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K. P. Mitton, P. Tummala, R. S. Mali, E. A. Guzman, M. N. Stewart, J. R. Sotzen, W. Gryc; Genome-Wide Maps of RNA Polymerase-II in Gene Promoters Identify Novel Gene Activation Events and Epigenetic Targets During Terminal Maturation of the Retina. Invest. Ophthalmol. Vis. Sci. 2010;51(13):1688.
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© ARVO (1962-2015); The Authors (2016-present)
To find gene expression changes during terminal maturation of the retina that escape detection with expression microarrays by mapping changes in RNA-Polymerase-II (Pol-II) binding around transcription start sites (TSS). Furthermore, to produce genome-wide maps of Pol-II data for correlation with epigenetic target modifications to DNA and Histones.
Mouse P-2 and P-25 neural retinal tissues were processed for Pol-II chromatin immunoprecipitation with subsequent analysis on the high resolution (35 bp) Affymetrix mouse promoter tiling GeneChip array, with 26,000 promoters. Coverage included 2,500 bp downstream of each TSS. Sample arrays were normalized to total genomic chromatin input using Tiling Array Software. Pol-II peak signal ratios (P-25/P-2) were calculated for each Pol-II active region, and mapped to specific genes. Quantitative-ChIP, and gene expression assays (Taqman) validated a minimal Pol-II peak signal ratio (P-25/P-2) for predicting gene activation increases with >95% accuracy. DNA methylation and H3K9ac levels were examined by Q-Chip.
Pol-II ChIP-on-Chip predicted increased activation for 822 genes in addition to those previously reported from expression microarray data. Over 300 have no known function and the remainder cluster into functions related to vision, gene expression, signal transduction, and neural development. Increased activation of over 30 test genes, predicted by a Pol-II peak signal ratio (P-25/P-2) >1.8, was confirmed by gene expression analysis and Q-Chip assays. DNA-methylation decreased and H3K9-acetylation increased in the Pol-II active region around the Rho and Rcvn TSS during developmental activation. Expression of the novel predicted genes Lpcat1 and Slc25a33 was diminished by 50% in Rd1 retina.
We generated genome-wide maps of Pol-II binding around transcription start sites that predict activation increases for specific genes with >95% accuracy. Hundreds of novel gene activation events are enriched for functions of vision, neural development and gene regulation. Pol-II active regions around the Rho and Rcvn TSS experience decreased DNA-methylation and increased H3K9-acetylation during activation. Some of the novel confirmed genes have human homologues within unidentified retinal disease regions, including Lpcat1 and Slc25a33. The first retinal Pol-II TSS maps were produced for examination in the UCSC Genome Browser, or its NEI version (EyeBrowse).
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