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Cynthia L. Montana, Guang-Hua Peng, Nicholas Tran, Shiming Chen, Joseph C. Corbo; Otx2, Crx, And RORβ2 Directly Regulate Nrl, A Photoreceptor Cell Fate Determinant. Invest. Ophthalmol. Vis. Sci. 2011;52(14):5415.
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© ARVO (1962-2015); The Authors (2016-present)
The transcription factor Nrl is a master regulator of rod photoreceptor differentiation, and mutations in NRL have been associated with human retinitis pigmentosa. Despite the developmental and clinical significance of this gene, however, little is known about the transcriptional regulation of Nrl itself. The goal of this research was to identify transcription factors and their binding sites that directly regulate Nrl transcription in the developing mouse retina.
We analyzed the mouse Nrl promoter region for important cis-regulatory elements via retinal explant electroporation. Promoter variants were cloned upstream of the fluorescent reporter DsRed, electroporated into retinal explants isolated from postnatal day 0 (P0) mice, and quantitatively assessed for expression level after eight days in explant culture. To confirm the identity of trans-acting Nrl regulators, a variety of techniques were used including electrophoretic mobility shift assays (EMSA), chromatin immunoprecipitation (ChIP), and quantitative RT-PCR (qRT-PCR).
We discovered a phylogenetically conserved, 30-nucleotide region that is critical for Nrl promoter activity. This region contains high affinity binding sites for the retinal transcription factors Crx and RORβ2, and point mutations in these sites abolished promoter activity. We demonstrated via EMSA that Crx, Otx2, and RORβ2 can bind to the critical region in vitro; mutations in the Crx site abolished Crx and Otx2 binding, while mutations in the RORβ2 site abolished RORβ2 binding. ChIP performed with antibodies to Crx, Otx2, and RORβ2 on P3 retinal tissue confirmed that these factors are bound to the critical region in vivo. Finally, qRT-PCR analysis of Nrl transcript levels in wildtype and Crx-/- retinas suggest that Crx is required mainly for the maintenance of Nrl expression rather than its initiation.
Our results indicate that Crx, Otx2, and RORβ2 directly regulate Nrl transcription by binding to specific sites within the Nrl promoter. We propose a model in which Nrl expression is primarily initiated by Otx2 and RORβ2 and later maintained at high levels by Crx and RORβ2.
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