Identification of the minimal melanocyte-specific promoter in the melanocortin receptor 1 gene.
BACKGROUND
The understanding of cutaneous pigmentation biology is relevant from the biologic and clinical point of view. The binding of alpha-melanocortin and its specific receptor, on the plasma membrane of melanin synthesising cells, plays a crucial role in melanins biosynthesis. Furthermore, loss of MC1R function is associated with an increased incidence of melanoma and non-melanoma skin cancer. The expression of the alpha-melanocortin receptor gene is highly controlled but, at the present, region responsible for tissue-specific activity of the gene promoter has not been identified.


METHODS
We have cloned the genomic sequences upstream the human MC1R coding gene. A DNA fragment of 5 kilobases upstream the human MC1R encoding sequence was placed in front of a reporter gene and several deletion mutants of such fragment have been prepared. These constructs have been tested for the ability to drive the melanocyte-specific gene expression of the reporter gene using transfection experiments in melanocyte and non-melanocyte cell lines. From these experiments we identified a DNA fragment with the ability to drive the gene transcription in a tissue-specific way and we used this small DNA fragment in DNA-protein interaction assays.


RESULTS
We show that the 150 base pairs upstream the MC1R gene initiation codon are able to drive the melanocyte-specific gene transcription. Furthermore, we provide experimental evidences suggesting that on such minimal melanocyte-specific gene promoter can assemble tissue-specific complexes.


CONCLUSIONS
The present results strongly imply that the transcriptional regulation of the melanocyte-specific MC1R gene requires an internal promoter located in the 150 base pairs upstream the initiation codon.
Miccadei
Stefania
S
Laboratory of Molecular Pathology and Ultrastructure, Regina Elena Cancer Institute, Rome, Italy. miccadei@ifo.it
Pascucci
Barbara
B
Picardo
Mauro
M
Natali
Pier Giorgio
PG
Civitareale
Donato
D
D000818:Animals; D018387:Codon, Initiator; D024202:Electrophoretic Mobility Shift Assay; D006367:HeLa Cells; D006801:Humans; D008544:Melanocytes / Q000378:metabolism*; D051379:Mice; D041681:NIH 3T3 Cells; D011401:Promoter Regions, Genetic / Q000235:genetics*; D044102:Receptor, Melanocortin, Type 1 / Q000235:genetics* / Q000378:metabolism; D014158:Transcription, Genetic; D014162:Transfection
D018387:Codon, Initiator; D044102:Receptor, Melanocortin, Type 1