HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

J. Biol. Chem., Vol. 282, Issue 3, 1838-1850, January 19, 2007

This Article Full Text Full Text (PDF) All Versions of this Article: 282/3/1838    most recent M609517200v1 Submit a Letter to Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Esumi, N. Articles by Zack, D. J. Search for Related Content PubMed PubMed Citation Articles by Esumi, N. Articles by Zack, D. J. Social Bookmarking                      What's this?

VMD2 Promoter Requires Two Proximal E-box Sites for Its Activity in Vivo and Is Regulated by the MITF-TFE Family^*

Noriko Esumi¹, Shu Kachi², Peter A. Campochiaro^¶3, and Donald J. Zack^¶||**4

From the The Guerrieri Center for Genetic Engineering and Molecular Ophthalmology, The Wilmer Eye Institute, the Departments of Ophthalmology, ^¶Neuroscience, and ^||Molecular Biology and Genetics, and ^**The 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 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).

Received for publication, October 10, 2006

^* This work was supported in part by research grants from the NEI, National Institutes of Health, and the Foundation Fighting Blindness, unrestricted funds from Research to Prevent Blindness, Inc., and generous gifts from Robert and Clarice Smith and The Guerrieri Family Foundation. 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.

² Present address: Dept. of Ophthalmology, Nagoya University School of Medicine, 65 Tsurumacho, Showa-ku, Nagoya, Aichi, 466-8550, Japan.

³ The George S. and Dolores Dore Eccles Professor of Ophthalmology and Neuroscience.

⁴ The Guerrieri Professor of Genetic Engineering and Molecular Ophthalmology at the Wilmer Eye Institute and recipient of a Research to Prevent Blindness Senior Investigator Award.

¹ To whom correspondence should be addressed: 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.

CiteULike    Complore    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this?

This article has been cited by other articles:

				J. M. Fustin, H. Dardente, G. C. Wagner, D. A. Carter, J. D. Johnston, G. A. Lincoln, and D. G. Hazlerigg Egr1 involvement in evening gene regulation by melatonin FASEB J, March 1, 2009; 23(3): 764 - 773. [Abstract] [Full Text] [PDF]

				N. Esumi, S. Kachi, L. Hackler Jr, T. Masuda, Z. Yang, P. A. Campochiaro, and D. J. Zack BEST1 expression in the retinal pigment epithelium is modulated by OTX family members Hum. Mol. Genet., January 1, 2009; 18(1): 128 - 141. [Abstract] [Full Text] [PDF]

				H. C. Hartzell, Z. Qu, K. Yu, Q. Xiao, and L.-T. Chien Molecular Physiology of Bestrophins: Multifunctional Membrane Proteins Linked to Best Disease and Other Retinopathies Physiol Rev, April 1, 2008; 88(2): 639 - 672. [Abstract] [Full Text] [PDF]

				R. F. Mullins, M. H. Kuehn, E. A. Faidley, N. A. Syed, and E. M. Stone Differential Macular and Peripheral Expression of Bestrophin in Human Eyes and Its Implication for Best Disease Invest. Ophthalmol. Vis. Sci., July 1, 2007; 48(7): 3372 - 3380. [Abstract] [Full Text] [PDF]