Human Molecular Genetics, 2001, Vol. 10, No. 16 1631-1638
© 2001 Oxford University Press
Functional analyses of two newly identified PITX2 mutants reveal a novel molecular mechanism for Axenfeld–Rieger syndrome
1Department of Ophthalmology and The Vision Science Research Program, University Health Network, The Toronto Western Hospital, Toronto, Ontario M5T 2S8, Canada, 2Departments of Ophthalmology and Medical Genetics, University of Alberta, Edmonton, Canada and 3Department of Ophthalmology, The Hospital for Sick Children, Toronto, Ontario, Canada
Received March 15, 2001; Revised and Accepted June 11, 2001.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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The specific role of PITX2 in the pathogenesis of anterior segment dysgenesis has yet to be clearly defined. We provide here new insight into PITX2 pathogenesis through mutational and functional analyses. Three PITX2 mutations were found in a screen of 38 unrelated individuals affected with anterior segment anomalies (8%). All three mutations were found among the 21 individuals affected with Axenfeld–Rieger syndrome (ARS). We have identified two novel mutations, a valine
leucine (V45L) missense mutation at position 45 within the PITX2 homeodomain, and a seven amino acid duplication (7aaDup) of residues 6–12 of the homeodomain. DNA-binding studies of the two mutant PITX2 proteins demonstrated a <10-fold reduction in the DNA-binding activity of the V45L mutant, and a >100-fold reduction in activity of the 7aaDup mutant. Luciferase reporter assays showed a >200% increase in PITX2 transactivation activity of the V45L mutant, while the 7aaDup mutant was unable to transactivate at detectable levels. Our analyses of the V45L PITX2 mutant reveal that the DNA-binding domain of PITX2 can influence transactivation activity independently of DNA binding. Furthermore, our findings expand the hypothesis that the amount of residual PITX2 activity underlies the variable severity of ocular phenotypes that result from PITX2 mutation. For the first time, we present evidence that increased PITX2 activity may underlie the severe ARS ocular phenotype. We conclude that increased activity of one PITX2 allele may be as physiologically disruptive as a mutation that nullifies a PITX2 allele, with either condition resulting in ARS. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Axenfeld (1) first described a patient with a prominent annular white line near the limbus at the level of Descemet’s membrane (embryotoxon posterior). Also noted were a number of delicate fibrillae (iris processes) from the iris stroma crossing the anterior chamber to this line. Rieger (2) subsequently described two patients with the findings observed by Axenfeld, with the addition of marked iris stromal atrophy and pupillary abnormalities (ectopia and dyscoria, respectively). Since then, Rieger syndrome has been further defined and refers to an association of anterior segment dysgenesis with non-ocular findings that often include dental anomalies, maxillary hypoplasia and failed involution of the umbilicus (3–5). Because of the phenotype and genotype overlap between Axenfeld and Rieger syndromes, it was recently proposed that these conditions could be referred to as one condition, Axenfeld–Rieger syndrome (ARS) (6). Axenfeld–Rieger anomaly (ARA) is defined as a separate condition with the presentation of ocular findings alone. Patients with Axenfeld–Rieger ocular malformations have a 50% increased risk of developing glaucoma by the second decade (6–8) posing the threat of visual loss and blindness.
ARS is an autosomal-dominant genetically heterogeneous disorder with four chromosomal loci identified on chromosomes 4q25 (9), 6p25 (10), 13q14 (11) and 16q24 (12). Two genes have thus far been characterized: PITX2 and FOXC1 on chromosomes 4q25 (13) and 6p25 (14,15), respectively. PITX2 encodes a bicoid-related homeodomain transcription factor, which is expressed in mouse embryos in the eye mesenchyme, dental lamina and umbilical cord (13). Mice heterozygous for null and hypomorphic alleles have ocular and umbilical abnormalities consistent with ARS (16,17). Mutations in PITX2 have been associated with ARS and other cases of anterior segment malformation, such as Peters’ anomaly (18), iris hypoplasia (IH) (18) and iridogoniodysgenesis syndrome (IGDS) (20). ARA has been linked to chromosome 6p25 (10), and can also result from mutation of FOXC1 (15), but there is no evidence for the involvement of PITX2 with ARA at this date.
Structural analysis using X-ray crystallography and NMR of the DNA-binding homeodomain proteins engrailed and antennapedia showed that the homeodomain consists of three helical regions in a tightly folded structure (21,22). The integrity of the homeodomain is essential for binding DNA and is critical for PITX2’s activity as a transcription factor. Many of the previously published mutations in PITX2 have been amino acid substitutions within the homeodomain region of the protein. Expression of mutant PITX2 recombinant proteins has been used to test the DNA-binding and transactivation capabilities of the altered homeodomains (23,24) and shed light on genotype–phenotype correlations (23). We assessed the incidence of PITX2 mutations in a patient population affected with anterior segment anomalies (including ARS, ARA, IH and Peters’ anomaly) and studied the functional consequences of two novel mutations within the PITX2 homeodomain.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Mutational analysis
Thirty-eight individuals with anterior segment dysgenesis were screened for PITX2 mutations. Of these individuals, 21 were affected with ARS, nine with ARA, five with IH and three with Peters’ anomaly. Single-strand conformation polymorphism (SSCP) screening of the coding sequence showed altered migration patterns in PCR amplicons containing the PITX2 homeobox, in three unrelated cases. Other alterations detected proved to be polymorphisms found in normal controls: exon 2, –30c/t; exon 3, C620G; exon 4, C1162G. Direct cycle sequencing identified three distinct sequence changes, two of which are described here for the first time. These mutations were not seen in 100 unaffected ethnically diverse controls. All three individuals with PITX2 mutations had been diagnosed with ARS. Their clinical features are summarized in Table 1. Out of 21 individuals with ARS, <15% (and only 8% of patients with anterior segment dysgenesis) showed a mutation in PITX2.
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The first sequence change, point mutation G854C, resulting in an R53P missense mutation of the homeodomain, has previously been reported in an individual with ARS (13). Two novel mutations were identified in the homeobox region: a point mutation, G830C, resulting in a V45L missense mutation in the third helix of the homeodomain (Fig. 1A), and an in-frame duplication of 21 bp causing a seven amino acid duplication of T44 to K50, residues 6–12 of the homeodomain (Fig. 1B).
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Immunofluorescence and western analysis
Recombinant mutant-PITX2 proteins were expressed by transfecting mammalian COS-7 cells following introduction, by site-directed mutagenesis, of the V45L and 7aaDup mutations into the PITX2 cDNA subcloned within a mammalian expression vector. Detection of the recombinant mutant-PITX2 proteins was made possible with an N-terminal Xpress‘ epitope encoded by the expression vector. Immunofluorescence with an Xpress‘ antibody of mammalian COS-7 cells transfected with the mutant PITX2 constructs demonstrated stable recombinant wild-type and mutant PITX2 proteins with full nuclear localization (Fig. 2). COS-7 cells transfected with a plasmid producing a LacZ–Xpress‘ fusion protein resulted in cytoplasmic, and not nuclear, localization of the fusion protein (Fig. 2), indicating that the Xpress‘ tag is not responsible for the nuclear localization of PITX2 molecules. Protein extracts of the transfected COS-7 cells were prepared and analyzed by western blot with the Xpress‘ antibody; stable recombinant mutant-PITX2 proteins of the proper size of 35 kDa were demonstrated (Fig. 3). Western results were also used to approximate the amount of recombinant PITX2 proteins within whole-cell extracts used in binding experiments below.
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Electrophoretic mobility shift assays (EMSAs)
EMSAs have been used previously to demonstrate that wild-type PITX2 can form DNA–protein complexes with the CE-3 oligonucleotide containing the DNA-binding site of Pitx1 (23). Untransfected COS-7 cell extracts are unable to bind the CE-3 probe (23), confirming that the CE-3 electrophoretic mobility shift seen with COS-7 cells transfected with PITX2 cDNA is due to the presence of recombinant PITX2, rather than a property endogenous to COS-7 cell proteins (23). Protein extracts from COS-7 cells transfected with a plasmid expressing a LacZ–Xpress‘ fusion protein also could not alter the mobility of the CE-3 probe, demonstrating that the Xpress epitope itself does not provide DNA-binding ability to the recombinant PITX2 proteins (23). The CE-3 binding probe was used in EMSAs to assess the DNA-binding ability of the V45L and 7aaDup PITX2 mutants of this study.
Less than 10-fold V45L mutant PITX2 protein extract was required to generate a binding shift equal to that of 1x wild-type (wt)PITX2 (Fig. 4). DNA binding equivalent to 1x wtPITX2 was seen with as little as 2x V5L PITX2 protein on some gels. 100-fold 7aaDup mutant was required to produce a detectable shift that was approximately five times weaker than the shift produced by 1x wtPITX2. The 7aaDup mutant PITX2–DNA complex migrated slower than the wtPITX2 complex, possibly a result of the mutation introducing seven additional amino acids (Fig. 4). These results were replicated using two different protein extracts twice in EMSAs, for each of the mutations.
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Transactivation studies
Co-transfection of the pGL3 CE-3 Luciferase reporter and the pcDNA4/HisMax PITX2 constructs demonstrated increased transactivation capability of the V45L mutant PITX2 protein, averaging at 228.5% wtPITX2 activity (Fig. 5B). The 7aaDup mutant PITX2 protein was unable to transactivate above baseline levels detected with the empty (no PITX2) pcDNA4/HisMax vector. The V45L mutant protein was unable to transactivate the pGL3-SV40 Promoter Luciferase reporter without the CE-3 binding site (Fig. 5C). Co-transfection of the wtPITX2 and V45L expression constructs with the CE-3 Luciferase reporter results in transactivation at levels between those of 2x wtPITX2 and 2x V45L (Fig. 5D).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Three PITX2 mutations were found in three of 38 unrelated individuals affected with anterior segment anomalies (8%). All three mutations were found among the 21 individuals affected with ARS, suggesting a PITX2 mutation frequency in ARS of just <15%. This frequency reflects the known genetic heterogeneity of ARS and is consistent with previous studies (9,21). Of the three homeodomain mutations, two are novel and one, a single nucleotide substitution resulting in R53P in the PITX2 homeodomain, has been identified previously (13) and characterized (23–25). The second mutation identified, V45L, changes an amino acid in the recognition helix of the homeodomain. The third mutation is a 21 bp in-frame duplication, introducing seven extra amino acids at the junction of the N-terminal arm and helix 1 of the homeodomain. This mutation is the first reported duplication within the homeodomain of PITX2.
The 7aaDup mutant results in a defective PITX2 protein with nominal levels of DNA-binding activity (Fig. 4) and no detectable transactivation of a Luciferase reporter (Fig. 5). The duplicated amino acid residues 6–12 of the PITX2 homeodomain are part of a proposed N-terminal arm that is thought to fit into the minor DNA groove. Interactions between the N-terminal arm and DNA supplement the contacts made by helix 3 for DNA recognition and high affinity DNA-binding (21,22). This duplication of the N-terminal arm may inhibit proper binding of PITX2 with the DNA target by interfering with the docking of helix 3 in the DNA major groove. The duplication may also alter the conformational folding of the homeodomain, thereby preventing DNA-binding.
The V45L mutation leads to an interesting PITX2 mutant. DNA-binding analysis with an oligonucleotide containing the Pitx1 binding site (CE-3) (23) and recombinant V45L mutant PITX2 protein demonstrated a <10-fold reduction (as little as 2-fold in some experiments) in DNA-binding ability (Fig. 4). Despite decreased DNA binding, transactivation studies with a CE-3 Luciferase reporter construct demonstrated that the V45L mutant showed a >200% increase in transactivation activity over that of wtPITX2 (Fig. 4). Our data indicate that, within the PITX2 homeodomain, the DNA-binding and transcriptional activities of PITX2 are separable as mutation of residue 45 has distinct effects on each of these activities. Separable DNA-binding and transactivation abilities have also very recently been proposed for the forkhead domain of FOXC1, a second transcription factor implicated in Axenfeld–Rieger malformations (26). In the case of FOXC1, proteins with either F112S or I126M missense mutations of the FOXC1 DNA-binding forkhead domain are able to bind DNA at nearly wild-type levels, but exhibit severe reductions in transcriptional activation potential. There is no evidence that the V45L mutant is binding to a site other than the CE-3 element within the CE-3 Luciferase reporter construct, ruling out this possibility to account for the increased transactivation by V45L. Control transactivation studies using the standard pGL3-SV40 Luciferase reporter construct (without the CE-3 insert) revealed that both wtPITX2 and V45L require the CE-3 binding site for transactivation, being unable to transactivate the empty pGL3-SV40 construct (Fig. 5C). In fact, it appears that wtPITX2 and V45L mutant PITX2 proteins are competing for binding sites, as co-transfection experiments of wtPITX2 and V45L constructs together result in transactivation levels somewhere between the two (Fig. 5D). Our results also indicate that the V45L PITX2 mutation is not likely to represent a dominant-negative mutation, as the addition of V45L protein does not reduce the ability (beyond competition for binding sites) of wtPITX2 protein to transactivate when the two are assayed together. WtPITX2 and V45L constructs also do not appear to interact synergistically, as co-transfection of wtPITX2 and V45L together does not result in transactivation levels above and beyond that of V45L.
Substitution of valine with leucine would generally be considered a conservative mutation; leucine differs from valine by containing one extra methyl group within its main chain. However, position 45 occurs within the hydrophobic core between helices 1 and 3 of the homeodomain, shown to be required for proper homeodomain folding (27), and is conserved in >97.5% of all homeodomains. Of the nearly 800 homeodomains entered into the NIH homeodomain database (http://genome.nhgri.nih.gov/homeodomain), only 19 examples (four of which are orthologs of other species) were found with a leucine at position 45 of the homeodomain (Fig. 6). Biochemical studies mutating different residues of the antennapedia homeodomain found that position 45 is under high levels of steric hindrance (28) and, therefore, extension of the main methyl chain may not be a conservative change at this position within most homeodomains. The 15 homeodomains with a leucine at position 45 are divergent from eukaryotic homeodomain sequences (29), particularly within the hydrophobic core (27), and at positions of significant steric hindrance (28), within helices 1 and 2. This may reflect homeodomain conformations with greater potential for accommodating the larger leucine side-chain at position 45 of helix 3.
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-helices are indicated above. Dashes indicate identity of amino acid residues with PITX2. Note that many of the residues of the homeodomains that naturally contain a leucine at position 45 are divergent (highlighted in gray) from the consensus for homeodomains of higher eukaryotes (29). These divergent residues tend to occur at residues of the hydrophobic core (indicated by circles) (27), and at positions determined to encounter steric hindrance (open and closed squares represent moderate and high levels of steric hindrance, respectively) (28). Divergence at residues of the hydrophobic core and at residues that encounter internal steric hindrance within helices 1 and 2 may represent conformations with greater potential for accepting the larger leucine side-chain at position 45.We believe that the substitution of valine with leucine at position 45 affects homeodomain conformation which intrinsically affects DNA binding and transactivation differently. The same conformational change could theoretically increase the ability of PITX2 to recruit transcriptional machinery, or increase the affinity of protein–protein interactions. Studies on the glucocorticoid receptor (GR) have demonstrated that substitution of leucine with a valine residue within the transactivation domain results in decreased transactivation (30). Further mutagenesis experiments of the GR transactivation domain indicated that the relative hydrophobicity and helix-forming propensity of residues of the transactivation domain are important to activity (30). The substitution of leucine with valine in the GR transactivation domain was surmised to be deleterious to activity by decreasing hydrophobicity and/or helical propensity (30). It is possible that introduction of a leucine residue at position 45 of the PITX2 homeodomain improves transactivation activity in a direct manner for PITX2 by increasing hydrophobicity and/or helix-forming propensity of a transactivation domain that overlaps the PITX2 homeodomain. However, conservation of the valine residue at position 45 may be indicative of its importance to homeodomain folding and structure that is perhaps required for optimal DNA-binding.
We suspect that the slight decrease in DNA binding seen with the V45L PITX2 mutant is not disease-causing in this ARS patient. Instead, our in vitro investigation of the V45L PITX2 mutant leads us to propose increased transcriptional activation as the mechanism underlying this case of ARS. Our proposal that increased PITX2 transcriptional activity is disease-causing is supported by recent studies demonstrating that three copies (with presumably increased amounts of protein activity) of FOXC1 and PAX6 also disrupt ocular development (31–33). Confirmation of our conclusions from these cell culture assays will have to come from studies of animals transgenic for the PITX2 V45L mutation in order to assay the effects of the V45L mutation in a developing mammal.
In patients heterozygous for PITX2 alleles found to be inactive (by DNA-binding and transactivation assays), such as the 7aaDup mutant of this study, having only 50% of the normal amount of PITX2 protein from the remaining wild-type PITX2 allele also results in ARS. Additional PITX2 activity, contributed by PITX2 alleles with some residual activity, appears to result in milder ocular phenotypes (23). On the basis of earlier data, we proposed that the variance in PITX2 activity, due to specific PITX2 mutations, underlies the different anterior chamber anomalies classified as IH, IGDS and ARS (23). We now extend this hypothesis and propose that PITX2 overactivity may be as physiologically disruptive to development of the eye as PITX2 mutations that eliminate activity. This report further defines the very narrow window for proper PITX2 activity, exemplifying the complex series of events involving PITX2 in the developmental pathways of the anterior segment of the eye.
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MATERIALS AND METHODS |
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Clinical analysis
This study was approved by the Research Ethics Board of the Hospital for Sick Children and the Toronto Western Hospital Human Subjects Review Committee. Clinical evaluations included a comprehensive eye exam and partial systemic exam relating to the findings of ARS. Proband and available family members were recruited. Twenty milliliters of blood were drawn for DNA extraction (34). DNA samples were stored at 4°C.
Mutational analysis
The coding sequence and intron–exon boundaries of PITX2 were PCR amplified and screened for mutations by single-strand conformational polymorphism (SSCP) and direct sequencing. Each PCR reaction contained 50 ng DNA, 200 µM of each dNTP, 5% dimethyl sulfoxide, 10 pmol of each primer, 1.5–2.0 mM MgCl2, GeneAmp PCR Buffer II and 1 U AmpliTaq polymerase (Applied Biosystems), in 20 µl total volume. Reactions were performed on the MJ Research PTC-100 or Stratagene Robocycler for 35 cycles with an initial denaturing step, variable annealing temperatures with a final 8 min extension at 72°C. PCR primers used were published previously (13,20).
The four PCR amplicons were screened by SSCP as described previously (35). Samples showing an altered migration pattern were sequenced using the Thermo Sequenase Cycle Sequencing Core Kit (US79610; Visible Genetics) using previously published methods (36). Sequence changes observed were compared to the literature or to the sequence of 100 unaffected, unrelated controls.
PITX2 cDNA constructs
Development of the wild-type (wt)PITX2 cDNA-pcDNA4/HisMax (Invitrogen, Carlsbad, CA) mammalian expression vector construct has been described previously (23). The V45L and 7aaDup mutations were introduced into the wtPITX2 cDNA construct using the QuikChange Site-Directed Mutagenesis Kit (Stratagene Cloning Systems, La Jolla, CA) with the following mutagenic primers (mutations in bold) as per the manufacturer’s instructions: V45L, forward 5'-C CTT ACG GAA GCC CGA CTC CGG GTT TGG-3', reverse 5'-CCA AAC CCG GAG TCG GGC TTC CGT AAG G-3'; 7aaDup, forward 5'-G CGG CAA AGG CGG CAG CGG ACT CAC TTT ACC AGC CAG CAG ACT CAC TTT ACC AGC CAG CAG CTC CAG GAG CTG GAG GCC-3', reverse 5'-GGC CTC CAG CTC CTG GAG CTG GCT GGT AAA GTG AGT CTG CTG GCT GGT AAA GTG AGT CCG CTG CCG CCT TTG CCG C-3'. Qiagen MaxiPrep columns (Qiagen Inc., Mississauga, ON) were used to isolate and purify plasmid DNA which was fully sequenced by standard [33P]ddNTP methods (Amersham Pharmacia Biotech Inc., Baie d’Urfé, PQ).
Protein expression
Expression and extraction of the recombinant V45L and 7aaDup mutant PITX2 proteins was performed as described previously (23). Wild-type and mutant PITX2 cDNA constructs within the pcDNA4/HisMax vector (500 ng) were introduced into mammalian COS-7 cells (2 x106 cells per 100 mm plate in 16 ml Dulbeco’s modified Eagle’s medium + 10% fetal bovine serum) with the FuGENE 6 transfection reagent (Roche Molecular Biochemicals, Indianapolis, IN) (24 µl). Cells were harvested by scraping 72 h post-transfection. Protein extracts were prepared by sonication in 200 µl of lysis buffer (25% glycerol, 20 mM HEPES pH 7.8, 150 mM NaCl, 0.2 mM EDTA, 0.5 mM dithiothreitol, 2.5 mM phenylmethylsulfonyl fluoride).
Western analysis
Whole-cell protein extracts were resolved by SDS–PAGE and transferred to PVDF membrane (Bio-Rad Laboratories, Hercules, CA). Recombinant PITX2 protein was detected with a mouse monoclonal antibody, Anti-Xpress‘ (1:5000 dilution), to the plasmid-encoded Xpress‘ epitope. The secondary antibody, goat-anti-mouse (1:2500 dilution), linked to horseradish peroxidase (Pierce Rockford, IL), was detected by SuperSignal' West Pico Chemiluminescent Substrate (Pierce).
Immunofluorescence
For immunofluorescence, COS-7 cell transfections were scaled down to one-eighth to accommodate smaller 6-well plates (30 x 10 mm) containing coverslips. Additional COS-7 cells were transfected with a LacZ–Xpress‘ control vector. Following 72 h growth, cells were fixed with 1% paraformaldehyde for 6 min, permeated by 0.05% TritonX-100 for 10 min, and blocked with 5% bovine serum albumin for 1 h (36). Recombinant protein was detected with Anti-Xpress antibody (Invitrogen; 1:400 dilution), and a secondary rabbit-anti-mouse antibody linked to Cy3 (Jackson ImmunoResearch Laboratories Inc., West Grove, PA) (1:400 dilution). Nuclei were counterstained with DAPI, and cells were examined by fluorescent microscopy (Leica Mikroskopie und Systeme GmbH, Wetzlar, Germany) with a TRITC filter (Chroma Technology Corp., Brattleboro, VT).
EMSAs
Whole-cell protein extracts from transfected COS-7 cells, standardized to levels of 1x wtPITX2 protein by western analysis results, were used in EMSAs with the 32P-labeled double-stranded oligonucleotide: 5'-CAGGTCAGTTCAGCGGATCCTGTCGACCAGGATGCTAAGCCTCTGTCAGGCGAATTCAGTGCAACTGCAGC-3' containing the CE-3 binding site of Pitx1 (underlined), as described previously (22). V45L and 7aaDup mutant PITX2 extracts were assayed from 1 to 20x and 1 to 100x, respectively, relative to 1x wtPITX2. Two different extract preparations representing separate transfections of COS-7 cells were independently assayed by EMSA, for both V45L and 7aaDup.
Transactivation studies
Transactivation experiments were carried out in mammalian HeLa cells with a pGL3-SV40 Promoter Luciferase reporter vector containing four copies of the CE-3 oligonucleotide (+++–) (sense and antisense orientations), constructed previously (23). The wtPITX2, V45L and 7aaDup expression constructs (1x = 500 ng DNA) and a ß-gal-CMV control vector (100 ng DNA), were co-transfected with the Luciferase reporter (10 ng DNA), as described previously (23). Amounts of luciferase were standardized to the ß-gal internal control [quantitated with a ß-galactosidase Enzyme Assay System (Promega, Madison, WI)]. Transfections were repeated eight times for the V45L mutants and seven times for the 7aaDup mutants. Transactivation with an empty pGL3-vector, not containing the CE-3 oligonucleotide, confirmed that the CE-3 element is required for transactivation. Experiments were also done co-transfecting wtPITX2 and V45L constructs together (each 1x DNA) with comparison to COS-7 cells separately transfected with double (2x) the amount of wtPITX2 or V45L DNA.
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ACKNOWLEDGEMENTS |
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The authors wish to thank Drs E.V.Semina and J.C.Murray (University of Iowa) for providing us with the PITX2 cDNA, Margaret Hughes for assistance with tissue culture, members of the Ocular Genetics laboratory, and Drs S.E.Andrew and D.A.Underhill (University of Alberta) for their critical discussion and review of this manuscript. This work was supported by funding from the Glaucoma Research Society of Canada (E.H.), CIBC World Market Research Fund of the Glaucoma Foundation USA (M.A.W.) and a studentship to K.K. from Fight for Sight, the Research Division of Prevent Blindness America. M.A.W. is an Alberta Heritage Fund for Medical Research Senior Scholar and a Canadian Institutes of Health Research Investigator.
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FOOTNOTES |
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+ To whom correspondence should be addressed at: Toronto Western Hospital, 399 Bathurst Street, Room 6-412, Toronto, Ontario M5T 2S8, Canada. Tel: +1 416 603 5418; Fax: +1 416 603 5126; Email: eheon@uhnres.utoronto.ca The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors
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REFERENCES |
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