Transcriptional Regulation of Cellular Retinaldehyde-binding Protein in the Retinal Pigment Epithelium A ROLE FOR THE PHOTORECEPTOR CONSENSUS ELEMENT

Breandán N. Kennedy; Steven Goldflam; Michelle A. Chang; Peter Campochiaro; Alberta A. Davis; Donald J. Zack; John W. Crabb

doi:10.1074/jbc.273.10.5591

Transcriptional Regulation of Cellular Retinaldehyde-binding Protein in the Retinal Pigment Epithelium

A ROLE FOR THE PHOTORECEPTOR CONSENSUS ELEMENT*

From the ^‡Adirondack Biomedical Research Institute, Lake Placid, New York 12946, the ^**Department of Anatomy and Cell Biology, University of Texas Southwestern Medical Center, Dallas, Texas 75235, and the ^¶Department of Ophthalmology, Wilmer Eye Institute, Johns Hopkins University School of Medicine, Baltimore, Maryland 21287

Abstract

Cellular retinaldehyde-binding protein (CRALBP) is abundantly expressed in the retinal pigment epithelium (RPE) and Müller cells of the retina, where it is thought to function in retinoid metabolism and visual pigment regeneration. Mutations in human CRALBP that destroy retinoid binding have been linked to autosomal recessive retinitis pigmentosa. To identify the DNA elements that regulate expression of the human CRALBP gene in the RPE, transient transfection studies were carried out with three CRALBP-expressing human RPE cell culture systems. The regions from −2089 to −1539 base pairs and from −243 to +80 base pairs demonstrated positive regulatory activity. Similar activity was not observed with cultured human breast, liver, or skin cells. Since sequence analysis of the −243 to +80 region identified the presence of two photoreceptor consensus element-1 (PCE-1) sites, elements that have been implicated in photoreceptor gene regulation, the role of these sequences in RPE expression was examined. Mutation of either PCE-1 site significantly reduced reporter activity, and mutation or deletion of both sites dramatically reduced activity. Electrophoretic mobility shift analysis with RPE nuclear extracts revealed two complexes that required intact PCE-1 sites. These studies also identified two identical sequences (GCAGGA) flanking PCE-1, termed the binding CRALBP element (BCE), that are also important for complex formation. Southwestern analysis with PCE-1/BCEcontaining probes identified species with apparent masses near 90–100 and 31 kDa. These results begin to identify the regulatory regions required for RPE expression of CRALBP and suggest that PCE-1-binding factor(s) may play a role in regulating RPE as well as photoreceptor gene expression.

Footnotes

↵* This work was supported in part by National Institutes of Health Grants EY06603, EY09769, EY01765, and EY05951; the Foundation Fighting Blindness; unrestricted funds from Research to Prevent Blindness, Inc.; and the Rebecca P. Moon, Charles M. Moon, Jr., and Dr. P. Thomas Manchester Research Fund. Preliminary reports of this work were presented at the 67th Annual Meeting of the Association for Research in Vision and Ophthalmology, Ft. Lauderdale, FL, May 14–19, 1995 (Kennedy, B. N., Goldflam, S., Chang, M. A., Hacket, S., Campochiaro, P., Davis, A., Zack, D. J., and Crabb, J. W. (1995) Invest. Ophthalmol. & Visual Sci. 36, S124 (Abstr. 608); Chang, M. A., Kennedy, B., Crabb, J. W., Hackett, S., Campochiaro, P., and Zack, D. J. (1995) Invest. Ophthalmol. & Visual. Sci. 36, S124 (Abstr. 609) and at the 68th Annual Meeting of the Association for Research in Vision and Ophthalmology, Ft. Lauderdale, FL, April 21–26, 1996 (Kennedy, B. N., Chang, M. A., Campochiaro, P., Zack, D. J., and Crabb, J. W. (1996)Invest. Ophthalmol. & Visual Sci. 37, S336 (Abstr. 1540).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵§ This work was submitted in partial fulfillment of the requirements for a doctoral degree in the Cell and Molecular Biology Program jointly administered by the University College Dublin (Dublin, Ireland) and the Adirondack Biomedical Research Institute (Lake Placid, NY).
↵‖ Present address: Jules Stein Eye Inst., UCLA, Los Angeles, CA 90024.
↵‡ Recipient of a career development award from Research to Prevent Blindness, Inc.
↵§§ To whom correspondence should be addressed: Protein Chemistry Facility, Adirondack Biomedical Research Inst., 10 Old Barn Rd., Lake Placid, NY 12946. Tel.: 518-523-1281; Fax: 518-523-1849; E-mail:jcrabb{at}cell-science.org.