Human Molecular Genetics Advance Access originally published online on October 10, 2008
Human Molecular Genetics 2009 18(1):128-141; doi:10.1093/hmg/ddn323
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BEST1 expression in the retinal pigment epithelium is modulated by OTX family members
1 The Guerrieri Center for Genetic Engineering and Molecular Ophthalmology at The Wilmer Eye Institute 2 Department of Ophthalmology, Johns Hopkins University School of Medicine, 832 Maumenee Building, 600 N, Wolfe Street, Baltimore, MD 21287-9289, USA 3 Department of Neuroscience, The Johns Hopkins University School of Medicine, Baltimore, MD 21287, USA 4 Department of Molecular Biology and Genetics, The Johns Hopkins University School of Medicine, Baltimore, MD 21287, USA 5 The McKusick-Nathans Institute of Genetic Medicine, The Johns Hopkins University School of Medicine, Baltimore, MD 21287, USA
* To whom correspondence should be addressed at. Tel: +1 4105025230; Fax: +1 4105025382; Email: nesumi{at}jhmi.edu
Received September 5, 2008; Accepted October 3, 2008
A number of genes preferentially expressed in the retinal pigment epithelium (RPE) are associated with retinal degenerative disease. One of these, BEST1, encodes bestrophin-1, a protein that when mutated causes Best macular dystrophy. As a model for RPE gene regulation, we have been studying the mechanisms that control BEST1 expression, and recently demonstrated that members of the MITF–TFE family modulate BEST1 transcription. The human BEST1 upstream region from –154 to +38 bp is sufficient to direct expression in the RPE, and positive-regulatory elements exist between –154 and –104 bp. Here, we show that the –154 to –104 bp region is necessary for RPE expression in transgenic mice and contains a predicted OTX-binding site (Site 1). Since another non-canonical OTX site (Site 2) is located nearby, we tested the function of these sites using BEST1 promoter/luciferase constructs by in vivo electroporation and found that mutation of both sites reduces promoter activity. Three OTX family proteins – OTX1, OTX2 and CRX – bound to both Sites 1 and 2 in vitro, and all of them increased BEST1 promoter activity. Surprisingly, we found that human and bovine RPE expressed not only OTX2 but also CRX, the CRX genomic region in bovine RPE was hypersensitive to DNase I, consistent with active transcription, and that both OTX2 and CRX bound to the BEST1 proximal promoter in vivo. These results demonstrate for the first time CRX expression in the RPE, and suggest that OTX2 and CRX may act as positive modulators of the BEST1 promoter in the RPE.
Present address: Department of Ophthalmology, Nagoya University School of Medicine, 65 Tsurumacho, Showa-ku, Nagoya, Aichi 4668550, Japan.