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G. Peng, J. Liu, S. Chen; Functional Analysis of NR2E3 Mutations Associated With Enhanced S–Cone Syndrome (ESCS) . Invest. Ophthalmol. Vis. Sci. 2005;46(13):3067.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Nr2e3 is an orphan nuclear receptor specifically expressed by rod photoreceptors. Nr2e3 mutations cause enhanced S–cone syndrome in humans and rd7 in mice, featuring excess S–cones but loss of rods. We reported previously that Nr2e3 activates the transcription of rod genes but represses cone genes by interacting with the homeodomain protein Crx. The goal of this study was to examine if genetically identified Nr2e3 mutations alter the ability of Nr2e3 to interact with Crx and regulate target gene transcription. Methods: Site–directed mutagenesis introduced specific mutations into Nr2e3–coding cDNA in a mammalian expression vector. Co–immunoprecipitation measured the physical interaction between mutant forms of Nr2e3 (Crx) and wild–type Crx (Nr2e3). Co–transfection in HEK293 cells analyzed the potency of mutant Nr2e3 to regulate opsin promoter activity. Chromatin immunoprecipitation (ChIP) on retinas of wild–type and mutant mice measured target occupancy of Nr2e3 and Crx in the presence and absence of each interacting partner. Results: Four Nr2e3 mutations were studied. A mutation in the DNA–binding domain (DBD), R97H, diminished Nr2e3 interaction with Crx. Similarly, a DBD mutation in Crx, E80A, also reduced Crx interaction with Nr2e3. Two mutations in Nr2e3–DBD, R76W and R97H, decreased the ability of Nr2e3 to modulate Crx (and/or Nrl)–mediated activation of rhodopsin and cone opsin promoters. Two ligand–binding domain (LBD) mutations, R311Q and W234S, diminished the potency of Nr2e3 to repress Crx–mediated activation of M–cone opsin, although they had little effect on rhodopsin. ChIP demonstrated that Nr2e3 is associated with Crx–regulated rod and cone genes in a Crx–dependent manner, while Crx binds to its targets independent of Nr2e3. Conclusions: Nr2e3 acts as a dual–function transcription regulator for the photoreceptor genes by interacting with Crx. Mutations in either Nr2e3 or Crx could affect their physical and/or functional interaction and result in transcriptional dysregulation of target genes. This study provides insights into the molecular mechanisms for photoreceptor degeneration linked to Nr2e3 or Crx.
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