title: Transcriptional regulation of rod photoreceptor homeostasis revealed by in vivo NRL targetome analysis.
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.
authors.affiliation: Neurobiology-Neurodegeneration and Repair Laboratory, National Eye Institute, National Institutes of Health, Bethesda, Maryland, United States of America.
authors.forename: Hong
authors.initials: H
authors.lastname: Hao
authors.forename: Douglas S
authors.initials: DS
authors.lastname: Kim
authors.forename: Bernward
authors.initials: B
authors.lastname: Klocke
authors.forename: Kory R
authors.initials: KR
authors.lastname: Johnson
authors.forename: Kairong
authors.initials: K
authors.lastname: Cui
authors.forename: Norimoto
authors.initials: N
authors.lastname: Gotoh
authors.forename: Chongzhi
authors.initials: C
authors.lastname: Zang
authors.forename: Janina
authors.initials: J
authors.lastname: Gregorski
authors.forename: Linn
authors.initials: L
authors.lastname: Gieser
authors.forename: Weiqun
authors.initials: W
authors.lastname: Peng
authors.forename: Yang
authors.initials: Y
authors.lastname: Fann
authors.forename: Martin
authors.initials: M
authors.lastname: Seifert
authors.forename: Keji
authors.initials: K
authors.lastname: Zhao
authors.forename: Anand
authors.initials: A
authors.lastname: Swaroop
mesh_terms: D000818:Animals; D050976:Basic-Leucine Zipper Transcription Factors / Q000235:genetics* / Q000378:metabolism*; D001665:Binding Sites; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D004195:Disease Models, Animal; D005136:Eye Proteins / Q000235:genetics* / Q000378:metabolism*; D055785:Gene Knockdown Techniques; D053263:Gene Regulatory Networks; D059014:High-Throughput Nucleotide Sequencing; D018398:Homeodomain Proteins / Q000235:genetics / Q000378:metabolism; D006706:Homeostasis / Q000235:genetics; D006801:Humans; D056484:Jumonji Domain-Containing Histone Demethylases / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D009474:Neurons / Q000378:metabolism; D012160:Retina / Q000378:metabolism / Q000502:physiology; D058499:Retinal Dystrophies / Q000235:genetics / Q000378:metabolism; D017948:Retinal Rod Photoreceptor Cells / Q000378:metabolism*; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism; D014158:Transcription, Genetic
chemical_list: D050976:Basic-Leucine Zipper Transcription Factors; D004268:DNA-Binding Proteins; D005136:Eye Proteins; D018398:Homeodomain Proteins; C084884:Nrl protein, mouse; D015534:Trans-Activators; D014157:Transcription Factors; C109288:cone rod homeobox protein; D056484:Jumonji Domain-Containing Histone Demethylases; C485614:Kdm5b protein, mouse