Biochemical and functional identification of cis-elements and trans-factors regulating IRBP gene expression

Purpose. The study of molecular mechanisms regulating the expression of the interphotoreceptor retinoid-binding protein (IRBP) gene can provide a better understanding of the function of the protein. The present study is to identify cis-elements and trans-factors that are involved in IRBP gene expression. Methods. For DNA-protein binding analyses, methylation interference, missing contacts, gel shift and footprinting were used. For functional assay, transient transfection of an immortalized retinal culture, (E1A-NR.3) was performed with a CAT gene directed by various candidate promoters. Results. Sequence and footprint analyses with purified proteins on 4 identified DNA-protein contact points suggested that they are: hormone response element (HRE), photoreceptor conserved element (PCE), SP1, and AP2. The HRE contains two direct repeats of the half-site A(CA/AT)TCA. Gel shift analysis using probes containing the HRE, but not their mutants, with either nuclear extracts or expressed RARα and RXRΓ showed specific bands that were supershifted by specific antibodies. In the transfected cells, the cotransfection of RARα and RXRΓ expression plasmids altered the activity of the CAT gene directed by the IRBP promoter. Conclusions. All of the identified cis-elements may be involved in the regulation of IRBP gene expression. Further functional studies using mutant constructs in transfections are underway.