Transcriptional Regulation of Neural Retina Leucine Zipper (Nrl), a Photoreceptor Cell Fate Determinant

Abstract

Background: The transcription factor Nrl is required for rod photoreceptor development, but mechanisms governing Nrl transcription remain largely unknown. Results: The transcription factors CRX, OTX2, and ROR regulate Nrl by binding directly to its promoter region. Conclusion: These three factors combinatorially control Nrl expression in the developing mouse retina. Significance: This study elucidates a critical link in the photoreceptor cis-regulatory network. The transcription factor neural retina leucine zipper (Nrl) is a critical determinant of rod photoreceptor cell fate and a key regulator of rod differentiation. Nrl / rod precursors fail to turn on rod genes and instead differentiate as cones. Furthermore, NRL mutations in humans cause retinitis pigmentosa. Despite the developmental and clinical significance of this gene, little is known about the transcriptional regulation of Nrl itself. In this study, we sought to define the cis-and trans-acting factors responsible for initiation and maintenance of Nrl transcription in the mouse retina. Utilizing a quantitative mouse retinal explant electroporation assay, we discovered a phylogenetically conserved, 30-base pair region immediately upstream of the transcription start site that is required for Nrl promoter activity. This region contains binding sites for the retinal transcription factors CRX, OTX2, and ROR, and point mutations in these sites completely abolish promoter activity in living retinas. Gelshift experiments show that CRX, OTX2, and ROR can bind to the critical region in vitro, whereas ChIP experiments demonstrate binding of CRX and OTX2 to the critical region in vivo. Thus, our results indicate that CRX, OTX2, and ROR directly regulate Nrl transcription by binding to critical sites within the Nrl promoter. We propose a model in which Nrl expression is primarily initiated by OTX2 and ROR and later maintained at high levels by CRX and ROR.