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D.C. Otteson, Y. Liu, D.J. Zack; The Transcriptional Repressor KLF15 Binds a C-Rich Sequence Present in Both Human and Bovine Rhodopsin Proximal Promoters . Invest. Ophthalmol. Vis. Sci. 2003;44(13):3541.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Ongoing studies designed to understand the molecular mechanisms regulating photoreceptor-specific gene expression have identified the zinc-finger gene KLF15 (Krüppel-like factor 15) as a potential transcriptional repressor of Rhodopsin and IRBP (interphotoreceptor retinoid binding protein) promoters. The present studies were designed to characterize the DNA binding specificity of KLF15. Methods: Electrophoretic mobility shift assays (EMSA) followed standard procedures, using affinity purified, GST-tagged fusion proteins and EDTA-free binding buffer supplemented with ZnCl2. Results: The KLF15 binding site was previously localized to a 29 bp DNA element (B-Rho29) located between -94 and -66 bp upstream of the transcription start site of bovine Rhodopsin. The corresponding region of the human Rhodopsin promoter (H-Rho29) differs at only 3 bases. In EMSA, the zinc-finger domain of KLF15 binds to both B-Rho29 and H-Rho29. Binding activity was abolished by addition of 50 mM of the divalent cation chelator EDTA, but not by addition of 250 mM of the Ca+2 chelator EGTA. In the presence of 50 mM EDTA, KLF15 binding activity was reconstituted by addition of Zn+2, but not Mg+2. Mutated oligonucleotides, tested as competitors against radiolabeled B-Rho29 or H-Rho29, revealed the core binding site CnCCCC. A repressor element within the IRBP promoter, previously identified as a binding site for the zinc-finger transcription factor MOK2 (Arranz et al. J Biol Chem 2001 276:11963), does not contain this consensus sequence and was unable to compete with B-Rho29 or H-Rho29 for KLF15 binding. Conclusions: The zinc-finger containing DNA binding domain of KLF15 binds in a Zn++ dependent manner to a CnCCCC sequence within the proximal promoters of both bovine and human Rhodopsin. KLF15 represses transactivation of IRBP promoter constructs in vitro, however this activity is not mediated via the previously identified MOK2 binding site. Multiple potential binding sites for KLF15 are present in the Rhodopsin and IRBP promoters. DNAase I footprinting studies are underway to further assess KLF15 binding to these sites.
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