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S. P. Yadav, J. Nellissery, A. Swaroop; Regulation of Rhodopsin Expression: Identification of Rhodopsin Enhanceosome Components From Bovine Retina. Invest. Ophthalmol. Vis. Sci. 2010;51(13):5971.
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A stringent control of rhodopsin expression is critical for structural and functional maintenance of rod photoreceptors. Recruitment of transcription factors NRL, CRX and NR2E3 to the rhodopsin promoter facilitates the binding of the general transcription factors and RNA Pol II. The goal of this study is to identify all proteins in the rhodopsin enhanceosome that contribute to rhodopsin expression in mature retina.
Nuclear fraction was obtained from bovine retinas by differential centrifugation. About 300µg of total nuclear protein was pre-cleared by salmon sperm DNA and poly (dI-dC). Biotinylated oligonucleotides spanning the critical promoter region upstream of the rhodopsin transcription start site were incubated overnight in the presence of protease and phosphatase inhibitors. Streptavidin-tagged Dynabeads were added to facilitate the binding of the oligonucleotides along with the bound proteins. After incubation, the beads were separated, and bound proteins were eluted in 1M KCl. The proteins were analyzed by SDS-PAGE, visualized by silver staining and further characterized by mass spectrometry and immunoblotting.
Immunoblot analysis of the rhodopsin promoter oligo-bound proteins showed the presence of NRL, CRX, CREB, CK2 and RNA Pol II, further establishing the close synergistic interaction of NRL and CRX in controlling the rhodopsin promoter. Our mass spectra data revealed that a majority of the proteins binding to rhodopsin promoter belong to the spliceosome complex, chromatin remodeling, DNA repair and transcription elongation. Additional studies are in progress to validation the interactions and their relevance to rhodopsin expression in vivo.
Our data supports the assertions that transcription is associated with chromatin remodeling and coupled to splicing and to DNA damage machinery. Further analysis of the rhodopsin enhanceosome should reveal new insights into tissue-specific gene regulation and provide new candidates for retinal degenerative diseases.
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