Arrest of amelogenin transcriptional activation in bromodeoxyuridine-treated developing mouse molars in vitro

An important issue in craniofacial biology is understanding the molecular mechanisms that regulate the transcription of genes during development. Low concentrations of the thymidine analogue, 5-bromodeoxyuridine (BrdU), have been used to perturb transcription of tissue-specific genes in a variety of tissue types, although the molecular mechanism for this inhibition has not been elucidated. The purpose of the present study was to examine the following: (1) if amelogenin transcription is inhibited in mouse molars cultured in the presence of BrdU, (2) if changes in methylation patterns of the amelogenin gene can be detected with terminal differentiation of ameloblasts in vivo and in vitro; and (3) if changes in methylation patterns of the amelogenin gene can be detected in mouse molars cultured in the presence of BrdU. Northern blot hybridization and RNA phenotyping analysis revealed that bromodeoxyuridine (BrdU) incorporation into the DNA of developing mouse mandibular first molars (M1) in vitro inhibited amelogenin transcription. Restriction endonuclease digestion of M1 genomic DNA followed by Southern blot hybridization analysis revealed that amelogenin transcriptional activity in vivo and in vitro did not correlate with changes in methylation of the amelogenin gene. These results suggested that, unlike several other developmentally regulated genes, transcriptional regulation of the amelogenin gene may not be associated with changes in DNA methylation patterns.