Zhang Zhichun, Tian Hua, Lv Ping, Wang Weiping, Jia Zhuqing, Wang Sainan, Zhou Chunyan, Gao Xuejun
Department of Cariology and Endodontology, School and Hospital of Stomatology, Peking University, Beijing, PR China.
Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Peking University, Beijing, PR China.
PLoS One. 2015 Mar 27;10(3):e0121288. doi: 10.1371/journal.pone.0121288. eCollection 2015.
Mutation of distal-less homeobox 3 (DLX3) is responsible for human tricho-dento-osseous syndrome (TDO) with amelogenesis imperfecta, indicating a crucial role of DLX3 in amelogenesis. However, the expression pattern of DLX3 and its specific function in amelogenesis remain largely unknown. The aim of this study was to investigate the effects of DLX3 on enamel matrix protein (EMP) genes. By immunohistochemistry assays of mouse tooth germs, stronger immunostaining of DLX3 protein was identified in ameloblasts in the secretory stage than in the pre-secretory and maturation stages, and the same pattern was found for Dlx3 mRNA using Realtime PCR. In a mouse ameloblast cell lineage, forced expression of DLX3 up-regulated the expression of the EMP genes Amelx, Enam, Klk4, and Odam, whereas knockdown of DLX3 down-regulated these four EMP genes. Further, bioinformatics, chromatin immunoprecipitation, and luciferase assays revealed that DLX3 transactivated Enam, Amelx, and Odam through direct binding to their enhancer regions. Particularly, over-expression of mutant-DLX3 (c.571_574delGGGG, responsible for TDO) inhibited the activation function of DLX3 on expression levels and promoter activities of the Enam, Amelx, and Odam genes. Together, our data show that DLX3 promotes the expression of the EMP genes Amelx, Enam, Klk4, and Odam in amelogenesis, while mutant-DLX3 disrupts this regulatory function, thus providing insights into the molecular mechanisms underlying the enamel defects of TDO disease.
远端缺失同源盒3（DLX3）的突变导致人类毛发-牙齿-骨综合征（TDO）并伴有牙釉质发育不全，这表明DLX3在牙釉质形成过程中起着关键作用。然而，DLX3的表达模式及其在牙釉质形成中的具体功能仍 largely未知。本研究的目的是探讨DLX3对牙釉质基质蛋白（EMP）基因的影响。通过对小鼠牙胚的免疫组织化学分析，发现分泌期成釉细胞中DLX3蛋白的免疫染色比分泌前期和成熟期更强，实时PCR检测Dlx3 mRNA也呈现相同模式。在小鼠成釉细胞系中，DLX3的强制表达上调了EMP基因Amelx、Enam、Klk4和Odam的表达，而DLX3的敲低则下调了这四个EMP基因的表达。此外，生物信息学、染色质免疫沉淀和荧光素酶分析表明，DLX3通过直接结合Enam、Amelx和Odam的增强子区域来反式激活它们。特别地，突变型DLX3（c.571_574delGGGG，与TDO相关）的过表达抑制了DLX3对Enam、Amelx和Odam基因表达水平和启动子活性的激活功能。总之，我们的数据表明DLX3在牙釉质形成过程中促进EMP基因Amelx、Enam、Klk4和Odam的表达，而突变型DLX3破坏了这种调节功能，从而为TDO疾病牙釉质缺陷的分子机制提供了见解。
