April 2009
Volume 50, Issue 13
Free
ARVO Annual Meeting Abstract  |   April 2009
The DNA-Binding Domain (DBD) Is Essential for NR2E3 Dimerization and Interaction With Crx
Author Affiliations & Notes
  • R. Roduit
    IRO-Inst. for Res. in Ophthalmology, Sion, Switzerland
  • P. Escher
    IRO-Inst. for Res. in Ophthalmology, Sion, Switzerland
  • M. Emery
    IRO-Inst. for Res. in Ophthalmology, Sion, Switzerland
  • N. Voirol
    IRO-Inst. for Res. in Ophthalmology, Sion, Switzerland
  • T. Favez
    IRO-Inst. for Res. in Ophthalmology, Sion, Switzerland
  • D. F. Schorderet
    IRO-Inst. for Res. in Ophthalmology, Sion, Switzerland
    EPFL-Ecole Polytechnique Fédérale de Lausanne, Lausanne, Switzerland
  • Footnotes
    Commercial Relationships  R. Roduit, None; P. Escher, None; M. Emery, None; N. Voirol, None; T. Favez, None; D.F. Schorderet, None.
  • Footnotes
    Support  PE is support by FNRS#3100A0-122269/1
Investigative Ophthalmology & Visual Science April 2009, Vol.50, 5459. doi:
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      R. Roduit, P. Escher, M. Emery, N. Voirol, T. Favez, D. F. Schorderet; The DNA-Binding Domain (DBD) Is Essential for NR2E3 Dimerization and Interaction With Crx. Invest. Ophthalmol. Vis. Sci. 2009;50(13):5459.

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

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Abstract

Purpose: : NR2E3 (PNR) is an orphan nuclear receptor essential for proper photoreceptor determination and differentiation. In humans, mutations in NR2E3 have been associated with the recessively inherited enhanced short wavelength sensitive (S-) cone syndrome (ESCS) and, more recently, with autosomal dominant retinitis pigmentosa (adRP). NR2E3 acts in concert with the transcription factors Crx and Nrl to repress cone-specific genes and activate rod-specific genes. NR2E3 and Crx have been shown to physically interact by their DNA-binding domain (DBD), which may also be implicated in the dimerization process of the nuclear receptor. However, neither NR2E3 homodimerization nor NR2E3/Crx complex formation has been investigated in detail.

Methods: : In this present work, we analyzed the dimerization of the NR2E3 protein and its interaction with Crx by bioluminescence resonance energy transfer (BRET2) which utilizes Renilla luciferase (hRluc) protein and its substrate DeepBlueC as an energy donor and a mutant green fluorescent protein (GFP2) as the acceptor. We investigated, on whole intact cells, the role of NR2E3 DBD-mutations in dimerization and association with Crx.

Results: : We clearly showed that NR2E3 formed homodimers in HEK-293T cells. Moreover, all causative NR2E3 mutations present in the DBD of the protein showed an alteration in dimerization, except for the R76Q and the R104W mutants. Interestingly, the adRP-linked G56R mutant was the only DBD-NR2E3 mutant that showed a correct interaction with Crx. Finally, we observed a decrease in rhodospin gene transactivation for all DBD-NR2E3 mutants tested and no potentiation for the adRP-linked G56R mutant. In addition, the p.G56R mutant enhanced the transrepression of M-opsin promoter, while all other DBD-NR2E3 mutants did not repress M-opsin transactivation.

Conclusions: : A defect, either in the dimer formation or in the interaction of NR2E3 with Crx, leads to abnormal transcriptional activity on rhodopsin and M-opsin promoter and to an atypical retinal development; while the titration of Crx by p.G56R-NR2E3 leads to low levels of rhodopsin and M-opsin expression and may be responsible for the strong adRP phenotype.

Keywords: photoreceptors • transcription factors • mutations 
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