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K. Petrukhin, C. Mandavia, F. Fonseca-Galea, I. P. Chernov; High Throughput Identification of Direct Target Genes for Photoreceptor-specific Orphan Nuclear Receptor NR2E3. Invest. Ophthalmol. Vis. Sci. 2009;50(13):485.
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NR2E3 (PNR/RNR) is a retina-specific orphan nuclear receptor believed to play a role in photoreceptor development, differentiation, and survival. Several reports identified multiple genes differentially expressed in the wild type and Nr2e3 -/- mouse retinas. Only a fraction of differently expressed genes is presumed to be a direct target for NR2E3 regulation. In this study we used two-dimensional electrophoretic mobility shift assay (2D-EMSA) to experimentally identify NR2E3 response elements in genomic fragments covering 70 genes whose expression was changed in the Nr2e3 -/- retina most significantly.
Fourteen pools of short 32P-labeled DNA fragments representing the full length of 70 human genes were incubated with the purified NR2E3 protein followed by separation in a non-denaturing plyacrylamide gel (EMSA; first dimension). The resulting lane was excised from the gel, incubated with SDS to disrupt NR2E3-DNA interaction and placed on the top of the second dimension gel. DNA fragments bound to NR2E3 were retarded during the first run, but their mobility was restored in the second dimension due to disruption of NR2E3-DNA complexes. The spots corresponding to fragments bound to NR2E3 during the first dimension were located below the diagonal representing unbound fragments which have the same mobility in both dimensions. These spots were excised from the gel, cloned, sequenced and verified in individual EMSA reactions.
2D-EMSA was used to analyze a total of 14 Mb of human genomic sequence covering 70 genes for the presence of DNA fragments capable of forming complexes with NR2E3. Approximately 60% of genes contain potential response elements which were found both proximally and distally from target promoters. The distance form potential NR2E3 response elements to transcription starts varied drastically from 500 bp to 100 kb indicating that short-range and long-range regulation may be important for regulation of different subsets of target genes.
Using 2D-EMSA we identified a subset of genes containing DNA fragments capable of forming a complex with NR2E3. This subset defined a group of presumed direct targets for NR2E3 regulation among the genes differentially expressed in the Nr2e3 -/- retina. 2D-EMSA is a novel high-though approach for identification of response elements for nuclear receptors and other transcription factors.
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