Functional analysis of paired box missense mutations in the PAX6 gene
Functional analysis of paired box missense mutations in the PAX6 geneHank Kejun Tang, Lian-Yu Chao and Grady F. Saunders*
Department of Biochemistry and Molecular Biology, Box 117, The University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030, USA
Received August 9, 1996;Revised and Accepted December 6, 1996
Mutations in the human PAX6 gene produce various phenotypes, including aniridia, Peters' anomaly, autosomal dominant keratitis and familial foveal dysplasia. The various phenotypes may arise from different mutations in the same gene. To test this theory, we performed a functional analysis of two missense mutations in the paired domain: the R26G mutation, previously reported in a case of Peters' anomaly, and an unreported I87R mutation, which we identified in a patient with aniridia. While both the R26 and the I87 positions are conserved in the paired boxes of all known PAX genes, X-ray crystallography has shown that only R26 makes contact with DNA. We showed that the R26G mutant failed to bind a subset of paired domain binding sites but, surprisingly, bound other sites and successfully transactivated promoters containing those sites. In contrast, the I87R mutant had lost the ability to bind DNA at all tested sites and failed to transactivate promoters. Our data support the haploid-insufficiency hypothesis of aniridia, and the hypothesis that R26G is a hypomorphic allele.
The PAX6 gene encodes a 422 amino acid protein that has paired-box and homeobox motifs (1 ). The paired-box motif, originally identified in the Drosophila segmentation gene paired (2 ), is conserved in the Pax family of transcription factors (3 ,4 ), which are important for organogenesis. The PAX6 genes are conserved in both vertebrates and invertebrates, including human (1 ,5 ), mouse (6 ,7 ), quail (8 ), chick (9 ), zebrafish (10 ,11 ), Drosophila (12 ), sea urchin (13 ), Lineus sanguineus (14 ) and Caenorhabditis elegans (15 ,16 ). In most species, the PAX6 genes are expressed primarily in the nervous tissues and sensory organs such as the eye. The involvement of the PAX6 genes in eye development was revealed by some of the naturally occurring PAX6 mutations, such as aniridia in humans (17 ,21 ), small-eye in rodents (22 -23 ) and eyeless in Drosophila (12 ). Mutations in the human PAX6 gene can also result in Peters' anomaly (24 ), autosomal dominant keratitis (25 ) and isolated foveal dysplasia (26 ).
Almost all of the PAX6 mutations identified so far cause deletion or truncation of the PAX6 protein (27 ). The truncations are distributed throughout the entire coding region, with one hot spot at codon 240. For unknown reasons, few missense mutations have been detected in PAX6. Of the three reported, one (R208W) was from a patient with classic aniridia (18 ), one (R128C) was from a patient with isolated familial foveal dysplasia (26 ) and one (R26G) was from a patient with Peters' anomaly (24 ). Aniridia is a disorder involving the entire eye; Peters' anomaly and keratitis involves only the anterior chamber and foveal dysplasia only the posterior chamber of the eye. The relative milder phenotype of the R26G mutation raised the possibility that it is a hypomorphic allele.
The R208W mutation disrupts nuclear translocation signaling (24 ,28 ). The R26G mutation presumably affects the DNA-binding function of the paired domain. The paired domain has a bipartite structure (29 ) with a highly conserved N-terminal subdomain and a less well conserved C-terminal subdomain. X-ray crystallography structural studies indicate that the N-terminal subdomain, where R26G is located, adopts a helix-turn-helix (HTH) structure and makes base-specific contacts with the DNA (30 ). The R26 residue contacts the DNA phosphate backbone. In contrast, the C-terminal subdomain does not interact with DNA, although that domain also bears a strong resemblance to the HTH motif.
The C-terminal subdomains of the paired regions of PAX5 (29 ) and PAX6 (31 ) have been shown to have a role in DNA binding in vitro. However, the C-terminal subdomain is not essential for the function of the Drosophila prd protein. Missense mutations identified in the C-terminal subdomains of the C.elegans pax6 gene (15 ) and the human PAX6 gene (26 ) indicate that the C-terminal subdomain is essential for the function of the PAX6 proteins in vivo. In the study reported here, we identified in a patient with aniridia a missense mutation, I87R, which was also located in the C-terminal subdomain. Functional analysis revealed that this mutation dramatically reduced the DNA-binding ability of the paired domain, indicating that the C-terminal subdomain is essential for the DNA binding of the PAX6 protein in vivo. Because paired missense mutations generally reduce rather than abolish DNA-binding ability, we examined the effect of the R26G mutation by functional analysis. Our data indicated that the effect of the R26G mutation on DNA binding varied according to the nucleotide sequence in the different DNA-binding sites, supporting the hypothesis that the R26G mutation changes the potential targets for this transcription factor (i.e. R26G is a hypomorphic allele).
We tested DNA samples from patients with aniridia and other anterior segment malformations for missense mutations in the PAX6 gene. The amino acid sequence of the PAX6 protein is highly conserved during evolution, suggesting that amino acid substitutions were not well tolerated during evolution. We therefore expected that aniridia or other eye abnormalities would be caused by missense mutations in PAX6. However, of the >40 PAX6 mutations reported, the vast majority were truncation mutations; only three were missense mutations. Heteroduplex analysis (data not shown) and subsequent sequencing of exon 6 in a sporadic case of aniridia revealed a T -> G transversion that resulted in missense mutation I87R (Fig. 1 ). The patient was a 1.5-year-old boy with aniridia, microcephaly, mild developmental delay and several minor dysmorphic features including a prominent metopic ridge with mild plagiocephaly and brachycephaly, somewhat large ears with an underdeveloped helix on the left ear, a small nose with a short columella and small alae nasi, a sacral dimple, a left supernumerary nipple, proximally placed thumbs and dorsal puffiness of the feet.
To assess the functional significance of the I87R mutation and another missense mutation, R26G, previously identified in a case of Peters' anomaly (24 ), we compared the mutant and wild-type PAX6 proteins in an in vivo transcriptional activation assay. The I87R and R26G mutant cDNAs were created by site-directed mutagenesis. We co-transfected NIH3T3 cells with PAX6, PAX6-I87R, and PAX6-R26G expression plasmids with a reporter plasmid in which the luciferase gene was placed downstream of a TATA box and three copies of the PAX6-binding sites [CD19-2(A-ins)] (13 ). Both mutants had lost the ability to activate transcription of the reporter gene (Fig. 3 A). We also tested a reporter construct in which the chloramphenicol acetyltransferase (CAT) gene was placed downstream of a minimal E1b promoter and six copies of the PAX6 consensus sequence P6CON (31 ). The I87R mutant had lost activation ability as expected (Fig. 3 B), but the R26G mutant produced normal activation in HeLa cells (data not shown), and greater than normal activation in NIH3T3 cells (Fig. 3 B). We think this was probably due to the different binding sites used for the reporters. Expression of the wild-type and mutant PAX6 constructs was verified by Western blotting with anti-PAX6 polyclonal antibodies (Fig. 3 C). We conclude that the I87R missense mutation results in a loss of the activator function of the PAX6 protein, which is in agreement with the haploid-insufficiency theory of aniridia.
To investigate the effect of the I87R and R26G mutations on DNA binding, we performed a quantitative electrophoretic mobility shift assay (EMSA). Full-length wild-type and mutant PAX6 proteins were synthesized by in vitro translation and quantitated with a PhosphorImager (Molecular Dynamics) (Fig. 4 ). Equivalent amounts of wild-type and mutant proteins were used for DNA-binding analysis with PAX6-binding sites P6CON and CD19-2(A-ins). The relative binding affinities were also assessed by PhosphorImager analysis. The I87R mutation reduced binding to P6CON by 14-fold, whereas the R26G mutation reduced binding by only 4-fold. These in vitro DNA binding data correlated with the in vivo results of the transcription assay in that R26G bound P6CON moderately well and activated the P6CON reporter. To measure the binding of the mutant proteins to the CD19-2(A-ins) sequence, we synthesized the probe P6/CD/P6, in which the consensus region in P6CON was replaced by that of CD19-2(A-ins) (Fig. 6 ). Both mutants showed a 14-fold reduction in binding to this probe (Fig. 5 ), in accordance with the in vivo data that both mutants had lost the ability to activate transcription of the CD19-2(A-ins) reporter (Fig. 3 A).
The paired domain is a bipartite DNA-binding domain in which each subdomain recognizes half of the consensus sequence. The N-terminal subdomain of the PAX6 paired motif, where R26G is located, should recognize the 5' half of P6CON, and the C-terminal subdomain, where I87R is located, should recognize the 3' half, as has been determined for PAX5 by DNA-binding assays (29 ) and for prd by crystallography (30 ). To determine which of the nucleotides that differ between P6CON and CD19-2(A-ins) were responsible for the different binding affinity of the R26G mutant, we replaced P6CON nucleotides with CD19-2(A-ins) sequences (Fig. 6 ). First, we replaced the 3' half of P6CON with the 3' half of CD19-2(A-ins) to create the probe P6/CD. The R26G mutant bound relatively strongly to P6/CD, as it did to P6CON and unlike the I87R mutant, which bound ~14-fold less strongly (Fig. 5 ). Next, the 5' half of P6CON was replaced with the 5' half of CD19-2(A-ins) to create the probe P6CON-GG (Fig. 6 ). Both mutants bound 14-fold less strongly to this probe (Fig. 5 ), suggesting that the strong binding of the R26G mutant to P6CON was determined by the two G nucleotides. To determine which of the two G nucleotides were responsible for the differential binding, we changed one G nucleotide at a time to give rise to the two probes, P6CON/TG and P6CON/GT. The R26G mutant bound 10-fold less strongly to these two probes than to the P6CON-GG probe (Fig. 5 ). Thus, both G nucleotides were necessary for the high binding affinity of the R26G mutant.
The PAX6 mutant phenotype is dosage dependent: it presents as various forms of aniridia in heterozygotes and as a lethal craniofacial malformation in homozygotes. The heterozygous mutant phenotype is variable and includes clinically distinct entities such as aniridia, Peters' anomaly, foveal dysplasia and keratitis. While these different phenotypes could result from different genetic mutations in the same gene, there are often instances in which one genotype produces different phenotypes. For example, the same genetic mutation of the PAX6 gene can result in variable degrees of aniridia that ranges from having a normal-sized iris to having no iris (19 ,20 ). The bases for the variable phenotypes can be studied by functional analysis of the appropriate mutants. The R26G mutation was reported previously in a case of Peters' anomaly and suspected as a hypomorphic allele because of the less severe phenotype in this family (27 ). The R26 residue is conserved in all known Pax genes and contacts the DNA phosphate backbone and the main-chain carbonyl of residue 63 in the crystal structure (residue 66 in PAX6) (30 ). Changing a charged residue to a non-charged polar residue at position 26 would obviously disrupt these contacts, but determining the exact effect of R26G requires functional analysis because paired box missense mutations may reduce rather than abolish DNA-binding ability. We demonstrated that the R26G mutant failed to bind a subset of paired domain binding sites but successfully bound and transactivated promoters containing other sites. This raises the interesting possibility that the R26G mutant may retain the ability to activate a subset of downstream target genes while failing to activate others. However, it remains to be determined whether these observations made in vitro reflect the in vivo situation. The phenotype of Peters' anomaly, in contrast to the phenotype of aniridia which involves the entire eye, is limited to the anterior chamber and is characterized by a defect in the posterior layers of the cornea, resulting in adhesions between the cornea, iris and lens.
The present study gives the first biochemical evidence of a PAX6 hypomorphic mutation. The genetic background is obviously important in patients with PAX6 mutations. Truncation mutations of PAX6 are not predictably one or another phenotype. Missense mutations of PAX6 lead to aniridia, Peters' anomaly and isolated foveal dysplasia, as well as congenital cataracts. All but isolated foveal dysplasia have also been attributed to mutations resulting in premature protein truncation. It remains to be determined if any other of the observed PAX6 mutations are hypormorphs and if some of the phenotypic variation already observed in PAX6 is the result of hypomorphic mutants versus amorph mutants.
The paired domain has a bipartite structure initially suggested by the in vitro DNA-binding studies of the PAX5 protein (29 ). Indeed, X-ray crystallography revealed that the paired domain consists of two discrete globular domains, both of which adopt a HTH structure resembling that of the homeodomain and Hin recombinase (30 ). Although only the N-terminal subdomain was shown to interact with the DNA in the crystal structure, the C-terminal subdomain was shown to have a role in DNA binding in vitro. First, the consensus binding site of the PAX5 paired domain was shown to consist of two half-sites, and each half-site is recognized by one subdomain of the paired motif (29 ). Secondly, an isoform of the PAX6 protein with a 14 amino acid insertion in the N-terminal subdomain recognizes a different consensus binding site through the C-terminal subdomain (31 ). Furthermore, missense mutations in the C-terminal subdomain were identified in C.elegans pax6 (16 ) and recently in human PAX6 (26 ), indicating the importance of this subdomain in vivo. We identified another C-terminal subdomain mutation, I87R, in the human PAX6 gene from a patient with aniridia. The positions of the three human paired box missense mutations are conserved in all known Pax genes including the Drosophila prd, which is separated from the human gene by >500 million years of evolutionary time. Although the C-terminal subdomain is not essential for DNA binding of the Drosophila prd protein, our functional analysis of the I87R mutant demonstrated that this C-terminal subdomain mutation decreased the DNA-binding ability of the paired domain. The exact role of I87 is unclear, but as I87 is not in the recognition helix of the HTH motif, it is thus likely to be important in protein conformation. It would be interesting to know whether changing a hydrophobic to a charged residue at this position would be deleterious to other Pax proteins, including the Drosophila prd protein.
Of the two C-terminal subdomain mutations in PAX6, one, R128C, resulted in isolated foveal dysplasia (26 ) and the other, I87R, resulted in aniridia. In contrast, the N-terminal subdomain mutation R26G resulted in Peters' anomaly. The idea of isoform-specific phenotypes has been proposed in which mutations in the C-terminal subdomain affect the PAX6-5a isoform, resulting in more posterior anomalies (toward the retina) and mutations in the N-terminal subdomain affect the PAX6 isoform, resulting in more anterior anomalies (in the anterior chamber). Our data showed that the C-terminal subdomain mutation I87R affected the DNA-binding ability of the entire paired domain.
To identify point mutations in the PAX6 gene, genomic DNA samples were isolated from the white blood cells of the aniridia patients and their relatives. Exon 6 was amplified by PCR with the primers described previously (5 ), with one cycle of 97oC for 4 min, followed by 35 cycles of 94oC for 50 s, 60oC for 1 min, and 72oC for 40 s. For heteroduplex analysis, the PCR products were then diluted, boiled for 2 min, slowly cooled down and kept at room temperature for >4 h and analysis was performed with MDE gels, (J.T. Baker, Inc., Phillipsburg, NJ) according to the manufacturer's instructions. For DNA sequencing, the PCR products were ligated into the vector PCR II using the TA cloning kit (Invitrogen). Clones selected representing each of the initial complementary strands were sequenced using the USB sequencing kit.
The CMV-PAX6 expression plasmid was constructed by PCR cloning. The forward primer used, 5'-GCCCAAGCTTCCAGCATGCAGAACAGTC-3', contains a HindIII site and a Kozak consensus site for translational initiation. The reverse primer, 5'-GGACTAGTCTTACTGTAATCTTGGCCAGTA-3', contains a SpeI site and a stop codon. The entire coding region of the PAX6 cDNA was amplified by PCR using the cDNA clone ph12 as template. The PCR products were digested with HindIII and SpeI and inserted into the HindIII-XbaI polylinker sites of the parental vector Rc-CMV (Invitrogen). Site-directed mutagenesis was performed by the recombinant PCR method (32 ). The two internal mutant oligos used for R26G were 5'-CCGGACTCCACCCGGCAGAAGATTGTAG-3' (sense) and 5'-TACAATCTTCTGCCCGGTGGAGTCCGG-3' (antisense). The internal mutant oligos used for I87R were 5'-GTTGTAAGCAAAAGAGCCCAGTATAAG-3' (sense) and 5'-TTATACTGGGCTCTTTTGCTTACAAC-3' (antisense). Both the wild-type PAX6 and the missense mutant constructs were assessed by automated sequencing to ensure that no random mutations were generated during PCR.
NIH3T3 cells were plated at a density of 4-6*105 cells per 60 mm Petri dish and transfected with plasmid DNA coated with the polycationic lipid lipofectamine (Life Technologies) according to the manufacturer's instructions. Each dish was transfected with 2.0 [mu]g of the reporter plasmid P6CON-CAT (31 ) or CD19-2(A-ins)- luciferase (13 ), 1 [mu]g of CMV effector plasmid and 0.6 [mu]g of pSV2[beta]gal (Promega) as internal control. Cell extracts were prepared after 48-72 h and assayed for [beta]-galactosidase and luciferase activity (Tropix) with a luminometer according to the manufacturers' instructions. The CAT assays were performed according to standard procedures (Molecular Cloning, CSH) and extracts were normalized by [beta]-gal activity. The percent acetylation was quantitated directly from thin-layer chromotography plates using a PhosphorImager (Molecular Dynamics).
The PAX6 cDNA was inserted into a prokaryotic expression vector (pRSET, Invitrogen) from which a fusion protein was translated in Escherichia coli. The PAX6 fusion protein starts with six histidine residues at the N-terminus followed by a 15 amino acid epitope tag, a specific protease cleavage site and amino acids 16-422 of PAX6. The fusion protein was purified by nickel column chromatography (NTA resin, Qiagen), verified on SDS-PAGE, and sent to the core facility to raise polyclonal antibodies in rabbits. The rabbit anti-PAX6 serum was diluted 1000-fold for Western blotting with the Amersham ECL kit according to the manufacturer's instructions.
The same CMV expression plasmids, which contained a T7 promoter in the 5' region, were used as templates for in vitro translation in the TNT T7-coupled reticulocyte lysate system (Promega) according to the manufacturer's instructions. The oligonucleotides were labeled with T4 kinase. The annealed oligonucleotides were incubated with in vitro-translated proteins at room temperature for 10 min in 15 [mu]l of 15 mM Tris-HCl (pH 7.5), 90 mM KCl, 0.7 mM EDTA, 0.2 mM dithiothreitol, 1 [mu]g/ml bovine serum albumin, 6.5% glycerol, and 1 [mu]g of poly(dI-dC). Protein-DNA complexes were separated on a 6% native polyacrylamide gel in 0.25* Tris-Borate-EDTA and detected with a PhosphorImager (Molecular Dynamics).
We gratefully acknowledge Drs Theresa Grebe for clinical material and M. Busslinger and R. Maas for the recombinant vectors. We also thank Ruby Desiderio and Kathy Tucker for their assistance in preparing this manuscript. This research was supported by grants from the National Institutes of Health (EY 09675, EY10608, and CA 16672) and Texas Advanced Research Program grant 000015-046.
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