Abstract
Abstract: :
Purpose: Several approaches led to the identification of a nuclear receptor which expression is restricted to the retina: Photoreceptor cell–specific Nuclear Receptor (PNR). PNR belongs to the orphan nuclear receptor category, it has no endogenous ligand identified. Several mutations in the human gene encoding PNR, also known as NR2E3, cause Enhanced S–Cone Syndrome (ESCS), a disease characterized by hypersensibility to blue light and excess of blue cones followed by photoreceptor degeneration. Our study was aimed to determine the effect of naturally occuring and artificial mutations on PNR Ligand Binding Domain, or PNR(LBD), repressive transcriptional property. Methods: Point mutations were introduced in PNR(LBD) fused to an heterologous DNA–Binding Domain (GAL4), by oligonucleotide–directed mutagenesis. Expression and DNA binding ability of transfected mutated GAL4–PNR(LBD) and wild–type were shown to be similar by Western–blot and EMSA. GAL4–PNR(LBD) transcriptional activity was measured in transient co–transfection assays on luciferase reporter gene controlled by a promoter containing GAL4 upstream activation sequences (GAL4–UAS). Results: Two naturally occuring mutations, R385P and M407K, abolish transcriptional repression by PNR. Four other naturally occuring mutations have no effect on transciptional repression. Strikingly, the artificial R385L has no effect on transcriptional repression, suggesting that R385P has a conformational effect. The deletion of H12 helix (N397stop), where M407K mutation is localized, also suppresses transcriptional repression. This is in agreement with the role of this helix in co–repressor interaction highlighted in other nuclear receptor. The fact that the majority of the mutations does not interfere with the transcriptional repression support the existence of a ligand for PNR.
Keywords: transcription factors • mutations • retinal degenerations: hereditary