Cloning and characterization of the murine ameloblastin promoter

The molecular mechanisms directing the highly restricted expression pattern of murine ameloblastin were characterized by cloning and functional analys is of the ameloblastin promoter. The transcription start site, mapped by pr imer extension, was located 19 base pairs (bp) 5' of the published cDNA. Th e promoter was analyzed in a mouse ameloblast-like cell line (LS8) and was compared with promoter activity in primary gingival fibroblasts and pulp fi broblasts. Sequential 5'-deletion mutants encompassing DNA sequences from - 1616 to -781 bp exhibited high promoter activity in LS8 cells, whereas the promoter activity decreased to 50% of the full-length construct in the -781 - and -477-bp regions. The -217-bp promoter region regained promoter activi ty that approached the activity of the full-length promoter construct, sugg esting that both positive and negative cis-acting regions may be involved i n ameloblastin transcriptional regulation. Activity of the ameloblastin pro moter in gingival and pulp fibroblasts was minimal and ranged from 8 to 30% of the activity in ameloblast-like cells. Several DNA-protein complexes we re formed between functionally important promoter fragments and nuclear ext racts from LS8 cells. The inactivity of promoter constructs in pulp and gin gival fibroblasts as well as the absence of similar DNA-protein complexes f rom these cells suggest that regulatory regions of the murine ameloblastin promoter may function in a cell-specific manner.