The upstream region of the Rpe65 gene confers retinal pigment epithelium-specific expression in vivo and in vitro and contains critical octamer and E-box binding sites

RPE65 is essential for all-trans- to Il-cis-retinoid isomerization, the hal lmark reaction of the retinal pigment epithelium (RPE). Here, we identify r egulatory elements in the Rpe65 gene and demonstrate their functional relev ance to Rpe65 gene expression. We show that the 5' flanking region of the m ouse Rpe65 gene, like the human gene, lacks a canonical TATA box and consen sus GC and CAAT boxes. The mouse and human genes do share several cis actin g elements, including an octamer, a nuclear factor one (NFI) site, and two E-box sites, suggesting a conserved mode of regulation. A mouse Rpe65 promo ter/beta-galactosidase transgene containing bases -655 to +52 (TR4) of the mouse 5' flanking region was sufficient to direct high RPE-specific express ion in transgenic mice, whereas shorter fragments (-297 to +52 or -188 to 52) generated only background activity. Furthermore, transient transfection of analogous TR4/luciferase constructs also directed high reporter activit y in the human RPE cell line D407 but weak activity in the non-RPE cell Lin es HeLa, HepG2, and HS27. Functional binding of potential transcription fac tors to the octamer sequence, AP-4, and NFI sites was demonstrated by direc ted mutagenesis, electrophoretic mobility shift assay, and cross linking. M utations of these sites abolished binding and corresponding transcriptional activity and indicated that octamer and E-box transcription factors synerg istically regulate the RPE65 promoter function. Thus, we have identified th e regulatory region in the Rpe65 gene that accounts for tissue-specific exp ression in the RPE and found that octamer and E-box transcription factors p lay a critical role in the transcriptional regulation of the Rpe65 gene.