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.
Esumi
Noriko
N
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
Oshima
Yuji
Y
Li
Yuanyuan
Y
Campochiaro
Peter A
PA
Zack
Donald J
DJ
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