May 2003
Volume 44, Issue 13
Free
ARVO Annual Meeting Abstract  |   May 2003
Trannscriptional Control of the Xenopus Rhodopsin Promoter
Author Affiliations & Notes
  • S.L. Whitaker
    Biochem & Molec Bio, SUNY Upstate Med Univ, Syracuse, NY, United States
  • B.E. Knox
    Biochem & Molec Bio, SUNY Upstate Med Univ, Syracuse, NY, United States
Investigative Ophthalmology & Visual Science May 2003, Vol.44, 3532. doi:
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      S.L. Whitaker, B.E. Knox; Trannscriptional Control of the Xenopus Rhodopsin Promoter . Invest. Ophthalmol. Vis. Sci. 2003;44(13):3532.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Abstract: : Purpose: To further understanding of control of cell specific visual pigment gene expression, the role of Otx and maf family transcription factors in regulating the rhodopsin promoter was investigated. Methods: Reporter constructs containing the 5’ immediate upstream region from the Xenopus rhodopsin gene, driving expression of either luciferase or EGFP, were used in cell and embryo transfections, and in transgenic Xenopus. Transcription factor plasmids were co-transfected or integrated to determine the effect that these proteins have on transcriptional activity of the rhodopsin promoter. Results: Recent data suggests the Xenopus transcription factor Xotx5b may play a role similar to mammlian Crx, in photoreceptors. Co-transfection of HEK293 cells with the proximal Xenopus rhodopsin promoter and Xotx5b or Crx showed similar levels of transcriptional activation (~6-fold). In promoter constructs in which either of two highly conserved ATTA binding sites were deleted, only a two-fold decrease (BAT1) or no decrease (Ret1) in transcriptional activity was observed. Thus, there appear to be redundant cis-acting elements for Xotx5b/Crx. The Xenopus homolog of Nrl has not yet been described, however a lens transcription factor, XL-maf, groups phylogenetically with Nrl. In situ hybridizations detect XL-maf in the photoreceptor layer in stage 48 tadpoles. In HEK transfections, XL-maf stimulates the Xenopus rhodopsin promoter (~15-fold). Xotx5b and XL-maf act synergistically on the Xenopus rhodopsin promoter (~150-fold). We identified nucleotides –84/-58 as the major cis-acting element necessary for stimulation by Nrl. This element is highly conserved with mammalian rhodopsin NRE. Removal of either of two ATTA binding sites increased stimulation by Nrl. The NRE was found to be essential for the synergistic stimulation by Crx and Nrl, while removal of the Ret1 region increased synergy (320-fold). In transfections of Xenopus embryos, mammalian Crx and Nrl stimulate expression in trunks. Neither mammalian Crx nor Nrl caused noticeable increase in stimulation in Xenopus embryo heads. In transgenic animals, mammalian Otx5b/Crx and XL-maf/Nrl were both able to drive early and extra-ocular expression from a Xenopus rhodopsin-GFP promoter. Conclusions: We showed that two Xenopus genes, Xotx5b and XL-maf, are functionally equivalent to the mammalian transcription factors, Crx and Nrl. High-levels of promoter activation require the NRE, with an additional negative regulatory activity mediated by Ret1. Appropriate activation of the rhodopsin gene during development appears to be, in part, determined by the expression pattern of Otx5/Crx and XL-maf/Nrl.

Keywords: transcription factors • transcription • transgenics/knock-outs 
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