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V. A. Mcilvain, S. E. Reks, T. V. Fedotova, B. E. Knox; Regulation of Rhodopsin Transcription by Rax Genes. Invest. Ophthalmol. Vis. Sci. 2010;51(13):1108.
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© ARVO (1962-2015); The Authors (2016-present)
Elucidating mechanisms of transcription in the adult retina is an important goal for understanding retinal maintenance, ageing and disease. Homeobox genes are important developmental regulators but have less well-studied roles in the mature retina. We investigated the role of Rax genes, which bind to highly conserved sequences in phototransduction gene promoters, as transcriptional activators. We studied their distribution, function and transcriptional specificity, including a novel clone related to RxL/Qrx.
Adult Xenopus retina was used to generate a cDNA library for a yeast one hybrid screen using a Ret1-like sequence as bait (5'-CCAATTAAGAGAT). Expression patterns were determined by RT-PCR and in situ hybridization. Morpholino oligonucleotides were injected into embryos with an EGFP tracer and harvested at stage 39 for real-time RT-PCR transcript analysis. Transcriptional activity was measured using transfected HEK293 cells, Xenopus transcription factors in pCS2 and Xenopus opsin promoter-luciferase reporters. In vitro translated proteins were used for electrophoretic mobility shift assays to determine DNA binding activity.
We identified a new gene, Rax2b, which has higher expression in the adult retina compared to its paralog RxL (Pan et al. 2006, IOVS 47, 4245). Morpholino-knockdown of Rax2b caused a decrease in rhodopsin mRNA but did not significantly affect Nrl or Crx transcript levels. Rax transcription factors, alone or in combination with either Nrl/Crx, did not affect transcription in cell-transfection assays. But they significantly increased activity when both Nrl/Crx were included. Mutational analysis of the Xenopus rhodopsin promoter identified Ret1 as a major target for Rax2b. Other conserved sites, Bat1 and Ret4, are targets for Crx.
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