| Summary: | 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.
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