From the Wilmer Eye Institute, Johns Hopkins University School of Medicine, Baltimore, Maryland 21231.
J Biol Chem. 2014 May 2;289(18):12908-21. doi: 10.1074/jbc.M114.556738. Epub 2014 Mar 14.
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.
视网膜色素上皮 (RPE) 具有支持视网膜光感受器的特殊功能，包括视觉色素的再生。视觉循环中的酶和载体蛋白依次发挥作用，以再生和持续供应 11-顺式视黄醛给视网膜光感受器细胞。然而，尚不清楚视觉循环基因的表达如何在转录水平上协调。在这里，我们显示六个视觉循环基因的近端上游区域包含染色质可及的性别决定区 Y 盒 (SOX) 结合位点，SOX9 和 LIM 同源框 2 (LHX2) 在成熟的 RPE 细胞核中共表达，并且 SOX9 与同源盒 2 (OTX2) 协同作用以激活 RPE65 和视黄醛结合蛋白 1 (RLBP1) 启动子，并与 LHX2 协同作用以激活视网膜 G 蛋白偶联受体 (RGR) 启动子。ChIP 揭示 SOX9 和 OTX2 结合到 RPE65、RLBP1 和 RGR 的启动子区域，LHX2 结合到牛 RPE 中的 RPE65 和 RGR 的启动子区域。用人类胎儿 RPE 细胞进行 ChIP 显示 SOX9 和 OTX2 也结合到人类 RPE65、RLBP1 和 RGR 启动子。在小鼠 RPE 中条件性失活 Sox9 导致几种视觉循环基因的表达减少，最显著的是 Rpe65 和 Rgr。此外，生物信息学分析预测多个常见的 microRNAs (miRNAs) 调节视觉循环基因，并且用荧光素酶报告构建体共转染 miRNA 模拟物验证了一些预测的 miRNAs。这些结果表明 SOX9 是视觉循环基因的关键调节剂，首次揭示了 LHX2 在 RPE 中的功能作用，并提示常见 miRNAs 可能调节视觉循环基因。
