Visual impairment in a vast majority of retinal and macular degenerative diseases can be attributed to dysfunction or death of photoreceptors 
. Despite the central role of cones in transduction of vision in humans, rods constitute 95% of all photoreceptors and are generally the first to die in retinal neurodegeneration. A relatively late onset of clinical manifestations in these diseases underscores the importance of stringently maintaining the function of highly metabolically active photoreceptors. The control of homeostasis must be exerted at multiple levels as quantitatively precise expression of phototransduction proteins and their transport to the modified sensory cilia (outer segments) are critical for photoreceptor survival. In addition to its essential role in photoreceptor differentiation, NRL has been implicated in the regulation of rod phototransduction genes, such as rhodopsin and cGMP phosphodiesterase α and β subunits 
. Here we identify global transcriptional targets of NRL and integrate our data with reported targets of CRX, another key regulator of photoreceptor genes. Our results show that NRL and CRX together control the expression of most, if not all, genes involved in rod phototransduction through a cis
-regulatory module, which also includes the binding sites for NR2E3, ESRRβ, RORβ and in some cases MEF2C. Equally important is the finding that non-CRX containing NRL cis
-regulatory modules fine-tune the expression of additional photoreceptor-expressed genes, which may contribute to high metabolic demand in rod photoreceptors.
ChIP–Seq has emerged as a cost effective, high-throughput technology for high-resolution genome-wide mapping of in vivo
locations for chromatin modifications and transcription factor binding 
. Despite the fundamental difference in sequencing chemistry and nucleotide base calling software between the Illumina and ABI/SOLiD sequencing platforms 
, our ChIP–Seq data from the two are remarkably comparable, further validating the in vivo
NRL binding events reported here. In addition to enrichment in promoter regions, a number of NRL ChIP–Seq peaks are detected in intronic regions of annotated genes; some of these might reflect alternative promoter usage in photoreceptors as reported recently for Mef2c
We previously proposed that photoreceptor precursors have a “default” S-cone fate and a “tug-of-war” among a selected few transcription factors specifies rod versus
cone cell type 
. NRL and TRβ2 respectively initiate the rod and M-cone pathways 
, with NRL being the dominant activator of rod genes and a suppressor of cone genes together with its target NR2E3 
. Enrichment of a distinct set of transcription factor binding sites in NRL ChIP–Seq peaks in genes that are down- or up-regulated in Nrl−/−
retina suggests specific and discrete cis
-regulatory modules for rod versus
cone photoreceptor expressed genes. CRX strongly activates the expression of both rod and cone genes 
. An overlap of CRX peaks in over 50% of NRL ChIP–Seq peaks is consistent with their synergistic function in activating rod-expressed genes. Indeed, all rod phototransduction genes were included in this group. Notably, CRX ChIP–Seq peaks are much smaller than NRL peaks at the same loci and loss of NRL leads to more significant decrease in gene expression than in Crx−/−
retina, suggesting a fundamental role of NRL in regulating rod genes. CRX likely enhances rod gene expression by altering the chromatin conformation via
recruitment of histone acetylases 
. In cone genes (up-regulated in Nrl−/−
retina), binding of both CRX and NRL is consistent with the common photoreceptor precursor hypothesis 
. Additional studies (e.g., histone modifications) are needed to clarify differential regulation of specific genes by NRL and CRX in rod versus
Like many key transcription factor nodes in GRNs 
, NRL likely auto-regulates its own expression as suggested by strong NRL ChIP–Seq peaks in Nrl
promoter and intronic regions. While the key role of NR2E3 as a secondary node downstream of NRL is to repress cone-specific genes 
, two newly reported NRL targets – ESRRβ and MEF2C – function as transcriptional regulators for activation and/or maintenance of rod gene expression 
. A new secondary node in rod GRN that our studies identified is KDM5B (also called Jarid1b), a Jumonji-domain containing histone demethylase, which is associated with chromatin remodeling and transcriptional repression 
. KDM5B reportedly activates the expression of self-renewal-associated genes by suppressing cryptic initiation and maintaining proper H3K4me3 gradient for productive transcriptional elongation 
. We observe a significant overlap between the genes altered by loss of NRL and KDM5B, indicating a broader role of KDM5B in regulating rod homeostasis downstream of NRL. We hypothesize that differential expression of KDM5B may contribute to chromatin organization and metabolic differences between rod and cone photoreceptors 
Retinal and macular diseases are genetically heterogeneous with over 200 mapped loci; of these, almost 150 genes have been identified (http://www.sph.uth.tmc.edu/Retnet/
). A catalog of genome-wide NRL targets with overlapping CRX binding sites, reported here, provides excellent candidate genes for mutation screening in patients with inherited retinal neurodegenerative diseases. We have listed almost 100 genes (see Table S5
) that map to retinal disease loci. Interestingly, knockdown of 16 target genes, reported in this study, resulted in photoreceptor cell death or abnormal morphology, highlighting the importance of NRL targets in maintaining normal physiology and the association of perturbed target gene expression with retinal diseases.
A key aspect of photoreceptor homeostasis is the daily renewal of almost 10% of outer segment membrane discs, which requires a stringent control of the synthesis of specific phototransduction proteins and lipid molecules. Therefore, the target gene, Lman1
, attracted our attention as its knockdown led to shorter photoreceptor outer segments and abnormal location of cell bodies (close to the sclera), which is characteristic of cone photoreceptors or late-born rods, whereas the early-born rods locate towards the vitreous side. LMAN1 participates in transport between the endoplasmic reticulum and Golgi 
. Our data suggests that LMAN1 performs critical roles in photoreceptor homeostasis by controlling lipid homeostasis and/or biogenesis of membrane discs. Abnormal location of nuclei to scleral side in photoreceptors after its knockdown by in vivo
electroporation could be due to rod to cone transformation in the absence of NRL, or delayed rod birth as a result of abnormal signaling for rod fate determination.
, another interesting target of NRL, encodes the Wnt1-inducible signaling pathway protein 1 that exerts cytoprotective and/or growth promoting effects 
by repressing p53 and activation of Akt kinase 
. WISP1 could therefore act as a survival or maintenance factor for photoreceptors. Further investigations on WISP1 may yield new targets for neuroprotective strategies in retinal degeneration.
Gene regulatory networks (GRN) control multiple pathways during development and homeostasis and provide conceptual framework for elucidating disease mechanisms 
. Transcription factors reside near the top of GRNs; their abnormal expression and/or activity can cause widespread changes in target genes 
. Our studies demonstrate a pivotal role of NRL in controlling rod homeostasis by modulating the expression of numerous target genes, which in turn maintain distinct aspects of cell function and survival. Elucidation of combinatorial regulation of genes by NRL and its co-regulators (specifically CRX) and identification of distinct downstream nodes (such as KDM5B) provide a framework to construct GRN for functional maintenance in mammalian rod photoreceptors ().
A simplified gene regulatory network in rod and cone photoreceptors.