Purchase this article with an account.
K Rengarajan, E Stodulkova, VT Do, JH Boatright, JM Nickerson; Identification of a mammalian IRBP tissue restricting transcription factor by EMSA and UV Photo-crosslinking and SDS-PAGE . Invest. Ophthalmol. Vis. Sci. 2002;43(13):3635.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
Purpose: The transcription of the interphotoreceptor retinoid binding (IRBP) gene is limited to photoreceptor cells of the retina and pinealocytes. The 5' flanking region between positions -70 and -156 of the IRBP gene confers this tissue-restricted promoter activity (Boatright et al. FEBS Letters 2001; 504: 27-30). Here, we sought to identify nuclear proteins that bind to this cis element. We used EMSAs and UV light irradiation to photo-crosslink DNA to protein to determine the size of DNA-binding protein by SDS-PAGE. Methods:Transient transfections with CAT reporter plasmids containing the -140 and -156 promoter fragment were carried out in WERI and Neuro 2A cells. EMSAs were carried out by standard methods. Nuclear extracts were prepared from WERI cells, Neuro 2A cells, and various mouse tissues. 20-mers containing the 140 to -156 cis- element were used as a radiolabeled probe, and unlabelled competitors of varying oligonucleotide sequences were added to define sequence specificity. Nucleic acid -protein complexes were cross-linked for 45 min under a 254 nm UV light. SDS disrupted non-covalently attached DNA-protein complexes. Results:Transfections showed that -140 to -156 reduced promoter activity in Neuro 2A cells, but allowed activity in WERI cells. The -140 to -156 DNA specifically bound nuclear protein complexes in EMSAs, differing in mobility between brain and retina. SDS-PAGE analysis of the cross-linked DNA protein complex revealed a band of ∼55 kDa. Without UV treatment no band appeared. Competition assays with unlabeled dsDNAs demonstrated sequence specificity in cross-linking of complexes from neural tissues but not other sources. Little was found in extracts from wild type retina, though signal was strong in rd-/rd- retinal extract. Conclusion:Transient transfections refined the restricting element to -140 to -156. The 55 kDa protein's presence only in neural tissue is consistent with a hypothetical role as an inhibitory transcription factor whose presence in neural tissue, but not photoreceptors, restricts IRBP expression. SDS-PAGE analysis sharply limits the scope of potential candidate proteins to a narrow size range near 55 kDa.
This PDF is available to Subscribers Only