degeneration of rods in the rd1 mouse model of RP results in the loss of expression of RdCVF by rods as well as by bipolar cells at the onset of cone degeneration [8,10]. The potential of RdCVF administration for promoting survival of the central vision attributable to cones in patients suffering from RP is well illustrated by the finding that injection of RdCVF protein in the P23H transgenic rat model of dominant RP both slows down secondary cone loss and, more importantly, preserves cone function as measured by electroretinography [11]. Both RdCVF and RdCVF2 are encoded by an unspliced transcript from their respective genes. NXNL1 and 2 also express a spliced mRNA that encodes the protein products RdCVFL and RdCVF2L, which unlike RdCVF and 2 contain an entire thioredioxin-fold [9]. This suggests that the mammalian nucleor- edoxin-like genes are bi-functional, with one mRNA encoding a cone survival factor and a second encoding an active thioredoxin enzyme involved in the defense against oxidative stress, similar to the situation reported for the RdCVFL homolog in the arthropod Carcinoscorpius [12,13]. The search for proteins targeted by the putative redox-controlling activity of RdCVFL has led to the identification of a functional interaction between RdCVFL and the microtubule association protein TAU [14]. The hyperphosphoryla- tion of TAU in the retina of the mouse carrying a deletion of the Nxnl1 gene likely plays a role in the degeneration of rods observed in that model and in also in their sensitivity to oxidative stress [15]. A key element in understanding nucleoredoxin-like signaling in the eye is the restricted expression of the Nxnl1 and 2 genes in the retina. Nxnl1 expression is fully restricted to the eye, where it is expressed prominently by photoreceptors but also by bipolar cells [8,10]. Although Nxnl2 is expressed in selected regions of the brain as well as in the eye [9], within the retina RdCVF2 and RdCVF2L are expressed exclusively by photoreceptor cells. In the rd1 mouse model of RP, the death of rods (which account for 97% of all photoreceptors) results in the loss of retinal expression of all the products of the nucleoredoxin-like genes, including expression of Nxnl1 from bipolar cells [10]. This highly regulated expression pattern of the Nxnl1 and 2 genes raised the interesting question as to what are the mechanisms that control its cellular specificity within the retina. Understanding the regulation of the RdCVFs is not only important for what it can teach us about fundamental questions of cell type-specific gene transcription and processing, but also because manipulating expression of members of the RdCVF family could have therapeutic potential. Work from a number of groups, using a variety of experimental approaches, has identified some of the transcription factors that mediate photore- ceptor gene expression. Among the factors identified are CRX, NRL, NR2E3, RX/RAX, PAX6, and QRX [16–24]. Interest- ingly, many of these factors are homeodomain proteins, and it seems that combinatorial use of a relatively small number of proteins can account for a significant degree of photoreceptor specificity. Also of interest, mutation of or modulation of the expression of many of these proteins can cause abnormalities of photoreceptor development and/or retinal degeneration. For example, the homeoprotein CRX (cone rod homeobox), which itself is expressed predominantly in photoreceptors, binds to the consensus sequence C/TTAATCC/A and has functionally important binding sites present in the upstream region of many photoreceptor-specific genes, including rhodopsin, arrestin, IRBP, and b-phosphodiesterase-6 [18,19,24]. Knock-out of CRX in mice does not significantly alter photoreceptor lineage decisions, but it does markedly affect photoreceptor differentiation both morpho- logically and at the gene expression level [25,26]. In humans, CRX mutations induce a variety of phenotypes of retinal degeneration [27]. CRX Regulates RdCVF In a first step towards understanding the regulation of RdCVF expression, we recently used a combined promoter and transcrip- tomic analysis to identify some of the transcription factors that modulate Nxnl1 expression, and among the factors identified were the homeoproteins CHX10/VSX2, VSX1, and PAX4, and the zinc finger protein SP3 [10]. Here, we have extended that analysis and focused on the cis-acting 59-upstream regulatory sequences in both the murine and human Nxnl1 and 2 genes. Since a bioinformatic evolutionary comparison analysis (phylogenic foot- printing) did not reveal significant sequence conservation between the 59-regions, we utilized an experimental approach consisting of transient transfection of luciferase reporter constructs in four distinct cell lines and ex vivo electroporation of mouse retinal explants with GFP reporter plasmids. The use of the retinal explants allowed analysis of cell type-specific elements. We mapped active proximal promoter regions in both species to small regions of the Nxnl1 and Nxnl2 genes. The expression pattern of the electroporated GFP-reporters showed that while the identified Nxnl1 promoter sequence specifies expression to photoreceptor cells, the identified Nxnl2 active promoter fragments are not sufficient to restrict reporter expression to photoreceptors. However, we did find that combining a minimal and inactive 134 base pair (bp) piece of the Nxnl1 promoter with an active 79 bp piece of the Nxnl2 upstream region could restore photoreceptor specificity. This suggested that the 134 bp Nxnl1 sequence contains sufficient information for photoreceptor spec- ificity. Furthermore, by mutation analysis we showed that a CRX binding element located within the 134 bp Nxnl1 fragment is necessary for promoter activity, thereby implicating CRX in the cell type-specific regulation of RdCVF expression. Results Mapping the transcriptional start sites of the murine and human NXNL1 and NXNL2 genes In order to delineate the 59 upstream regions of the mouse and human NXNL1 and NXNL2 genes, we determined the position of their putative transcriptional start sites (TSSs) by rapid amplifica- tion of cDNA ends (RACE), using RNA purified from mouse and human retinas. As negative controls, we performed the reactions using RNA isolated from the retina of the Nxnl1 and Nxnl2 knock- out mice since the strategy used to generate these animal models results in the elimination of their respective mRNAs [15] and Jaillard et al., (submitted). Additionally, as control for human retinal tissue, we used HeLa cells (ATCC CCL-2) since northern blot analysis showed that NXNL1 transcripts are not expressed by these cells in Audo et al., (manuscript in preparation). As expected, RACE-PCR products for both Nxnl1 and 2 were present with wild-type (wt) retina, while with Nxnl1-/- RNA only a product for Nxnl2 was generated, and with Nxnl2-/- RNA only a product for Nxnl1 was generated (Figure 1A). In addition, we confirmed that both NXNL1 and NXNL2 are expressed in the human retina and that HeLa cells express NXNL2 but not NXNL1. The products of the RACE reactions were cloned, analyzed by gel electrophoresis and representative clones were sequenced (Figure 1B). Three and four retinal putative TSSs were identified for the mouse Nxnl1 and Nxnl2 genes, respectively, three for human retinal NXNL1, and only one for human retinal NXNL2 (Figure 1C). For nomenclature purposes, we have chosen to label the nucleotides in each gene based on the position of its major putative TSS (+1) (since Nxnl1 had two major TSSs of similar strength, one at 268 bp and one 252 bp, we arbitrarily chose to use the one at 252 bp for labeling). Interestingly, while as noted above NXNL2 is expressed by HeLa cells, its major putative TSS in HeLa cells is at 272 bp, PLoS ONE | www.plosone.org 2 October 2010 | Volume 5 | Issue 10 | e13075