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Sharda P. Yadav, Jacob Nellissery, Anand Swaroop; Nono Modulates The Activity Of Rhodopsin Promoter. Invest. Ophthalmol. Vis. Sci. 2011;52(14):11.
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© ARVO (1962-2015); The Authors (2016-present)
To elucidate the transcriptional regulation of rhodopsin expression using proteomic approaches to identify proteins that interact with distal enhancer sequence elements in the rhodopsin promoter.
Bovine nuclear extract was prepared by differential centrifugation. Biotin tagged oligo from rhodopsin distal enhancer region (DER) was used to isolate the bound proteins. Isolated proteins were further separated on SDS-PAGE and differentially bound protein bands were analyzed by mass spectrometry. Transcriptional activities of the candidate proteins on rhodopsin promoter were tested using luciferase assay in HEK-293 cells.
We identified several candidate proteins binding to rhodopsin DER by mass data analysis. Non-pou domain containing octamer-binding protein (NONO/P54nrb) for which we have got maximum number of peptides was selected for its detailed study on rhodopsin promoter. Transcriptional analysis of mouse Nono on bovine rhodopsin promoter revealed that it can specifically modulate NRL and CRX activity several fold while has no effect on Nrl and Nr2e3 promoters suggesting its specificity towards rhodopsin promoter. To further evaluate specificity of Nono we used NRE containing Tcp10a promoter and observed 22-fold increase in its activity while a control reporter lacking NRE, has no effect. These results suggest that the activity of Nono on rhodopsin promoter might be NRE dependent.
We have identified Nono as a transacting factor binding to rhodopsin DER and it can synergistically activate NRL and CRX mediated transctivation of rhodopsin promoter. Further analysis of Nono should reveal new insights into rhodopsin gene regulation.
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