Lhx8募集Suv39h1表观遗传调控成牙本质细胞增殖分化平衡的功能与机制

Elucidation of the exact molecular mechanism controlling odontoblast differentiation process is one of fundamental works to fully understand the tooth development and to successfully achieve dentin regeneration. Our previous work has demonstrated that homeobox gene Lhx8 plays a critical role in the maintenance of cell proliferation and progenitor status via epigenetic suppression of key odontoblast differentiation genes, such as Dspp. Our further pilot studies revealed that Suv39h1, a key histone epigenetic modification enzyme, displayed a highly similar expression pattern with that of Lhx8 in the dental mesenchyme. Importantly, we found that Lhx8 showed strong physical interaction with Suv39h1, and knock-down of Suv39h1 significantly blocked the odontoblast terminal differentiation suppression induced by Lhx8. Based on these previous results, we hypothesize that Lhx8 epigenetically regulates the balance of cell proliferation and differentiation via recruiting Suv39h1. In this project, we will further investigate whether Lhx8 interact with Suv39h1 and determine whether Lhx8 can mediate recruitment of Suv39h1 to the promoters of key genes (Dspp, Col1a1, and Msx1/2) controlling odontoblast terminal differentiation and induce tri-methylation of H3K9 of these genes, which therefore will sustain the suppression and the progenitor status of dental mesenchyme cells. Such epigenetic suppression will therefore control the delicate balance between proliferation and differentiation of dental mesenchyme cells. Our study will provide a novel epigenetic insight into molecular mechanisms of tooth development.

阐明成牙本质细胞分化的分子机制是认识牙发育过程和实现牙本质再生的关键。申请人前期研究发现同源盒基因Lhx8通过表观修饰抑制Dspp等成牙本质细胞终末分化关键基因表达，维持细胞增殖和未分化的前体状态。申请人进一步预实验显示：组蛋白甲基转移酶Suv39h1在牙源性间充质中的表达谱与Lhx8高度相似，且与Lhx8具有强相互作用；干涉Suv39h1显著阻断Lhx8对成牙本质细胞终末分化的抑制。据此，我们提出科学假说：Lhx8通过募集Suv39h1在表观遗传水平调控牙发育进程。本项目将通过牙胚原位杂交、牙胚重组和细胞学等实验，明确牙发育早期，Lhx8是否通过与Suv39h1相互作用，将其募集到Dspp，Col1a1，Msx1/2等成牙本质细胞终末分化关键基因的启动子区，导致局部H3K9me3修饰和基因表达抑制，维持牙源性间质细胞的前体细胞状态和增殖分化平衡。本研究有望揭示牙发育的表观遗传新机制。

牙发育是受基因网络精细调控的过程，前期研究显示Lhx8在牙间充质发育尤其是成牙本质细胞定向分化过程中发挥重要作用，但具体机制仍不清楚。因此，本项目旨在探讨Lhx8影响牙/骨源性间充质干细胞定向命运决定和增殖分化平衡的分子机制。.本项目研究发现，在牙髓干细胞中Lhx8和Suv39h1存在相互作用，在小鼠胚胎E12.5-16.5期间，Lhx8 和Suv39h1在牙间充质细胞中共表达，且早期上皮信号FGF8和晚期间充质信号BMP2的表达模式与上述两者在牙间充质中的表达高度相关；进一步研究发现，FGF8和BMP2分别在转录水平激活和抑制Lhx8的表达，牙间充质早期Lhx8高表达通过与Suv39h1使细胞维持干性未分化状态，而敲低Suv39h1则显著影响Lhx8和FGF8的功能，FGF8和BMP2 通过Lhx8/Suv39h1复合体发挥对牙间充质发育的调控作用，而Lhx8/Suv39h1通过使成牙本质细胞终末分化相关基因的启动子区发生H3K9三甲基化，精细调节牙间充质增殖和分化的平衡。.为进一步探讨间充质干细胞增殖分化平衡的关键基因Lhx8及BMP2的精确时空调控在体内骨组织再生过程中的作用，我们借助光遗传手段，构建调控Lhx8和BMP2的光控表达系统，蓝光可在转染的间充质干细胞中实现诱导BMP2过表达和干涉Lhx8的效果，光控表达BMP2和shLhx8能有效促进间充质干细胞的体外成骨作用；将转染的间充质干细胞加载到PLGA支架上移植入到大鼠颅骨缺损区域后，发现蓝光照射后可显著促进颅骨缺损部位的骨再生。.本项目的研究结果揭示了牙间充质增殖分化平衡的表观遗传新机制，以及Lhx8/Suv39h1和FGF8、BMP2在其中发挥的作用，为认识牙发育提供了新依据；此外，通过光遗传手段的研究结果进一步验证了Lhx8和BMP2精确时空调控对体内骨修复再生的影响，为发展精准再生医学策略提供了新思路。