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Noriko Esumi, Tomohiro Masuda, Jun Wan, Karl Wahlin, Jared Iacovelli, Natalie Wolkow, Joshua Dunaief, Donald Zack, Jiang Qian; Transcriptional and post-transcriptional regulation links RPE65, RLBP1, and RGR in the RPE. Invest. Ophthalmol. Vis. Sci. 2013;54(15):2612.
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RPE cells perform many specialized functions that require the precisely regulated expression of a number of RPE-specific genes. We have been studying the mechanisms that regulate gene expression in the RPE, with particular interest in examining the hypothesis that genes involved in the same pathway are controlled by a common regulatory network. Here, we aim to test this hypothesis using the visual cycle as a model system.
In this study, we focused on three visual cycle genes, RPE65, RLBP1, and RGR. For transcriptional regulation, we used RT-qPCR and immunohistochemistry for gene expression, DNase I hypersensitivity assay for defining proximal promoters, cell transfection for promoter function, and chromatin immunoprecipitation (ChIP) for binding of transcription factors. Sox9 conditional knockout mice were used to study SOX9’s role in RPE gene expression. For post-transcriptional regulation, we used bioinformatics to predict microRNA (miRNA) binding sites in the 3’-untranslated region (3’UTR), and cotransfection of miRNA mimics with 3’UTR-luciferase constructs to test miRNA-target mRNA interaction.
Conditional knockout of Sox9 in mature RPE resulted in significantly reduced expression of several visual cycle genes including Rpe65, Rlbp1, and Rgr. Promoter analyses by cotransfection showed that SOX9 and OTX2 activate the RPE65 and RLBP1 promoters, whereas SOX9 and LHX2 activate the RGR promoter. ChIP revealed that SOX9, OTX2, and LHX2 all bind to the proximal upstream region of RPE65, RLBP1, and RGR in bovine RPE cells. The expression of SOX9 and OTX2 in the RPE is well known; however, LHX2 has not been reported to be involved in RPE gene regulation. We found that LHX2 is highly expressed in mature RPE, with similar expression patterns to those of SOX9 in that both genes are expressed exclusively in Muller glia and RPE cells in adults. The 3’UTR of the three genes, particularly RPE65 and RLBP1, share multiple predicted miRNA sites, and cotransfection of a miR-137 mimic significantly decreased luciferase activity of the constructs containing the 3’UTR of RPE65 and RLBP1.
These results indicate the important role of SOX9 as a central link in the transcriptional regulation of RPE65, RLBP1, and RGR. In addition, RPE65 and RLBP1 are targets of the same miRNA(s), suggesting that these genes share a common core regulatory structure.
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