PRC2在牙发育过程中的功能及机制研究

Gene expression is regulated by transcription factors and epigenetic regulatory factors. At present, there is a relative lack of research on epigenetic regulation of tooth development. One key factor of epigenetic regulation is Polycomb repressive complex 2 (PRC2), which has the activity of histone methyltransferase and is involved in the regulation of stem cell fate and the development of organs. But the role of PRC2 on tooth development is still unclear. This project intends to use Ezh2, Eed conditional knockout mice as models to study the sequence changes in the development of teeth, as the gene knockout can simulate the inhibition of PRC2 function. In vivo mice experiments to explore the role of PRC2 on tooth development, in vitro cell and tissue experiments to explore PRC2 impact on tooth development. The roles and mechanisms of PRC2 complex were finally revealed in tooth development, which helps to expand the understanding of epigenetic regulatory factors in tooth development, improve the theory of tooth developmental biology, and provide basic research on the mechanism of tooth developmental abnormalities.

基因表达受到转录因子和表观遗传调控因子的调控，目前从表观遗传调控方面研究牙发育过程的研究相对匮乏。表观遗传调控的关键因子多梳抑制复合体2（Polycomb repressive complex 2, PRC2）具有组蛋白甲基转移酶的活性，参与干细胞命运决定的调控和器官的生长发育，但其对牙发育过程的调控作用依然不清楚。本课题拟利用Ezh2、Eed条件性敲除小鼠模拟PRC2功能抑制后研究牙发育过程中的序列变化，小鼠体内实验探究PRC2对牙发育的作用，体外细胞和组织实验探究PRC2影响牙发育的机制，最终揭示PRC2复合体在牙发育过程的作用及其机制，有利于扩展表观遗传调控因子调控牙发育的认识，完善牙发育生物学的理论，对牙发育异常的机制提供基础研究。

牙齿是口腔中重要的咀嚼器官，牙冠表面由高度矿化的牙釉质被覆并暴露于口腔，小鼠磨牙随着牙冠萌出露出釉质的过程中伴随着成釉器的消失，表明牙上皮干细胞分化形成成釉器后存在干细胞耗竭情况来保证牙冠的正常发育。此过程伴随着一系列基因的时空性表达并受多层机制调控，其中包括遗传学与表观遗传学机制。作为一种被广泛研究的表观遗传调节因子，抑制性H3K27me3修饰已被证明在干细胞的命运决定中不可或缺。已有研究报道K14-Cre介导的Ezh2（H3K27me3的主要甲基转移酶）上皮特异性缺失的转基因小鼠中，未能发现釉质发育的紊乱。另有研究提出Eed作为PRC2复合核心亚基直接调控H3K27me3水平而对表观遗传调控起更重要的作用。因此，在该项研究中，本课题组利用单细胞测序技术首先证明了釉质发育过程存在干细胞生理耗竭情况，且发现Eed/H3K27me3信号变化与此趋势最为一致。使用CRISPR/Cas9技术构建了Eed flox小鼠后与K14-Cre小鼠杂交，以在胚胎牙上皮中条件性敲除Eed。转基因小鼠K14-Cre;Eedfl/fl出现磨牙釉质缺失表型。机制上Eed/H3K27me3缺失抑制Sox2、Ki67的表达，使磨牙干细胞提前出现耗竭，无法维持足够的干细胞产生成釉细胞分泌釉质并维持牙冠正常发育。体外实验恢复Eed/H3K27me3信号可挽救牙上皮干细胞(DESCs)的干性及增殖能力。此外，Eed可通过维持Ezh2水平对DESCs中H3K27me3修饰起调节作用。该项目探索了小鼠磨牙成釉器发育过程中干细胞生理性耗竭的情况，Eed/H3K27me3通过调控磨牙干细胞龛细胞数量及多能性来维持成釉器及釉质的正常形成，丰富了PRC2调控牙发育过程的作用和机制研究。