Identification of a KRAB-zinc finger protein binding to the Rpe65 gene promoter.

PURPOSE: We wish to identify transcriptional factors involved in regulation binding to the proximal promoter region of the RPE65 gene that confers RPE-specific expression. METHODS: We incubated human D407 RPE cell nuclear extract with double-stranded (sense 5-prime biotinylated) oligonucleotides, based on the RPE65 proximal gene promoter, bound to streptavidin-Dynabeads. Bound nuclear proteins were eluted, separated on SDS-PAGE, and analyzed by mass spectrometry. Peptide sequence was used to identify cDNA clones that were subcloned into pCDNA3.1 for expression and co-transfection into D407 cells to assess transcriptional activation of mouse Rpe65 gene promoter/reporter constructs. SiRNA interference was used to suppress ZNF492 expression. RESULTS: We identified a D407 nuclear protein binding to biotinylated-DNA/streptavidin beads as the product of clone KIAA1473 encoding a protein named ZNF492. ZNF492 has an open reading frame of 531 amino acids with a truncated N-terminus and lacks the usual Krüppel-associated box-A (KRAB-A) while KRAB-B remains intact and has 12 C2H2 zinc-fingers in tandem arrangement. Co-expression in D407 cells of ZNF492 protein did not activate TR1, a mouse Rpe65 gene promoter/reporter construct with 49-bp 5-prime flanking sequence, but did activate construct TR2, containing 188-bp 5-prime flanking sequence, by 2.5-fold, and the longer constructs TR4, containing 655-bp 5-prime flanking sequence, and TR5, containing 1240-bp 5-prime flanking sequence, by about 2-fold. SiRNA-mediated suppression of ZNF492 in D407 resulted in decreased Rpe65 promoter activity. CONCLUSIONS: We have identified ZNF492, a KRAB-zinc finger protein, by its interaction with immobilized RPE65 promoter DNA sequence. This KRAB-zinc finger protein serves as a moderate transcriptional factor for Rpe65 gene upregulation. In ZNF492, absence of KRAB-A might reduce or prevent co-repressor binding to account for the modest upregulation of Rpe65 gene expression.

Mutation of key residues of RPE65 abolishes its enzymatic role as isomerohydrolase in the visual cycle.

RPE65 is essential for isomerization of vitamin A to the visual chromophore. Mutations in RPE65 cause early-onset blindness, and Rpe65-deficient mice lack 11-cis-retinal but overaccumulate alltrans-retinyl esters in the retinal pigment epithelium (RPE). RPE65 is proposed to be a substrate chaperone but may have an enzymatic role because it is closely related to carotenoid oxygenases. We hypothesize that, by analogy with other carotenoid oxygenases, the predicted iron-coordinating residues of RPE65 are essential for retinoid isomerization. To clarify RPE65's role in isomerization, we reconstituted a robust minimal visual cycle in 293-F cells. Only cells transfected with RPE65 constructs produced 11-cis-retinoids, but coexpression with lecithin:retinol acyltransferase was needed for high-level production. Accumulation was significant, amounting to >2 nmol of 11-cis-retinol per culture. Transfection with constructs harboring mutations in residues of RPE65 homologous to those required for interlinked enzymatic activity and iron coordination in related enzymes abolish this isomerization. Iron chelation also abolished isomerization activity. Mutating cysteines implicated in palmitoylation of RPE65 had generally little effect on isomerization activity. Mutations associated with Leber congenital amaurosis/early-onset blindness cause partial to total loss of isomerization activity in direct relation to their clinical effects. These findings establish a catalytic role, in conjunction with lecithin:retinol acyltransferase, for RPE65 in synthesis of 11-cis-retinol, and its identity as the isomerohydrolase.