Abstract
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.
Highlights
Molecular mechanisms underlying neuronal differentiation and generation of complex sensory and behavioral circuits in the mammalian central nervous system are still poorly elucidated
Loss of rod photoreceptors is an early clinical manifestation in most retinal neurodegenerative diseases that eventually result in cone cell death and blindness
The transcription factor NRL is a key regulator of rod photoreceptor cell fate and gene expression
Summary
Molecular mechanisms underlying neuronal differentiation and generation of complex sensory and behavioral circuits in the mammalian central nervous system are still poorly elucidated. As perturbations in homeostatic mechanisms (e.g., during aging and disease) can cause dysfunction or death of neurons [4,5], a better understanding of GRNs that control neuronal homeostasis would augment the design of therapies for neurodegenerative diseases. The rod and cone photoreceptors in mammalian retina are highly specialized neurons that transduce visual signals under dim and bright light conditions, respectively [6]. Functional impairment or loss of rod photoreceptors is an early clinical manifestation in most retinal neurodegenerative diseases that eventually results in cone cell death and blindness [9,10,11]. The GRNs that dictate homeostatic responses in mature rod photoreceptors have not been elucidated
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