Potential regulators of CRALBP gene expression

Purpose. To identify DNA elements regulating expression of the human cellular retinaldehyde-binding protein (CRALBP) gene. CRALBP is expressed in retinal pigment epithelium (RPE), but not photoreceptors, and may help control routing of 11-cis-retinoid in the visual cycle. Methods. Activity of human CRALBP gene promoter-Luciferase fusion constructs was assayed by transfection of the human ARPE cell Line (Dunn et al., 1995 IOVS 36, S766, #3540). Electrophoretic mobility shift assays (EMSA) were used to compare binding of nuclear factors to wild type and mutant oligonucleotide probes spanning the proximal CRALBP promoter in a region containing two copies of the photoreceptor consensus element (PCE) and two adjacent elements that we term BCE. Results. Transfection results from the ARPE cell line mimicked results from primary human RPE cells and the D407 human RPE cell line (Kennedy et al., 1995 IOVS 36, S124, #608). EMSA with 29nt CRALBP probes and RPE nuclear proteins demonstrated two strong signals with wild type probe and little binding to PCE or BCE mutant probes. The PCE mutant oligonucleotide competes away one of the complexes and the BCE mutant oligonucleotide competes away both complexes. Conclusions. Transfections in the ARPE cell line corroborated previous in vitro results. The PCE and BCE sites in the CRALBP promoter appear to interact with RPE nuclear proteins, perhaps in a cooperative fashion. The potential role of these elements in regulating the restricted expression of CRALBP is currently under investigation by transient transfection assays and transgenic mouse approaches.