Analysis of the VMD2 promoter and implication of E-box binding factors in its regulation.
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.
The Guerrieri Center for Genetic Engineering and Molecular Ophthalmology, Wilmer Eye Institute, and Department of Ophthalmology, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21287-9289, USA. nesumi@jhmi.edu
Noriko
N
Esumi
Yuji
Y
Oshima
Yuanyuan
Y
Li
Peter A
PA
Campochiaro
Donald J
DJ
Zack
D024506:5' Flanking Region; D000818:Animals; D001483:Base Sequence; D000075002:Bestrophins; D002417:Cattle; D018118:Chloride Channels; D004268:DNA-Binding Proteins / Q000502:physiology; D024721:E-Box Elements / Q000502:physiology*; D005123:Eye / Q000166:cytology; D005136:Eye Proteins / Q000235:genetics*; D005786:Gene Expression Regulation; D006801:Humans; D007473:Ion Channels; D051379:Mice; D008822:Mice, Transgenic; D051739:Microphthalmia-Associated Transcription Factor; D008969:Molecular Sequence Data; D010857:Pigment Epithelium of Eye / Q000378:metabolism; D011401:Promoter Regions, Genetic; D012313:RNA / Q000302:isolation & purification; D014157:Transcription Factors / Q000502:physiology; D014162:Transfection
C113133:BEST1 protein, human; C491856:Best1 protein, mouse; D000075002:Bestrophins; D018118:Chloride Channels; D004268:DNA-Binding Proteins; D005136:Eye Proteins; D007473:Ion Channels; C497234:MITF protein, human; D051739:Microphthalmia-Associated Transcription Factor; C497235:Mitf protein, mouse; D014157:Transcription Factors; D012313:RNA