May 2008
Volume 49, Issue 13
Free
ARVO Annual Meeting Abstract  |   May 2008
Dissection of Rhodopsin Kinase (GRK1) Enhancer/Promoter: the Role of Zinc Finger Transcription Factors
Author Affiliations & Notes
  • S. C. Khani
    Ophthalmology, SUNY at Buffalo, Buffalo, New York
  • E. Kasperek
    Ophthalmology, SUNY at Buffalo, Buffalo, New York
  • J. E. Young
    Ophthalmology, SUNY at Buffalo, Buffalo, New York
  • Footnotes
    Commercial Relationships  S.C. Khani, None; E. Kasperek, None; J.E. Young, None.
  • Footnotes
    Support  NIH Grant EYR01-13600
Investigative Ophthalmology & Visual Science May 2008, Vol.49, 5400. doi:https://doi.org/
  • Views
  • Share
  • Tools
    • Alerts
      ×
      This feature is available to authenticated users only.
      Sign In or Create an Account ×
    • Get Citation

      S. C. Khani, E. Kasperek, J. E. Young; Dissection of Rhodopsin Kinase (GRK1) Enhancer/Promoter: the Role of Zinc Finger Transcription Factors. Invest. Ophthalmol. Vis. Sci. 2008;49(13):5400. doi: https://doi.org/.

      Download citation file:


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

      ×
  • Supplements
Abstract

Purpose: : Human rhodopsin kinase (RK) is driven by a conserved photoreceptor-specific enhancer/promoter that is active in both cones and rods across multiple species. The RK enhancer/promtoer has been used as the promoter of choice to drive therapeutic gene expression in retinitis pigmentosa animal model photoreceptors. Knowledge of the modulators of the RK enhancer/promoter activity would add to the utility of this particular enhancer by allowing titration of the activity and potentially the specificity of a construct. The goal of the studies is to evaluate the role of conserved core sequences and the role of zinc finger transcription factors in modulating the activity of enhancer.

Methods: : Double-stranded DNA segments carrying deletions or substitutions in the central enhancer/promoter core were synthesized and incorporated into luciferase expression plasmids. The activities of resultant luciferase plasmids were analyzed in retinoblastoma transient transfection assays and in HEK293 cells by contransfection with Sp1, Sp3, and Sp4 alone or together with other transcription factors. Yeast one-hybrid, gel retardation and southwestern binding assays were used to identify interactions and cognate transcription factors.

Results: : Mutagenesis of the highly conserved core sequences including the Crx and TA-rich TBP binding site led to 50-70% decline in activity. Sequences immediately surrounding the highly conserved core were essential for sensitivity to zinc finger factors. Sp1 and Sp4 led to greater than 10 and 5 fold increase in enhancer activity respectivity. Only modest stimulation was seen with Sp3. Gel retardation and southwestern blot analysis demonstrated interaction of G-rich candidate DNA sequences with high molecular weigh retinal nuclear proteins resembling Sp proteins. Yeast one hybrid analysis led to identification of retina ZNF216, a retina enriched zinc finger protein, but none of the above candidate proteins. Cotransfection with ZNF216 expression plasmid did not stimulate the basal enhancer activity but led to 2 fold increase in the Crx-stimulated activity.

Conclusions: : The RK enhancer/promoter is composed of a conserved core photoreceptor-specific enhancer immediately surrounded by zinc finger sensitive regions. The Sp transcription factors however may not be the high affinity cognate for this region. The above studies are prelude to identification of the factors modulating the activity of the enhancer/promoter and development of targeting constructs with a range of activities across photoreceptors.

Keywords: photoreceptors • gene/expression • transcription factors 
×
×

This PDF is available to Subscribers Only

Sign in or purchase a subscription to access this content. ×

You must be signed into an individual account to use this feature.

×