Identification of the downstream targets of Nrl: the key regulator of rod photoreceptor differentiation

Abstract

Abstract: : Purpose:The neural retina leucine zipper (Nrl) is currently the earliest known marker of rods. In the mature retina it regulates the expression of genes encoding rod phototransduction proteins. Its pivotal role in rod differentiation was identified by studies in mouse, where loss of Nrl results in the functional transformation of all rods into cones. Our goal is to generate retinal expression profiles of Nrl–knockout mouse to identify transcripts that are specifically associated with rods or cone photoreceptors. Methods:RNA samples from P2, P10 and 2M retinas of Nrl null and wild type control mice were labeled using standard protocols and hybridized to Affymetrix MGU74Av2 GeneChips. Four replicates were performed for each time–point. Data were normalized using the Robust Multichip Average algorithm. Differentially expressed genes were identified and ranked using a novel multicriteria statistical methodology. Data were confirmed using quantitative real–time PCR. The promoter regions of candidate genes were tested by luciferase reporter assays in cell culture and/or by chromatin immunoprecipitation (ChIP). Results:Microarray analysis revealed more than 300 differentially expressed genes in the Nrl knockout retina. Real–time PCR confirmed over 85% of >50 genes tested. In addition to Rho, Pde6a and Pde6b (known direct targets of Nrl), ChIP revealed that Nrl apparently directly regulates a significant proportion of the candidate genes examined. These downstream targets include rod transducin alpha–subunit, cyclic nucleotide gated channel alpha 1 (ion channel), and phosducin. In addition several Nrl target genes of unknown function were identified. Luciferase assays confirmed some of these findings but in a few cases revealed no significant level of activation by Nrl, probably because of the lack of appropriate transcriptional machinery necessary for the expression of these targets. Conclusions:Using a combination of microarray analysis with methods of validation such as reporter assays and ChIP, we have identified many putative downstream targets of Nrl. This information is necessary for understanding the transcriptional regulatory hierarchy governing the differentiation and functional maintenance of rod photoreceptors. Elucidation of such networks and pathways should assist in the identification of novel therapeutic targets to combat retinal diseases.

Keywords: gene microarray • gene/expression • transcription factors