VMD2 promoter requires two proximal E-box sites for its activity in vivo and is regulated by the MITF-TFE family.
The retinal pigment epithelium (RPE) is crucial for the function and survival of retinal photoreceptors. VMD2 encodes bestrophin, an oligomeric chloride channel that is preferentially expressed in the RPE and, when mutated, causes Best macular dystrophy. Previously, we defined the VMD2 upstream region from -253 to +38 bp as being sufficient to direct RPE-specific expression in the eye, and we suggested microphthalmia-associated transcription factor (MITF) as a possible positive regulator. Here we show that in transgenic mice the -154 to +38 bp region is sufficient for RPE expression, and mutation of two E-boxes, 1 and 2, within this region leads to loss of promoter activity. A yeast one-hybrid screen using bait containing E-box 1 identified clones encoding MITF, TFE3, and TFEB, and chromatin immunoprecipitation with antibodies against these proteins enriched the VMD2 proximal promoter. Analysis using in vivo electroporation with constructs containing mutation of each E-box indicated that expression in native RPE requires both E-boxes, yet in vitro DNA binding studies suggested that MITF binds well to E-box 1 but only minimally to E-box 2. MITF knockdown by small interfering RNA (siRNA) in cell culture revealed a strong correlation between MITF and VMD2 mRNA levels. Sequential transfection of a luciferase construct with expression vectors following MITF siRNA revealed that TFE3 and TFEB can also transactivate the VMD2 promoter. Taken together, we suggest that VMD2 is regulated by the MITF-TFE family through two E-boxes, with E-box 1 required for a direct interaction of MITF-TFE factors and E-box 2 for binding of the as yet unidentified factor(s).
Esumi
Noriko
N
The Guerrieri Center for Genetic Engineering and Molecular Ophthalmology, The Wilmer Eye Institute, the Departments of Ophthalmology, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21287-9289, USA. nesumi@jhmi.edu
Kachi
Shu
S
Campochiaro
Peter A
PA
Zack
Donald J
DJ
D000818:Animals; D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors / Q000502:physiology; D000075002:Bestrophins; D004268:DNA-Binding Proteins / Q000737:chemistry; D005136:Eye Proteins / Q000235:genetics* / Q000502:physiology*; D005786:Gene Expression Regulation; D007473:Ion Channels; D051379:Mice; D008822:Mice, Transgenic; D051739:Microphthalmia-Associated Transcription Factor / Q000502:physiology*; D009154:Mutation; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D034741:RNA, Small Interfering / Q000378:metabolism; D015533:Transcriptional Activation
D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; C491856:Best1 protein, mouse; D000075002:Bestrophins; D004268:DNA-Binding Proteins; D005136:Eye Proteins; D007473:Ion Channels; D051739:Microphthalmia-Associated Transcription Factor; C497235:Mitf protein, mouse; D034741:RNA, Small Interfering; C484068:Tcfeb protein, mouse; C068486:Tcfe3 protein, mouse