Higher order chromatin structure of the tyrosinase gene locus

An important enhancer/MAR (matrix attachment region) regulatory element in the mouse tyrosinase locus, located 15 kb upstream of the transcription initiation site, is responsible for imparting high level, position-independent tyrosinase transgene expression in neural crest-derived melanocytes. As MARs often coincide with the domain boundaries of gene loci, this MAR may also be the 5' domain boundary of the tyrosinase locus. General DNase I sensitivity assays of genomic DNA from cultured Cloudman S91 mouse melanoma cells showed that the region immediately flanking the upstream side of the MAR demonstrates a sensitivity approaching that of bulk chromatin, indicating that the MAR may be acting as a structural transition point between the open chromatin structure of the tyrosinase gene and the more closed architecture synonymous with heterochromatin. Four novel melanocytespecific DNase I hypersensitive sites (DHS) were mapped in the human tyrosinase gene locus of cultured SK-Mel-28 human melanoma cells. Three DHS were found upstream of the transcription start site: one at -10.5 kb which may represent the human homologue to the -15 kb enhancer/MAR in the mouse tyrosinase locus, one co-localizing with the promoter, and one representing a -2 kb enhancer element that is essential for high level human tyrosinase expression. A DHS located 15 kb downstream of exon 5 was also found that maps to no known cw-regulatory element. In addition, transient transfection analyses using recombinant luciferase reporter genes showed that the human tyrosinase coding sequences exhibit repressor activity, an observation that might explain discrepancies of coat pigmentation of transgenic mice generated in different studies. As well, the SK-N-SH human neuroblastoma cell line was used to investigate the temporal activation of the human tyrosinase gene locus.Medicine, Faculty ofPathology and Laboratory Medicine, Department ofGraduat