Shh基因组蛋白二价修饰在牙上皮分化中的作用机制研究

Enamel, the hardest mineralized tissue in vertebrates, is composed of tightly packed hydroxyapatite crystals and confers the protection and masticatory function in human body. Enamel formation relies on sequentially ameloblast differentiation throughout the dental epithelial sheet on enamel organ. The labial enamel of mouse incisor forms continuously throughout the lifetime. This relies on the epithelial stem cells reside at the cervical loop site of incisor tooth germ. This continuous growth of incisor enamel is regulated by sequential and reciprocal interactions between the epithelial and mesenchymal tissues. These spatial temporal signals most likely are overseen by mechanisms on multiple layers both genetically and epigenetically. While the importance of the transcriptional control of the cell fate determination is well documented, it has become increasingly evident that epigenetic regulation of gene transcription plays a key role in organogenesis. Posttranslational modifications of histone proteins are thought to be important epigenetic events that are intimately associated with transcription regulation in cell fate determination and differentiation. Histones are subject to various modifications, including methylation, acetylation, phosphorylation, ubiquitination and ribosylation. Among them, histone methylation is one of the most widely studied epigenetic modifications. Prominent histone modifications include H3K4 methylation, which has been implicated in transcriptional activation and deposited by Trithorax group proteins, and H3K27 methylation, which has been implicated in transcriptional repression and deposited by Polycomb group proteins. Many developmental regulatory gene loci are marked with both H3K4 and H3K27.methylation, the so-called ‘bivalent marks’. The combination of the seemingly ‘conflicting’marks suggests that these genes are kept silenced by H3K27methylation while remaining ‘poised’ for expression events that are presumably dependent upon H3K4 methylation. These ‘bivalent’ chromatin domains often mark lineage-regulatory genes and have garnered wide attention because they might contribute to the precise unfolding of gene expression programs during pluripotency and differentiation. It is possible that bivalent domains convey temporal and spatial precision to the expression of lineage control genes during pluripotency and differentiation...In previous study, we have characterized the spatiotemporal status of histone H3 lysine 4 trimethylation (H3K4me3) and histone H3 lysine 27 trimethylation (H3K27me3) epigenetic marks and histone methylation/demethylation transferases in the mouse developing first molar. The results indicated a possible epigenetic regulatory mechanism during mouse tooth development. In this current study, we explore the spatiotemporal status of H3K4me3 and H3K27me3 epigenetic marks, and the existence of bivalent marks during this process. Histone methylation or demethylation transferases in enamel formation and ameloblast differentiation will be measured via quantitative polymerase chain reaction (qPCR), immunohistochemistry, and western blot. The bivalent modification on enamel formation related gene SHH will be characterized by ChIP or ChIP-sequence. ChIP-qPCR and co-IP will be performed to investigate the mechanism of histone methylation or demethylation transferases in participating the bivalent modification. By carrying out this study, we target to explore the mechanism of bivalent histone modification in playing a critical role during enamel formation and ameloblast diffentiation.

牙齿是重要的咀嚼器官，牙冠表面由高度矿化的牙釉质被覆并暴露于口腔。小鼠切牙釉质的形成与持续更新同颈环处上皮干细胞的自我更新及定向分化密不可分。该过程伴随着一系列基因的时空性表达并受多层机制调节，包括遗传学与表观遗传学机制。组蛋白甲基化修饰是近年来研究广泛的表观遗传学调控机制，已被发现参与生物发育过程及干细胞分化。本课题组前期研究发现小鼠牙胚中组蛋白甲基化修饰状态存在时空性特点。本项目拟研究在小鼠切牙成釉系细胞分化过程中组蛋白修饰状态的时空性特征，探索成牙关键基因Shh启动子区域是否存在组蛋白二价修饰标记（H3K4me3/H3K27me3），并研究组蛋白甲基化/去甲基化转移酶参与调节组蛋白二价修饰状态的作用及机制。通过本课题研究初步探索在小鼠切牙成釉系细胞分化过程中的表观遗传学调控机制，进一步阐明组蛋白二价修饰在牙釉质形成过程中的存在状态及参与调控釉质形成的形式。

牙齿是重要的咀嚼器官，牙冠表面由高度矿化的牙釉质被覆并暴露于口腔。小鼠切牙釉质的形成与持续更新同颈环处上皮干细胞的自我更新及定向分化密不可分。该过程伴随着一系列基因的时空性表达并受多层机制调节，包括遗传学与表观遗传学机制。组蛋白甲基化修饰是近年来研究广泛的表观遗传学调控机制，已被发现参与生物发育过程及干细胞分化。课题前期研究显示，小鼠牙胚中组蛋白甲基化修饰状态存在时空性特点。本课题发现了在牙上皮干细胞成釉向谱系定向过程中，存在组蛋白二价修饰状态的改变，H3K4me3修饰状态普遍存在于分化各个阶段，而H3K27me3在暂时性扩增细胞及前成釉细胞显著减弱；Shh是组蛋白二价修饰的靶向调控基因，Ezh2是介导这一过程的关键组蛋白酶。体外构建BMP4诱导牙上皮干细胞成釉向分化模型，证实了EZH2能够通过调节Shh启动子区域的H3K27me3的修饰水平调控Shh的表达，该过程依赖于其自身的甲基转移酶活性；且EZH2/H3K27me3通过Shh/Ptch1/Gli1信号通路调控成釉分化关键基因Amelogenin及牙上皮多能性基因Sox2的表达，进而决定牙上皮干细胞走向成釉向分化还是维持干细胞状态。该研究探索了小鼠切牙成釉系细胞分化过程中，组蛋白甲基化转移酶EZH2通过调控成釉向分化关键基因Shh启动子区H3K27me3的修饰状态，调控牙上皮分化的表观遗传学调控机制，完善了组蛋白二价修饰在牙釉质形成过程中的调控作用理论。