Elements regulating the transcription of human interstitial retinoid-binding protein (IRBP) gene in cultured retinoblastoma cells

Purpose. To identify cis-acting elements and trans-acting factors involved in the expression of human IRBP gene. Methods. Transient transfection of WERI-Rb1 and HeLa cells, DNase 1 footpri nting, gel mobility-shift assay and yeast one-hybrid system were used to st udy the regulatory elements that are involved in the expression of human IR BP gene. Results. A region between -1620 and -1411 was shown to have enhancer proper ties. Using nuclear extracts from WERI-Rb1 and HeLa cells, four footprints were identified in the proximal promoter region (-206 to +68). The core pro moter element IP1 binds to OTX2 in the yeast one-hybrid system. By cotransf ecting HeLa cells, OTX2 could transactivate the irbp promoter. The function s of IP2 (from -119 to -86) and IP3 (from -183 to -147) remain to be determ ined. The region containing the HeLa cell-specific footprint IP4 (from -202 to -180) could silence the OTX2 transactivation of the irbp promoter. Conclusion. The 5'-flanking region of irbp contains an enhancer sequence. T he possible silencer upstream from the core promoter may serve to suppress expression of irbp in HeLa cells. When the proximal promoter is used to ide ntify binding proteins in a human retina library by the yeast one hybrid sy stem, nine of the identified clones contained the cDNA sequence for the hom eodomain protein OTX2. Since no clones for the homeodomain protein CRX were found, and since OTX2 can transcriptionally activate irbp in normally non- expressing HeLa cells, it is possible that OTX2 rather than CRX is the tran scriptional activator for irbp in human photoreceptors.