Abstract
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 RORβ, and point mutations in these sites completely abolish promoter activity in living retinas. Gel-shift experiments show that CRX, OTX2, and RORβ 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 RORβ 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 RORβ and later maintained at high levels by CRX and RORβ.
Highlights
The transcription factor neural retina leucine zipper (Nrl) is required for rod photoreceptor development, but mechanisms governing Nrl transcription remain largely unknown
A 30-Nucleotide Region Is Critical for Nrl Promoter Activity— To identify the cis- and trans-acting factors required for regulation of Nrl, we undertook a detailed analysis of its proximal promoter region
We have shown that OTX2, CRX, and ROR directly regulate Nrl expression in developing mouse rod photoreceptors
Summary
The transcription factor Nrl is required for rod photoreceptor development, but mechanisms governing Nrl transcription remain largely unknown. To conduct the Nrl promoter analysis in the living mouse retina, we utilized a retinal explant electroporation assay [20] This assay has been used previously to quantify the activity of photoreceptor-specific cis-regulatory regions [21] and represents a much more realistic assay for analyzing cis-regulation than traditional mammalian cell culture approaches. Using this system, we have found that three retinal transcription factors, OTX2, CRX, and ROR, activate Nrl transcription by binding directly to a highly conserved region of the Nrl promoter
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