A Single Cis-acting Element in a Short Promoter Segment of the Gene Encoding the Interphotoreceptor Retinoid-binding Protein Confers Tissue-specific Expression

doi:10.1074/jbc.270.3.1289

A Single Cis-acting Element in a Short Promoter Segment of the Gene Encoding the Interphotoreceptor Retinoid-binding Protein Confers Tissue-specific Expression (*)

From the (1)Institute of Biology and Genetics, University of Genova, Viale Benedetto XV 6, 16132 Genova, Italy, the
(2)Department of Genetics, Biology and Medical Chemistry, University of Torino, Via Santena 5 bis, 10126 Torino, Italy, and the
(3)National Institute for Research on Cancer, Viale Benedetto XV 10, 16132 Genova, Italy

^§ To whom correspondence should be addressed:
IBiG-University of Genova, Viale Benedetto XV 6, 16132 Genova, Italy
. Tel.: 39-10-353-8981; Fax: 39-10-353-8978.

Abstract

Interphotoreceptor retinoid-binding protein (IRBP) is the major protein component of the interphotoreceptor matrix. IRBP has a highly restricted tissue-specific expression in retinal photoreceptor cells and in a subgroup of pinealocytes. With the purpose of understanding how transcriptional regulation contributes to the expression of human IRBP, we have studied a short promoter fragment (from −123 to +18, relative to the transcription start site). We demonstrate, by analysis of the expression of the lacZ reporter gene fused to this short promoter fragment in transgenic mice, that it is sufficient to confer tissue-specific expression in retinal photoreceptors and in pinealocytes. DNA/protein binding assays, performed to identify binding sites for tissue-specific trans-acting factors, have shown that an element between −45 and −58 binds a factor present only in nuclear extracts of retinoblastoma-derived cell lines, which express IRBP. An element further upstream, between −86 and −106, binds apparently ubiquitous factors. Site-directed mutagenesis was performed to disrupt a GATTAA motif included in the −45 to −58 binding site and a second inverted GATTAA motif present shortly upstream. In transgenic mice bearing the mutated version of the promoter fragment, the expression of the reporter gene was completely abolished, thus suggesting that this element is essential for tissue-specific expression. A GATTAA motif appears in the 5′-flanking regions of several photoreceptor-specific genes, suggesting that this could be the recognition site for a photoreceptor-specific factor.

Footnotes

↵* This work was supported by a grant from the Associazione Italiana per la Ricerca sul Cancro to (R. R. and A. A.), a grant from Consiglio Nazionale delle Ricerche “Ingegneria Genetica” (to D. N.), and by a Telethon grant. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

IRBP

interphotoreceptor retinoid-binding protein

bp

base pair(s)

X-gal

5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside

lacZ

gene encoding β-galactosidase

GRA

gel retardation assay.
↵²N. Bobola, A. Albini, D. Noonan, and R. Ravazzolo, manuscript in preparation.
- Received July 21, 1994.