The Cis-regulatory Logic of the Mammalian Photoreceptor Transcriptional Network

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

Transcriptional Regulation of Rod Photoreceptor Homeostasis Revealed by In Vivo NRL Targetome Analysis

A stringent control of homeostasis is critical for functional maintenance and survival of neurons. In the mammalian retina, the basic motif leucine zipper transcription factor NRL determines rod versus cone photoreceptor cell fate and activates the expression of many rod-specific genes. Here, we report an integrated analysis of NRL-centered gene regulatory network by coupling chromatin immunoprecipitation followed by high-throughput sequencing (ChIP–Seq) data from Illumina and ABI platforms with global expression profiling and in vivo knockdown studies. We identified approximately 300 direct NRL target genes. Of these, 22 NRL targets are associated with human retinal dystrophies, whereas 95 mapped to regions of as yet uncloned retinal disease loci. In silico analysis of NRL ChIP–Seq peak sequences revealed an enrichment of distinct sets of transcription factor binding sites. Specifically, we discovered that genes involved in photoreceptor function include binding sites for both NRL and homeodomain protein CRX. Evaluation of 26 ChIP–Seq regions validated their enhancer functions in reporter assays. In vivo knockdown of 16 NRL target genes resulted in death or abnormal morphology of rod photoreceptors, suggesting their importance in maintaining retinal function. We also identified histone demethylase Kdm5b as a novel secondary node in NRL transcriptional hierarchy. Exon array analysis of flow-sorted photoreceptors in which Kdm5b was knocked down by shRNA indicated its role in regulating rod-expressed genes. Our studies identify candidate genes for retinal dystrophies, define cis-regulatory module(s) for photoreceptor-expressed genes and provide a framework for decoding transcriptional regulatory networks that dictate rod homeostasis

RETINA-Specific Expression of Kcnv2 Is Controlled by Cone-Rod Homeobox (Crx) and Neural Retina Leucine Zipper (Nrl)

Cone dystrophy with supernormal rod response (CDSRR) is an autosomal recessive disorder that leads to progressive retinal degeneration with a distinct electroretinogram (ERG) phenotype. CDSRR patients show reduced sensitivity to dim light, augmented response to suprathreshold light and reduced response to flicker. The disorder is caused by mutations in the KCNV2 gene, which encodes the Kv11.1 subunit of a voltage-gated potassium channel. Here, we studied the retina-specific expression and cis-regulatory activity of the murine Kcnv2 gene using electroporation of explanted retinas. Using qRT-PCR profiling of early postnatal retinas, we showed that Kcnv2 expression increased towards P14, which marks the beginning of visual activity in mice. In vivo electroporation of GFP-Kcnv2 expressing plasmids revealed that Kv11.1 localizes to the inner segment membranes of adult P21 photoreceptors. Using bioinformatic prediction and chromatin immuno-precipitation (ChIP), we identified two Crx binding sites (CBS) and one Nrl binding site (NBS) in the Kcnv2 promoter. Reporter electroporation of the wild type promoter region induced strong DsRed expression, indicating high regulatory activity, whereas shRNA-mediated knockdown of Crx and Nrl resulted in reduced Kcnv2 promoter activity and low endogenous Kcnv2 mRNA expression in the retina. Site-directed mutagenesis of the CBS and NBS demonstrated that CBS2 is crucial for Kcnv2 promoter activity. We conclude that nucleotide changes in evolutionary conserved CBS could impact retina-specific expression levels of Kcnv2