The bioinformatic analysis of the shortest active NXNL1 promoter constructs revealed the existence of a putative CRX binding element (256 to 250 bp in NXNL1 and 254 to 248 bp in Nxnl1). Because of the essential role of the homeodomain protein CRX in the maturation of photoreceptors and its implication in inherited photoreceptor degeneration [18,38,39,25], the role of this binding element was further analyzed by mutagenesis. The integrity of the CRX binding element was found to be essential for the activity of the Nxnl1 promoter in vitro and ex vivo (Figures 7A and B). While we cannot rule out the possibility that another promiscuous homeodomain protein regulates the expression of RdCVF through this ‘‘CRX’’ element, CRX it is the most likely candidate to be mediating the observed biological activity. The deletion analysis of the human NXNL2 gene identified a 79 bp promoter element that showed promoter activity in both the cell line and retinal explant assay systems (Figures 2C and 3C). The activity of this element is not restricted to photoreceptor cells (Figure 5C). Interestingly, this 79 bp sequence is active when fused to an inactive 134 bp NXNL1 minimal proximal promoter sequence (Figure 6B and C), resulting in a reporter expression pattern that shows restriction to photoreceptors (Figure 6D). These findings demonstrate that the NXNL2 79 bp sequence from 2393 to 2315 bp contains positive regulatory activity that can act independently of its own basal promoter and also strongly suggest that the NXNL1 277 to +57 bp proximal promoter region contains sufficient cell type-specific information, when supple- mented with a less specific transcriptional activator, to restrict expression to photoreceptor cells. The identity of the biologically active regulatory elements within the 79 bp NXNL2 sequence, and the transcription factor(s) that bind to them, are presently unknown. For the cell type restricting activity of the 134 bp NXNL1 region, the putative CRX binding site discussed above appears to be important. Mutation of the CRX element abolished promoter activity of the NXNL2/NXNL1 fusion construct in Y79 cells (Figure 7A). Although mutation of the CRX site did not significantly reduce promoter activity in the explant system, it did lead to loss of photoreceptor specificity (Figure 8). These results, taken together, suggest that the CRX site within the NXNL1 proximal promoter region plays a dual function depending on its context. Within the native NXNL1 promoter it appears to act as a positive transcriptional regulator whose activity is essential for expression. Without its activity there is no detectable expression so in this context its possible role in influencing cell type specificity cannot be assessed. In the context of the NXNL2/NXNL1 fusion construct, presumably because an element, or elements, within the 79 bp NXNL2 sequence is able to replace its transcriptional activating role, its function in restricting expression to photore- ceptors is unmasked. Future studies will hopefully provide mechanistic insight into how CRX carries out these dual roles, and also provide information on the other regulatory sites and factors responsible for the photoreceptor expression of RdCVF and RdCVF2. Materials and Methods Animals No animal experimentation was performed. The animals used in this study were obtained from Charles River France (L’Arbresle). Retinal tissues were obtained from newborn C57BL/6 mice. Animals were sacrificed the day of birth and eyes were dissected to recover neural retinal tissues (Authorization 75– 1208 delivered on July 21 2008 by the Minister of Agriculture). The methods used to secure animals and tissues complied with the ARVO Statement fro the Use of Animals in Ophthalmic and CRX Regulates RdCVF Vision Research. No animal experimentation was performed. The animals used in this study were obtained from Charles River France (L’Arbresle). Rapid Amplification of cDNA Ends (59 RACE) The rapid amplification of cDNA ends (59 RACE) was carried out using total RNA purified from mouse retina, human retina and cultured HeLa cells by CsCl2 ultracentrifugation. A First- ChoiceTM RLM-RACE Kit (Ambion) was used to identify transcription start sites according to manufacturer’s instructions. Briefly, total RNA was treated with calf intestinal phosphatase to remove the 59-phosphate. The RNA was then treated with tobacco acid pyrophosphatase to remove the cap structure of the mRNA. A synthetic RNA adapter was then ligated to the RNA population to allow ligation to only RNA having been capped. After reverse transcription reaction, cDNA was obtained by nested-PCR using two reverse primers for each gene (human NXNL1: 59 CCGCCCGCAGTACATAGAACTCA 39 and 59 TCTGTGAGCCGCACGAAGAAGTC 39; mouse Nxnl1: 59 TC- AGCACGTCCCCACCAG GCTTAA 39 and 59 TCAGCACG- TCCCCACCAGGCTTAA 39; human NXNL2: 59 GCGCAT- GAAGTCCAGCATCTCC 39 and 59 TCGGCTGACACGAA- GACCACTTC 39; mouse Nxnl2: 59 CGTTCTGCTTGATGAC- CACCAGC 39 and 59 GGGGATGGCGGTGAT TTCGTA 39) and the two forward primers provided with the kit. PCR products were analyzed on 2% agarose gels and the reaction products were cloned into pGEM T Easy (Promega). A subset of recombinant clones were analyzed on agarose gel and the plasmid displaying different insert sizes were sequenced from both directions. Theses sequences were aligned to the genomic sequences. Cell culture Human Y79 cells from an immortalized cell line derived from a human retinoblastoma (ATCC HTB-18) were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum. Cells were maintained in suspension or for transfection purposes were grown as a monolayer. For cell attachment, plates were treated with poly (D-lysine) (0.2 mg/ml, Sigma) and laminin (0.1 mg/ml, Sigma) for 3 hours at 37uC. Human HEK 293 cells (ATCC CRL- 1573) and COS-1 cells (ATCC CRL-1650) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS, Invitrogen). 661W cells (murine cone photoreceptor derived cells) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS, Invitrogen) and 2mM L-glutamine (Invitro- gen). HeLa cells (ATCC CCL-2) were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum. Cell media were changed every 2–3 days, and cells were split weekly. Promoter reporter plasmids construction The sequence upstream ATG of both murine and human Nxnl1 and Nxnl2 were cloned in the promotorless pGL4.17 (expressing a firefly luciferase, Promega) and peGFP (Clontech) plasmid using the gateway technology (Invitrogen). The vectors were converted into a gateway destination vector by introducing a cassette containing the attR sites flanking the ccdB gene into the multiple cloning site. To identify DNA sequences that regulate murine and human Nxnl1 and Nxnl2 transcription, a series of deletions from the 59 end of each gene was created. Primers were designed to amplify different lengths of the 59end regions of each gene, from approximately 100 base pairs (bp) to 4.5 kb and the attB sites were incorporated into the forward (attB site 59 GGG GAC AAG TTT GTA CAA AAA AGC AGG CTT A 39) and the reverse (attB site 59 GGG GAC CAC TTT GTA CAA GAA AGC TGG PLoS ONE | www.plosone.org 13 October 2010 | Volume 5 | Issue 10 | e13075