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D.C. Otteson, H.L. Lai, Y. Liu, D.J. Zack; KLF15 Binds to Known Repressor Elements in Bovine Rhodopsin and IRBP promoters and Requires N–terminal Domain for Repressor Activity. . Invest. Ophthalmol. Vis. Sci. 2004;45(13):2254.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Continuing research designed to understand the molecular mechanisms regulating photoreceptor–specific gene expression have identified KLF15, a zinc–finger transcription factor, as a repressor of the rhodopsin and interphotoreceptor retinoid binding protein (IRBP) promoters in vitro. The current studies seek to identify KLF15 binding sites in the proximal promoter regions of rhodopsin (RPPR) and IRBP and to localize repressor elements within KLF15. Methods: Standard DNAse I footprinting of bovine RPPR and IRBP promoters used bacterially expressed, GST–fusion proteins containing the DNA binding domain of human KLF15. Transactivation assays compared luciferase expression in 293 cells following transient transfection with full length or truncated KLF15 expression vectors and reporters containing the full (bRho225) or minimal (bRho130) bovine RPPR. Analysis used general linear models (SAS) with p<0.05 considered significant. Results: In DNAse I footprinting assays, KLF15 protected six sites in the RPPR and three in the IRBP promoter. Two KLF15 binding sites coincided with previously described repressor elements: the CRS–1 site (RPPR) and the G–rich repressor (IRBP). Deletion of the N–terminal 67 amino acids did not alter KLF15 repression of bRho130 or bRho225. N–terminal deletions of 160 or more amino acids abolished repressor activity and resulted in statistically significant increases in luciferase expression. Deletion of the C–terminal, DNA binding domain inactivated KLF15 resulting in no changes in luciferase levels compared to controls. Conclusions: KLF15 binds to multiple sites in the bovine rhodopsin and IRPB promoters including the previously described CRS–1 and G–rich repressor elements. These sites are bound by nuclear proteins present in both retinal and non–retinal cells and have been implicated in repression of photoreceptor genes in non–photoreceptor cells. KLF15 is expressed in multiple tissues and represses both the rhodopsin and IRBP promoters in vitro, making it an attractive candidate to participate in repression of inappropriate temporal and cellular expression of photoreceptor–specific genes in vivo.
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