Functional analysis of NR2E3 in retinal degenerations

Abstract

The vertebrate retina is a model system of the development of the central nervous system. Because of its rapid development and the availability of many molecular tools the zebrafish is an established model of retinal development. Development and maintenance of the retina is driven by a complex regulatory network of transcription factors. For instance, the development of photoreceptors is initiated by cone-rod homeobox (Crx), and neural leucine zipper (Nrl), and the nuclear receptors Trβ2 and PNR/Nr2e3 further determine proper rod and cone development. Recessive mutations in NR2E3 cause Goldmann-Favre syndrome also called enhanced S-cone syndrome and a unique dominant mutation located in the DNA-binding domain p.G56R causes severe retinitis pigmentosa. In the present study, the first aim consisted of generating a transgenic zebrafish line expressing hemizygously the dominant NR2E3p.G56R mutant protein to study the mechanism underlying retinitis pigmentosa. The human mutant protein was expressed in the retina of transgenic zebrafish but a severe clinical phenotype could not be recapitulated. However, dysregulation of opsin gene expression was observed. A second transgenic zebrafish line expressing the fluorescent mCherry protein under the control of a potential nr2e3 promoter was generated. This promoter fragment was sufficient to drive retina-specific mCherry expression in larvae and adult and might become a valuable tool to drive transgene expression in a spatio-temporal manner in the retina. In addition, we studied the coregulation and protein interactions between NR2E3 and the nuclear receptor Rev-erb α (NR1D1) as well as with the photoreceptor-specific transcription factors CRX and NRL. Finally, we performed functional and structural analysis of the NR2E3 ligand-binding domain (LBD) and identified dimerization potential as a new molecular mechanism in Goldmann-Favre syndrome. Notably, we identified the first genotype-phenotype correlation for two mutations located in the NR2E3 LBD.