Identification of regulatory elements in a conserved upstream region of the gene encoding interphotoreceptor retinoid-binding protein (IRBP)

IRBP is a retinoid and fatty-acid-binding protein synthesized in the photoreceptor cells and secreted into the interphotoreceptor matrix. The regulation of IRBP gene expression is crucial for the function of the IRBP gene during the visual cycle and retinal development.;To test our hypothesis, 5' end nested deletional analysis in the upstream region of the IRBP gene was conducted in transient transfection assays using a CAT reporter gene to initially localize the possible cis-elements. Deletional analysis was followed by in vitro DNA/Protein binding analysis to define transcription factor binding sites. Site-directed mutagenesis studies were then performed on the identified protein binding site to examine the function of the element. The effect of the orientation of the element on the gene expression was examined to characterize the enhancer activity of the defined element.;The results of deletional analysis showed that CAT activity from the construct lacking the distal homologous subregion is ∼75% lower than that containing the entire distal homologous region. The result of footprinting analysist indicated a Sp1 binding site around the G-rich motif in the distal homologous subregion, which also could be bound by nuclear extract from RB cells. Mutational analysis by changing four key nucleotides of the G-rich motif reduced reporter gene activity by ∼45%. Changing the orientation of the G-rich region did not affect reporter gene activity.;Our results suggest that there is a regulatory cis-element in the distal homologous subregion which is highly conserved in diverse species. This element contains a functional G-rich motif which is a likely Sp1 binding site and can function in either orientation. While the regulatory elements within the proximal homologous subregion are necessary for photoreceptor-specific IRBP gene expression, the identified G-rich regulatory element may be responsible for normal IRBP gene expression within the photoreceptors.;Previous work in this laboratory and others, using transgenic mice, has identified the regulatory region of the IRBP gene that is necessary for photoreceptor-specific transgene expression. Although sequence within 200 bp in the proximal homologous region is sufficient for photoreceptor-specific regulation, higher transgene activities are observed from mice harboring longer upstream sequences. A sequence comparison of 2 kb of the upstream region of the IRBP gene in diverse species revealed two homologous subregions: a proximal subregion within 300 bp of the transcriptional start site and a distal subregion between --1564 and --1244. We hypothesize that additional, possibly far-upstream elements located within the distal homologous subregion, may be important for IRBP gene regulation.