Regulation of the human tyrosinase gene in retinal pigment epithelium cells: the significance of transcription factor orthodenticle homeobox 2 and its polymorphic binding site.

OBJECTIVE

Tyrosinase is the rate-limiting enzyme responsible for melanin biosynthesis in the retinal pigment epithelium (RPE) of the eye. Melanin has an important role in retinal development, function, and protection against light-induced oxidative stress, and melanin levels are associated with age-related macular degeneration (AMD). Because the levels of and protection afforded by melanin seem to decline with increasing age, proper regulation of the human tyrosinase gene (TYR) in the RPE is an important but insufficiently understood process. Our purpose was to obtain detailed information on regulation of the TYR gene promoter in the human RPE and to specify the role of orthodenticle homeobox 2 (OTX2) and microphthalmia-associated transcription factor (MITF).

METHODS

We used luciferase reporter constructs to study regulation of the human TYR gene promoter in cultured human RPE cells. We further studied the role of OTX2 and MITF, their binding sites, and endogenous expression by using mutagenesis, electrophoretic mobility shift assay, yeast two-hybrid assay, RNA interference, and gene expression analyses.

RESULTS

In the RPE, OTX2 activated the human TYR gene promoter via direct trans-activation of novel OTX2 binding elements. In addition, we found that indirect activation by OTX2 via more proximal MITF binding sites, even in the absence of OTX2 sites, took place. These results are consistent with the physical interaction observed between OTX2 and MITF. Overexpression or knockdown of OTX2 in RPE cells resulted in corresponding changes in tyrosinase mRNA expression. Finally, we found that a single nucleotide polymorphism (SNP rs4547091) at the most proximal OTX2 binding site is associated with altered nuclear protein binding and a remarkable decrease in TYR promoter activity in RPE cells. This single nucleotide polymorphism (SNP) is more common in the European population in which AMD is also more prevalent.

CONCLUSIONS

In the RPE, OTX2 activates the human TYR gene promoter by direct DNA binding and by interaction with MITF. Such synergistic interaction highlights the role of OTX2 as a potential coregulator of numerous MITF target genes in the eye. Genetic differences in OTX2 binding sites affect tyrosinase regulation. Collectively, these findings emphasize the role of OTX2 in regulating the human TYR gene, with implications for inter-individual differences in melanin synthesis, retinal development, and function as well as susceptibility to retinal degeneration associated with aging.