Cell type-specific regulation of the chicken tyrosinase promoter

Master Thesis


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University of Cape Town

Melanin, the pigment found in the eyes and coats of vertebrates, is synthesised by two main cell types: melanocytes and retinal pigment epithelial (RPE) cells. These two cell populations. which arise from distinct embryological origins, differ with respect to the rate at which they produce melanin and the ways in which they respond to melanogenic stimuli. Tyrosinase is the rate-limiting enzyme in the melanin synthesis pathway, and the regulation of tyrosinase gene expression in mammalian melanocytes has been extensively studied. In contrast, regulation oftyrosinase gene expression in RPE cells has received little attention. In the present study, the chicken tyrosinase gene promoter was used to investigate possible differences in the regulation of tyrosinase expression in melanocytes and RPE cells. Transient transfection experiments were carried out in which reporter constructs, consisting oftyrosinase promoter deletion fragments linked to a luciferase reporter gene, were introduced into melanocytes, RPE cells and a non-pigmented cell line. The following results were obtained. (1) Reporter expression obtained with the longest (2.1kb) promoter fragment was significantly higher in pigmented cells (both melanocytes and RPE cells) than in non-pigmented cells, demonstrating the pigment cell-specificity of the chicken tyrosinase promoter. (2) Reporter expression obtained with a 0.5kb promoter fragment, containing conserved core regulatory elements ( an lnr, M-box and Sp 1 binding site), was higher in melanocytes than in RPE cells. This result suggests that the core elements are sufficient for high levels of tyrosinase expression in melanocytes, but not in RPE cells. (3) Reporter activity obtained with a 248bp promoter fragment containing no elements implicated in initiating tyrosinase transcription was strikingly high in RPE cells, and very low in melanocytes. This result suggested the presence of RPE-specific regulatory elements in the tyrosinase promoter. To determine which portion of the 248bp promoter fragment contained the element(s) responsible for this RPE-specific activity, three additional deletion constructs were cloned. Transient transfection experiments with these new constructs revealed that the RPE-effect observed with the 248bp construct was a serendipitous / unfortunate experimental artefact brought about by the ligation of 203bp of proximal promoter with 45bp of distal promoter. Examination of the sequence generated by this ligation revealed the presence of an element similar to PCE-1, an element recently implicated in RPEspecific gene regulation. Factors present in RPE cells, but not in melanocytes, may bind to this element to initiate transcription. Further investigation of the mechanism mediating this RPEspecific effect could contribute to the understanding ofRPE-specific gene regulation. In conclusion, the results of the present study strongly suggest that expression of the chicken tyrosinase gene is regulated differently in RPE cells and melanocytes, and begin to identify regions in the chicken tyrosinase promoter that might be responsible for mediating such differences.