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K. P. Mitton, P. Tummala, R. S. Mali, X. M. Zhang, A. E. Guzman; Chip-on-Chip Analysis for Genes Activated in Maturing Neural Retina: New Genes and Discovery of the Alternate Promoter Driving Retinal Mef2c Expression. Invest. Ophthalmol. Vis. Sci. 2009;50(13):3997.
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© ARVO (1962-2015); The Authors (2016-present)
We have demonstrated that FIZ1 (Flt-3 Interacting Zinc-finger) interacts with the NRL and CRX transcription factors and is recruited to the promoter complex on photoreceptor-specific genes (Rho, Pde6b, S-opsin, M-opsin). We pursued an initial analysis of 28,000 gene promoters to find genes activated during post-natal maturation of the mouse neural retina, in vivo, through direct mapping of RNA-Polymerase-II (Pol-II) binding and recruitment of FIZ1.
Mouse P2 and P25 neural retinal tissues were processed for ChIP-on-Chip with antibodies to Pol-II and FIZ1. GeneChip tiling arrays provided 10,000 bp coverage for each of 28,000 gene promoters, with 35 bp probe spacing. This included 2,500 bp down-stream of the transcription start site. Data files were processed with Tiling Array Software and TransPath analysis to identify active gene promoters. Quantitative-ChIP, and Taqman gene expression assays established a Pol-II peak signal ratio that predicted gene activation. Genes were grouped by function using DAVID gene ontology tools.
ChIP-on-Chip analysis of Pol-II binding predicted that over 1,100 genes increased expression from P2 to P25. 65% of these genes were not reported in two previous expression array studies. 898 genes were strictly activated after P2. FIZ1 was recruited to 27% of these genes. 194 of these genes have no known function. Of 704 annotated genes, most matched biological processes relevant to retinal development: Vision (89), Gene Expression (110), Signal Transduction (110), Synaptic Transmission (11), Phototransduction (10), Neuron / Nervous System Development (21), Chromatin Modification / Organization (15), Ion Transport (33). Pol-II and FIZ1 binding revealed specific activation of an alternate promoter for the Mef2c gene in adult neural retina, which was confirmed by transcript specific assays.
Pol-II ChIP-on-Chip accurately predicts genes activated during retinal maturation. These genes represent key biological functions involving gene regulation, vision, neural development and neuron function. Many genes were not reported in previous expression studies. FIZ1 was recruited to a substantial number of genes required for adult retinal function. Activation of a retina-preferred alternate promoter was discovered for the Mef2c transcription factor gene.
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