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V. Marigo, F. Vetrini, F. Giordano, R. Tammaro, E.M. Surace; Regulation of Ocular Albinism Type 1 (OA1) Gene Expression by the Microphthalmia Transcription Factor . Invest. Ophthalmol. Vis. Sci. 2003;44(13):2285.
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Purpose: Ocular Albinism type 1 (OA1) is an X-linked form of albinism isolated to the eye. The disease causes a severe visual handicap in affected males, manifesting foveal hypoplasia, horizontal and rotatory nystagmus, strabismus, photophobia and lack of stereoscopic vision. Contrary to other forms of albinism, melanin is synthesized but melanosome biogenesis appears to be impaired. The murine Oa1 gene is specifically expressed in pigmented cells in the retinal pigment epithelium (RPE) and in the skin. In order to understand how the transcription of the Oa1 gene is regulated, we performed promoter studies. Methods: Several murine Oa1 promoter fragments were cloned upstream of the luciferase reporter gene. To test the tissue specific activity of the promoter, these plasmids were transiently transfected into either melanocytes or fibroblasts. Promoter activities were measured by luciferase assay. Binding of Microphthalmia transcription factor (Mitf) to the Oa1 promoter was confirmed by Electrophoretic mobility shift assay (EMSA). Finally, in vivo control of Oa1 transcription by Mitf was analyzed by RT-PCR on RNA extracted from Mitf mutant eyes. Results: Using in vitro promoter analysis we found that the tissue specific regulation of Oa1 in pigmented cells (melanocytes) lays within the first 600bp upstream to the mRNA transcription start site. In this fragment a Mitf binding site was identified. A dose response to Mitf was measured by misexpressing Mitf in NIH3T3 fibroblasts. EMSA showed a specific binding of Mitf to this site. Finally, by RT-PCR on RNA extracted from Mitf mutant RPE, we demonstrated that Mitf is required for Oa1 transcription in vivo. Conclusions: Our study demonstrates that Oa1 expression is dependent upon Mitf. The upstream elements regulating the basic and tissue specific promoter activity are contained within the first 600bp. The promoter studies identified other positive and negative regulatory elements. Further work will be done to identify the transcription factors binding to these elements.
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