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

Noriko Esumi, Yuji Oshima, Yuanyuan Li, Peter A Campochiaro, Donald J Zack

Research output: Contribution to journalArticle

Abstract

The retinal pigment epithelium (RPE) is crucial for the normal development and function of retinal photoreceptors, and mutations in several genes that are preferentially expressed in the RPE have been shown to cause retinal degeneration. We analyzed the 5′-upstream 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.

Original languageEnglish (US)
Pages (from-to)19064-19073
Number of pages10
JournalJournal of Biological Chemistry
Volume279
Issue number18
DOIs
StatePublished - Apr 30 2004

Fingerprint

Retinal Pigments
Retinal Pigment Epithelium
Microphthalmia-Associated Transcription Factor
Luciferases
Nucleic Acid Regulatory Sequences
Assays
Vitelliform Macular Dystrophy
Genes
Electrophoretic mobility
Retinal Degeneration
Mutation
Vertebrate Photoreceptor Cells
Electrophoretic Mobility Shift Assay
Genetic Promoter Regions
Transgenic Mice
Transfection
Cells
Cell Line
Testing

ASJC Scopus subject areas

  • Biochemistry

Cite this

Analysis of the VMD2 Promoter and Implication of E-box Binding Factors in Its Regulation. / Esumi, Noriko; Oshima, Yuji; Li, Yuanyuan; Campochiaro, Peter A; Zack, Donald J.

In: Journal of Biological Chemistry, Vol. 279, No. 18, 30.04.2004, p. 19064-19073.

Research output: Contribution to journalArticle

@article{9cc57b3c48de4a33b1f8a78b2a81730e,
title = "Analysis of the VMD2 Promoter and Implication of E-box Binding Factors in Its Regulation",
abstract = "The retinal pigment epithelium (RPE) is crucial for the normal development and function of retinal photoreceptors, and mutations in several genes that are preferentially expressed in the RPE have been shown to cause retinal degeneration. We analyzed the 5′-upstream 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.",
author = "Noriko Esumi and Yuji Oshima and Yuanyuan Li and Campochiaro, {Peter A} and Zack, {Donald J}",
year = "2004",
month = "4",
day = "30",
doi = "10.1074/jbc.M309881200",
language = "English (US)",
volume = "279",
pages = "19064--19073",
journal = "Journal of Biological Chemistry",
issn = "0021-9258",
publisher = "American Society for Biochemistry and Molecular Biology Inc.",
number = "18",

}

TY - JOUR

T1 - Analysis of the VMD2 Promoter and Implication of E-box Binding Factors in Its Regulation

AU - Esumi, Noriko

AU - Oshima, Yuji

AU - Li, Yuanyuan

AU - Campochiaro, Peter A

AU - Zack, Donald J

PY - 2004/4/30

Y1 - 2004/4/30

N2 - The retinal pigment epithelium (RPE) is crucial for the normal development and function of retinal photoreceptors, and mutations in several genes that are preferentially expressed in the RPE have been shown to cause retinal degeneration. We analyzed the 5′-upstream 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.

AB - The retinal pigment epithelium (RPE) is crucial for the normal development and function of retinal photoreceptors, and mutations in several genes that are preferentially expressed in the RPE have been shown to cause retinal degeneration. We analyzed the 5′-upstream 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.

UR - http://www.scopus.com/inward/record.url?scp=2442567892&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=2442567892&partnerID=8YFLogxK

U2 - 10.1074/jbc.M309881200

DO - 10.1074/jbc.M309881200

M3 - Article

VL - 279

SP - 19064

EP - 19073

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

IS - 18

ER -