KLF15 (Krüppel-like factor15) Represses Transactivation of the Rhodopsin and IRBP Promoters and Inhibits their Transactivation by Crx in vitro

Abstract

Abstract: : Purpose: To characterize transcriptional regulation of the photoreceptor-specific genes, rhodopsin and IRBP (interphotoreceptor retinoid-binding protein) by the transcription factors, KLF15 and Crx (cone rod homeobox). Methods: HEK293 cells were transiently transfected with expression plasmids containing full length cDNA’s for KLF15 (0.25-2.5 µg/plate), Crx (0.25 µg/plate), Nrl (Neural retinal leucine zipper, 0.25 µg/plate) and one of two luciferase-reporter constructs: (1) bRHO130-luc containing 130 bp of the bovine rhodopsin proximal promoter or (2) bIRBP300-luc containing 300 bp of the bovine IRBP proximal promoter. Cell lysates were assayed for luciferase activity, with changes in relative luciferase activity analyzed using linear mixed models with SAS. Results: Transfection with KLF15 decreased basal levels of luciferase activity driven by bRHO130-luc and by bIRBP300-luc by 32-51% (p<0.009) and by 69-72% (p<0.0001), respectively, at all levels KLF15 tested. As previously reported, transfection with Crx increased luciferase activity with both promoter constructs, and transfection with an expression plasmid for the bZIP transcription factor Nrl also increased activity with bRHO130-luc. Co-transfection with Crx and increasing amounts of KLF15 resulted in a linear decrease in bRHO130-luc driven luciferase activity, with a 4-fold excess of KLF15 decreasing luciferase activity 41% (p=0.0026 vs. Crx alone). With bIRBP300-luc, co-transfection with Crx and increasing amounts of KLF15 (0.25-2.5 ug/plate) decreased luciferase activity at all concentrations tested, with 4-fold excess of KLF15 reducing luciferase expression 73% (p<0.0001 vs Crx alone). In similar co-transfection experiments with Nrl, KLF15 was also able to significantly inhibit Nrl-mediated transactivation of bRHO130. Conclusion: The zinc-finger containing transcription factor KLF15 (a.k.a. KKLF/kidney-enriched Krüppel-like factor) was identified as a potential transcriptional regulator by its ability to bind a 29-bp element within the bovine rhodopsin proximal promoter in a yeast one-hybrid assay. The ability of KLF15 to repress transactivation of the rhodopsin and IRBP proximal promoters suggests a role for KLF15 in inhibition of expression of photoreceptor-specific genes in non-rod photoreceptor cells in the retina.

Keywords: 605 transcription factors • 604 transcription • 476 molecular biology