Molecular Mechanism Of Bmp2-deficiency-leading Dentinogenesis Defects And DNA Methylation Regulatory Mechanism In Odontoblastic Differentiation

1.Molecular mechanism of Bmp2-deficiency-leading dentinogenesis defectsObjective:Study of tooth development is an important objective in the field of dental research,and differentiation of odontoblast is a fundamental part of tooth development,study of the odontoblastic differentiation will help us to gain a better understanding of tooth development.BMP signaling pathway is very important in tooth development,and BMP2 has been reported to affect tooth development and odontoblastic differentiation in many researches but the regulatory mechanism remains elucive.This part of our study is based on the Bmp2 conditional knockout mouse and aimed to provide new evidence for the further understanding of Bmp2's roles in tooth development and odontoblastic differentiation.Methods:By using Osx-cre and Bmp2^flox/flox transgenic mice,we generate Bmp2 conditional knockout mice.After checking the tooth phenotype in the Bmp2-c KO mouse with micro-CT,the RNA sequencing of dental papilla cells was performed.With the help of bioinformatics tools,the regulatory networks were established.We screened out the networks according to the logic of miRNA and selected the candidate miRNAs family.After analyzing and confirming the effects of the candidate miRNA,we established parts of the framework based on Bmp2 in odontoblastic differentiation.Results:After generation of the Bmp2 conditional knockout mice,we analyzed the molars and incisor of 1 month and 3 months Bmp2-c KO mice with micro-CT.The resulted showed that the deficiency of Bmp2 would lead to a dentinogenesis defects phenotype including decrease of the dentin volume,thinner dentin layer and enlarged pulp cavity.But we found no histological changes in the tooth germ of Bmp2-c KO mice at embryonic day E13.5,E14.5 and E18.5.After analyzing the data of RNA-seq based on the dental papilla cells from Bmp2-c KO mice of embryonic day E18.5,majority of the differentially expressed genes were found to be enriched in the field of tooth development,mineralized tissue differentiation and mesenchymal cell differentiation.Further we expanded the list of differentially expressed genes by adjusting the threshold of fold change and found that 322 genes were decreased in Bmp2-c KO group while 248genes were increased in Bmp2-c KO group.We extracted the sequence of the 3'UTR region of the down-regulated differentially expressed genes and performed enrichment analysis on their miRNA binding sites,and conducted joint analysis with the microarray results.Through this strategy,mir-181 family was screened out as candidate miRNA family subjected in the following study.The mir-181 family is consist of three clusters and having four different kinds of mature miRNAs including mir-181a,mir-181b,mir-181c and mir-181d.All the mir-181 family members were tested in the Bmp2 deficient dental papilla cells,mir-181a/mir-181b were found to be up-regulated in Bmp2-c KO group while mir-181c were slightly down-regulated and mir-181d showed no signal.Then we tested the expression of mir-181a/mir-181b/mir-181c in the odontoblastic differentiation process of mouse dental papilla cells and we found that mir-181a/mir-181b have a decreasing pattern along with the differentiation process.Further we found that overexpression of mir-181a/mir-181b could obviously suppress odontoblastic differentiation of mouse dental papilla cells and treatment of rh BMP2 could inhibit mir-181a/mi-181b's expression.Bioinformatics analysis show Tgfbr1 and Acsl4 to be the potential targets of mir-181a/mir-181b.By using luciferase assay,we confirmed that Tgfbr1 is the target of mir-181a/mir-181b while Acsl4 is the target of mir-181b.We also found an up-trend expression pattern of Acsl4 along with the process of odontoblastic differentiation of mouse dental papilla cells,and we also found that treatment of rh BMP2 could up-regulate both Tgfbr1 and Acsl4 in mouse dental papilla cells.On the other hand,we test the effects of rh BMP2 on DNA methyltransferases including DNMT1,DNMT3a and DNMT3b,and this treatment showed consistent suppression on all these three DNA methyltransferases,and inhibition of DNMT1 could obviously enhance odontoblastic differentiation.Conclusion:We confirmed that Bmp2 deficiency could up-regulate mir-181a and mir-181b,and the up-regulation of mir-181a/mir-181b could suppress Tgfbr1 and Acsl4 which could contribute to the retard of the odontoblastic differentiation.Otherwise Bmp2 could regulate the expression of DNA methyltransferases and could probably participate odontoblastic differentiation through DNA methylation mechanism.2.DNA methylation regulatory mechanism within the odontoblastic differentiationObjective DNA methylation is a critical epigenetic modulation in regulating gene expression in cell differentiation process,however,its detailed molecular mechanism during odontoblastic differentiation remains elusive.We aimed to study the global effect of DNA methylation on odontoblastic differentiation and how DNA methylation affects the transactivation of transcription factor(TF)on its target gene.Methods Promoter DNA methylation microarray and motif enrichment assay were performed to predict the most DNA-methylation-affected TF motifs during odontoblastic differentiation.The enriched target sites and motifs were further analyzed by methylation specific PCR(MS-PCR)and sequencing.The functional target sites were validated in vitro with Luciferase assay.The regulatory effect of DNA methylation on the enriched target sites in primary human dental pulp cells and motifs were confirmed by in vitro methylation assay.Results By analysing the data of DNA methylation array,we found 7507 D0 specific DNA methylated peaks which were de-methylated in D9,while 6725 DNA regions that were methylated after odontoblastic differentiation.To further characterize these D0-specific or D9-specific regions,de novo motif enrichment analysis was performed in each group.The most enriched motif were revealed to be CCCCGCCC and the best matched transcription factor was found to be SP1.In our previous study,we found that SP1 could transactivate Klf4 in odontoblastic differentiation of mouse dental papilla cells and inhibition of DNA methyltransferase could enhance odontoblastic differentiation of mouse dental papilla cells and increase the expression of Klf4 and Klf4's promoter region showed de-methylation in the data of DNA methylation array.All these evidences suggested that SP1 could probably regulate Klf4 in a DNA methylation dependent manner during odontoblastic differentiation.On the other hand,we also found three CpG islands located in the promoter region of human's KLF4 gene.This finding made us wondering whether it's a conservative phenomenon that the promoter region of KLF4 would show change of methylation state and SP1 could transactivate KLF4.So we test the DNA methylation change of KLF4's promoter region in odontoblastic differentiation of human dental pulp cells.We found that only the first out of the three CpG islands showed methylation change after odontoblastic differentiation.After analysing the sequence of the first CpG island,we found three SP1 binding sites.Then we used Bis-seq to test the methylation change of all these three SP1 binding sites and found that all of these three SP1 binding sites showed methylation changes after odontoblastic differentiation.Mutation of SP1 binding site at-75 within KLF4's promoter region significantly decreased the luciferase activity which indicated that the-75 site was the most important binding site for SP1,and we further confirmed the binding of SP1 on this-75 site with ChIP-qPCR.At last we found that in vitro methylation of KLF4's promoter decreased the transactivation of SP1 on KLF4.Conclusion We confirmed that SP1 regulates KLF4 through binding site lying in a CpG island in KLF4's promoter region which demethylated during odontoblastic differentiation thus enhancing the efficiency of SP1's binding and transcriptional regulation on KLF4.