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Tissue-specific gene expression is subjected to epigenetic and genetic regulation. Posttranslational modifications of histone tails alter the accessibility of nuclear proteins to DNA, thus affecting the activity of the regulatory complex of nuclear proteins. Methylation at histone 3 lysine 9 (H3K9) is a crucial modification that affects gene expression and cell differentiation. H3K9 is known to have 0-3 methylation states, and these four methylated states are determined by the expression of sets of histone methyltransferases. During development, teeth are formed through mutual interactions between the mesenchyme and epithelium via a process that is subjected to the epigenetic regulation. In this study, we examined the expression of all H3K9 methyltransferases (H3K9MTases) during mouse tooth development. We found that four H3K9MTases-G9a, Glp, Prdm2, and Suv39h1-were highly expressed in the tooth germ, with expression peaks at around embryonic days 16.5 and 17.5 in mice. Immunohistochemical and in situ hybridization analyses revealed that all four H3K9MTases were enriched in the mesenchyme more than in the epithelium. Substrates of H3K9MTases, H3K9me1, H3K9me2, and H3K9me3 were also enriched in the mesenchyme. Taken together, these data suggested that coordinated expression of four H3K9MTases in the dental mesenchyme might play important roles in tooth development.

The enzyme alkaline phosphatase (AP) (EC 3.1.3.1) in three different calcification areas was studied by means of a spectrophotometric micro method using p-nitrophenylphosphate as a substrate. Rat maxillary incisor odontoblasts and enamel organ from the zones of matrix formation and maturation and tissue from rabbit metatarsal cartilage were allowed to react with the substrate in glycine-NaOH buffer at room temperature. The reaction was found to be linear for a minimum of 20 min. The pH optima for AP from these tissues were in the pH range of 10.0-10.3. In order to compare AP from the four calcification areas different parameters were studied. Heating at 56 degrees C or 60 degrees C for varying times revealed that the enzymes were almost completely inactivated after 10 min. Mg2+ ions activated the enzymes by about 25% at concentrations of 2.5 mM (enamel organ 1.25 mM); while only higher concentrations of Mg2+ had an inactivating effect, Ca2+ and PO3-4 ions were inactivating at varying concentrations. F- ions showed no effect on AP activity at concentrations below 250 mM (enamel organ 125 mM) but caused inactivation of the enzymes at about 50% at 1 M. EDTA was found to be a very effective AP inactivator at concentrations above 0.06 mM, whereas urea did not noticeably affect the enzyme reactions at concentrations below 1 M. At higher concentrations, inactivation was observed. In order to determine AP localization in the epiphyseal plate successive 40-mum-thick, freeze-sectioned slices were analyzed. The activity was highest nearest the zone of cartilage calcification and decreased towards the reserve cell zone. It was concluded that the same AP isoenzyme is present in these quite different calcification loci.