Expression of Rod-Derived Cone Viability Factor: Dual Role of CRX in Regulating Promoter Activity and Cell- Type Specificity Sophie Lambard1,2,3, Sacha Reichman1,2,3, Cynthia Berlinicke4, Marie-Laure Niepon1,2,3, Olivier Goureau1,2,3, Jose´ -Alain Sahel1,2,3, Thierry Le´ veillard1,2,3*, Donald J. Zack1,2,3,4,5 1 Department of Genetics, INSERM, U968, Paris, France, 2 INSERM UPMC Univ Paris 06, UMR_S 968, Institut de la Vision, Paris, France, 3 CNRS, UMR_7210, Paris, France, 4 Department of Ophthalmology, Johns Hopkins University School of Medicine, Baltimore, Maryland, United States of America, 5 Department of Molecular Biology and Genetics, Department of Neuroscience, and Institute of Genetic Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland, United States of America Abstract Background: RdCVF and RdCVF2, encoded by the nucleoredoxin-like genes NXNL1 and NXNL2, are trophic factors with therapeutic potential that are involved in cone photoreceptor survival. Studying how their expression is regulated in the retina has implications for understanding both their activity and the mechanisms determining cell-type specificity within the retina. Methodology/Principal Findings: In order to define and characterize their promoters, a series of luciferase/GFP reporter constructs that contain various fragments of the 59-upstream region of each gene, both murine and human, were tested in photoreceptor-like and non-photoreceptor cell lines and also in a biologically more relevant mouse retinal explant system. For NXNL1, 59-deletion analysis identified the human 2205/+57 bp and murine 2351/+51 bp regions as having promoter activity. Moreover, in the retinal explants these constructs drove expression specifically to photoreceptor cells. For NXNL2, the human 2393/+27 bp and murine 2195/+70 bp regions were found to be sufficient for promoter activity. However, despite the fact that endogenous NXNL2 expression is photoreceptor-specific within the retina, neither of these DNA sequences nor larger upstream regions demonstrated photoreceptor-specific expression. Further analysis showed that a 79 bp NXNL2 positive regulatory sequence (2393 to 315 bp) combined with a 134 bp inactive minimal NXNL1 promoter fragment (277 to +57 bp) was able to drive photoreceptor-specific expression, suggesting that the minimal NXNL1 fragment contains latent elements that encode cell-type specificity. Finally, based on bioinformatic analysis that suggested the importance of a CRX binding site within the minimal NXNL1 fragment, we found by mutation analysis that, depending on the context, the CRX site can play a dual role. Conclusions/Significance: The regulation of the Nucleoredoxin-like genes involves a CRX responsive element that can act as both as a positive regulator of promoter activity and as a modulator of cell-type specificity. Citation: Lambard S, Reichman S, Berlinicke C, Niepon M-L, Goureau O, et al. (2010) Expression of Rod-Derived Cone Viability Factor: Dual Role of CRX in Regulating Promoter Activity and Cell-Type Specificity. PLoS ONE 5(10): e13075. doi:10.1371/journal.pone.0013075 Editor: Michael J. Pazin, National Institute on Aging, National Institutes of Health, United States of America Received June 1, 2010; Accepted September 6, 2010; Published October 7, 2010 Copyright: 2010 Lambard et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by funds from Agence Nationale de la Recherche (ANR, Chaire d’excellence), Inserm, Conventions Industrielles de Formation par la Recherche (CIFRE), National Institutes of Health (NIH), Foundation Fighting Blindness, and European Vision Institute FP6 programme acronym GENORET. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: T.L. and J-A.S. are patent-holders on the use of RdCVF and RdCVF2 for the treatment of neurological disease. This does not alter the authors’ adherence to all the PLoS ONE policies on sharing data and materials. * E-mail: thierry.leveillard@inserm.fr Introduction Retinitis pigmentosa (RP) is a genetically and clinically heterogeneous group of inherited retinal degenerative diseases that causes severe visual impairment in as many as 2 million patients worldwide [1,2]. Most identified forms of RP are caused by mutations in genes whose expression is enriched in or restricted to rod photoreceptor cells [3,4]. The loss of rods in RP is generally followed by a secondary degeneration of cone photoreceptors [5,6]. It is this secondary loss of cones that is most significant clinically because cones are required for high acuity, color, and most daylight vision. In the vast majority of cases, RP and related forms of retinal degeneration are untreatable, and all that can be offered to patients are supportive measures. Given the clinical importance of cone loss in retinal degener- ative disease, in our effort to develop broad-based therapies for RP that go beyond single gene replacement, we have been studying the mechanisms of cone cell loss. We and our colleagues have demonstrated that rods secrete factors that promote cone survival, reviewed in [7]. Furthermore, through expression and homology cloning approaches, we have identified a novel family of rod- secreted factors that are trophic for cones. These factors, Rod- Derived Cone Viability Factor (RdCVF) and RdCVF2, are encoded, respectively, by the nucleoredoxin-like genes NXNL1 and NXNL2 [8,9]. The cone-directed protective activity of retinal explant conditioned media is significantly reduced after immuno- depletion of RdCVF, suggesting that a major part of retinal derived cone survival activity in vivo is due to RdCVF [8]. The PLoS ONE | www.plosone.org 1 October 2010 | Volume 5 | Issue 10 | e13075