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J. Biol. Chem., Vol. 279, Issue 18, 19064-19073, April 30, 2004

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Analysis of the VMD2 Promoter and Implication of E-box Binding Factors in Its Regulation^*

From the ^aThe Guerrieri Center for Genetic Engineering and Molecular Ophthalmology at the Wilmer Eye Institute, the Departments of ^bOphthalmology, ^fNeuroscience, and ^hMolecular Biology and Genetics, and ⁱThe McKusick-Nathans Institute of Genetic Medicine, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21287-9289

The retinal pigment epithelium (RPE) is crucial for the normal development and function of retinal photo-receptors, and mutations in several genes that are preferentially expressed in the RPE have been shown to cause retinal degeneration. We analyzed the 5'-up-stream region of human VMD2, a gene that is preferentially expressed in the RPE and, when mutated, causes Best macular dystrophy. Transgenic mouse studies with VMD2 promoter/lacZ constructs demonstrated that a-253 to +38 bp fragment is sufficient to direct RPE-specific expression in the eye. Transient transfection assays using the D407 human RPE cell line with VMD2 promoter/luciferase reporter constructs identified two positive regulatory regions, -585 to -541 bp for high level expression and -56 to -42 bp for low level expression. Mutation of a canonical E-box located in the -56 to -42 bp region greatly diminished luciferase expression in D407 cells and abolished the bands shifted with bovine RPE nuclear extract in electrophoretic mobility shift assays. Independently a candidate approach was used to select microphthalmia-associated transcription factor (MITF) for testing because it is expressed in the RPE and associated with RPE abnormalities when mutated. MITF-M significantly increased luciferase expression in D407 cells in an E-box-dependent manner. These studies define the VMD2 promoter region sufficient to drive RPE-specific expression in the eye, identify positive regulatory regions in vitro, and suggest that MITF as well as other E-box binding factors may act as positive regulators of VMD2 expression.

Received for publication, September 5, 2003 , and in revised form, January 30, 2004.

^* This study was supported in part by grants from the National Eye Institute, Foundation Fighting Blindness, Steinbach Foundation, Macula Vision Foundation, and by a generous gift from Robert and Clarice Smith. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^d Present address: Dept. of Ophthalmology, Faculty of Medicine, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka, 812-8582 Japan.

^e Present address: Laboratory of Neurogenetics, Kennedy Krieger Inst., The Johns Hopkins University School of Medicine, Baltimore, MD 21287.

^g The George S. and Dolores Dore Eccles Professor of Ophthalmology and Neuroscience.

^j The Guerrieri Professor of Genetic Engineering and Molecular Ophthalmology and recipient of a Research to Prevent Blindness Senior Investigator Award.

^c To whom correspondence should be addressed: The Guerrieri Center for Genetic Engineering and Molecular Ophthalmology at the Wilmer Eye Inst., The Johns Hopkins University School of Medicine, 832 Maumenee Bldg., 600 N. Wolfe St., Baltimore, MD 21287-9289. Tel.: 410-502-5230; Fax: 410-502-5382; E-mail: nesumi{at}jhmi.edu.

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