An unexpected finding has emerged from the molecular biology laboratory at Tokushima University, Japan. drg. Ivan Arie Wahyudi, M.Kes., Ph.D., and his research team discovered that the Sp6 gene, known as an important regulator of tooth development in mice, has not just one but three distinct promoter regions. One of them was found in a completely unexpected location: inside an intron, a segment of DNA traditionally regarded as a “non-coding” region. The research was published in The Indonesian Journal of Dental Research in 2010, making it one of the early contributions by Indonesian scientists to the mapping of the genetic mechanisms underlying tooth development.
A Small Gene with a Major Role in Tooth Development
To understand why this finding matters, it is useful to start with the basics. The development of every tooth in the mouth is far from a simple process. It involves complex communication between two layers of tissue: the dental epithelium, the outer layer that will eventually form tooth enamel, and the mesenchyme, the inner tissue that forms dentin and pulp. These two tissues communicate with one another through molecular signals.
At the center of this molecular conversation, the Sp6 gene serves as one of the key coordinators. The gene produces the SP6 protein, a member of the SP/Krüppel-like family of transcription factors. Mice lacking the Sp6 gene exhibit abnormalities in their teeth, hair, and limb buds. In an earlier study, the same research team also found that overexpression of Sp6 in ameloblasts—the cells responsible for producing tooth enamel—suppressed expression of the follistatin gene, indicating that Sp6 has specific targets during enamel development.
However, a more fundamental question remained unanswered: how is Sp6 expression itself regulated? This is where the study by drg. Ivan and his team comes in.
Dissecting the Genetic “Switches” of the Sp6 Gene
The researchers used 5′ RACE (Rapid Amplification of cDNA Ends) on RNA extracted from the mandibles of 18.5-day-old mouse embryos. The results were surprising. They identified two different transcription start ends in Sp6 transcripts, which were subsequently designated exon 1a and exon 1b, each controlled by a different promoter—the first and second promoters, respectively.
What made the finding particularly interesting was the difference in activity between the two promoters. When tested using a luciferase reporter assay—a standard method for measuring gene promoter activity in which greater luminescence indicates stronger promoter activity—the first promoter produced only weak activity. By contrast, the second promoter showed much stronger activity, reaching 82–129 times that of the control, particularly in dental epithelial cells.
“Our findings may provide new insights into the mechanisms regulating Sp6 gene expression and link cytokine regulation to inductive interactions between the epithelium and mesenchyme.” — drg. Ivan Arie Wahyudi, M.Kes., Ph.D., and colleagues
Then came an unexpected discovery. When the team analyzed intron 2, a DNA region conventionally regarded as inactive in protein coding, they detected significant promoter activity. A construct containing only the intron 2 region still demonstrated approximately 110-fold activity, roughly one-third of the highest promoter activity measured. This led the researchers to identify it as a potential third promoter.
BMP and Wnt Signals: External Amplifiers
The findings did not stop at mapping the promoters. The team also investigated how two key signaling molecules involved in tooth development—BMP2 (bone morphogenetic protein 2) and Wnt1 (wingless)—affected Sp6 promoter activity.
The results provided an increasingly complex picture. BMP2 treatment of G5 cells increased Sp6 mRNA levels by up to sixfold, while Wnt1 increased them by approximately 2.5-fold. More interestingly, the response to BMP2 was detected only in constructs containing the third promoter, whereas Wnt1 affected both the second and third promoters. This suggests that each promoter has a different degree of sensitivity to signals originating outside the cell.
These findings strengthen the direct connection between the BMP and Wnt signaling pathways, which have long been recognized as key players in epithelial–mesenchymal interactions, and the regulation of Sp6 transcription during tooth development.
A New Window into Dental Biology
The research was not yet completely conclusive. The team acknowledged that definitive confirmation of the third promoter requires identification of the precise transcription start sites, an issue that remains for future research. The question of why the first promoter showed weak activity in dental epithelial cells also remains unresolved, with several possible explanations ranging from the epigenetic status of the cells to the absence of specific transcription factors such as Ctip2/Bcl11b.
Nevertheless, the contribution of this research is already clear. The regulatory map of Sp6 generated by the study provides a pathway toward understanding why developmental abnormalities of the teeth occur at the molecular level. Ultimately, understanding how genes “switch themselves on and off” during tooth development forms a foundation for future regenerative dentistry, in which one day missing teeth may potentially be regenerated not from artificial materials, but from the body’s own genetic instructions.
Authors: Achmad Zam Zam Aghasy, DDS, M.Kes.; Hazra Alifia Muharam
Photo: Freepik