The photoreceptor nuclear receptor Nr2e3 interacts with Crx and plays a dual role in regulating the transcription of rod and cone genes.

Abstract

Abstract: : Purpose: Nr2e3 is an orphan nuclear receptor expressed specifically by retinal photoreceptor cells. Nr2e3 mutations cause Enhanced S–cone Syndrome in humans and retinal degeneration in mice, with increased number of blue cones and photoreceptor degeneration. Using yeast two–hybrid assays, we identified the cone–rod homeobox (Crx) transcription factor as an interacting partner of Nr2e3. The goal of this study was to determine the importance of the Crx–Nr2e3 interaction in regulating the transcription of rod and cone genes. Methods: Yeast two–hybrid assays and co–immunoprecipitation were used to analyze physical interactions. Co–transfection assays using HEK293 cells and luciferase reporters harboring the promoters of rhodopsin and blue opsin measured the effect of the interaction on promoter activity. Immunohistochemistry on retinal sections was used to detect the expression of the two proteins. Chromatin immunoprecipitation (ChIP) detected binding of the two factors to their photoreceptor gene targets. Results: Interaction of Nr2e3 with Crx was detected using both yeast two–hybrid and in vitro co–immunoprecipitation assays. Interaction was mediated by the DNA binding domain of Nr2e3. In co–transfection assays, Nr2E3 enhanced Crx (and/or Nrl)–dependent activation of the rhodopsin promoter, but repressed Crx activation of the blue opsin promoter. Immunohistochemistry with antibodies specific to Nr2e3 and Crx localized Nr2e3 in the nucleus of rod photoreceptors and Crx in the nucleus of both rods and cones. Based on ChIP assays, in addition to opsins, both proteins bind in vivo to the promoters of several other rod and cone genes. Conclusions: Nr2e3 acts as a dual transcription regulator by interacting with Crx. It enhances rhodopsin, but represses blue opsin promoter activity. Nr2e3 also appears to regulate other Crx–targeted rod and cone genes. The interaction of Nr2e3 with Crx may contribute to the development (maintenance) of rod photoreceptors by directing photoreceptor precursors to become rods instead of blue cones.

Keywords: transcription factors • photoreceptors