The Photoreceptor Specific Nuclear Receptor NR2E3 (PNR) Is a Direct Target of NRL and Can Induce Rod Differentiation

Abstract

Abstract: : Purpose: NR2E3, also known as photoreceptor specific nuclear receptor (PNR), is expressed in rod photoreceptors and synergistically activates rod specific genes together with NRL, CRX and NR1D1 (Rev–erbα). Mutations in the human NR2E3 gene cause enhanced S–cone syndrome, characterized by increased numbers of S–cone photoreceptors and retinal degeneration. Nrl is shown to be essential for rod differentiation and Nr2e3 expression is undetectable in the Nrl–knockout mice. Our goal is to delineate the physiological function of Nr2e3 during photoreceptor development and define the relationship between Nrl and Nr2e3 in transcriptional regulation. Methods: Chromatin immunoprecipitation (ChIP) of mouse retina was performed using rabbit anti–NRL polyclonal antibody. Transient transfections were performed in HEK293 cells and luciferase reporter assays were used to measure promoter activity. Nr2e3 transgenic mice were generated in the Nrl^–/– mice background, where Nr2e3 expression was driven by Nrl, S–opsin or Crx promoter. Southern blotting was used to confirm the transgene incorporation. RT–PCR, immunoblotting and immunohistochemistry were used to examine the expression of photoreceptor genes. Retinal function was studied with ERG. Results: ChIP assay showed that Nrl is bound to the Nr2e3 promoter in vivo. Nr2e3 promoter was stimulated by co–expression of Nrl and/or Crx. Ectopic expression of Nr2e3 in the Nrl–knockout mice resulted in expression of rhodopsin and other rod–specific genes, with corresponding changes in photoreceptor morphology. No rod ERG, however, was detected. Conclusions: Our results suggest that Nr2e3 is downstream of Nrl in rod differentiation pathway and can activate many rod–specific genes even in the absence of Nrl.

Keywords: photoreceptors • transcription factors • retinal development