Cloning and characterization of the human RPE65 gene: A model for regulation of gene expression in the retinal pigment epithelium.

Abstract

The retinal pigment epithelium (RPE) is an integral part of the multi-cellular vertebrate retina. The RPE performs multiple support functions that are necessary for normal vision, and the importance of the RPE for photoreceptor cell viability is apparent in both normal retinal development and the progression of retinal diseases. To develop a model system for studying the mechanisms that regulate the development and differentiation of the RPE, the gene encoding the RPE-specific protein, RPE65, was cloned and characterized. RPE65 was identified using monoclonal antibodies generated against bovine RPE membrane preparations. Bovine cDNA sequences were then used to identify human cDNA and genomic clones. The human and bovine RPE65 cDNA sequences are highly conserved, but are not homologous to any other database sequences. Expression of RPE65 was detected only in the RPE, and was found to be rapidly down-regulated in cultured cells. The human gene structure was determined, and the gene was mapped to chromosome 1p31. This led to the identification of mutations in RPE65 that cause childhood-onset severe retinal dystrophy, emphasizing the importance of RPE-specific genes for supporting the function of the neural retina. To identify cis-regulatory elements that are important for the regulation of gene expression in the RPE, the RPE65 5$\sp\prime$-flanking region was cloned and characterized. Functional analysis indicates that promoter activity is conferred by sequences proximal to the transcription start site, and that the factors required for high level RPE65 expression are not present in most cultured cells. DNA-protein binding sites were identified in the proximal promoter region, including an extended AP-1 element that is a potential binding site for the retina-specific transcription factor NRL. Further studies revealed that NRL is expressed in the RPE, and can bind to and transactivate the RPE65 promoter. These results suggest that transcription factors common to the RPE and neural retina may play a role in coordinating gene expression in these two closely related tissues.