Biochemistry/Molecular Biology - ARVO

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Biochemistry/Molecular BiologyHEK 293 cells with a reporter containing opsin gene upstreamregions. Finally, we analyzed the retina of a Gtf2ird1-null mouseusing molecular, histological, and electrophysiological approaches.Results: YOH screening identified several TFs that bind to the LCR.Among those was Gtf2ird1, whose role in the retina has not beenstudied. Gtf2ird1 expression in M cones is first detectable at P10 andis maintained through adulthood. ChIP assays showed that Gtf2ird1interacts with the LCR and promoter regions of the M and S opsingenes. siRNA-mediated Gtf2ird1 knockdown suppressed M opsinexpression; transient co-transfection assays showed that Gtf2ird1synergistically enhanced L opsin promoter activity in the presence ofCRX. On the other hand, Gtf2ird1 abolished S opsin promoteractivity enhanced by CRX with RORα or RORβ. Consistent withthese results, M opsin gene expression in the Gtf2ird1-null mouseretina was significantly suppressed, while S opsin gene expressionwas significantly increased. Histological studies of Gtf2ird1-nullmouse retina showed that S opsin immuno-positive cells wereincreased in the dorsal retina, where S cones are normally scarce.Interestingly, most of them were also M opsin immuno-positive. Onthe other hand, there was no significant change between Gtf2ird1-nulland wild-type mouse retinas in both the number of cells and thedorsal-ventral distribution pattern of M opsin immuno-positive cells.ERGs reflected these cone changes.Conclusions: Our results indicate that Gtf2ird1 is a dual-functiontranscription factor that maintains M cone function by promoting Mopsin expression and suppressing S opsin expression in M cones.Commercial Relationships: Donald J. Zack, Alcon (C), Merck (F),Allergan (C); Anitha Yerrabelli, None; Xiaodong Zhang, None;Elizabeth A. Conner, None; Snorri S. Thorgeirsson, None; AnandSwaroop, None; Shiming Chen, None; Tomohiro Masuda, NoneSupport: the National Eye Institute, Guerrieri Family Foundation,Foundation Fighting Blindness, and Research to Prevent Blindness,Inc.Program Number: 2614Presentation Time: 9:00 AM - 9:15 AMTranscription and splicing associated protein NonO/p54nrbregulates rod specific genes including rhodopsin and theirregulators required for rod differentiation and homeostasisSharda P. Yadav, Hong Hao, Marie-Audrey I. Kautzmann, MatthewBrooks, Jacob Nellissery, Anand Swaroop. National Eye Institute,National Institutes of Health, Bethesda, MD.Purpose: We have identified Non pou domain containing octamerbinding protein (NonO) from bovine retinal extracts using therhodopsin distal enhancer region. The purpose of current study is toelucidate the role of NonO in regulating the expression of rhodopsin.Methods: To understand role of NonO in regulation of rhodopsinexpression we performed shRNA knockdown of NonO using in vivoelectroporation. P0 CD1 pups were co-electroporated with Rhodopsinpromoter driving DsRed reporter along with either control or NonOshRNA. To visualize the electroporated cells, both shRNA constructscontained GFP reporter driven by signal recognition particle alphapromoter. GFP and DsRed positive cells in outer nuclear layer of thecontrol and NonO shRNA electroporated retinas were examined. Toexplore in vivo role of NonO in rod photoreceptors of P21 retinas,chromatin immunoprecipitation was performed followed by deepsequencing (Chip-Seq). We choose zeitgeber (ZT) 6 and ZT18 asgenes implicated in rod outer segment disc renewal exhibitdifferential expression at these times. Occupancy of NonO on genepromoters was analyzed using Genomatix software and UCSCgenome browser.Results: Knockdown of NonO by shRNA resulted in death ofdeveloping rod photoreceptors. The rod death could be partiallyrescued by a truncated NonO, which contained C-terminal 224 to 473amino acids. Luciferase reporter assays showed that C-terminalregion of NonO is sufficient to activate Rhodopsin promoterluciferase reporter. Chip-Seq data suggested that NonO occupiedgenes are mostly involved in phototransduction pathway. NonOoccupancy on the phototransduction genes is higher at ZT6 comparedto ZT18. These results suggest that NonO is involved in circadianregulation of rhodopsin as well as other phototransduction genes andtheir regulators in mouse rod photoreceptors.Conclusions: NonO is critical for rod development and itsknockdown leads to rod cell death. C-terminal 224 to 473 aminoacids of NonO are sufficient to activate rhodopsin promoterluciferasereporter expression in vitro. Chip-Seq results suggest thatNonO plays an important role in modulating the levels ofphototransduction genes during the circadian cycle. Further analysisof NonO function in mice retina should provide new insights into theregulation of rod outer segment biogenesis.Commercial Relationships: Sharda P. Yadav, None; Hong Hao,None; Marie-Audrey I. Kautzmann, None; Matthew Brooks,None; Jacob Nellissery, None; Anand Swaroop, NoneSupport: National Eye InstituteProgram Number: 2615Presentation Time: 9:15 AM - 9:30 AMCorrelating Whole Genome DNA Methylation Patterns withRetinal Expression and Alternative SplicingJiang Qian, Jun Wan, Verity F. Oliver, Donald J. Zack, Shannath L.Merbs. Department of Ophthalmology, Johns Hopkins School ofMedicine, Baltimore, MD.Purpose: DNA methylation is a major epigenetic modification thatplays an important role in multiple cellular processes. It is generallybelieved that DNA methylation inhibits gene expression. However,how exactly the DNA methylation regulates tissue-specific geneexpression and splicing remains unclear. In this study, we performedgenome-wide DNA methylation profiling in retina and brain toevaluate the correlation between DNA methylation and tissuespecificgene regulation.Methods: DNA methylation profiling on two adult mouse tissues,retina and brain, was conducted using the comprehensive highthroughputarray for relative methylation (CHARM) NimbleGentiling array. Affymetrix exon microarrays were also used to measurethe gene expression and splicing differences between retina andbrain. Bioinformatics methods were developed and employed toanalyze the data and correlate the DNA methylation, gene expressionand splicing.Results: Numerous tissue-specific differentially methylated regions(tDMRs) were identified. These tDMRs located in various genomicregions, including promoter, exon, intron and intergenic regions.They were enriched in CpG island shores, but depleted in CpGislands. We then integrated these tDMRs with the transcriptome datameasured in the corresponding tissues by exon microarray. DMRsthat negatively correlated with downstream gene expression tended tobe close to transcription start sites. Tissue-specific genes were morelikely to be regulated by differential methylation than expected. Alarge number of DMRs located in exons or introns correlated with theexpression of an adjacent exon, suggesting a potential role of DNAmethylation in tissue-specific splicing.Conclusions: Our work provides a large-scale survey of differentialDNA methylation of retina-specific genes and lays the foundation forfurther mechanistic studies of the biological role of DNA methylationin tissue-specific gene regulation.©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.