Abstract
The retinal pigment epithelium (RPE) performs specialized functions to support retinal photoreceptors, including regeneration of the visual chromophore. Enzymes and carrier proteins in the visual cycle function sequentially to regenerate and continuously supply 11-cis-retinal to retinal photoreceptor cells. However, it is unknown how the expression of the visual cycle genes is coordinated at the transcriptional level. Here, we show that the proximal upstream regions of six visual cycle genes contain chromatin-accessible sex-determining region Y box (SOX) binding sites, that SOX9 and LIM homeobox 2 (LHX2) are coexpressed in the nuclei of mature RPE cells, and that SOX9 acts synergistically with orthodenticle homeobox 2 (OTX2) to activate the RPE65 and retinaldehyde binding protein 1 (RLBP1) promoters and acts synergistically with LHX2 to activate the retinal G protein-coupled receptor (RGR) promoter. ChIP reveals that SOX9 and OTX2 bind to the promoter regions of RPE65, RLBP1, and RGR and that LHX2 binds to those of RPE65 and RGR in bovine RPE. ChIP with human fetal RPE cells shows that SOX9 and OTX2 also bind to the human RPE65, RLBP1, and RGR promoters. Conditional inactivation of Sox9 in mouse RPE results in reduced expression of several visual cycle genes, most dramatically Rpe65 and Rgr. Furthermore, bioinformatic analysis predicts that multiple common microRNAs (miRNAs) regulate visual cycle genes, and cotransfection of miRNA mimics with luciferase reporter constructs validated some of the predicted miRNAs. These results implicate SOX9 as a key regulator of visual cycle genes, reveal for the first time the functional role of LHX2 in the RPE, and suggest the possible regulation of visual cycle genes by common miRNAs.
Highlights
The visual cycle is an enzymatic cascade that regenerates the visual chromophore
We found that RPE65, retinaldehyde binding protein 1 (RLBP1), retinal G protein-coupled receptor (RGR), and RBP1 contain at least one consensus OTX site in the proximity of the identified sex-determining region Y box (SOX) sites
The results indicate that these visual cycle genes share the presence of chromatin-accessible SOX binding sites in retinal pigment epithelium (RPE) cells in vivo, suggesting that one or more SOX proteins may be involved in coordinately regulating the expression of visual cycle genes and that the putative SOX regulatory protein(s) may act through interaction with an OTX family member
Summary
The visual cycle is an enzymatic cascade that regenerates the visual chromophore. Results: Visual cycle gene expression is regulated by SOX9 in combination with OTX2 or LHX2 and can be modulated by common microRNAs. Enzymes and carrier proteins in the visual cycle function sequentially to regenerate and continuously supply 11-cis-retinal to retinal photoreceptor cells It is unknown how the expression of the visual cycle genes is coordinated at the transcriptional level. Major RPE-expressed components of the visual cycle are three enzymes (RPE65, LRAT, and RDH5), two retinoid carrier proteins (RLBP1 and RBP1), and a modulator (RGR), which work sequentially to regenerate and continuously supply 11-cis-retinal to retinal photoreceptors. Injection of sodium iodate (NaIO3), an oxidizing agent known to induce selective RPE cell death followed by retinal degeneration, resulted in down-regulation of RPE65, LRAT, and RLBP1 within 2 days [9, 10] These findings support the idea that pan-down-regulation of visual cycle genes may be a general response of RPE cells to stress [8]. We show that the 3Ј UTRs of the visual cycle genes share binding sites for common miRNAs, suggesting that visual cycle genes may be coordinately regulated at the posttranscriptional level
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