TET1介导的DNA去甲基化调控FAM20C促进牙髓干细胞成牙本质分化

Human dental pulp stem cells (DPSCs) possess the capacity to differentiate into odontoblast-like cells in respond to exogenous stimuli and play an important role in reparative dentinogenesis. DNA demethylation is a crucial epigenetic modification which regulates the proliferation and differentiation of several cell types; however, its role in the odontogenic differentiation of DPSCs is still unknown. Ten-eleven translocation 1 (TET1) is a recently discovered DNA methyldioxygenase that plays important roles in promoting DNA demethylation and transcriptional regulation. Our previous study demonstrated that TET1 could enhance the capability of odontogenic differentiation of dental pulp cells. The genome-wide DNA methylation changes of dental pulp cells were explored and family with sequence similarity 20 C (FAM20C) was found to show a significant decrease in local DNA methylation during the odontogenic differentiation. Our preliminary experiments also indicated that FAM20C expression increased during the odontogenic induction of the cells. Accordingly, we hypothesize that TET1 enhances the odontogenic differentiation of DPSCs by regulating the DNA methylation status of FAM20C. This project will firstly explore the effects of TET1 and FAM20C on the physiological properties of DPSCs following overexpression and knockdown of these genes. The research then investigates the potential mechanism of TET1-dependent DNA demethylation of FAM20C and its effect on the odontogenic differentiation of the cells. Finally, the dentinogenesis experiment in vivo will be performed to demonstrate the capacity of dental pulp repair by TET1 or FAM20C modified DPSCs. This project may illuminate the regulatory effect of TET1-dependent DNA demethylation on the odontogenic differentiation of DPSCs and may open new avenues for research into dental pulp repair and regeneration.

牙髓干细胞（dental pulp stem cells，DPSCs）受外界刺激时能向成牙本质向分化，形成修复性牙本质。DNA甲基化/去甲基化是重要的表观遗传修饰形式，可调控多种组织发育及细胞分化，但其对DPSCs分化的影响尚未知。本课题组前期研究表明，DNA去甲基化酶TET1（Ten-Eleven-Translocation 1）可促进牙髓细胞成牙本质分化；通过DNA甲基化芯片筛选出细胞成牙本质分化过程中甲基化水平下调的Family with sequence similarity 20 C（FAM20C）。为探索TET1能否通过调控FAM20C的甲基化水平促进DPSCs成牙本质分化，本项目拟构建TET1及FAM20C过表达、沉默DPSCs模型，研究二者对DPSCs分化性能的影响，同时阐明TET1通过何种机制调控FAM20C甲基化。本项目可望从表观遗传学角度为牙髓修复再生研究提供新思路。

牙髓干细胞（dental pulp stem cells，DPSCs）受外界刺激时能向成牙本质向分化，形成修复性牙本质。DNA去甲基化是重要的表观遗传修饰形式，可调控多种组织发育及细胞分化，但其对DPSCs分化的影响尚未知。本课题组的研究表明，DNA去甲基化酶TET1（Ten-Eleven-Translocation 1）可促进牙髓细胞成牙本质分化；通过DNA甲基化芯片筛选出细胞成牙本质分化过程中甲基化水平下调的Family with sequence similarity 20 C（FAM20C）。为探索TET1能否通过调控FAM20C的甲基化水平促进牙髓细胞成牙本质分化，本项目从成人健康牙髓组织中分离培养牙髓细胞，发现TET1 和FAM20C均表达于牙髓细胞且表达量随矿化诱导时间的延长而升高。采用慢病毒载体成功构建稳定敲低TET1及FAM20C的牙髓细胞株，检测细胞增殖和成牙本质向分化能力，结果显示，敲低TET1可抑制牙髓细胞增殖及成牙本质分化能力；敲低FAM20C可抑制细胞成牙本质分化，过表达FAM20C则促进牙髓细胞成牙本质分化。诱导TET1沉默的牙髓细胞成牙本质向分化，检测TET1通过调控FAM20C等靶基因影响细胞分化的机制，结果发现，TET1沉默可导致全基因组5mC水平增加、5hmC降低，矿化诱导后 FAM20C、 DSPP、DMP1表达下调；FAM20C重组蛋白可部分挽救TET1沉默导致的DSPP、DMP1表达下降；TET1敲低后FAM20C启动子部分区域5hmC水平降低，TET1蛋白可结合于该5hmC变化区域附近，且矿化诱导后富集增加，同时发现TET1未富集于DSPP、DMP1等成牙本质分化因子的基因启动子区。以上结果提示，在牙髓细胞成牙本质向分化过程中，TET1蛋白可通过结合于FAM20C基因启动子区，介导其发生DNA羟甲基化依赖的转录激活，从而促进细胞分化。本项目成功阐明了TET1通过调控FAM20C去甲基化促进牙髓细胞成牙本质分化的机制，从表观遗传学角度为牙髓修复再生研究提供了新思路。