ARREST OF AMELOGENIN TRANSCRIPTIONAL ACTIVATION IN BROMODEOXYURIDINE-TREATED DEVELOPING MOUSE MOLARS IN-VITRO

An important issue in craniofacial biology is understanding the molecu lar mechanisms that regulate the transcription of genes during develop ment. Low concentrations of the thymidine analogue, 5-bromodeoxyuridin e (BrdU), have been used to perturb transcription of tissue-specific g enes in a variety of tissue types, although the molecular mechanism fo r 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 ch anges in methylation patterns of the amelogenin gene can be detected w ith terminal differentiation of ameloblasts in vivo and in vitro; and (3) if changes in methylation patterns of the amelogenin gene can be d etected in mouse molars cultured in the presence of BrdU. Northern blo t hybridization and RNA phenotyping analysis revealed that bromodeoxyu ridine (BrdU) incorporation into the DNA of developing mouse mandibula r first molars (M1) in vitro inhibited amelogenin transcription. Restr iction endonuclease digestion of M1 genomic DNA followed by Southern b lot hybridization analysis revealed that amelogenin transcriptional ac tivity in vivo and in vitro did not correlate with changes in methylat ion of the amelogenin gene. These results suggested that, unlike sever al other developmentally regulated genes, transcriptional regulation o f the amelogenin gene may not be associated with changes in DNA methyl ation patterns.