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SA Padove, VT Do, HT Nguyen, JM Nickerson, JH Boatright; Characterization of DNA/protein complexes that restrict IRBP expression . Invest. Ophthalmol. Vis. Sci. 2002;43(13):3657.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Interphotoreceptor Retinoid Binding Protein (IRBP) is expressed primarily in photoreceptor cells and pinealocytes. The -156 to -70 region of the mouse IRBP gene (relative to transcription start) is required for proper, cell-type restricted activity. In this study, we sought to (1) precisely define cis-elements in this region and (2) identify and characterize proteins that bind these elements. Methods: Three approaches were used: Electrophoretic shift mobility assay (EMSA), DNA-protein UV cross-linking with SDS-PAGE, and yeast one-hybrid screening. EMSAs were used to precisely define fragments within the mouse IRBP -156 to -70 region that bind protein complexes from nuclear extract of several tissues and cell lines. Mutant probes were used to assess sequence specificity of binding. Sizes of the EMSA-visualized proteins were estimated by UV cross-linking them to probes and separating on SDS-PAGE. Size and target sequence data were compared to protein databases. A yeast one-hybrid system, using these precisely-defined fragments, was used to screen retina and brain fusion libraries for putative transcription factors. Results: Four distinct 6-12 base regions produced banding patterns that were not present when mutated. For one fragment, 4 different sized proteins bound, though only 3 were sequence-specific. The same 4-banded pattern was seen in mouse brain, mouse rd/rd retina, human retinoblastoma and mouse neuroblastoma cells. However, binding was negligible with non-neuronal or wild-type retina extracts. Positive clones were obtained from yeast one-hybrid library screenings. Database comparison indicated consistency with other tissue-specific regulatory mechanisms in which inhibitory and stimulatory transcription factors compete for the same DNA target sequence. Conclusion: Very specific, short regions of the IRBP restriction element were identified as binding domains. One fragment appears to bind neuronal, but not photoreceptor, proteins. This fragment may bind both inhibitor and stimulator transcription factors. To identify these factors, we are continuing with yeast one screening and amino-acid sequencing of complexes isolated via our UV cross-linking assay.
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