QRX, a novel homeobox gene, modulates photoreceptor gene ...retina/publications/Wang2004.pdf · Southern blot analysis was carried out with genomic DNA from eight species [human,

QRX, a novel homeobox gene, modulatesphotoreceptor gene expression

Qing-liang Wang1,2, Shiming Chen5, Noriko Esumi2, Prabodh K. Swain6,{, Heidi S. Haines8,

Guanghua Peng5, B. Michele Melia2, Iain McIntosh1, John R. Heckenlively9,

Samuel G. Jacobson10, Edwin M. Stone8, Anand Swaroop6,7 and Donald J. Zack1,2,3,4,*

1Program in Human Genetics and Molecular Biology, McKusick–Nathans Institute of Genetic Medicine, 2Department

of Ophthalmology, 3Department of Molecular Biology and Genetics, 4Department of Neuroscience, Johns Hopkins

University School of Medicine, Baltimore, MD, USA, 5Department of Ophthalmology and Visual Science, Washington

University School of Medicine, St Louis, MO, USA, 6Department of Ophthalmology and Visual Sciences and 7Human

Genetics, University of Michigan, Ann Arbor, MI, USA, 8Department of Ophthalmology, University of Iowa College of

Medicine, Iowa City, IA, USA, 9Jules Stein Eye Institute, University of California–Los Angeles School of Medicine,

Los Angeles, CA, USA and 10Department of Ophthalmology, University of Pennsylvania, Philadelphia, PA, USA

Received January 8, 2004; Revised and Accepted March 10, 2004

A novel paired-like homeobox gene, designated as Qrx, was identified by a yeast one-hybrid screen using thebovine Rhodopsin promoter Ret-1 DNA regulatory element as bait. Qrx is preferentially expressed in both theouter and inner nuclear layers of the retina. Its homeodomain is nearly identical to that of Rx/Rax, atranscription factor that is essential for eye development, but it shares only limited homology elsewhere.Although Qrx and Rx/Rax show similar DNA binding properties in vitro, the two proteins demonstrate distincttarget selectivity and functional behavior in promoter activity assays. QRX synergistically increases thetransactivating function of the photoreceptor transcription factors Crx and NRL and it physically interactswith CRX. Qrx is present in the bovine and human genomes, but appears to be absent from the mousegenome. Nonetheless, a 5.8 kb upstream region of human QRX is capable of directing expression inpresumptive photoreceptor precursor cells in transgenic mice. These results indicate that Qrx may beinvolved in modulating photoreceptor gene expression. In addition, the finding of rare heterozygous QRXsequence changes in three individuals with retinal degeneration raises the possibility that QRX may beinvolved in disease pathogenesis.

INTRODUCTION

The neuroretina has been studied as a model of neuronalfunction, development and degeneration (1). Photoreceptorcells, consisting of rods and cones, carry out phototransductionand pass their signals onto bipolar, retinal ganglion cells, andeventually to higher centers within the brain to generate vision.An interesting challenge in developmental neurobiology isdeciphering the mechanisms that orchestrate how the manyspecialized retinal cell types develop and differentiate from alimited number of common progenitor cells (2–4).

Retinal development is a function of differential generegulation, defined by precise temporal and spatial patterns of

gene expression. Significant progress has been made indeciphering some of the complex regulatory network thatfunctions to define the profile of gene expression in particularretinal cells. Among the cis-acting DNA elements involved areRet-1/PCE-1, Ret-4, BAT-1 and NRE (5–10); the trans-actingtranscription factors include CRX, NRL, RX/RAX, PAX6 andCHX10 (8,9,11–17). Rx/Rax, which belongs to the Q50 typepaired-like class of homeodomain proteins, is one of the earliestexpressed transcription factors in the developing eye, appears tobe functionally conserved and is essential for normal eyedevelopment within vertebrates (15,18–22). Rx/Rax has alsobeen implicated in determining the fate of Muller cells (19) andregulating photoreceptor-specific gene expression (23).

*To whom correspondence should be addressed at: Johns Hopkins University School of Medicine, Maumenee Bldg 809, 600 N. Wolfe St, Baltimore,MD 21287, USA. Tel: þ1 4105025230; Fax: þ1 4105025382; Email: [email protected]{Present address: National Brain Research Center, Gurgaon, India, 122001.

Human Molecular Genetics, 2004, Vol. 13, No. 10 1025–1040DOI: 10.1093/hmg/ddh117Advance Access published on March 17, 2004

Human Molecular Genetics, Vol. 13, No. 10 # Oxford University Press 2004; all rights reserved

Mutations of a number of the transcription factors involved inthe proliferation and differentiation of retinal cells can give riseto human retinal disease. As an example, Crx, the cone rodhomeobox gene, is responsible for controlling the differentia-tion of photoreceptor cells by modulating the expression ofphotoreceptor genes, including Rhodopsin and Crx itself(12,13,24–26). Mutations of CRX have been found to causecone-rod dystrophy (14,27), Leber congenital amaurosis(28,29) and retinitis pigmentosa (30). Another important factorin the regulation of Rhodopsin and in maintaining normalphotoreceptor function is the neural retina leucine zipperprotein, Nrl, which acts synergistically with Crx through adirect interaction with Crx’s homeodomain (12,31,32).Missense mutations of NRL can also cause autosomaldominant retinitis pigmentosa (33). Nrl is preferentiallyexpressed in the rod photoreceptors (34), and its loss in miceresults in the failure of rod photoreceptor development and anexcess of functional S-cones (35).

Here, we report the identification and characterization of anovel retinal homeobox gene, designated as Qrx (Q50-typeretinal homeobox gene), which shares a nearly identicalhomeobox with Rx/Rax, plays a role in modulating theexpression of Rhodopsin and possibly other photoreceptorgenes, and may be associated with retinal degeneration.

RESULTS

Qrx encodes a novel paired-like homeodomain protein

A bovine retinal cDNA-Gal4 activation domain fusion librarywas screened in a yeast one-hybrid assay using a bait thatcontains the bovine Rhodopsin promoter Ret-1/PCE-1 element(�148 to �126 bp, referred to as Ret-1 below). In the primaryscreen of 2.07 million clones, 72 positives were identified bygrowth on histidine-deficient medium plates. After plasmidrecovery, retransformation, and testing for both growth onhistidine-deficient medium plates and positive activity in a b-galactosidase assay, 55 of these clones were confirmed as ‘true’positives. Partial sequence analysis of these clones revealed that42 clones matched Chx10 (36), five matched Vsx1 (a Chx10homologue) (37–39), and eight represented novel sequences.Among the eight, two encoded the same 184 amino acidresidue Q50-type paired-like homeodomain containing protein,which we designate as Qrx. In an independent screen using theBAT-1 element as bait (�112 to �84 bp), three additional Qrxclones were identified. Clones encoding the human QRXorthologue were obtained by screening a lgt10 human retinalcDNA library with the bovine Qrx cDNA as probe.

Human and bovine Qrx are 91% identical at the amino acidlevel, with 100% conservation in the homeodomain (Fig. 1A).The homeodomain begins at residue 27. A putative mono-partite nuclear localization signal (NLS), PKKKHRR, that ishomologous to the SV-40 T antigen NLS (CPKKKRKV) (40)is located four amino acids N-terminal to the beginning of thehomeodomain. In addition, the Qrx homeodomain contains asequence (QNRRAKWRRQE) that is homologous to a Crxsequence (KNRRAKCRQQR) that has been reported to beimportant for its nuclear localization (41).

BLAST analysis (42,43) indicated that the closest homologueof Qrx is Rx/Rax, to which it is 93% identical in thehomeodomain. Outside the homeodomain, however, the onlyother region of significant homology is within the OARdomain, which is part of the C-terminal tail, in which a 20amino acid stretch shows �60% identity. The OAR domain hasbeen suggested to be involved in both transcriptional activation(44) and repression (45), but its role in Rx/Rax is unknown.Besides Qrx, 14 other Rx/Rax or Rx/Rax-related genes fromnine different species have been identified to date. Amino acidsequence comparison and phylogenetic analysis indicate thathuman and bovine Qrx are the most distantly related membersof the family and the most homologous member is theDrosophila Rx/Rax (Fig. 1D). Rx/Rax from human, mouse andrat seem to be more closely related to each other than to othermembers of the family. Rx1/Rax1 from zebrafish, astyanax andchicken are clustered together with the Rx2 from oryzias andzebrafish, while the chicken Rx/Rax2 (C-Rx2) is mosthomologous to the xenopus Rx and Rx2.

Qrx appears to be deleted from theMus musculus genome

Multiple efforts were made to clone a murine (Mus musculus)Qrx ortholog, including screening of murine (M. musculus)retinal cDNA and genomic libraries with a human QRXcDNA probe, screening an arrayed murine genomic librarywith a human QRX cDNA and oligomer probes, and areverse transcription PCR (RT–PCR) approach with severaldegenerate primers based on areas of conservation betweenthe human and bovine sequences. Although several murineRx/Rax clones and a number of other sequences wereobtained, no clone representing a murine Qrx ortholog wasidentified, suggesting the possibility that there might not be amurine Qrx.

To further address this issue, we pursued both experi-mental and bioinformatic comparative genomic approaches.Southern blot analysis was carried out with genomic DNAfrom eight species [human, bovine, M. musculus (C57BL6and 129SvJ), Mus casteneus, Mus spretus, rat, zebrafish andDrosophila] using a human QRX coding region fragment asthe probe. The human QRX probe detected the Qrx gene inboth human and bovine as a single band (data not shown). Inaddition, several weak bands were identified in M. casteneus,M. spretus, zebrafish and Drosophila, but not in M. musculusor rat. Although the identities of these other bands areunknown, they could represent orthologs of QRX or someother homologous gene. However, since the murine Rx/Raxgene was not identified under the hybridization conditionsemployed, they are unlikely to represent Rx/Rax orthologs.

Bioinformatic comparison of the human QRX locus onchromosome 19p13.3 (see mapping data below) to theconserved syntenic region in M. musculus (10C1, 79 kb fromthe centromere) provided additional evidence to support theabsence of a Qrx gene in mouse. The murine region (derivedfrom the publicly available NCBI Human–Mouse HomologyMap and the Celera database, which was generated with datafrom the 129X1/SvJ, DBA/2J and A/J strains, with reported6-fold coverage and >99% accuracy) shows significant overallconservation with the human region (Fig. 1E) (46–48).

1026 Human Molecular Genetics, 2004, Vol. 13, No. 10

The genes flanking QRX in human (the centromeric ZN-FINGER RNA BP, and MATK and the telomeric MRP154, andAPBA3) are preserved in mouse in terms of position andorientation. However, the interval between them is only 2 kb inmouse compared to 10 kb in human, and sequence analysis

does not reveal evidence of any homeobox-like sequence, noreven any substantial open reading frame, within the interval.In addition, BLAST analysis of human QRX against mousegenome databases did not reveal any significant matches exceptto the homeodomain itself.

Figure 1. Qrx belongs to the paired-like class of homeobox genes. (A) Amino acid sequence alignment of bovine Qrx (B-Qrx), human QRX (H-QRX), and homeo-domains of RX (RX-HD), PAX6 (PAX6-HD) and CRX (CRX-HD). Residues identical to human QRX are represented as dashed lines. The homeodomains areshaded in a box with the arrowhead depicting the position of the second intron and the asterisk marking the residues defining the paired-like class. The predictedsequence changes identified in the three human patients are shown at the corresponding positions with underlined letters above the wild-type human QRX aminoacid sequence. (B) Human QRX gene structure. The QRX gene is composed of three exons. Its coding region is encoded by exons 2 and 3. HD represents the homeo-domain. The numbers mark the nucleotide positions of intron 2 and the start and stop codons within the QRX cDNA. (C) Intron–exon boundary sequence of thehuman QRX gene. The 50 and 30 intron sequences match the canonical 50-gt/30-ag sequence as underlined. (D) Phylogenetic analysis of Rx/Rax and Rx/Rax-relatedgenes. Amino acid sequences corresponding to human QRX (H-QRX-p, protein accession number AAP41547), bovine Qrx (B-Qrx-p, AAP41546), DrosophilaRx/Rax (D-Rx-p, NP_611526), zebra fish Rx3 (Z-Rx3-p, NP_571302), oryzias Rx3 (O-Rx3-p, CAC69975), mouse Rx/Rax (M-Rx-p, NP_038861), humanRX/RAX (H-RX-p, NP_038463), rat Rx (R-Rx-p, NP_446130), xenopus Rx2 (X-Rx2-p, 042567), xenopus Rx (X-Rx-p, O42201), chicken Rx2/Rax2(C-Rx2-p, Q9PVX0), zebra fish Rx1 (Z-Rx1-p, O42356), astyanax Rx1 (A-Rx1-p, Q9I9D5), chicken Rx1/RaxL (C-Rx1-p, Q9PVY0), oryzias Rx2 (O-Rx2-p,Q9I9A2) and zebra fish Rx2 (Z-Rx2-p, NP_571301) were used to generate the phylogenetic tree using the Geneworks program. The length of the horizontal linesrepresents the relative relatedness of the gene groups. (E) Schematic physical maps of the human QRX locus (19p13.3) (top panel) and conserved syntenic region inM. musculus (10C1.78–79) (bottom panel). The centromeric genes encoding ZN-FINGER RNA BP and MATK (megakyracytes-associated tyrosine kinase), as wellas the telomeric genes encoding APBA3 and MRP154, are conserved in the same orientation in human and M. musculus. The physical distance between the flan-king genes of QRX is �10 kb in human and 2 kb in mouse. This data was generated through the use of the Celera Discovery System and Celera’s associateddatabases.

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Qrx is preferentially expressed in outer andinner nuclear layers of retina

The expression pattern of Qrx in adult tissues was examined bynorthern blotting (Fig. 2A) and RT–PCR (data not shown)analyses. Among the tissues tested with northern blotting[retina, retinal pigment epithelium (RPE), brain, heart, liver,muscle and testis], Qrx is only expressed in the retina, with asingle abundant transcript of �2 kb. However, by RT–PCRanalysis, Qrx expression was also detected weakly in the brain,testis and spleen. Based on RT–PCR analysis of the earliestbovine embryonic retinal tissue available, Qrx expressionduring development is evident as early as 4.5 months gestation(data not shown). For comparison, bovine Rhodopsin isminimally detectable at 5.2 months gestation and is highlyexpressed by 6.3–7.4 months gestation (7,49). By in situhybridization analysis, Qrx is expressed in cells of the outernuclear (ONL) and inner nuclear layers (INL), with strongerstaining in the ONL (Fig. 2B and C). The expression of QRX inadult human retina has a similar pattern, which overlaps largelywith that of RX/RAX (data not shown).

A human QRX ‘promoter’ fragment is sufficient to driveGFP expression in presumptive photoreceptorprecursors in transgenic mice

Given the absence of Qrx in mice, we were interested inwhether the human QRX promoter would still function in mice(i.e. whether the regulatory information was evolutionarilyconserved). Transgenic mice were generated using a 8.1 kbgenomic DNA fragment containing the human QRX locus(including 5.8 kb upstream sequence). To aid expressionstudies, an IRES-EGFP cassette was added to the downstreamend of the genomic fragment. Analysis of postnatal day 1retinas from four out of five analyzed independent transgeniclines revealed strong GFP expression in cells in the outer regionof the neuroblast layer, presumptive developing photoreceptorcells of the forming outer nuclear layer (Fig. 2D and E). Also ofpotential interest is moderate GFP expression in isolated cellsin the middle part of the neuroblast layer, perhaps migratingprogenitor cells. These results suggest not only the strongconservation of the upstream regulatory complexes betweenhuman and mouse retina gene expression but also thelikelihood of the involvement of QRX in the developmentand function of photoreceptors.

Qrx and Rx/Rax bind to both the Ret-1 andBAT-1 elements

Electrophoretic mobility shift assay (EMSA) and DNase Ifootprint analyses were carried out to characterize the DNAbinding properties of QRX. Since the full-length QRX fusionprotein was insoluble, recombinant QRX homeodomain GSTfusion protein (QRX-HD-WT) containing six amino acids N-terminal to the homeodomain and five amino acids C-terminal toit, was expressed and purified from bacteria. As expected basedon the initial one-hybrid results, QRX-HD-WT binds to bothRet-1 and BAT-1 oligos in EMSA (Fig. 3A). With largeramounts of QRX-HD-WT protein, faint additional bands oflower mobility were also evident (Fig. 3A, lanes 3 and 4),

possibly due to the binding of QRX-HD-WT multimers to theDNA target. The binding specificity of the fusion protein to Ret-1 and BAT-1 sites was assessed by cold competitor assay. Whenwild-type cold DNA oligomer was added to either Ret-1(Fig. 3A, lanes 5 and 6) or BAT-1 binding reactions (Fig. 3A,lanes 17 and 18), the binding of QRX-HD-WT to the labeledprobes was competed away by the cold competitor. Whenthe putative binding site in Ret-1 was mutated (MUT for Ret-1),the oligomer could no longer compete effectively with the probe(Fig. 3A, lanes 7 and 8). In the case of the BAT-1 site, when onlyone of the two putative QRX-HD-WT binding sites within theelement was mutated, the cold competitor could still competeaway the binding (Fig. 3A, MUT-1 and MUT-2, lanes 19–22).When both sites in the BAT-1 element were mutated (MUT-3),the mutant competitor showed minimal, if any, ability tocompete with the labeled wild-type probe (Fig. 3A, lanes 23 and24). A non-target sequence (NTS) cold competitor derived fromanother region of the Rhodopsin promoter showed no competi-tion for the labeled Ret-1 probe (Fig. 3A, lanes 9 and 10). Inaddition to the EMSA analysis with the purified QRX home-odomain GST fusion protein (QRX-HD-WT), we also examinedthe interaction of native Qrx from bovine retinal nuclear extractwith both Ret-1 and BAT-1 using an affinity purified rabbitpolyclonal anti-Qrx antibody (6760) in the supershift assay.It appears that the anti-Qrx antibody largely eliminated theinteraction of retina-enriched factors, presumably includingboth Qrx and Qrx-Crx complex in the retinal nuclear extract,with both Ret-1 and BAT-1 (Fig. 3B, lanes 3, 4 and 11, 12),

Figure 2. Expression analysis of Qrx. (A) Northern blot analysis of bovine tis-sues using bovine Qrx as probe. The top panel shows the image of a northernblot with 10 mg of total RNA per lane isolated from bovine retinal pigmentepithelium (P), retina (R), brain (B), heart (H), liver (L), muscle (M) and testis(T) hybridized with a 32P-labeled bovine Qrx cDNA probe. The bottom panelshows the same membrane stained with ethidium bromide under UV lightbefore hybridization. (B) In situ hybridization of bovine Qrx in bovine retina.Expression of Qrx is seen in outer nuclear layer (ONL) and inner nuclear layer(INL), with 100 ng of antisense Qrx probe. The bar in the images represents25 mm. (C) Parallel experiments as in (B) except that the same amount of senseprobe was used. The bar in the images represents 25mm. (D) DAPI staining ofthe retina from a human QRX-IRES-EGFP transgenic mouse at P1 stage. RPE(retinal pigment epithelium), NBL (neuroblast layer), GCL (ganglion celllayer). (E) Strong GFP expression in the developing neuroblast layer of retinaof the same mouse as in (D).


whereas it did not alter the non-specific interaction present in the293 cell nuclear extract (Fig. 3B, lanes 7, 8, and 15, 16). Inaddition, the pre-immune serum (Fig. 3B, lanes 5 and 13) fromthe corresponding rabbit did not show any blockage of thebinding activity. This is not surprising because the amino acidsused as the peptide antigen (codon 88 to 107) to generate theanti-Qrx antibody (6760) are located in close vicinity to theC-terminus of the Qrx homeodomain (codon 27–86) and arelikely to be required for the DNA binding activity of Qrx.

To further explore the DNA binding properties of QRX in thecontext of the Rhodopsin promoter, and to compare it withother factors implicated in Rhodopsin regulation, DNase Ifootprinting analysis was performed with a probe spanning theRhodopsin promoter from �225 to þ70 bp. Consistent withthe EMSA results, the Ret-1 and BAT-1 sites were efficiently

protected by QRX-HD-WT (Fig. 4, lanes 1–5 and 14–18).As might be predicted from the high degree of homologybetween the QRX and Rx/Rax homeodomains, the Rx/Raxhomeodomain pattern of protection is essentially identical tothat of QRX (Fig. 4, lanes 6–9 and 19–22). However, thebinding affinity of Rx/Rax may be somewhat lower in that �4-fold more protein was required to achieve a similar degree ofprotection. Of potential significance, the protection pattern ofQRX and Rx/Rax is similar to, but distinct from, that of Crx(12) (Fig. 4, lanes 10–13 and 23–26). The Ret-1 and BAT-1protected sequences are overlapping but not identical, and Crxalso protects the Ret-4 site. In addition, the patterns ofhypersensitive sites are also distinct, presumably reflectingdifferent DNA-protein conformations (Fig. 4, see arrows andarrowheads).

Figure 3. QRX binds specifically to the Ret-1 and BAT-1 elements in EMSA. (A) EMSA with Ret-1 or BAT-1 probe in the absence (lanes 1–4 and lanes 11–16) orpresence of cold oligo DNA competitors (lanes 5–10 and lanes 17–24). Lanes 1 and 11 do not contain protein. Lanes 2–4 contain increasing amounts of QRX-HD-WT (1, 2 and 4 ng). Lanes 12–16 contain increasing amounts of QRX-HD-WT (1, 2, 4, 8 and 16 ng). Lanes 5–10 contain a constant amount of QRX-HD-WT(2 ng). Lanes 5, 7 and 9, contain a 200-fold excess of cold competitor Ret-1WT, Ret-1MUT, NTS (non-target sequence), respectively. Lanes 6, 8 and 10, a 10 000-fold excess of cold competitor Ret-1WT (WT), Ret-1MUT (MUT), NTS (non-target sequence), respectively. Lanes 17–24 contain a constant amount of QRX-HD-WT (4 ng). Lanes 17, 19, 21 and 23 contain a 200-fold excess of cold competitor BAT-1WT (WT), BAT-1MUT1 (MUT1), BAT-1MUT2 (MUT2) or BAT-1MUT3(MUT3), respectively. Lanes 18, 20, 22 and 24, contain 10 000-fold excess of cold competitor BAT-1WT (WT), BAT-1MUT1 (MUT1), BAT-1MUT2 (MUT2) orBAT-1MUT3 (MUT3), respectively. (B) EMSA with Ret-1 or BAT-1 probe using nuclear extracts from either bovine retina or 293 cells in the presence (lanes 3, 4,7, 8, 11, 12, 15, 16) or absence (lanes 2, 6, 10, 14) of the anti-Qrx antibody (6760). In lanes 5 and 13, the same volume (as lanes 3 and 11) of preimmune serum (PI)was used rather than the 6760 antibody.


QRX specifically transactivates the Ret-1element in the presence of Crx and NRL

To assess the transcriptional activity of QRX, we utilized atransient transfection assay with HEK293 cells. Using thebovine Rhodopsin �130 to þ70 and �225 to þ70 bp luciferasereporter that we employed previously to assess the transactivat-ing activity of Crx and Nrl (8,9), we did not observe significanttransactivating activity with QRX, either alone or in combina-tion with Crx and/or NRL (data not shown). To develop apotentially more sensitive assay, and one that would be specificfor individual DNA binding sites, thereby eliminating potentialneighboring negative regulatory elements, luciferase reporterconstructs were generated in which multimers of the Ret-1,BAT-1, Ret-4 or NRE/Ret-4 element were placed upstream of aminimal CMV promoter. Co-transfection of a QRX expressionplasmid with each of these promoter elements again revealedminimal transactivating activity. This result was distinct fromthat with Rx/Rax, which could activate the BAT-1 construct(Fig. 5B) and has been reported to activate a Ret-1/PCE-1 sitefrom the Arrestin promoter (23).

The multimer reporters demonstrated a number of specificpatterns in terms of their abilities to be activated by the varioustranscription factors that were tested. When used alone, Crxactivated the Ret-1 and Ret-4 constructs (Fig. 5A and D), and

NRL selectively stimulated the NRE/Ret-4 containing reporter(Fig. 5C). However, when QRX was co-transfected withCrx and NRL, it further stimulated the expression of theRet-1 construct by more than 10-fold over the effect of Crxand NRL combined in a dose dependent manner (Fig. 5E).The level of activation achieved with QRX was significantlyhigher (5-fold more) than what could be achieved simplyby increasing the amount of Crx or NRL (data not shown).The transactivating effect peaks at 0.3 mg of QRX expressionplasmid (Fig. 5E), which is relatively low for this type ofassay. Furthermore, as little as 0.1 mg of QRX expressionplasmid can achieve more than 5-fold activation. Thistransactivation activity of QRX is binding site specific, eventhough as noted above QRX binds to both the Ret-1 and BAT-1sites, its ability to stimulate the activity of Crx and NRL is onlyobserved with Ret-1 (BAT-1 data not shown). Rx/Rax alsostimulates the combined activity of Crx and NRL, but only toabout 50% the level of QRX (Fig. 5F). In order to dissect therelative contributions of Crx and NRL interactions inthe observed QRX stimulation, QRX was tested with eitherof the two factors individually. QRX stimulates Crx alonesignificantly, to �50% the activity seen with Crx and NRL,whereas QRX stimulation of NRL alone is only minimal(Fig. 5F), suggesting that Crx is the key contributor in thisthree factor interaction.

Figure 4. QRX homeodomain (QRX-HD-WT), as well as RX homeodomain (RX-HD) binds specifically to the Ret-1 and BAT-1 elements within the Rhodopsinpromoter in DNase I footprint analysis. QRX-HD and RX-HD show identical pattern of binding to their target elements in the Rhodopsin promoter (Ret-1 and BAT-1). Their patterns of binding are distinct from that of the CRX homeodomain (CRX-HD), as indicated by the arrows (for top strand) and arrowheads (for bottomstrand). In addition, unlike CRX-HD, QRX-HD and RX-HD do not bind to the Ret-4 element. The target elements are bracketed and labeled accordingly. 0.25mg ofeach protein was used in lanes 2, 6, 10, 15, 19 and 23; 1mg in lanes 3, 7, 11, 16, 20 and 24; 4 mg in lanes 4, 8, 12, 17, 21 and 25; 16mg in lanes 5, 9, 13, 18, 22 and 26.


QRX physically interacts with CRX, but not NRL

The finding that Crx is essential for most of the transactivat-ing function of QRX suggested that the QRX protein mightphysically interact with Crx, and that this interaction might beresponsible for the observed synergy between the factors. Totest this hypothesis, we performed co-immunoprecipitationand GST pull-down analyses. An anti-CRX antibody

precipitated radioactively labeled in vitro transcribed andtranslated QRX protein only in the presence of CRX (Fig. 6A,lane 1). The same antibody did not yield precipitate whenused with the empty QRX expression vector (data not shown),nor did it precipitate QRX in the absence of CRX (Fig. 6A,lane 5). In addition, using recombinant proteins expressed inHEK 293 cells, QRX (QRX-Xp) was co-immunoprecipitatedwith Flag tagged CRX (CRX-Flag) (Fig. 6B, lane 1).

Figure 5. QRX specifically transactivates the Ret-1 element synergistically with Crx and NRL, whereas other factors show distinct target selectivity and QRXvariants differentially affect its transcriptional activity. The boxes in the plots represent the experimental data points falling between the 25th and 75th percentileof each group. The bars on either side of the boxes depict the range of the data. The medians of each group are represented by a triangle. *P-value� 0.0001 and**0.0001�P-value� 0.0025 as compared to the reference group (the reference group is the vector group in A–E, the QRX group in F and the QRX-WT group atthe corresponding DNA concentrations in G). (A) Effect of individual factors (1mg/plate) on Ret-1CMV. (B) Effect of individual factors (1 mg/plate) on BAT-1CMV. (C) Effect of individual factors (1mg/plate) on NRE þ Ret-4. (D) Effect of individual factors (1 mg/plate) on Ret-4. (E) The effect of QRX on Ret-1CMV in the presence of Crx and NRL at increasing DNA concentrations. (F) Effect of Rx on Ret-1CMV in the presence of Crx and NRL and QRX’s effecton Ret-1CMV in the presence of either Crx or NRL (shown in % activity of QRX with Crx and NRL). (G) Effect of the mutations of QRX at different DNAconcentrations on the synergistic transactivation activity of QRX with Crx and NRL on Ret-1CMV.


Consistent with these results, we found in a yeast two-hybridassay that the Crx homeodomain interacts with Qrx (data notshown). In contrast, QRX failed to interact with NRL in aGST pull-down assay (Fig. 6C, lane 4). This is in contrast tothe finding that both Rx and Crx interact with NRL (Fig. 6C,lanes 8 and 12), and that deletion of the transactivationdomain containing amino-terminus of NRL (GdN), which hasbeen shown to be involved in its interaction with Crx (32),reduced its interaction with both Rx and Crx (Fig. 6C, lanes7 and 11).

Mapping of QRX to human chromosome 19p13.3, andidentification of QRX sequence variants in cone-roddystrophy and age-related macular degeneration patients

Human QRX was mapped to chromosome 19p13.3 by radiationhybrid mapping, in which it was closely linked to the marker WI6480 with a lod score of >3. The result was confirmed by somaticcell hybrid mapping to chromosome 19 and subsequently also bydata from the human genome project. The cosmid F16403, whichspans the entire QRX cDNA fragment, also maps to the same

Figure 5. Continued.


locus. By comparing the sequence of the human QRX cDNA tothe cosmid F16403, the genomic structure of QRX wasdetermined. The QRX cDNA is composed of three exons, withthe coding region encoded by exons 2 and 3 (Fig. 1B). All theintron–exon boundary sequences match the canonical GT-AGconsensus sequence (Fig. 1C).

To test the hypothesis that defects of QRX and its proteinproduct could result in retinal disease, we screened a cohort ofpatients with retinopathies for QRX mutations: 322 cone-roddystrophy (CORD), 107 Leber congenital amaurosis (LCA),92 age-related macular degeneration (AMD), 14 autosomalrecessive retinitis pigmentosa (ARRP) and 14 autosomaldominant retinitis pigmentosa (ADRP) patients, as well as apool of 94 normal individuals. Three different heterozygoussequence variations were identified in patients with retinaldisease that would be expected to alter the QRX protein(Table 1). In contrast, no amino acid altering sequencevariations were observed in the pool of 94 normal controlindividuals. A 67-year-old patient with the clinical diagnosis ofAMD was found to harbor a heterozygous G to A change atcodon 87, which would be expected to result in a change fromarginine to glutamine (CGG to CAG in Fig. 7A). Thisreplacement of a bulky, positively charged amino acid by aneutral one occurs at the c-terminal border of the homeodomain(Fig. 1A), and suggested that it might affect the protein’s DNAbinding activity. When this possibility was directly tested usinga GST tagged recombinant QRX-HD-R87Q mutant protein, wefound the R87Q alteration to cause an apparent increase inbinding affinity for the Ret-1 and BAT-1 elements (Fig. 7C andD). This increase was not due to different amounts of themutant or wild-type proteins used in the assay (Fig. 7B).

A 78-year-old patient with macular degeneration and electro-retinographic evidence of peripheral retinal involvement (which weinterpret as CORD) was found to harbor a missense change incodon 137 (GGC to CGC). This change would be expected toresult in the substitution of arginine for glycine (Table 1).Unfortunately, no family members of either of these two patientswere available for interview, clinical examination or DNA analysis.

A third heterozygous QRX sequence variation, a 6 bp(CCCGGG) insertion between codons 137 and 141, wasidentified in an unrelated CORD patient. This change would beexpected to result in an in-frame insertion of a proline and aglycine (Fig. 1A). Since this region of the gene containsmultiple repeats of the CCCGGG sequence, insertion orduplication of the CCCGGG sequence at multiple positionsbetween codons 137 and 141 could result in the same finalsequence. For simplicity, we designate this mutation as140P_141Gdup (50). The same sequence change was alsoidentified in the proband’s mother and two siblings. Althoughthese relatives are said to have ‘normal’ vision, they all live inPakistan and are not available for ophthalmologic exam andtesting. Hence, the possibility that they have mild CORDcannot be excluded. If the relatives in fact do not have CORD,then this would suggest either incomplete penetrance, perhapsdue to modifier or environmental influence, or that the140P_141Gdup sequence change is not disease-associated.

The R87Q, G137R and 140P_141Gdup sequence variantswere expressed and tested in vitro to assess their effect on theinteraction between QRX and CRX, and their functionalconsequence in the context of other transcription partnersin vivo. In co-immunoprecipitation analyses, the G137R and140P_141Gdup (140PGIns) proteins demonstrated decreasedbut not absent interaction with CRX, while the R87Q proteindid not show decreased interaction (Fig. 6A). In transienttransfection assays, all three sequence variants demonstratedsubtle but statistically significant (P< 0.0001) and reproducible

Figure 6. QRX physically interacts with CRX, which also interacts with NRL,and QRX variants demonstrate reduced interaction between QRX and CRX(A) In vitro transcribed and translated 35S-labeled QRX (WT and mutants) isco-immunoprecipitated with CRX using an anti-CRX antibody (lanes 1–5).Both the G137R (lane 3) and the 140P_141Gdup mutation (140PGIns in lane4) resulted in reduced amount of QRX being precipitated suggesting decreasedinteraction between CRX and these mutant QRX proteins. However, the R87Qmutation resulted in little, if any, change compared to the wild-type (lane 2). Inthe absence of CRX, QRX is not precipitated (lane 5). Lanes 6–9 demonstratethat an approximately equal amount of each input protein was used. (B)Recombinant QRX (QRX-Xp) synthesized in HEK293 cells is co-precipitatedwith CRX-Flag. Immunoprecipitation using anti-Flag-M2 resin containingeither CRX-Flag, or its vector control, with QRX-Xp was performed asdescribed in experimental procedures. Lanes 1, 2, 4 and 5 are elutes (20ml)from the resin with the indicated protein extracts; lanes 3, 6 and 7 are the inputs(10ml) as indicated. Lanes 1, 2, 3 were probed with anti-Xpress (Invitrogen,1 : 1000) and goat anti-mouse IgG-HRP and lanes 5, 6, 7 with anti-CRXP261 antibody (1 : 250) and anti-rabbit IgG-HRP. Arrows with labeling indicatethe running positions for QRX-Xp and CRX-flag. (C) GST pull-down assayswith in vitro transcribed and translated 35S-labeled QRX (QRX*), Rx/Rax(Rx*) or Crx (Crx*). QRX* did not show evidence of binding to columns withGST-NRL (GNRL, lane 4), GST-NRL with transactivation domain deleted(GdN, lane 3) nor with GST alone (lane 2). In contrast, both Rx and Crx interactwith GNRL (lanes 8 and 12) and GdN (lanes 7 and 11) but not with GST alone(lanes 6 and 10).


difference from the QRX-WT in their ability to transactivate theRet-1 element in the presence of Crx and NRL (Fig. 5G). Atthe DNA concentration that showed peak QRX-WT activity(0.3 mg/plate), R87Q and 140P_141Gdup resulted in increasedtransactivation activity of 30 and 50%, respectively, whileG137R demonstrated 40% reduced transactivating activity at aconcentration of 1 mg/plate.

DISCUSSION

The transcriptional network implicated in the regulation ofphotoreceptor gene expression is becoming increasinglycomplex, and now includes Crx (12–14,51), Nrl (8,9,31,32,34,52), Erx (53), Rx (15,16,19,21,23), Mash1 (17,54),Mok2 (55), nuclear receptor NR2E3 (35,56), thyroid hormonereceptor beta 2 (57), the Sp family (58), and, at least inDrosophila, Pax6 (59–61). The combinatorial interplay of thesefactors is important not only for orchestrating normal

development and function, but also in mediating retinalpathology, as shown by reports that mutations in many oftheir genes have been associated with photoreceptor disease(14,27,33). Here, we have presented the characterization of anew transcription factor, Qrx, which appears to be involved inmodulating the expression of photoreceptor specific genes andmay play a role in retinal degeneration. Based on its structure,Qrx is a member of the Q50-type paired-like homeobox genefamily, and its closest homolog is Rx/Rax. Rx/Rax and itsrelated genes have been identified from nine different species,and some species express as many as three distinct familymembers (15,21,62).

Unfortunately, however, relatively little functional informa-tion is available for these Rx/Rax-related genes, and whetherQrx represents a true ortholog of any of them is unclear. Onefinding, however, of potential relevance to Qrx is thatexpression of a putative dominant negative allele of a chickenRx/Rax-related gene, cRaxL (C-Rax1), causes a decrease inexpression of early photoreceptor markers (63). An interesting,and puzzling, aspect of the evolution of Qrx is that it appears tobe absent from the M. musculus genome. Qrx seems to havebeen deleted from the M. musculus genome during evolution,rather than having evolved more recently than the branch pointof M. musculus. Although it had been thought that the orderRodentia, to which M. musculus belongs, is more distantlyrelated to the primate lineage than the order Certartiodactyla, towhich bovine belongs (64), more recent molecular phyloge-netic analysis indicates that M. musculus is a closer relative tohuman than is bovine (65,66). If so, a mechanism explainingthe absence of a murine Qrx ortholog as being due to morerecent evolution would necessitate an independent origin for

Table 1. Summary of QRX sequence variants found in patients with retinaldisease

DNAchange

Proteinchange

Disease Genetic class

CGG>CAG R87Q Age-related maculardegeneration

Heterozygous

GGC>CGC G137R Cone-rod dystrophy HeterozygousCCCGGG

insertion140P_141Gdup Cone-rod dystrophy Heterozygous

Figure 7. The R87Q sequence change results in an apparent increase of the binding affinity of QRX to the Ret-1 and BAT-1 elements. (A) Heterozygous DNAsequence change (CGG to CAG) resulting in R87Q mutation. (B) Coomassie blue staining of QRX-HD-WT and QRX-HD-R87Q showing equivalent protein quan-tities (in nanograms). (C) EMSA comparing QRX-HD-WT and QRX-HD-R87Q DNA binding to the Ret-1* probe. The amount of protein (in nanograms) used ineach reaction is indicated at the top of each lane. The quantity of probe shifted with QRX-HD-R87Q, compared to QRX-HD-WT, was increased 12.8-, 1.8- and2.3-fold with 4, 8 and 16 ng of protein, respectively. (D) EMSA comparing QRX-HD-WT and QRX-HD-R87Q DNA binding to the BAT-1* probe. The amount ofprotein (in nanograms) used in each reaction is indicated at the top of each lane. The quantity of probe shifted with QRX-HD-R87Q, compared to QRX-HD-WT,was increased 4.6-, 4.8- and 2.3-fold with 4, 8 and 16 ng of protein, respectively.


the gene in human and bovine ancestors, and this seemsunlikely given the high degree of nucleic acid sequencehomology between the bovine and human orthologs. A morelikely mechanism involves a small deletion mediated byrecombination between the flanking repetitive elements. Ofpotential significance, multiple genes immediately flanking Qrxare conserved between mouse and human, but there are localdisruptions of the nearby synteny (Fig. 1E). Perhaps related tothese findings, compared to the human genome, the mousegenome is evolutionarily more volatile (67), giving mice theability to rapidly adapt to changing environments. It is thereforeinteresting that, as demonstrated by the transgenic experiments,the regulatory information from the human QRX promoterregion is still largely recognized by the mouse. As a practicalside benefit, a QRX promoter/GFP transgene may turn out to bea useful early marker of developing photoreceptor cells.

The absence of murine orthologs for human genes, and viceversa, is not rare. It has been estimated to occur for as many as3–5% of genes in both genomes (48). Furthermore, homeoboxgenes appear to have a higher than average frequency of suchvariation. In human, of the 302 homeobox loci we identifiedfrom a NCBI Locuslink search, 139 harbor already character-ized homeobox genes. The other 163 represent novel homeo-box genes predicted from their nucleotide sequences orexpressed sequence tag (EST) clusters. Within the group ofpreviously characterized 139 homeobox genes, nine (6.5%)are absent from the mouse genome based on the search of genesymbols, NCBI HomoloGene data and nucleotide sequenceBLAST of both the NCBI and the Celera databases. Analysis ofthe known murine homeobox genes revealed a similarfrequency for which human orthologs could not be identified.These ‘missing’ homeobox genes are likely to be importantcontributors in the processes that account for the biological,morphological, and functional difference between mouse andman. In the eye, for example, variation in properties such as thenumber of types of cones, the relative frequency of cones androds and the presence or absence of a macular region couldpartly be determined by differences in the pattern of homeo-domain proteins expressed in the retina.

The absence of a murine Qrx has made functional anddevelopmental analysis more difficult. Nonetheless, severallines of evidence do provide insights into the role of Qrx withinthe retina. Although results of transient transfection experi-ments must always be interpreted with caution in terms of theirrelevance to the in vivo situation, the data presented suggestthat Qrx may play a role in modulating the expression ofRhodopsin and perhaps other photoreceptor genes. While QRXdid not demonstrate significant transactivating activity onits own, when used in combination with Crx and NRL, itstimulated reporter gene expression more than 10-fold. Thiseffect was observed only with the Ret-1 site and not with theRet-4 or BAT-1 sites. In the same assay with the same site, Rx/Rax showed less than half the activity demonstrated by QRX,even though in the DNA binding assays it demonstratedessentially identical sequence specificity. Rx/Rax was the mostpotent activator of the BAT-1 reporter construct. These findingsare noteworthy since although homeodomain proteins demon-strate in vivo specificity, they often show significant promis-cuity in in vitro binding studies and cell transfection assays. Aschematic model of Rhodopsin regulation that integrates these

results, as well as previous studies, is shown in Figure 8. Anunusual aspect of the model is that NRL is suggested tofunction by two mechanisms. With its already identifiedbinding site, NRE, Nrl is shown to function in the standardmanner as a DNA binding transcription factor (9). However, wesuggest that with the Ret-1 site, NRL functions more as a co-activator or modulator, acting through protein–protein interac-tion with Crx without its direct DNA binding. This hypothesisis based on the finding that the addition of NRL with the Ret-1reporter approximately doubles the activity observed with Crxand QRX alone (Fig. 5F), yet NRL does not demonstratedetectable binding to the Ret-1 sequence. NRL is shown asinteracting directly with Crx, but not with QRX, based on theco-immunoprecipitation and GST pull-down assays shown inFigure 6. A second potential co-activator shown in the model isp300/CBP, as has been reported previously that it can bothphysically interact with Crx and enhance its transactivatingfunction (68). An additional level of complexity, not indicatedin Figure 8, is that negative acting transcription factors maybind the Rhodopsin promoter and repress expression in non-photoreceptor retinal cells or modulate Rhodopsin promoteractivity even in rods (51,52). Since Qrx does not exist in mousegenome, presumably its activity in regulating Rhodopsinexpression could be compensated by other factors, includingRx, in mouse.

The identification of QRX sequence changes in patients withretinal diseases is also consistent with a role for QRX inphotoreceptor function and/or survival. Although the absenceof existing or available relatives for the R87Q and G137Rprobands, as well as the presence of 140P_141Gdup in theproband’s presumably normal relatives, make it difficult toconclude that any of the identified sequence changes trulyrepresent disease-causing mutations, the nature and positions ofthe sequence changes and the biochemical studies with themutated proteins, are all supportive of this possible association.Each of the sequence changes led to an alteration that could bedetected in our functional assays. The G137R mutation resultedin decreased interaction with CRX and decreased transactiva-tion activity, the 140P_141Gdup led to decreased interactionwith CRX but increased transactivation, and the R87Qmutation increased both transactivation and DNA bindingactivity. The possibility that increased transcription factoractivity can cause eye disease is not novel, having beenreported previously with both NRL (33) and PAX6 (69).Although this suggestion that different, and sometimesopposite, types of transcription factor alterations can result insimilar ocular phenotypes may at first seem paradoxical, itprobably reflects the growing consensus that the retinal geneexpression network is so tightly regulated that a variety ofdifferent perturbations can all result in retinal degeneration.

MATERIALS AND METHODS

Yeast one hybrid assay

Library screening was performed essentially as described inChen et al. (12) except the target elements used here were Ret-1(five copies) or BAT-1 (three copies).


Radiation hybrid mapping

The MIT Genebridge radiation hybrid panel was analyzedusing primers corresponding to human QRX cDNA sequences(forward: 50-CAGTCTGGGCCAGCGCCACCTC-30, reverse:50-GGCTGGAGTGCAGCAGTGTG-30) that amplify the494 bp PCR products. Analysis of the resulting PCR patternwas submitted to the database (http://www-genome.wi.mit.edu/cgi-bin/contig/rhmapper), which indicated close linkage to themarker WI6480 with a LOD score of more than 3.

Northern blot analysis

Total RNA was isolated from different fresh bovine tissues(RPE, retina, brain, heart, liver, muscle and testis) using theTRIZOL reagent (Invitrogen). Approximately 10 mg of totalRNA from each tissue was used. After UV crosslinking, themembrane was hybridized to a probe generated from thefull-length bovine Qrx cDNA followed by washing andovernight autoradiography.

In situ hybridization

In situ hybridization experiments were performed essentially aspreviously described (70), except that DIG-labeled bovine Qrxriboprobes were generated by in vitro transcription of either T3(antisense probe) or T7 (sense probe) promoter driven plasmidvector containing partial bovine Qrx cDNA (191 basepairs),which has been digested with either EcoRI (sense probe) orBamHI (antisense probe). Eight mm thick cryosections ofbovine retina, fixed in fresh 4% paraformaldehyde, were usedin the hybridization with buffer containing 100 ng/ml of eitherantisense or sense DIG-labeled riboprobe.

Generation and analysis of the QRX-IRES-EGFPtransgenic mice

A polylinker containing EcoRI–Af lII–SmaI–AgeI–BamHI siteswas inserted into the EcoRI and BamHI sites of the pIRES2-EGFP vector (BD Biosciences #632306). The 8.1 kb humanQRX fragment generated by digesting the cosmid F16403 withBsaAI and Age1 enzymes was directionally cloned into theSmaI and AgeI sites of the above modified pIRES2-EGFPvector. The resulting construct was further digested with Af lIIto release the 9.6 kb QRX-IRES-EGFP fragment for micro-injection into the mouse embryos of SJL/J mice at the Johns

Hopkins Transgenic Core Facility. The resulting transgenepositive mice were genotyped by PCR analysis and further bredfor multiple generations to eliminate the rd1 locus fromthe background. Out of a total of 12 transgenic founders, nineindependent lines were established. GFP expression in theperinatal retinas of five of these nine lines have been analyzed.Ten micrometer cryosections of the eyecups from transgenepositive mice were generated for standard histological pro-cessing and viewed with a Nikon Eclipse E1000 microscopeusing DAPI (excitation: 330–380 nm; dichroic mirror: 400 nm;barrier: 435–485 nm) or FITC (excitation: 465–495 nm;dichroic mirror: 505 nm; barrier: 515–555 nm) filter sets.

Expression and purification of GST-taggedQRX-HD-WT, QRX-HD-R87Q,Rx-HD, Crx-HD

Bovine Qrx cDNA fragment corresponding to codons 20–91 ormouse Rx/Rax cDNA fragment corresponding to codons 130–200 was cloned in frame into the BamHI (50) and EcoRI (30)sites of the pGEX-4T-2 vector (Pharmacia). Since the aminoacid sequence within this region is 100% conserved betweenhuman and bovine Qrx and also between human and mouseRx/Rax, we designate the constructs as QRX-HD-WT and RX-HD, respectively. The QRX-HD-WT was also used as thetemplate for the generation of QRX-HD-R87Q mutant constructusing QuikChange site-directed mutagenesis kit (Strategene).CRX-HD was the same as previously described (12). All theexpression constructs were transformed and expressed inbacteria strain BL21 (Pharmacia). The GST fusion proteinswere purified following protocol provided by Pharmacia. Theconcentrations and quality of purified proteins were determinedby both SDS–PAGE/Coomassie blue staining and spectro-photometry.

Generation of polyclonal anti-Qrx antibody (6760)

Peptide antigens corresponding to the region of codon 88–107of both human (LESGSGAVAAPRLPEAPALP) and bovine(LESGSGAVAAPRLPEVPALP) Qrx were synthesized accord-ing to standard procedure at Genemed Synthesis Inc. Theantigens were injected as a mixture into two rabbits accordingto standard immunization protocol and antisera were obtainedand affinity-purified using the peptide antigens.

Figure 8. Model of combinatorial network regulating Rhodopsin expression. The primary target of QRX within the bovine Rhodopsin promoter appears to bethe Ret-1 element. Upon binding to Ret-1 and CRX, which is associated with NRL through protein–protein interaction, QRX forms a heterotrimeric complexwith CRX and NRL and together they are able to recruit other co-activators and/or components of the basal transcription machinery (PolII complex) to activatetranscription. RX, bound to the BAT-1 element, may be forming a second complex with NRL and CRX, bound to the adjacent NRE and Ret-4 elements, respec-tively. However, whether these two complexes function coordinately is unclear. For simplicity, putative repressors, are not included in the diagram.


EMSA

For the study of purified QRX-HD-WT GST fusion protein, fill-inlabeled radioactive (32P) double stranded short DNA probes(10 000 cpm/ml) corresponding to the Ret-1WT (50-GGCCCACCTGGAAGCCAATTAAGCC-30) and BAT-1WT (50-GCAGCAGTGAGGATTAATATGATTAATAACGCC-30) elements wereused to mix with purified GST-tagged QRX-HD-WT protein inthe presence or absence of appropriate amount of non-radioactivecompetitors. The DNA sequence for the mutant non-radioactivecompetitors are as following: Ret-1MUT (MUT: 50-GGCCCACCTGGAAGCACCGGCAGCC-30), NTS (non-target sequence:50-GGCCTCTGCTCTTTCCCAGGGTCCCC-30), BAT-1MUT1(MUT1: 50-GCAGCAGTGAGGATGCCGATGATTAATAACGCC-30), BAT-1MUT2 (MUT2: 50-GCAGCAGTGAGGATTAATATGCGGCATAACGCC-30), BAT-1MUT3 (MUT3: 50-GCAGCAGTGAGGATGCCGATGCGGCATAACGCC-30). Assays werecarried out as previously described (71). The analysis of nativeQrx protein in nuclear extract from bovine retina and the control293 cell nuclear extract were carried out essentially the same asabove except that 4 mg of nuclear extract, either from retina or293 cells, and 0.1 mg of Poly(dI-dC) were used in each reaction.Nuclear extracts were added 20 min before the addition of 1.5 ml(Fig. 3B, lanes 3, 7, 11, 15) or 4.5 ml (Fig. 3B, lanes 4, 8, 12, 16)of the anti-Qrx antibody (6760). As controls, 1 ml of unpurifiedpreimmune serum (PI) was added instead in lanes 5 and 13 ofFigure 3B.

DNase I footprinting

DNase I footprintings were performed essentially the same aspreviously described (71), except that up to 16 ng of purifiedGST-tagged QRX-HD-WT, RX-HD, CRX-HD proteins wereused to mix with either top (forward) or bottom (reverse) strandlabeled DNA probes corresponding to bovine Rhodopsinpromoter �225 to þ70 bp.

Human subjects

Informed consent was obtained from participants afterexplanation of the studies. The research procedures were inaccordance with institutional guidelines and the Declaration ofHelsinki. Clinical ocular examinations and visual functiontesting were performed in the patients with retinal degenera-tions that had candidate gene screening.

Screening of QRX mutations using single-strandconformational polymorphism (SSCP) andautomated sequencing

Samples from patients and normal controls were screened in anidentical manner for mutations in the 50UTR and codingsequences of the QRX gene using single-strand conformationalpolymorphism (SSCP) and sequencing analyses. Four pairs ofoligonucleotide primers (exon1/forward: 50-ACCTTGAGCTAATCCTCCCC-30, exon1/reverse: 50-CACCCTGAGGGGATCTCTG-30; exon2B/forward: 50-CCAAGAAGAAGCACCGGA-30, exon2B/reverse: 50-AAAGATGTGTGTGTTGGGGG-30; exon3A/forward: 50-TCAGACGGGAGTCAGAACCC-30,exon3A/reverse: 50-TCCAGGGGCAGCGACATG-30; exon3B/

forward: 50-CTGGAGTCAGGCTCGGGT-30, exon3B/reverse:50-CGGTTGCTCCATGACCTC-30) were used to amplify allbut 90 base pairs of the coding sequence [the sequenceencoding codons 1–30 is so GC rich (72%) that we were notable to amplify it despite using four different oligonucleotideprimer pairs and numerous different reaction conditions]. Therest of exons 1 and 2 and the first portion of exon 3 (3A) wereamplified with the Qiagen Taq DNA Polymerase Kit containingCoenzyme Q. The second portion of exon 3 (3B) was amplifiedwith MasterAmp 2X PCR PreMix L from Epicentre. PCRproducts were denatured for 3 min at 94�C and electrophoresedon 6% polyacrylamide/5% glycerol gels at 25 W for �3 h atroom temperature. Following electrophoresis, gels were stainedwith silver nitrate (72). PCR products exhibiting an electro-phoretic shift in SSCP analysis were sequenced. Mutationswere identified by the approximately equal peak intensity oftwo fluorescent dyes at the mutant base. The 140P_141Gdupsequence was further confirmed by cloning the mutant PCRproducts into the pGEM T-EASY vector (Promega) andsequencing multiple clones to identify both the mutant andnormal allele sequences. All sequencing was bi-directional.

Generation of target element containing reporterconstructs, QRX wild-type and mutant expressionconstructs and transient transfection assays

Ret-1 (10 copies), BAT-1 (three copies), Ret-4 (four copies) andRet-4 þ NRE (four copies) were individually and directionallycloned into the XhoI site of the modified PGL2-promoter vector(Clontech), in which the SV40 promoter has been substitutedby a minimal CMV promoter. QRX wild-type cDNA was in-frame cloned into the BamHI (50) and EcoRI site of thepcDNA3.1HisB vector (Invitrogen). The mutant clones (R87Q,G137R and 140P_141Gdup) were generated usingQuikChange site-directed mutagenesis kit (Strategene) usingthe wild-type QRX construct as the template in the initial PCRreactions. The resulting mutant constructs were sequenced andcorrect inserts were cut out and pasted into the pcDNA3.1HisBvector to eliminate any potential secondary mutations. TheDNA of the resulting subclones were purified twice throughCsCl gradient. The Crx and NRL expression constructs, thetransfection of 293 cells and luciferase and b-gal assays wereessentially as previously described (12).

In vitro translation and GST pull-down assay

QRX, Rx/Rax and Crx were translated and radio-labeled with35S-methionine using coupled in vitro transcription/translationsystem (Promega) and Glutathione Sepharose bound GST, GST-DNRL and GST-NRL were prepared as described earlier (32).Equal amounts (�30%) of each in vitro translated protein (QRX,Rx/Rax and Crx) were incubated separately with GST, GST-DNRLor GST-NRL (�100mg of protein). After the final wash, theGlutathione Sepharose bound protein was solubilized in 2� SDSsample buffer by heating for 5 min at 100�C. Proteins labeled with35S-methionine were analyzed by SDS–PAGE followed byautoradiography. Along with the pull-down proteins, 10% of thetotal protein used for binding was analyzed as input to check thetranslation efficiency of the individual protein.


In vitro and in vivo expression and co-immunoprecipitationanalysis of CRX and wild-type and mutant QRX proteins

Co-immunoprecipitation experiments using in vitro translatedwild-type and mutant QRX (35S-labeled) and Crx were carriedout using anti-Crx antibody P261, essentially as described (73)with minor modifications. The coupling time with Protein-Abeads was reduced to 3 h instead of overnight, and a cocktail ofprotease inhibitors (Roche Molecular Biochemicals) was addedto each reaction.In vivo expression of recombinant QRX-Xp (Xpress tagged

hQRX) and CRX-Flag (Flag-tagged hCRX) were carried outby individually transfecting HEK293 cells cultured on 100 mmplates with 5 mg of hCRX-Flag and hQRX-pcDNA3.1/HisB,respectively. Pull-down assays were performed with the above293 cell extracts after the cells were washed twice with PBS(pH 7.4) and lysed 48 h post transfection in 1 ml of lysis buffercontaining 50 mM Tris–Cl (pH 7.4), 150 mM NaCl, 1 mM

EDTA, 0.1% NP40 and 10 ml of a protease inhibitor cocktail(Sigma) at 4�C for 20 min. Soluble cell extracts (950 ml)containing CRX-Flag, or empty vector, were incubated with40 ml of anti-Flag M2 affinity resin equilibrated with TBS(50 mM Tris–Cl, pH 7.4, 150 mM NaCl) at 4�C for 3 h withgentle agitation. The bound resin was spun down and washedthree times with 0.5 ml of TBS followed by incubation with cellextracts containing QRX-Xp for an additional 3 h at 4�C. After3 washes with 1 ml TBS, proteins bound to the resin wereeluted with 100 ml of 3� Flag peptide (150 ng/ml in TBS) andanalyzed using SDS–PAGE and western blots with primaryantibodies specific to CRX (73) or Xpress (the expression tagfor hQRX), and secondary goat anti-mouse IgG-HRP (for anti-Xpress) or anti-rabbit IgG-HRP (for anti-CRX), respectively.ECL plus western blotting detection reagents and Hyperfilm forECL were used for detection.

Statistical analysis of transient transfection data

Relative luciferase activities from each experiment wereanalyzed and compared among transcription factors using alinear mixed model. This is analogous to analysis of variance(ANOVA), except that rather than averaging correlated observa-tions prior to analysis as required by ANOVA, all data points areused in the analysis and the correlation in observations arisingfrom using the same solution mix to transfect two tissue cultureplates was modeled directly, using the assumption that the twotissue culture plates were exchangeable experimental units. Noformal adjustment for multiple comparisons of transcriptionfactors was made; however, only P-values� 0.0025 wereconsidered statistically significant. All analyses were performedusing SAS statistical software (SAS Inc.).

ACKNOWLEDGEMENTS

The authors wish to thank Drs Peter H. Mathers for providingthe human Rx/Rax cDNA clone and for useful discussion andsuggestions on the manuscript; Jeremy Nathans for the humanretinal cDNA lgt10 phage library; Ching-Hwa Sung for thebovine retinal cDNA-Gal4 activation domain fusion library;Debora B. Farber and Mark Verardo for the Y79 cells; Adrian M.Timmers for the fetal bovine retinal RNA samples for initial

RT–PCR analysis and Chen-wei Wang for technical assistance.The research is supported in part by grants from the NationalEye Institute, Steinbach Foundation, Foundation FightingBlindness and Macula Vision Foundation, and generous giftsfrom Mr and Mrs Stevie and Marshall Wishnack and Mr andMrs Robert and Clarice Smith. D.J.Z. is the Guerrieri Professorof Genetic Engineering and Molecular Ophthalmology, and therecipient of a Research to Prevent Blindness Senior InvestigatorAward.

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QRX, a novel homeobox gene, modulates photoreceptor gene ...retina/publications/Wang2004.pdf · Southern blot analysis was carried out with genomic DNA from eight species [human,