Zinc-finger domains of the transcriptional repressor KLF15 bind multiple sites in rhodopsin and IRBP promoters including the CRS-1 and G-rich repressor elements
© Otteson et al; licensee BioMed Central Ltd. 2005
Received: 26 April 2005
Accepted: 17 June 2005
Published: 17 June 2005
In the retina, many of the genes that encode components of the visual transduction cascade and retinoid recycling are exclusively expressed in photoreceptor cells and show highly stereotyped temporal and spatial expression patterns. Multiple transcriptional activators of photoreceptor-specific genes have been identified, but little is known about negative regulation of gene expression in the retina. We recently identified KLF15, a member of the Sp/Krüppel-like Factor family of zinc-finger containing transcription factors, as an in vitro repressor of the promoters of the photoreceptor-specific genes rhodopsin and IRBP/Rbp3. To gain further insight into the mechanism of KLF15-mediated regulation of gene expression, we have characterized the binding characteristics and specificity of KLF15's DNA binding domains and defined the KLF15 binding sites in the rhodopsin and IRBP promoters.
In EMSA and DNAseI footprinting assays, a KLF15-GST fusion protein containing the C-terminal zinc-finger domains (123 amino acids) showed zinc-dependent and sequence-specific binding to a 9 bp consensus sequence containing a core CG/TCCCC. Both the bovine rhodopsin and IRBP promoters contained multiple KLF15 binding sites that included the previously identified CRS-1 and G-rich repressor elements. KLF15 binding sites were highly conserved between the bovine, human, chimp and dog rhodopsin promoters, but less conserved in rodents. KLF15 reduced luciferase expression by bRho130-luc (containing 4 KLF15 sites) and repressed promoter activation by CRX (cone rod homeobox) and/or NRL (neural retina leucine zipper), although the magnitude of the reduction was smaller than previously reported for a longer bRho225-luc (containing 6 KFL15 sites).
KLF15 binds to multiple 9 bp consensus sites in the Rhodospin and IRBP promoters including the CRS-1 and G-rich repressor elements. Based on the known expression pattern of KLF15 in non-photoreceptor cells, we hypothesize an in vivo role for KLF15 in repressing photoreceptor-specific gene expression in the inner retina.
Photoreceptors are the highly specialized sensory receptors of the retina and express a unique array of genes that enable them to convert light energy into a neural signal. Many of these genes, including those encoding components of the phototransduction cascade [e.g. rhodopsin (Rho), transducin, arrestin, α– and β-phosphodiesterase (PDE)] and those involved in retinoid recycling [e.g. interphotoreceptor retinoid binding protein (IRBP/RBP3)], are expressed only in photoreceptor cells in the retina and in a subset of cells in the pineal gland [1–5]. In addition, these genes have highly stereotyped temporal and spatial patterns of expression during retinal development [6–8] that are transcriptionally regulated [7, 9, 10]. We are interested in understanding the transcriptional networks that regulate photoreceptor-specific gene expression, not only as a model for cell-specific gene regulation, but to gain insight into the mechanisms that regulate photoreceptor differentiation and neuro-degenerative retinal disease.
The proximal promoter regions of both Rho (-225 to +70 base pairs relative to the transcription start site) [11–13] and IRBP (-123 to +18 bp) [6, 14] are sufficient to drive photoreceptor-specific expression of reporter genes in transgenic mice. Using DNAse I protection assays, binding sites for both retina-enriched and more widely expressed proteins have been identified on these promoters [15–22] and include both positive and negative regulatory elements [17–19, 23–29]. Among the positive regulatory elements, Ret-1, BAT-1 and PCE-1 each contain a core ATTA sequence that can be bound by homeodomain containing transcription factors including CRX , RX/RAX , ERX , QRX  and OTX2 [18, 34]. Binding sites for the basic-leucine zipper transcription factor NRL have been identified in the promoters of rhodopsin [35, 36] as well as other photoreceptor-specific genes including arrestin  and the rod-specific α- and β-PDE [37, 38]. Combinatorial effects by CRX and NRL result in synergistic increases in Rho promoter activation in vitro that likely contribute to the high levels of expression in rod photoreceptors in vivo [29, 30, 39] Both Crx and Nrl play key roles in photoreceptor development and survival: in Crx knock-out mice, rod photoreceptors fail to elaborate outer segments and degenerate and the mice lack detectible visual function ; in Nrl knock-out mice, rod photoreceptors apparently fail to differentiate (although there is an increase in cone-like cells) and mice have severely reduced visual function . In addition, mutations in both CRX and NRL have been identified in patients with a variety of inherited retinal degenerative diseases including autosomal dominant and recessive retinitis pigmentosa, Leber congenital amaurosis and cone-rod dystrophy [42–45].
Although transcriptional repression plays an important role in development and cell fate determination in the central nervous system and inappropriate de-repression of gene expression can contribute to disease [46, 47], relatively little is known about negative regulation of photoreceptor-specific gene expression. Negative regulatory elements, including the CRS-1 site in the rhodopsin promoter  and the G-rich repressor in the IRBP promoter  are bound by nuclear proteins present in both retinal and non-retinal tissues, although the identities of the specific transcription factors have not been established. The zinc-finger transcription factor MOK2 binds to sites in the promoter and intron 1 of IRBP and represses transactivation of reporter constructs containing these elements in vitro . Other proteins interact with CRX (e.g. ATXN-7  and BAF ) or NRL (e.g. FIZ-1 ) and can reduce their ability to activate transcription, but these appear to repress via protein/protein interactions rather than binding directly to the promoter. We recently identified KLF15, a member of the Sp/KLF family of zinc-finger containing transcription factors, as a repressor of both the rhodopsin and IRBP promoters in vitro .
KLF15 is expressed in both retinal and non-retinal tissues and, like other members of the Sp/KLF family, is characterized by the presence of three Krüppel-type zinc-finger domains at the C-terminus [52–54]. Members of this gene family have been characterized as regulators of both tissue-specific and ubiquitous genes and can function as either transcriptional activators, repressors or both depending on promoter context [55, 56] KLF15 appears to be bi-functional, as it represses the Rho and IRBP promoters  and the kidney-specific Clc-K1 and Clc-K2 promoters  but activates the Glut4 (glucose transporter 4) promoter . KLF1/EKLF [57, 58], KLF8  and KLF15  all bind a core CACCC site in vitro, although this is shorter than the 9 bp site predicted for a three zinc-finger containing protein and is likely to provide only limited specificity. To gain further insight into the mechanism of KLF15-mediated regulation of gene expression, we have characterized the binding characteristics and specificity of KLF15 and defined the KLF15 binding sites in the Rho and IRBP promoters.
EMSA analysis of KLF15 binding
Comparison of the proximal promoter regions showed that the 29 bp fragment of the bovine Rho promoter (bRho29) used as bait in the yeast one-hybrid assay that identified KLF15  shared high sequence identity with the equivalent element from the human rhodopsin promoter (hRho29), differing by only 5 bp. In EMSA, KLF15-ZF fusion protein shifted oligomers for both bRho29 (Fig. 1B) and hRho29 (Fig. 1C) and these could be supershifted by addition of antibodies against KLF15 (generous gift of S. Uchida ) (Fig. 1D) or GST (Fig. 1E). The lower molecular weight band that was supershifted by the anti-KLF15 but not by anti-GST antibodies most likely resulted from low levels of protein that had lost the N-terminal, GST-tag, but retained the C-terminus of the protein containing the DNA binding domain and the anti-KLF15 antibody recognition site.
Zinc dependence of KLF15 binding
Sequence specificity of KLF15 binding
KLF15 binding sites in rhodopsin and IRBP promoters
Effects of KLF15 on a minimal bovine rhodopsin promoter
We previously identified KLF15 as a transcriptional repressor of the rhodopsin and IRBP promoters in vitro and report here the characterization of KLF15's DNA binding properties. For these analyses, we analyzed protein binding specificity on bovine promoters using a truncated human KLF15-GST fusion protein containing the C-terminal DNA binding domain, but lacking the N-terminal repressor domain . Since zinc-finger domains typically function as modular DNA binding motifs that can bind their target sequences independently of other protein domains [67, 68], the binding specificity of the truncated KLF15 fusion protein is predicted to be similar, if not identical, to the full length protein. Previous studies showed that recombinant GST fusion proteins containing the full length human or rat KLF15 bound to an element in the CLC-K1 promoter  and we found that our truncated KLF15 fusion protein also bound the same element in EMSA (data not shown). The amino acid sequence of the zinc finger domains of bovine KLF15 differs from human, chimp, dog, mouse and rat at only a single amino acid residue (isoleucine to valine) and the flanking regions that were included in the KLF15-ZF fusion protein differ at only 11 amino acid residues between human and bovine KLF15. We would predict, therefore, that the DNA binding specificity would show only minor differences (if any) between these species. Supporting this prediction, the human KLF15-GST fusion protein shifted oligos containing either the bovine or human KLF15 consensus site. Using EMSA, we have confirmed that proteins present in retinal extracts can bind specifically to the KLF15 binding site. However, this result does not necessarily mean that the observed binding activity is attributable to KLF15. Other members of the KLF family recognize similar G/C rich binding motifs [54, 57–59] and MAZ (myc-associated zinc-finger protein) can also recognize the KLF15 core binding site . Thus, it remains a distinct possibility that, in addition to KLF15, there are other retinal proteins that can recognize this site.
Several of the KLF15 binding sites that we identified in DNAse I footprinting correspond to previously identified promoter elements. In EMSA, both retina-specific and ubiquitous nuclear proteins bind a 20 bp probe (-55 to -36) containing the Ret-4 element (including the KR-b site) in the rhodopsin promoter . Interestingly, mutations in the G-rich sequence within the Ret-4 element that would be predicted to disrupt the KLF15 binding site eliminated binding by the ubiquitously expressed proteins . In DNAse I footprinting assays of the bovine rhodopsin proximal promoter, nuclear extracts from bovine retina  and from Y79 and WERI retinoblastoma cells  protected the BAT-1 (-103 to -84) site. Two additional sites protected by bovine retinal proteins are Ret-1 (-138 to -126) and CRS-1 (-177 to -199) [16, 20]. Three of these sites correspond to sites protected by KLF15 in our DNAse I footprinting, KR-6/Ret-4, KR-c/BAT-1 and KR-e/CRS-1. In addition to being protected by nuclear proteins from retinal extract, the CRS-1 site is protected by proteins present in nuclear extracts from non-retinal tissues . KLF15 is expressed in the multiple tissues including the retina [52–54] and can repress rhodopsin promoter constructs in transient transfections . Although we do not yet know the relative importance of these different KLF15 binding sites in regulation of target gene expression in vivo, our in vitro assays showed that KLF15 more effectively repressed a rhodopsin promoter construct (bRho225) containing six KLF15 binding sites (including KR-e/CRS-1) than a shorter promoter construct (bRho130) that contains only 4 (including the KR-b/Ret-4 and KR-c/BAT-1 sites), suggesting that both the proximal and distal sites influence KLF15's repressor activity.
The G-rich repressor element is located between -156 and -70 bp in IRBP promoter and overlaps the KI-c binding site for KLF15. Deletion of the G-rich element increases reporter gene expression in retinoblastoma cells [14, 19, 34] and results in inappropriate expression of GFP reporter constructs in non-retinal tissues in Xenopus laevis . In DNAse I footprinting, this site is protected by proteins present in nuclear extracts from both retinoblastoma WERI-Rb1 and non-retinal HeLa cells . Based on the ability of oligos containing the Sp1 consensus site to compete with the G-rich element in EMSA, it was proposed that Sp1 or Sp1-related proteins bind this site in vivo . KLF15 represses transactivation of an IRBP promoter construct in vitro, is expressed in both retinal and non-retinal tissues, and is structurally and phylogenetically related to the Sp family of transcription factors , making it an attractive candidate to bind the G-rich repressor element in vivo.
Potential KLF15 binding sites corresponding to the CRS-1 and G-rich sites are present in the bovine, human, chimp and dog promoters. It was, however, somewhat surprising that no consensus KLF15 binding sites were identified at corresponding locations in the mouse or rat promoters. As the zinc-finger domains are identical between these species, it seems unlikely that the lack of identifiable binding sites at these locations reflects species-specific differences in KLF15's binding specificity. Although we cannot formally exclude this possibility, there are several possible alternative interpretations of this observation. The simplest explanation is that the location or number of KLF15 binding sites differs between species, possibly reflecting the accumulation of mutations during evolution. A comparison of well characterized promoters of 51 genes found substantial differences between human and mouse, with 32–40% of the binding sites identified in the human promoters containing sufficient sequence changes in the mouse promoters to render them non-functional . In addition, despite our evidence showing that KLF15 binds the CRS-1 and G-rich repressor elements in vitro, there may be species-specific differences in the identity and/or sequence specificity of the transcription factor(s) that bind this site in vivo.
Changes in transcriptional regulation are thought to play a significant role in generating phenotypic differences between species  and there are clearly morphological and developmental differences between cows, primates, dogs and rodents. In the retina, differences include the time course of embryonic/fetal development and cellular differentiation, as well as the proportions and absolute numbers of different neuronal subtypes (e.g. rods vs. cones) present in the mature retina. Along with these morphological differences, there are species-specific variations in the regulation of photoreceptor-specific gene expression. In the bovine retina, transcripts for rhodopsin, IRBP, arrestin, rod α-transducin and rod α-PDE all begin to accumulate relatively late in retinal development, at the time when the first outer segments are detected . In contrast, in mice and rats there is considerable variation in the developmental stage when different photoreceptor-specific genes are first expressed [8, 9, 72]. Interestingly, the CRS-1/KR-e site was initially proposed to play a role in repressing premature rhodopsin expression during retinal development, based on the observation that protection of the CRS-1 site by fetal bovine retinal proteins diminishes throughout development  in a temporal pattern that is coordinated with increases in rhodopsin transcription and mRNA accumulation . Further emphasizing the extent of the differences between species, a large scale analysis of expressed genes in the mouse retina found that out of nearly 2000 known genes that could be assigned map locations in the mouse genome, 25% had no identifiable human ortholog . Although the identification and characterization of conserved genes and regulatory mechanisms has provided significant contributions to our understanding of retinal development and disease, there are likely to be novel and potentially valuable insights that can be obtained through comparative analysis of the basis of species-specific differences.
Several KLF proteins have been shown to bind a core CACCC element [57–59] and Gray and colleagues  demonstrated that 5'-CACCC-3' is sufficient for KLF15 binding in EMSA. A single zinc finger typically makes contact with 3 adjacent bases  therefore the three zinc-fingers of KLF15 would be predicted to have a 9 bp binding site, consistent with our EMSA results. We found that mutations outside of the 9 bp consensus site had no effect on DNA binding in EMSA; however, when we used TargetExplorer to re-analyze the KLF15 protected sequences identified in DNAse I footprinting, making no a priori assumptions as to the actual size of the consensus binding site, a longer 12–13 bp consensus sequence was identified. The functional significance of the extended consensus site is unclear, but it is interesting to note that the KR-b, KR-c and KR-d sites respectively overlapped the Ret-4, BAT-1 and Ret-1 sites, all containing known binding sites for homeodomain containing proteins including CRX [30, 31]. The KR-b site is located at the 3' end of the Ret-4 site within a G-rich sequence is a binding site for nuclear proteins present in both retinal and non-retinal tissues . The highly conserved KR-d site is within a GC-rich sequence that functions to facilitate binding of retinal nuclear proteins to the adjacent Ret-1 site . One interpretation is that KLF15 binding sites are preferentially located adjacent to binding sites for other transcription factors, possibly CRX or other OTX-related factors. In EMSA using total retinal proteins, mutation of the KLF15 binding site increased binding of some retinal proteins to the Δ10 oligo, as shown by the increased intensity of KLF15-independent bands (Fig. 5). As mutation of the KLF15 binding site does not alter the core homeodomain binding site present in these oligos, homeodomain proteins (e.g. CRX; RX/RAX; OTX2) present in retinal extracts likely bind to both the wild-type and mutant oligos. Although mutation of the KLF15 site may have inadvertently created a novel protein binding site, several of the bands showing increased intensity with the Δ10 oligo were also present using the wild-type bRho29 oligo, raising the possibility that occupancy of the KLF15 site can affect protein/DNA interactions of other DNA binding proteins. The proximity of KLF15 and CRX binding sites and our findings that (1) KLF15 can repress transactivation of the rhodopsin promoter by CRX in vitro and (2) both KFL15 and CRX are expressed in non-photoreceptor cells in the inner nuclear layer  are consistent with a possible in vivo role for KLF15 in repressing inappropriate activation of the rhodopsin promoter by CRX in non-photoreceptor cells.
A similar correlation between KLF15 and CRX binding sites was not observed in the IRBP promoter. However, the KI-c site/G-rich repressor element in the bovine IRBP promoter is adjacent to a CpG dinucleotide (-115) that becomes hypomethylated specifically in retinal cells at the time IRBP expression is first detected [74, 75]. The G-rich repressor element is bound by proteins present in non-retinal tissues where the CpG dinucleotide remains methylated; therefore, it is tempting to speculate that KLF15 may participate in regulating methylation at this site. Other KLF genes are known to regulate chromatin remodeling. The transcriptional activator KLF1/EKLF recruits a SWI/SNF-related chromatin remodeling complex that is thought to be involved in regulation of the locus control region of the β-globin gene . The transcriptional repressor KLF14/BTEB3 interacts with a co-repressor (mSin3A) and the histone deacetylase protein HDAC-1 though repressor domains within the N-terminus to mediate transcriptional repression through chromatin remodeling . Interestingly, in our footprinting analysis of the rhodopsin promoter we observed several sites of increased DNAse I hypersensitivity adjacent to KLF15 protected regions, possibly reflecting alterations in DNA configuration and potentially lending additional support for involvement of KLF15 in chromatin remodeling.
The goal of these studies was to determine the binding characteristics and specificity of KLF15 and define the KLF15 binding sites in the Rho and IRBP promoters in order to gain further insight into the mechanism of KLF15-mediated regulation of retinal gene expression. We found that KLF15 binds to multiple 9 bp consensus sites in the rhodopsin and IRBP promoters including the previously identified CRS-1 and G-rich repressor elements. The presence of multiple KLF15 binding sites suggests that transcriptional repression is likely to play an important role in regulation of photoreceptor-specific genes. Based on the known expression of KLF15 in retinal and non-retinal tissues and the absence of KLF15 expression in photoreceptors, we hypothesize an in vivo role for KLF15 in repressing photoreceptor-specific gene expression in non-photoreceptor cells.
To generate KLF15-ZF-GST, the 3' end of the human KLF15 coding region (369 bp) and the stop codon was amplified from pCRII-hKLF15  using a combination of Taq (Invitrogen; Carlsbad, California) and PFU polymerase (Promega; Madison, WI), subcloned into the BamHI/EcoRI sites of pGEX (Amersham/Pharmacia; Piscataway, NJ) and confirmed by sequencing. KLF15-ZF fusion protein with an N-terminal, glutathione-S-transferase (GST) tag was purified from whole cell lysates of IPTG-induced (0.4 mM) E. coli (BL21S; Invitrogen; Carlsbad, California) using sepharose 4B (Amersham/Pharmacia; Piscataway, NJ) according to maunfacturer's instructions. Purified protein was quantified using bicinchoninic acid protein assay kit (BCA; Sigma; St. Louis, MO) and analyzed using standard 10% SDS-polyacrylamide gel electrophoresis and Western blot with anti-GST antibodies (Amersham/Pharmacia; Piscataway, NJ) diluted 1:8000.
Mice used for protein isolation were handled in accordance with the Association for Research in Vision and Ophthalmology Statement for the Use of Animals and methods were approved by the Animal Care and Use Committee of Johns Hopkins University. For total protein extracts, retinas were dissected from adult mice and placed directly into ice cold PBS containing protease inhibitors (CompleteMini Tablets, EDTA-free; Roche, Indianapolis, IN), sonicated for 10–15 seconds (Setting 5, 50% Duty Cycle) using a Branson Model 250 Sonifier (Branson Ultrasonics Corp., Danbury, CT) with a micro-tip, quantified by BCA and stored at -80C.
Electrophoretic mobility shift assays (EMSA)
EMSA assays followed previously published protocols [24, 78] and used a binding buffer optimized for the KLF15-ZF-GST fusion protein (4 mM HEPES, 5 mM EGTA; 100 mM KCl, 0.25 mM ZnCl2; 0.02% NP40; 0.1 M DTT; 4% glycerol). For analysis of Zn+2 specificity, ZnCl2 was eliminated from the binding buffer except as indicated. Probes with 5'-GG dinucleotide overhangs were generated by annealing single-stranded oligos with their reverse complement and end-labeled with 32P-dCTP using Klenow fragment of DNA polymerase (New England Biolabs, Beverly MA). Cold competitors were end-filled with Klenow using cold dCTP. Binding reactions were incubated for 90 to 120 minutes on ice; for supershift experiments antibodies against GST (Amersham/Pharmacia; Piscataway, NJ) or KLF15 C-terminal peptide [a generous gift of S. Uchida; ] were added after an initial 60 minute incubation. Following polyacrylamide gel electrophoresis on a 5% non-denaturing gel, dried gels were exposed to storage phosphor screens; peaks for each band and DLU (Digital Light Units) for the areas under the peaks were determined using Optiquant software (Packard Instruments). For each lane, the percent of oligo shifted was calculated as [(DLU shifted band/ total DLU) × 100]. For competition assays, "percent competed" was calculated as the difference between the percent oligo shifted without competitor and the percent oligo shifted with competitor.
DNAse I footprint analysis
DNAse I footprinting followed previously published procedures [24, 78] using 33P-end-labeled promoter fragments generated by PCR, KLF15-ZF-GST or GST fusion proteins and EMSA binding buffer optimized for KLF15-ZF. For bovine Rho promoter fragments, the template was a plasmid containing the bovine upstream Rho promoter (-2174 to +32 bp) ; for bovine IRBP promoter fragment (-300 to +132 bp), the template was a plasmid containing the corresponding region of the bovine IRBP promoter . Primers used for PCR amplification of promoter fragments were as follows: for bovine Rho (-225 to +70 bp): forward: 5'-AGGCCTCTGCTCTTTCCC-3'; reverse: 5'-CGCCGGCGGCGCGAACCCG-3'; for bovine Rho (-315 to -31 bp): forward: 5'-AGAGGGAAGTGGGCCTAGAG-3', reverse 5'- GAAGTGACCTCCCCTCCCTA-3'; for bovine IRBP: forward: 5'-CAGATGAGACCCCAACATAC-3'; reverse: 5'-ACAGAAGCTCTCTTGACACC-3'. The amount of fusion protein used varied from 16 to 250 ng per reaction and DNAse I digestions were carried out at room temperature for 1 minute.
Consensus sequence analysis
To identify consensus sequences for protein binding sites, sequences protected in DNAse I footprinting were analyzed using Target Explorer http://trantor.bioc.columbia.edu/Target_Explorer/, a program that uses CONSENSUS (version 6c) and WCONSENSUS (version 5c) to determine consensus patterns in unaligned sequences using an algorithm based on a matrix representation of a consensus pattern. WCONSENSUS determines the width of the pattern being sought without a priori knowledge of the length of the consensus sequence.
Promoter transactivation assays used transient transfections of human embryonic kidney cells (293) with expression vectors, hKLF15-pcDNA3.1 , Crx-pcDNA3.1 , Nrl-pED (gift of A. Swaroop ) and luciferase reporter plasmid bRho130-luc  following previously published methods [30, 52]. The total amount (μg) of DNA in each plate was kept constant by addition of the corresponding expression vector(s) lacking a cDNA insert. Basal activity of each reporter construct in 293 cells was defined as the relative luciferase expression in control plates transfected with reporter constructs and "empty" expression vectors. For each experiment, at least two independent transfections were performed on separate days, and for each combination of expression vectors tested, at least 4 DNA precipitates prepared with each precipitate divided equally to transfect two plates for a total of≥8 transfections per condition.
For each condition the fold change in relative luciferase relative to control transfections was calculated and analyzed as previously described . Statistical analysis used linear mixed models and comparisons of bRho130-luc and bRho225-luc reporter constructs used Wilcoxon Two Sample test (SAS; Cary, NC).
List of abbreviations
barrier to autointegration factor 1
basic transcription element binding protein 3
kidney-specific chloride channel
erythroid-specific Krüppel-like factor
empty spiracles-related homeobox
Flt3 interacting zinc finger protein 1
neural retina leucine zipper
Q50-type retinal homeobox
retinoid binding protein 3
retina and anterior neural fold homeobox.
Supported in part by grants F32EY13499 (DCO) and EY09769 (DJZ) from the NEI/NIH; the Foundation for Fighting Blindness Foundation and generous gifts from Marshall and Stevie Wishnack, and Robert and Clarice Smith; DJZ is the Guerrieri Professor of Genetic Engineering and Molecular Ophthalmology and is a recipient of the Senior Investigator Award from Research to Prevent Blindness.
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