Histone methylation assumes a crucial role in the intricate process of enamel development. Our study has illuminated the substantial prevalence of H3K4me3 distribution, spanning from the cap stage to the late bell stage of dental germs. In order to delve into the role of H3K4me3 modification in amelogenesis and unravel the underlying mechanisms, we performed a conditional knockout of Ash2l, a core subunit essential for the establishment of H3K4me3 within the dental epithelium of mice. The absence of Ash2l resulted in reduced H3K4me3 modification, subsequently leading to abnormal morphology of dental germ at the late bell stage. Notably, knockout of Ash2l resulted in a loss of polarity in ameloblasts and odontoblasts. The proliferation and apoptosis of the inner enamel epithelium cells underwent dysregulation. Moreover, there was a notable reduction in the expression of matrix-related genes, Amelx and Dspp, accompanied with impaired enamel and dentin formation. Cut&Tag-seq (cleavage under targets and tagmentation sequencing) analysis substantiated a reduction of H3K4me3 modification on Shh, Trp63, Sp6, and others in the dental epithelium of Ash2l knockout mice. Validation through real-time polymerase chain reaction, immunohistochemistry, and immunofluorescence consistently affirmed the observed downregulation of Shh and Sp6 in the dental epithelium following Ash2l knockout. Intriguingly, the expression of Trp63 isomers, DNp63 and TAp63, was perturbed in Ash2l defect dental epithelium. Furthermore, the downstream target of TAp63, P21, exhibited aberrant expression within the cervical loop of mandibular first molars and incisors. Collectively, our findings suggest that ASH2L orchestrates the regulation of crucial amelogenesis-associated genes, such as Shh, Trp63, and others, by modulating H3K4me3 modification. Loss of ASH2L and H3K4me3 can lead to aberrant differentiation, proliferation, and apoptosis of the dental epithelium by affecting the expression of Shh, Trp63, and others genes, thereby contributing to the defects of amelogenesis.

中文翻译：

---

ASH2L，H3K4 甲基化复合物的核心亚基，调节釉质生成。

组蛋白甲基化在牙釉质发育的复杂过程中发挥着至关重要的作用。我们的研究阐明了 H3K4me3 分布的广泛流行，从牙菌的帽状阶段到钟状晚期。为了深入研究 H3K4me3 修饰在釉质形成中的作用并揭示其潜在机制，我们对 Ash2l 进行了条件敲除，Ash2l 是在小鼠牙上皮内建立 H3K4me3 所必需的核心亚基。Ash2l的缺失导致H3K4me3修饰减少，随后导致晚期牙胚形态异常。值得注意的是，敲除 Ash2l 会导致成釉细胞和成牙本质细胞极性丧失。内釉质上皮细胞的增殖和凋亡发生失调。此外，基质相关基因 Amelx 和 Dspp 的表达显着减少，并伴有牙釉质和牙本质形成受损。Cut&Tag-seq（靶点切割和标记测序）分析证实 Ash2l 敲除小鼠牙上皮细胞中的 Shh、Trp63、Sp6 和其他细胞上的 H3K4me3 修饰减少。通过实时聚合酶链反应、免疫组织化学和免疫荧光进行的验证一致证实了 Ash2l 敲除后观察到的牙上皮细胞中 Shh 和 Sp6 的下调。有趣的是，Trp63 异构体 DNp63 和 TAp63 的表达在 Ash2l 缺损牙上皮中受到干扰。此外，TAp63 的下游靶标 P21 在下颌第一磨牙和门牙的颈环内表现出异常表达。总的来说，我们的研究结果表明 ASH2L 通过调节 H3K4me3 修饰来协调关键的釉质形成相关基因的调节，例如 Shh、Trp63 等。ASH2L和H3K4me3的缺失可通过影响Shh、Trp63和其他基因的表达导致牙上皮的异常分化、增殖和凋亡，从而导致釉质生成缺陷。