PROMOTER ANALYSIS OF RPE65, THE GENE ENCODING A 61-KDA RETINAL-PIGMENT EPITHELIUM-SPECIFIC PROTEIN

PURPOSE. To identify the functional promoter region and cis-acting ele ments that regulate the expression of RPE65, the retinal pigment epith elium (RPE)-specific gene responsible for certain forms of autosomal r ecessive childhood-onset severe retinal dystrophy. METHODS. A human ge nomic DNA clone containing the 5'-flanking region of RPE65 was isolate d and, 4.0 kb proximal to the transcription start site, was sequenced and analyzed for the presence of transcription factor-binding sites. P romoter activity was assayed by transient transfection of luciferase r eporter constructs containing nested deletions of the upstream sequenc e in the human RPE cell lines ARPE19 and D407, as well as in the SK-Me l-28 and HeLa cell lines. Specific DNA protein-binding sites present i n the 340 bp upstream of the transcription start site were identified by DNase I footprint analysis. RESULTS. Sequence analysis places the p olymorphic marker, D1S2803, within the RPE65 upstream region and ident ifies a number of sequences homologous to the gene encoding the cellul ar retinaldehyde-binding protein. Functional analysis indicates that b asal promoter activity is conferred by the sequence from -83 to +39 an d is approximately equivalent in all cell lines tested, with no other control elements detected in 3.6 kb of the upstream sequence. At least eight protected regions are identified in DNase I footprint assays, i ncluding sequences corresponding to the predicted TATA box, AP-4, and nuclear factor-1 DNA protein-binding sites. CONCLUSIONS. These finding s localize the basal promoter activity of RPE65, identify potential ci s-acting elements that act as positive regulators of gene expression, and suggest that additional regulatory elements are likely to be invol ved in restricting gene expression to the retinal pigment epithelium. Identification of promoter elements and genetic markers in the upstrea m sequence will enable the screening of patients with retinal degenera tion for possible mutations that affect RPE65 expression.