Biochemistry/Molecular Biology - ARVO

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Biochemistry/Molecular BiologyCommercial Relationships: Jiang Qian, None; Jun Wan, None;Verity F. Oliver, None; Donald J. Zack, Alcon (C), Merck (F),Allergan (C); Shannath L. Merbs, NoneSupport: NIH Grants EY021897 EY018703 and Unrestricted fundsfrom RPBProgram Number: 2616Presentation Time: 9:30 AM - 9:45 AMHistone marks predict cell plasticity of the adult human retinalpigment epitheliumTimothy A. Blenkinsop 1 , Alvaro Rada-Iglesias 2 , Joanna Wysocka 2 ,Sally Temple 1 . 1 Macular Degeneration Program, Neural Stem CellInstitute, Rensselaer, NY; 2 Developmental Biology, StanfordUniversity, Stanford, CA.Purpose: Retinal pigment epithelial (RPE) cells are one of the fewcell types well recognized to change fate in lower vertebrates. Werecently showed adult human RPE as old as 99 years differentiatedown mesenchymal and neural lineages using commercially availabledifferentiation media. Addressing whether this was due to themultipotency of the RPE cells or to a contaminating cell type, forexample MSCs, in the cultures was critical. Therefore, wedemonstrated that single, cloned RPE cells exhibit this ability,irrefutably demonstrating that they are multipotent cells. Wetherefore sought to understand the basis for this observed plasticity.Histone marks at promoter and enhancer sites can reveal both genesthat are actively being transcribed - i.e. active, and genes that arepoised - i.e. currently inactive, but can be activated by underappropriate conditions. Recently histone H3K27me3 mark has beenshown to be enriched in regions several kilobases upstream from thepromoter around enhancer regions that are poised in humanembryonic stem cells (hESCs). In contrast, histone mark H3K27acmarks similar locations when the downstream gene is active.Therefore, we hypothesized that these two marks can predict genechanges from being poised to active.Methods: We cultured human retinal pigment epithelium intopolarized layers and verified their terminal differentiation throughtransepithelial resistance (TER) in Ωcm2. These monolayersexpressed TER of > 200Ωcm2 similar to native RPE. We alsoconfirmed their identity immunohistochemically, by quantitativePCR. We then conducted H3K27ac and H3K27me3 Chromatinimmunoprecipitation-sequencing (ChIP-seq) on adult human RPEmonolayers.Results: We found RPE specific genes posses H3K27ac marksupstream of their promotor regions, whereas the genes we have foundto increase expression during differentiation, for example RUNX2during osteogenic conditions, possess the poised mark H3K27me3upstream of their promotor.Conclusions: These results suggest the histone marks can predict notonly the cell identity, but also the poised mark can predict RPEplasticity.Commercial Relationships: Timothy A. Blenkinsop, None; AlvaroRada-Iglesias, None; Joanna Wysocka, None; Sally Temple,Athghin Biotech (I)Support: Ey022079-01Program Number: 2617Presentation Time: 9:45 AM - 10:00 AMThe MLL1 Histone Methyltransferease is Essential forDevelopment of Photoreceptor FunctionDiana S. Brightman, Ray Suzuki, Shiming Chen. Ophthalmology andVisual Sciences, Washington University in St. Louis, St. Louis, MO.Purpose: Development and maintenance of retinal photoreceptorfunction requires precisely controlled gene expression. This isregulated by both photoreceptor-specific and general transcriptionregulators, including histone modification enzymes. Members of theMLL family of histone H3K4 methyltransferases are expressed inmouse photoreceptors. The most prominent of these is MLL1 whoseexpression increases between postnatal day 2 (P2) and P14, a criticalperiod for photoreceptor terminal differentiation. MLL1 expressiondepends on the key photoreceptor-specific transcription factor CRX,suggesting a potential role of MLL1 in the CRX regulatory pathway.We determined the role of MLL1 in photoreceptor gene expression,development and survival using a loss-of-function approach.Methods: To avoid embryonic lethality, Cre-loxP-mediatedconditional knockout (CKO) was used. Mice with Mll1 floxed alleles(Mll1 fl/fl ) were crossed with Crx-Cre mice to create Mll1 CKO in thedeveloping retina. Mll1 CKO mice show morphological changes inthe retina by H&E staining, immunohistochemistry (IHC) andelectron microscopy (EM), and visual function changes byelectroretinogram (ERG).Results: Cre activity was found in all cell layers of the developingMll1 CKO retina, in a superior-to-inferior gradient. As a result, Mll1expression was uniformly depleted in the superior region, where allretinal layers were significantly thinner by morphometry. IHC andEM analyses show abnormal superior outer plexiform layer (OPL)synapses. The intensity of the presynaptic markers V-GLUT1 andCTBP2 were markedly decreased at the OPL. Calbindin-positivehorizontal cells were also reduced, indicative of postsynaptic defects.Consistent with these defects, both dark and light-adapted ERGs of 1-month-old Mll1 CKO mice were significantly decreased, suggestingdefects in rod, cone, and inner retina functions. The morphologicaland functional changes are stable to 6 months of age, suggesting adevelopmental origin, not degeneration.Conclusions: MLL1 is required for the development of appropriateretinal structure and function. Additional Mll1 CKO usingphotoreceptor and INL cell-type-specific Cre lines are in progress todetermine cell autonomy and underlying molecular mechanisms.These studies will shed light on how general epigenetic modulatorscontribute to the regulation of cell-type specific gene expression anddevelopment.Commercial Relationships: Diana S. Brightman, None; RaySuzuki, None; Shiming Chen, NoneSupport: NIH Grant EY012543 (to SC), EY02687 (to WU-DOVS),RBP Lew R. Wasserman Award (to SC) and unrestricted funds (toWU-DOVS)Program Number: 2618Presentation Time: 10:00 AM - 10:15 AMRole of 5-hydroxymethylcytosine during postnatal retinaldevelopmentStylianos Michalakis 1 , Arshan Perera 1 , Susanne Koch 1 , MirkoWagner 2 , Lukas Windhager 3 , Kerstin Nagel-Wolfrum 4 , Tim M.Strom 5, 6 , Ralf Zimmer 3 , Thomas Carell 2 , Martin Biel 1 . 1 Center forIntegrated Protein Science at the Department of Pharmacy-Center forDrug Research, Ludwig-Maximilians-Universität München, Munich,Germany; 2 Center for Integrated Protein Science at the Department ofChemistry, Ludwig-Maximilians-Universität München, Munich,Germany; 3 Institute for Informatics, Ludwig-Maximilians-UniversitätMünchen, Munich, Germany; 4 Department of Cell and MatrixBiology, Institute of Zoology, Johannes Gutenberg University-Mainz,Mainz, Germany; 5 Institute of Human Genetics, TechnischeUniversität München, Munich, Germany; 6 Institute of HumanGenetics, Helmholtz Zentrum München, German Research Center forEnvironmental Health, Neuherberg, Germany.Purpose: 5-hydroxymethylcytosine (5hmC), also known as the sixthbase of the genome, is a recently discovered oxidative product of 5-©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. 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