Background Elucidation of gene regulation in developing, mature, and aging rod photoreceptors would allow better understanding of molecular mechanisms underlying their dysfunction in inherited degenerative diseases and assist in better design of targeted therapies. We have adopted a multi-pronged approach that centers on the rod photoreceptor-specific transcription factor NRL that is essential for rod differentiation and function. Results 1. Global expression profiling of rod photoreceptors We have generated transgenic mice that expresses green fluorescent protein (GFP) under control of the Nrl promoter in new-born and mature rod photoreceptors, which can then be flow-sorted from dissociated retinal preparations for further studies. Our photoreceptor expression profiling spans to 17 time points that reflect distinct stages in rod specification, maturation and aging. Our analyses reveal significant alterations (Fold change of >2) in the expression of 2,728 genes during the postnatal day (P) 0 to P21 developmental period, consistent with the role of NRL, its interacting proteins and targets during photoreceptor cell fate determination and functional maturation. The number of genes with significant (>2 fold) changes in expression during the aging period (from 3 months to 18 months) is limited to 154 genes. Our studies reveal a potential role for miR124 and other miRs in photoreceptor aging. The interaction between these miRs and their potential targets showing opposite expression pattern is being investigated (see also EY000451-04). We have compared NGS-derived retinal transcriptome profiling (RNA-Seq) to microarray and qRT-PCR methods using 21-day old wild type (WT) and Nrl-/- retinal mRNA. We found that RNA-seq data has a linear relationship with qRT-PCR for over four orders of magnitude and a goodness of fit (R2) of 0.8798. Hierarchical clustering of differentially expressed genes between Nrl-/- and WT retina has revealed a number of as yet uncharacterized genes that may contribute to rod or cone function. We have also initiated a project to investigate the retinal methylome from early development to postnatal, adult and aged rod photoreceptors (see also EY000451-04). Finally, we have shared our expertise and collaborated with other groups to establish gene networks: 1) in Muller glia cells associated with inflammation and gliosis in the retina (Dr. Vijay Sarathy, Northwestern University);2) in microglia that contribute to aging-associated diseases (Dr. Wai Wong, NEI);3) in a subclass of regulatory T cells (Dr. Vipin Kumar, Torre Pines Institute, San Diego);and 4) in the lens upon ablation of the Notch2 receptor (Peggy Zelenka, NEI). 2. Genes/pathways that guide rod differentiation and homeostasis 2.1. We performed an integrated analysis of NRL-centered transcriptional regulatory network by coupling ChIP-seq (chromatin immunoprecipitation followed by NGS) data with expression profiling and in vivo knockdown studies. We have identified 334 direct transcriptional targets of NRL and observed a specific enrichment of binding sites for the homeodomain protein CRX in NRL-ChIP-seq peaks. Twenty NRL target genes are associated with human retinal dystrophies, whereas 101 genes map to retinal disease loci. Evaluation of 26 sequence elements spanning NRL-ChIP-seq regions confirmed their enhancer function in reporter assays. In vivo knockdown of 18 NRL target genes resulted in death or abnormal morphology of rod photoreceptors. Of these, histone demethylase Kdm5b appears to function as a secondary node in NRL transcriptional hierarchy. Our studies highlight a cis-regulatory module for rod-specific genes and provide a framework for decoding photoreceptor regulatory networks. 2.2. Bioinformatics analysis, rapid analysis of cDNA ends (5′-RACE), reverse transcription coupled with qPCR using splice-site specific oligonucleotides, and ChIP suggest that Mef2c is expressed in the mature retina from an alternative promoter. In the retina, Mef2c expression from its alternative promoter is regulated by NRL. Our studies thus define another node in NRL-transcriptional hierarchy, implicating MEF2C as a transcriptional co-regulator of homeostasis in rod photoreceptor cells. 2.3. In a proteomic approach, we used retinal nuclear extract and an oligonucleotide derived from conserved bovine Rhodopsin distal regulatory region, to identify factors binding to rhodopsin promoter. One of the candidate binding proteins, the Non-pou domain containing octamer-binding protein (Nono) shows a dosage-dependent trans-activation of a 2.2kb Rhodopsin promoter sequence. ShRNA knock down of Nono in mouse retina by in vivo electroporation affects Rhodopsin expression and leads to the death of rod photoreceptors. 3. Pathways upstream of NRL 3.1. By in silico analysis and in vivo electroporation into the mouse retina of Nrl promoter-reporter constructs, we have identified a 0.9-kb sequence upstream of the Nrl transcription initiation site. This sequence contains cis-control elements essential and sufficient for appropriate Nrl expression in rod photoreceptors. Furthermore, we have established that retinoid-related orphan nuclear receptor β(RORβ) is a direct transcriptional regulator of Nrl and that cone rod homeobox (CRX), orthodenticle homolog 2 (OTX2), and cyclic AMP response element-binding protein (CREB) are implicated in modulating Nrl expression. These data have indicated the design of an adeno-assiociated virus (AAV) construct containing 0.3 kb of Nrl promoter/enhancer that can specifically direct target genes to either developing or mature rod photoreceptors (see also EY000474-03). 3.2. We have shown that GSK3-dependant phosphorylation of NRL leads to its degradation by the proteasome. A mutation at Ser50, associated with ADRP in human, leads to loss of GSK3-mediated phosphorylation and consequently an increase of NRL protein stability. Furthermore, the observation that NRL protein is quickly degraded in C57Bl6 mice after bright light exposure has prompted the investigation of circadian regulation of NRL protein expression (collaboration with Drs Felder and Hicks in France). To further investigate the role of phosphorylation and kinases in retinal development, we are using conditional Gsk3 knockout mice. We analyzed Gsk3bf/f;Rx-Cre mice and Gsk3bf/f;Rx-Cre mice carrying only one floxed allele for Gsk3a, to study the effect of the absence of Gsk3s during the early phases of retina development. In the absence of Gsk3b expression, we have observed a large pool of proliferating cells at postnatal ages (P6-P14) when most retinal progenitors have normally exited the cell cycle, as well as the presence of a large number of displaced ganglion cells in the inner nuclear layer of adult retinas. Since double mutants expressing Gsk3s under Rx promoter in early retinal progenitors (from embryonic day 8 (E)8) die embryonically, due to the ectopic activation of the gene in the forebrain. To circumvent this problem, new animals are currently being generated, that use the aPax6 promoter to restrict expression to the developing retina. PIAS3 (protein inhibitor of activated STAT3) is an E3-SUMO ligase implicated in photoreceptor differentiation that can be immunoprecipitated with NRL. We generated PIAS3 conditional knockout mice in collaboration with Dr Dong at NEI-Genomic Core. We recently confirmed by western blot the complete deletion of PIAS3 in Cre-positive animals. Further studies are in progress. Significance Our studies provide new insights into photoreceptor biology and disease. Components of the regulatory networks can be modulated for treatment of retinal diseases. Transcription networks will be exploited to manipulate stem cells in vitro to induce photoreceptor phenotypes, with potential applications for therapies of retinal degenerative diseases.
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