Regulatory Dynamics of Enamelin Expression by Msx2 controlling Enamel Formation and Biomineralization

Enamel is the hardest substance in the human body. The developmental process of enamel formation is called amelogenesis and is facilitated by cells called ameloblasts. Through stages of secretion, transition, and maturation, the ameloblasts deposit, modify, and mature the enamel’s mineral content, and thus regulates its overall hardness and thickness.  Enamel thickness has been considered a unique trait to understand fossils, as it gives us information about different species’ diets and way of life. Herbivorous animals have thinner enamel compared to omnivorous animals for example.  More importantly, the speed and incremental mode of enamel’s deposition allow for comparison between extinct and living species.  Teeth though serve as a record of information, making them excellent models for studying evolution, developmental biology mechanisms and genomic studies. In that context, earlier studies have indicated that among different but key, enamel-specific proteins, such as amelogenin, ameloblastin, etc., enamelin, stands out as it is under positive natural selection acting specifically on tooth enamel thickness in humans and is essential for the biomineralization of tooth enamel. They also found that enamelin gene, is under positive natural selection changes in its promoter regulatory region, a sequence slightly upstream in the DNA where several transcription factors bind.

Msx2 is a transcription factor that when its function is eliminated in mice (Msx2-deficient mice) results in the formation of teeth with thin or no enamel, indicating the essential role of Msx2 in enamel formation, thickness and biomineralization process.  These findings highlighted the urgent need to conduct detailed studies on Msx2’s interaction with the enamelin promoter for understanding its role during enamel development.  To this end, a new international collaborative study in Developmental Dynamics Journal conducted by a team of Harvard researchers led by Dr. Marianna Bei, an Assistant Professor of Surgery from the Harvard Medical School & Massachusetts General Hospital, and collaborators from Tokushima and Hiroshima Universities in Japan and the University of Southern California in California US investigated the genetic mechanisms that control the formation of tooth enamel, potentially leading to better diagnostic and therapeutic strategies for related conditions. In their studies, the researchers used both cell cultures and the Msx2 -deficient mouse model to investigate the expression and regulation of enamelin (Enam) in normal and Msx2-deficient environments. Specifically, they analyzed the presence and levels of Enam mRNA and protein during the pre-secretory and secretory phases of ameloblast development. The expression levels of Enam in these knockout mice were compared to wild-type controls using quantitative PCR and in situ hybridization techniques.  The team conducted ChIP experiments to confirm the physical binding of Msx2 to the Enam promoter in the cells. This involved using antibodies specific to Msx2 to pull down the DNA-protein complex and subsequently identify the segments of DNA bound by Msx2 using PCR. They used promoter assays where they constructed reporter plasmids containing segments of the Enam promoter linked to a luciferase reporter gene. These constructs were transfected into ameloblast-derived cell lines to observe the effect of Msx2 overexpression on promoter activity. To quantify the expression levels of Enam in different experimental setups (Msx2 overexpression, knockout, and control samples), the researchers used real-time PCR which allowed for precise measurement of mRNA levels of Enam and other related genes in ameloblasts.

The studies revealed a significant reduction in Enam expression in the pre-secretory and secretory ameloblasts of Msx2-deficient mice compared to wild-type mice. This indicated that Msx2 is vital for the normal expression of Enamelin during the early stages of enamel development. The promoter assays and ChIP experiments confirmed that Msx2 directly binds to specific sites on the Enam promoter regulatory region and that this binding was shown to repress the transcriptional activity of Enam, as evidenced by the decrease in luciferase activity in the reporter assays upon Msx2 overexpression.  Contrary to what might be expected for a gene essential for enamel formation, Msx2 was found to act as a repressor rather than an activator of Enam expression. The experimental data suggested that Msx2’s role might involve complex interactions with other transcription factors to finely tune Enam optimum dosage and onset of its full expression, spatially and temporally.  This complex network of interactions ensures the proper spatial and temporal expression of Enam, critical for correct enamel formation and biomineralization.

Overall, the authors’ findings on the transcriptional regulation of enamelin by Msx2 in enamel formation, thickness and biomineralization is significant for multiple reasons.  The research deepens our understanding of how enamelin, that is under positive natural selection acting specifically on tooth enamel thickness in humans, is influenced by transcription factors such as Msx2 that bind to its promoter regulatory region. This enriches our general knowledge of tooth development and contributes significantly to the field of developmental biology by demonstrating how specific genetic elements drive complex biological processes during development and evolution. Furthermore, since enamel is the hardest substance in the human body and essential for dental health, understanding Msx2’s role in regulating enamelin expression aids in grasping the structural integrity, biomineralization process and resilience of tooth enamel. The results can guide future studies aimed at enhancing enamel properties in dental materials and treatments. Additionally, the findings have practical implications in diagnosing and treating enamel formation defects, such as amelogenesis imperfecta and ectodermal dysplasia disorders which can severely affect dental health and quality of life.