The Cis-regulatory Logic of the Mammalian Photoreceptor Transcriptional Network

Abstract

The photoreceptor cells of the retina are subject to a greater number of genetic diseases than any other cell type in the human body. The majority of more than 120 cloned human blindness genes are highly expressed in photoreceptors. In order to establish an integrative framework in which to understand these diseases, we have undertaken an experimental and computational analysis of the network controlled by the mammalian photoreceptor transcription factors, Crx, Nrl, and Nr2e3. Using microarray and in situ hybridization datasets we have produced a model of this network which contains over 600 genes, including numerous retinal disease loci as well as previously uncharacterized photoreceptor transcription factors. To elucidate the connectivity of this network, we devised a computational algorithm to identify the photoreceptor-specific cis-regulatory elements (CREs) mediating the interactions between these transcription factors and their target genes. In vivo validation of our computational predictions resulted in the discovery of 19 novel photoreceptor-specific CREs near retinal disease genes. Examination of these CREs permitted the definition of a simple cis-regulatory grammar rule associated with high-level expression. To test the generality of this rule, we used an expanded form of it as a selection filter to evolve photoreceptor CREs from random DNA sequences in silico. When fused to fluorescent reporters, these evolved CREs drove strong, photoreceptor-specific expression in vivo. This study represents the first systematic identification and in vivo validation of CREs in a mammalian neuronal cell type and lays the groundwork for a systems biology of photoreceptor transcriptional regulation.

A typology of photoreceptor gene expression patterns in the mouse

“…Next, we performed ISH on a subset of the dysregulated genes to validate the microarray (8,38, and this study) results. We selected genes which were found to be up-or down-regulated in Nrl Ϫ/Ϫ , Nr2e3 Ϫ/Ϫ , or Crx Ϫ/Ϫ by microarray.…”

“…In contrast, we predict that Type II cone genes will show an increase of considerably lower magnitude in Nr2e3 Ϫ/Ϫ than in Nrl Ϫ/Ϫ . To test this hypothesis, we performed ISH for 37 cone-enriched genes on the Nr2e3 Ϫ/Ϫ retina (8,38) Of these, 24 probes gave a sufficiently strong signal to classify the Nr2e3 derepression pattern as Type I or II (Table 1 and SI Table 6). Remarkably, when these 24 genes are ranked according to the ratio of their Nrl Ϫ/Ϫ and Nr2e3 Ϫ/Ϫ microarray scores (Nrl:Nr2e3 ratio), the ratios were perfectly predictive of the pattern of expression.…”

“…One interpretation of this result is that a significant number of the up-regulated genes represent a response to injury in this retinopathic background. However, this is unlikely to account for the magnitude of the difference because there are only 691 up-regulated genes in the Crx mutant retina by the same criteria (38). Given that the Crx mutation results in a greater degree of retinal abnormality and more rapid photoreceptor cell death than the Nrl mutant (5,20), one would expect, if anything, a greater up-regulation of injury-response genes in that background.…”

“…Interestingly, Vax2os is markedly down-regulated in both Nrl Ϫ/Ϫ and Crx Ϫ/Ϫ (SI Table 4 and ref. 38), whereas Vax2 itself is unchanged in these backgrounds. To our knowledge, Vax2os represents the first rod-enriched gene known in the mouse to be asymmetrically expressed along the D-V axis.…”

New meaning in the message: Noncoding RNAs and their role in retinal development

“…Both CrxOS and Otx2OS are expressed primarily in amacrine and ganglion cells in the adult retina, while Crx and Otx2 are expressed in photoreceptor and bipolar cells, and thus show essentially complementary patterns of expression (Alfano et al, 2005). The only other HOST whose cellular expression pattern has been examined in retina is Vax2OS, which was independently identified as a transcriptional target of both Crx and Nrl in a recent microarray-based screen Hsiau et al, 2007). Vax2OS is selectively expressed in rod photoreceptors and, uniquely for rod-enriched transcripts, at higher levels in ventral than in dorsal retina.…”