protein biosynthesis and transport,all of which were evidenced by a diminished ability of odontoblastic differentiation,a significant reduction in intracellular proteins,and an even greater decline in extracellular supernatant proteins.Dentin matrix protein 1(Dmp1),dentin sialophosphoprotein(Dspp),and transmembrane protein 30B(Tmem30b)were identified as direct transcriptional regulatory targets.TMEM30B was intensively expressed in the differentiated odontoblasts,and exhibited a significant decline in both CREB3L1-deficient odontoblasts in vivo and in vitro.Deletion of Tmem30b impaired the ability of odontoblastic differentiation,protein synthesis,and protein secretion in mDPCs.Moreover,overexpressing TMEM30B in CREB3L1-deficient mDPCs partially rescued the extracellular proteins secretion.Collectively,our findings suggest that CREB3L1 participates in dentinogenesis and facilitates odontoblastic differentiation by directly enhancing the transcription of Dmp1,Dspp,and other differentiation-related genes and indirectly promoting protein secretion partially via TMEM30B.

Odontoblasts are primarily responsible for synthesizing and secreting extracellular matrix proteins,which are crucial for dentinogenesis.Our previous single-cell profile and RNAscope for odontoblast lineage revealed that cyclic adenosine monophosphate responsive element-binding protein 3 like 1(Creb3l1)was specifically enriched in the terminal differentiated odontoblasts.In this study,deletion of Creb3l1 in the Wnt1+lineage led to insufficient root elongation and dentin deposition.Assay for transposase-accessible chromatin with high-throughput sequencing(ATAC-seq)and RNA sequencing were performed to revealed that in CREB3L1-deficient mouse dental papilla cells(mDPCs),the genes near the closed chromatin regions were mainly associated with mesenchymal development and the downregulated genes were primarily related to biological processes including cell differentiation,protein biosynthesis and transport,all of which were evidenced by a diminished ability of odontoblastic differentiation,a significant reduction in intracellular proteins,and an even greater decline in extracellular supernatant proteins.Dentin matrix protein 1(Dmp1),dentin sialophosphoprotein(Dspp),and transmembrane protein 30B(Tmem30b)were identified as direct transcriptional regulatory targets.TMEM30B was intensively expressed in the differentiated odontoblasts,and exhibited a significant decline in both CREB3L1-deficient odontoblasts in vivo and in vitro.Deletion of Tmem30b impaired the ability of odontoblastic differentiation,protein synthesis,and protein secretion in mDPCs.Moreover,overexpressing TMEM30B in CREB3L1-deficient mDPCs partially rescued the extracellular proteins secretion.Collectively,our findings suggest that CREB3L1 participates in dentinogenesis and facilitates odontoblastic differentiation by directly enhancing the transcription of Dmp1,Dspp,and other differentiation-related genes and indirectly promoting protein secretion partially via TMEM30B.

Genome-wide association studies (GWASs) are the most widely used method to identify genetic risk loci associated with orofacial clefts (OFC). However, despite the increasing size of cohort, GWASs are still insufficient to detect all the heritability, suggesting there are more associations under the current stringent statistical threshold. In this study, we obtained an integrated epigenomic dataset based on the chromatin conformation of a human oral epithelial cell line (HIOEC) using RNA-seq, ATAC-seq, H3K27ac ChIP-seq, and DLO Hi-C. Presumably, this epigenomic dataset could reveal the missing functional variants located in the oral epithelial cell active enhancers/promoters along with their risk target genes, despite relatively less-stringent statistical association with OFC. Taken a non-syndromic cleft palate only (NSCPO) GWAS data of the Chinese Han population as an example, 3664 SNPs that cannot reach the strict significance threshold were subjected to this functional identification pipeline. In total, 254 potential risk SNPs residing in active cis-regulatory elements interacting with 1 718 promoters of oral epithelium-expressed genes were screened. Gapped k-mer machine learning based on enhancers interacting with epithelium-expressed genes along with in vivo and in vitro reporter assays were employed as functional validation. Among all the potential SNPs, we chose and confirmed that the risk alleles of rs560789 and rs174570 reduced the epithelial-specific enhancer activity by preventing the binding of transcription factors related to epithelial development. In summary, we established chromatin conformation datasets of human oral epithelial cells and provided a framework for testing and understanding how regulatory variants impart risk for clefts.
Chromatin ; Cleft Lip/genetics* ; Cleft Palate/genetics* ; Epithelium ; Genome-Wide Association Study ; Humans