Identification of a Dominant Negative Homeodomain Mutation in Rieger Syndrome*

Irfan SaadiDagger , Elena V. Semina§, Brad A. Amendt||, David J. Harris**, Kenneth P. MurphyDagger Dagger , Jeffrey C. MurrayDagger §§§, and Andrew F. RussoDagger ¶¶

From the Dagger  Genetics Program, Departments of § Pediatrics, Dagger Dagger  Biochemistry, §§ Biological Sciences, and  Physiology and Biophysics, University of Iowa, Iowa City, Iowa 52242 and ** Children's Mercy Hospital, Kansas City, Missouri 64108

Received for publication, September 20, 2000, and in revised form, February 15, 2001

    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Mutations in the PITX2 bicoid-like homeobox gene cause Rieger syndrome. Rieger syndrome is an autosomal-dominant human disorder characterized by glaucoma as well as dental hypoplasia, mild craniofacial dysmorphism, and umbilical stump abnormalities. PITX2 has also been implicated in the development of multiple organs and left-right asymmetry in the body plan. The PITX2 homeodomain has a lysine at position 50, which has been shown to impart the bicoid-type (TAATCC) DNA binding specificity to other homeodomain proteins. A mutation (K88E), found in a Rieger syndrome patient, changes this lysine to glutamic acid. We were intrigued by the relatively pronounced phenotypic consequences of this K88E mutation. In the initial analyses, the mutant protein appeared to simply be inactive, with essentially no DNA binding and transactivation activities and, unlike the wild type protein, with an inability to synergize with another transcription factor, Pit-1. However, when the K88E DNA was cotransfected with wild type PITX2, analogous to the patient genotype, the K88E mutant suppressed the synergism of wild type PITX2 with Pit-1. In contrast, a different PITX2 homeodomain mutant, T68P, which is also defective in DNA binding, transactivation, and Pit-1 synergism activities, did not suppress the wild type synergism with Pit-1. These results describe the first dominant negative missense mutation in a homeodomain and support a model that may partially explain the phenotypic variation within Rieger syndrome.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The PITX2 gene was cloned based on its linkage in Rieger syndrome (1). Rieger syndrome is an autosomal-dominant human disorder characterized by ocular anterior chamber anomalies causing glaucoma in more than 50% of affected individuals as well as dental hypoplasia, mild craniofacial dysmorphism, and umbilical stump abnormalities (1, 2). Other features associated with this syndrome include abnormal cardiac, limb, and pituitary development. PITX2 has also been shown to be a downstream target in left-right asymmetry pathways and organogenesis (3, 4). It is expressed asymmetrically to the left side during early development in the lateral plate mesoderm (LPM) and later in most of the organs derived from LPM, including the heart and the gut (4, 5).

The PITX2 protein is a 33-kDa protein that has a homeodomain similar in sequence to other bicoid binding homeodomain proteins, i.e. a characteristic lysine at residue 50 of the homeodomain (1). Bicoid-type homeodomains have a TAATCC consensus binding site, and PITX2 has been shown to specifically bind to this consensus sequence (6-8). In addition, PITX2 transactivates a reporter gene with multimers of the bicoid binding site (6). Amendt et al. (6) show that PITX2 interacts with Pit-1, an important transcription factor regulating expression of thyroid-stimulating hormone, growth hormone, and prolactin and the terminal differentiation of the cell types in which they are expressed (9, 10). Transient transfection assays with the prolactin promoter and both PITX2 and Pit-1 showed a strong synergistic effect on transactivation (6).

It has been demonstrated that residue 50 of a given homeodomain interacts with base pairs 5 and 6 of the hexanucleotide consensus (11-14). Interestingly, a mutation found in a Rieger syndrome patient changes the lysine (K) of position 50 in PITX2 homeodomain to a glutamic acid (E), a mutation that has marked phenotypic consequences. The mutation is referred to as K88E (position 88 is the location of Lys-50 in PITX2 isoform "a"). In this study we have examined the functional properties of the K88E mutant PITX2 protein and compared them to a previously described Rieger syndrome mutation that changes the threonine at position 30 of the homeodomain to a proline (T68P) (6). The T68P mutant has been shown to be defective in DNA binding and transactivation properties (6, 15). T68P mutant also failed to show synergistic transactivation of the prolactin promoter when cotransfected with Pit-1 (6). Similarly, the K88E mutant protein had reduced DNA binding and transactivation activities. K88E was also unable to synergize with the transcription factor, Pit-1. Unexpectedly, in contrast to T68P, K88E suppressed the Pit-1 synergism of wild type PITX2. These results support a model in which a dominant negative mutant protein can contribute to phenotypic variation within the haplo-insufficiency Rieger syndrome.

    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Clinical Data-- The proband was the six-pound, six-ounce infant born to a G1 P0 30-year old mother following a pregnancy complicated by asthma and gestational diabetes. At birth, the proband was noted to have midline abdominal defects, hyperflexive joints, and an abnormal ocular exam. The initial eye exam was interpreted as possible bilateral Peter's anomaly with bilateral iris hypoplasia and posterior embryotoxin, a normal-appearing lens and macula and several small corneal opacities with radial striae. Abdominal exam showed diastasis recti, a protuberant umbilicus, and a small amount of bowel present in the underlying protuberant area. There was a bifid uvula and submucous cleft palate. Renal ultrasound showed no evidence of abnormalities. Subsequent multiple exams under anesthesia show a spectrum consistent with anterior segment dysgenesis bilaterally that was more severe than those seen in many Rieger patients. The abdominal abnormalities gradually resolved with time, and the primary teeth were noted to be small and dysplastic. Physical parameters to age five years include normal growth parameters for height, weight, and head circumference and normal cognitive, gross, and fine motor milestones. The parents are not affected with any ocular, umbilical, or dental anomalies, and one subsequent sibling is also unaffected. The combination of ocular, umbilical, and dental anomalies is consistent with the spectrum of abnormalities seen in Rieger syndrome, and there are no clinical abnormalities yet that suggest growth hormone deficiency, hydrocephalus, or congenital heart disease.

Electrophoretic Mobility Shift Assays-- The human PITX2 cDNA (6), PITX2 T68P cDNA (6), and PITX2 K88E cDNA were cloned into pGex6p-2 (Amersham Pharmacia Biotech). The wild type and mutant cDNAs correspond to PITX2 isoform a. The K88E point mutation was created by polymerase chain reaction-based site-directed mutagenesis (16) using Pfu DNA polymerase (Stratagene) and confirmed by sequencing. Protein preparations were done similar to those described (6). The glutathione S-transferase moiety was removed by PreScission protease (Amersham Pharmacia Biotech). Proteins were quantitated by Bradford assays (Bio-Rad) and visualized on SDS-polyacrylamide gels.

The wild type PITX2 (6), T68P (6), and K88E cDNAs were also cloned into pcDNA3.1 MycHisC expression vector (Invitrogen) containing the T7 promoter and an in-frame C-terminal c-Myc epitope. These vectors were used in in vitro transcription and translation (TNT)1 reactions. One microgram of DNA was used in TNT reactions that were carried out according to the protocol supplied with the TNT kit (Invitrogen).

The 32P-labeled probe for electrophoretic mobility shift assays (EMSAs) was generated by annealing complementary oligonucleotides containing the sequence gatccGCACGGCCCATCTAATCCCGTGggatc and end-filled using Klenow polymerase with [32P]dATP as described (17). Standard binding assays were carried out by incubating the oligonucleotide probe (1.0 pmol) in a 20-µl reaction containing binding buffer (20 mM Hepes, pH 7.5, 5% glycerol, 50 mM NaCl, 1 mM dithiothreitol), 0.1 µg of poly(dI-dC), and 100-300 ng of protein on ice for 15 min. The samples were electrophoresed for 3.5 h at 280 V on 8% polyacrylamide gels as described (6).

Computer Modeling-- The structure of Antp homeodomain-DNA complex was obtained from the Protein Data Bank (accession code 1AHD (18)) (14). The computer program SYBYL (Tripos, Inc.) was used to model the wild type and K88E PITX2-DNA complexes by substituting all the necessary amino acid residues in the homeodomains and nucleotide base pairs. The structures were minimized (dielectric constant = 80; Delta  energy for termination = 0.05 kcal/mol), the complex was reannealed around the residue change (dielectric constant = 80; Delta  energy for termination = 0.05 kcal/mol; reannealing "hot" radius = 6 Å, "interesting" radius = 12 Å), and the entire complex was minimized (dielectric constant = 80; Delta  energy for termination = 0.005 kcal/mol). Total energy, E, of the entire complex was calculated using the Tripos force field, represented by the equation: E = Sigma Estr + Sigma Ebend + Sigma Eoop + Sigma Etors + Sigma Evdw + (Sigma Eele + Sigma Edist  c Sigma Eang  c + Sigma Etors  c + Sigma Erange  c + Sigma Emulti + Sigma Efield fit), where str = bond stretched or compressed from its equilibrium bond length, bend = bending bond angles from their equilibrium values, oop = bending planar atoms out of the plane, tors = torsion or twisting about bonds, vdw = van der Waals nonbonded interactions, ele = electrostatic interactions, dist  c = distance constraints, tors  c = torsion angle constraints, range  c = range constraints, and multi = multifit.

Cell Culture and Reporter Gene Assays-- The wild type PITX2 (6), T68P (6), and K88E cDNAs cloned into pcDNA3.1 MycHisC expression vector (Invitrogen) contain the cytomegalovirus (CMV) promoter and an in-frame C-terminal c-Myc epitope. The TK-Bic reporter contains the herpes simplex virus thymidine kinase minimal promoter with four bicoid elements (6). The prolactin-luciferase reporter contains 2500 base pairs of the rat prolactin enhancer/promoter (19). A CMV beta -galactosidase (6) or an SV40 beta -galactosidase (Promega) reporter plasmid was used as a control for transfection efficiency. The rat Pit-1 expression plasmid is under the control of the Rous sarcoma virus promoter (6). The rat Pit-1 Delta 8-128 (20), Pit-1 Delta POU (21), and Pit-1 W261C (22) expression plasmids were provided by S. J. Rhodes. The MEK kinase (amino acids 380-672) expression vector was obtained from Stratagene.

COS7 and CHO cells for Figs. 4-7 were cultured in 60-mm dishes and electroporated as described (23). Transfection assays used 5-10 µg of the Myc-tagged PITX2 plasmids along with either the TK-Bic or prolactin reporter in COS7 (260 mV; 960 microfarads) or CHO (340 mV; 960 microfarads) cells. 1 µg of CMV-beta -galactosidase (COS7) or pSV-beta -galactosidase (CHO) was also added to each sample. For Figs. 8-11, 2 × 105 COS7, CHO, or GH3 cells were cultured in 12-well plates and transfected by lipofection in the absence of antibiotics. FuGene 6 (Roche Molecular Biochemicals) was used for COS7 (9 µl) and CHO cells (3 µl), whereas LipofectAMINE 2000 (Life Technologies, Inc.) was used for GH3 cells (4 µl). Assays used 0.5 µg of each construct for CHO and GH3 cells, whereas 1.5 µg of each construct were used for COS7 cells. The DNA was incubated with the corresponding transfection reagent in the absence of serum according to the manufacturer's protocol. The DNA transfection reagent mixture was added to cells growing in antibiotic-free media in the presence of serum and incubated for 16-20 h. After incubation, the cells were scraped, lysed, and assayed using luciferase assay reagents (Promega) and beta -galactosidase assay reagents (Tropix Inc.). Luciferase activities were corrected for both transfection efficiency and protein levels (Bradford assays) (Bio-Rad). For Western blots, equal amounts of cell extracts from transfection assays were electrophoresed on a 12.5% SDS-polyacrylamide gel, transferred to polyvinylidine difluoride filters (Millipore), and immunoblotted using a c-Myc antibody (Santa Cruz Labs) and ECL reagents (Amersham Pharmacia Biotech). Statistical significance was calculated using a simple t test for means of two samples.

    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Identification of the K88E Mutation-- The pedigree of the affected proband and family with a de novo mutation is shown in Fig. 1a. The proband presented the characteristic Rieger syndrome features including small and dysplastic primary teeth (Fig. 1b), protuberant umbilicus, and ocular anomalies. The initial eye exam was interpreted as possible bilateral Peter's anomaly; however, subsequent exams revealed a spectrum consistent with bilateral anterior segment dysgenesis more severe than those seen in many patients with Rieger syndrome. Single strand conformation polymorphism analysis of PITX2 exons in proband genomic DNA identified a heterozygous A right-arrow G change in codon 88 (Fig. 1c), resulting in a lysine (AAG) to glutamic acid (GAG) substitution.


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Fig. 1.   Identification of the K88E mutation in a Rieger patient. a, the pedigree of the affected proband and family showing a de novo mutation. b, the maxillary occlusal radiograph on the left shows conically shaped primary central and lateral incisors. The radiodensity of the enamel suggests either hypoplastic or hypomineralized enamel. The primary canines have bulbous crowns. There is no evidence of the developing permanent incisor crowns, suggesting that they are congenitally missing. The mandibular occlusal radiograph on the right shows small primary central and lateral incisors with hypoplastic or hypomineralized enamel. The developing permanent incisor crowns are conical in shape. The lower right permanent central incisor is congenitally missing. c, sequence of the PITX2 fragment from genomic DNA of unaffected (1) and affected (2) members of the family. Nucleotide substitution of Ala to Gly in one allele from affected individual results in a change of a codon for lysine (AAG) to a codon for glutamic acid (GAG) at position 88.

K88E Is Defective in DNA Binding-- We evaluated the DNA binding activity of the mutant PITX2 protein. Wild type PITX2 has been shown to bind TAATCC bicoid consensus sequence in an EMSA (Fig. 2a) (6). In contrast, the K88E mutant showed little or no binding to bicoid probe (Fig. 2a). Similar amounts of bacterial-synthesized wild type PITX2 and K88E mutant proteins were used (Fig. 2b). EMSA was also done with similar amounts of wild type and mutant PITX2 proteins obtained from in vitro TNT reactions (Fig. 2, c-d). As seen with the bacterial-generated proteins, the K88E mutant protein had little or no binding to the bicoid probe. For comparison, the T68P mutant protein also showed greatly reduced binding under these conditions, in agreement with previous reports (6, 15).


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Fig. 2.   K88E mutant protein does not bind the bicoid DNA element. a, increasing amounts (100, 200, and 300 ng) of WT PITX2 and K88E mutant proteins were incubated with a probe containing the bicoid consensus sequence (TAATCC) in an EMSA. The bound complex (Comp) is indicated. Competitor lanes indicate incubation of 200 ng of protein with a 33-fold molar excess of unlabeled probe before incubation with labeled probe. The autoradiograph was purposefully overexposed to maximize potential detection of a K88E·DNA complex. b, Coomassie-stained gel of the PITX2 and K88E protein preparations used in panel a (after PreScission protease cleavage to remove the glutathione S-transferase moiety), with prestained molecular weight standards (Life Technologies, Inc.) indicated. The wild type and K88E proteins are the same size. c, 5 µl of wild type PITX2, K88E, and T68P in vitro TNT products were incubated with the bicoid probe in an EMSA. The bound complex is indicated. d, an autoradiograph of a gel electrophoresed with 2 µl of wild type PITX2, K88E, and T68P TNT products showing comparable expression. The molecular weight markers (Life Technologies, Inc.) are indicated. The wild type, K88E, and T68P TNT products are the same size.

To get a better understanding of how the change from a lysine to a glutamic acid can result in significantly reduced DNA binding, we used a computer-modeling program to predict the PITX2 homeodomain-DNA complex structure (Fig. 3). There have been only three homeodomain-DNA complex structures determined through crystallization or NMR spectroscopy (Protein Data Bank codes 1AHD, 1APL, 1HDD (18)). Of the three, we decided to model the PITX2 homeodomain after the Antennapedia (Antp) homeodomain-DNA complex primarily because it is one of the best characterized homeodomain proteins, the homeodomain is 43% identical to PITX2, and its DNA binding consensus sequence (TAATGG) closely matches that of PITX2 (TAATCC) (7, 14). The Antp homeodomain contains a glutamine at position 50 (Gln-50), which was shown to positively interact with positions 5 and 6 of its hexanucleotide consensus sequence (GG) (14). We used the computer program SYBYL to substitute all the mismatched residues in the Antp homeodomain-DNA complex to those of the PITX2 homeodomain-DNA complex, including the substitution of Gln-50 to Lys-50 and TAATGG to TAATCC, with recalculated local annealing and energy minimization. In the case of wild type PITX2, Lys-50 does not clash with positions 5 and 6 (Fig. 3). However, the glutamic acid at position 50 causes an observable change in the spatial orientation of GG bases on the antisense strand of the TAATCC consensus sequence (Fig. 3). The overall calculated energy for the K88E mutant homeodomain-DNA complex is 38 kcal/mol higher than wild type PITX2 complex. The greatest contributions to the difference were from angle-bending energy (Delta Ebend = 24.5 kcal/mol) and torsional energy (Delta Etor = 21.4 kcal/mol).


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Fig. 3.   Computer model of the K88E mutant homeodomain-DNA complex. The PITX2 homeodomain-DNA complex was modeled upon the NMR solution structure of an antennapedia homeodomain-DNA complex using the SYBYL program. The homeodomain (yellow) and DNA (dark green) are displayed as a ribbon-tube except for lysine (wild-type PITX2) or glutamic acid (K88E mutant) at position 50 of the homeodomain and GG dinucleotide, representing the antisense bases to positions 5 and 6 of the hexanucleotide DNA binding sequence (TAATCC).

K88E Is Defective in Transactivation-- Given the decreased DNA binding activity, we predicted that the K88E mutant protein would also have reduced transactivation activity. The transactivation activity of K88E protein was measured by transient transfection assays in COS7 and CHO cells, which do not express the endogenous PITX2 gene. Wild type PITX2 transactivated a luciferase reporter gene containing the thymidine kinase promoter with four TAATCC binding sites (TK-Bic) ~5-fold (Fig. 4a). Wild type PITX2 specifically activates this reporter and does not transactivate the parental TK-luciferase or the CMV-beta -galactosidase reporter genes (6). The K88E mutant did not appreciably transactivate the TK-Bic reporter (~1.3-fold) (Fig. 4a). This is consistent with the reduced binding to the bicoid element in vitro. We then asked whether this defective transactivation was also manifested with a natural target, the prolactin promoter (Pro-Luc). Wild type PITX2 activated the prolactin promoter ~8-fold in COS7 (6) and 13-fold in CHO (Fig. 4b) cells. In contrast, the K88E only weakly activated the prolactin promoter (~2-fold) in CHO cells (Fig. 4b). There were approximately equivalent amounts of wild type and K88E mutant PITX2 proteins expressed in the transfected cells (Fig. 4c). These results demonstrate that the K88E mutant protein has defective transactivation activity that is similar to its reduced binding activity.


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Fig. 4.   K88E mutant protein has little or no transactivation activity. a, COS7 cells were transfected with a luciferase reporter plasmid containing the thymidine kinase promoter with four upstream bicoid consensus sites (TK-Bic) alone or in combination with PITX2 or K88E. The luciferase activity is shown as the mean fold-activation compared with reporter alone ± S.E. from 5 independent experiments. *, significantly different from wild type PITX2 (p < 0.018). b, the transfections in a were repeated in CHO cells with a luciferase reporter plasmid containing the prolactin promoter. The data from four independent experiments are shown as the mean fold-activation compared with reporter alone ± S.E. *, significantly different from wild type PITX2 (p < 0.001). c, Western blot using anti-c-Myc antibody of equal amounts of cell extracts (20 µg) from one of the cotransfection experiments in CHO cells. The cells were transfected with the indicated amounts of either wild type and/or K88E PITX2 expression vector DNAs. The prestained molecular weight standards (Life Technologies, Inc.) and PITX2 bands are indicated. The wild type PITX2 and K88E proteins are the same size.

K88E Does Not Synergize with Pit-1-- We also looked at the transactivation activity of the K88E mutant in the presence and absence of the POU homeodomain transcription factor, Pit-1. It has previously been shown that Pit-1 and PITX2 can synergistically activate the prolactin promoter (6). The significance of the PITX2 interaction with Pit-1 is supported by their coexpression in early pituitary development and the phenotype of Pitx2 knockout mice (24, 25). Coexpression of Pit-1 with wild type PITX2 yielded about 350-fold transactivation of the prolactin reporter plasmid in CHO cells (Fig. 5, lane 4). In contrast, the K88E mutant did not synergize with Pit-1 (Fig. 5, lane 7). This is similar to the T68P mutant, which also failed to synergize with Pit-1 (Fig. 5, lane 10) (6).


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Fig. 5.   K88E mutant protein prevents wild type PITX2 synergism with Pit-1. CHO cells were transfected with the prolactin-luciferase reporter DNA alone or with PITX2, K88E, and/or T68P in the presence and absence of Pit-1 (10 µg) as indicated. Cotransfection of equal amounts of K88E mutant with wild type (WT) PITX2 (5 µg each) yielded decreased activation relative to 5 µg of WT PITX2 alone (*, p < 0.001) or 10 µg of PITX2 (p < 0.006). The activity is shown as mean fold-activation compared with reporter alone ± S.E. from five independent experiments.

K88E Suppresses Wild-type PITX2 Synergism with Pit-1-- To explain the marked phenotype of the patient carrying the K88E mutation, we tested the possibility that the mutant protein might interfere with the wild type protein activity. Indeed, the presence of one wild type and one mutant allele in Rieger syndrome can be viewed as an autosomal dominant as well as haplo-insufficiency disorder. To simulate this situation, we co-transfected equal amounts of wild type PITX2 and K88E mutant DNA with either the TK-Bic (Fig. 4a) or prolactin (Fig. 4b) reporter plasmids and found no significant effect on wild type activity. We then repeated the same experiments in the presence of Pit-1. Surprisingly, the K88E mutant protein suppressed the synergistic effect of wild type PITX2 with Pit-1 significantly (Fig. 5, lane 8). In contrast, the T68P mutant did not suppress the synergism of wild type PITX2 with Pit-1 (Fig. 5, lane 11). As a control for the increased amount of transfected DNA, transfection with additional wild type PITX2 DNA yielded similar activity as seen with 5 µg of wild type PITX2 (Fig. 5, lane 5). As additional controls, K88E did not suppress the co-transfected CMV beta -galactosidase or SV40 beta -galactosidase reporters. Titration of the K88E mutant DNA in the co-transfection assay indicated that approximately equimolar amounts of wild type and K88E DNAs are required for significant inhibition of wild type PITX2 activity (Fig. 6). Again, titration of the T68P mutant DNA did not result in any inhibition of wild type synergism (Fig. 6). To confirm the dose-dependent nature of the dominant-negative effect, we transfected additional wild type PITX2 DNA to the equimolar amounts of wild type and K88E (Fig. 7). The additional amount of wild type DNA was able to significantly rescue the dominant-negative effect (Fig. 7, lane 3; p < 0.015) to the same level (70%) as predicted by the titration curve in Fig. 6 (lane 3).


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Fig. 6.   K88E mutant protein suppresses wild type PITX2 synergism with Pit-1 in a dose-dependent manner. CHO cells were cotransfected with prolactin-luciferase reporter (5 µg), Pit-1 (10 µg), WT PITX2 (5 µg), and the indicated amounts (µg) of additional WT PITX2, K88E, or T68P DNA. Cotransfection of increasing amounts of K88E mutant DNA with WT PITX2 shows a significant decrease in activation relative to 5 µg of WT PITX2 alone (*, p < 0.028; **, p < 0.006). The data represent four independent experiments with the mean fold-activation ± S.E.


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Fig. 7.   Wild type PITX2 can rescue the dominant negative effect of the K88E mutant. CHO cells were cotransfected with prolactin-luciferase reporter (5 µg), Pit-1 (5 µg), and the indicated amounts (µg) of WT PITX2, or K88E DNA. Cotransfection of equal amounts of K88E mutant DNA with WT PITX2 in the presence of Pit-1 (5 µg each) (lane 2) shows a significant decrease in activation relative to 5 µg of WT PITX2 alone with Pit-1 (lane 1) (*, p < 0.006). Cotransfection of 10 µg of WT PITX2 with 5 µg of K88E in the presence of Pit-1 (lane 3) increased the activation significantly higher than the activation with equal amounts of WT PITX2 and K88E (lane 2) (**, p < 0.015). The data represent three independent experiments with the mean fold-activation ± S.E.

We wanted to know if K88E could suppress Pit-1-independent activation of the prolactin promoter. We used a constitutively active MEK kinase, which stimulates the mitogen-activated protein kinase signal transduction pathway, to activate the prolactin promoter in CHO cells (Fig. 8, lane 2). The K88E mutant was not able to suppress the MEK kinase-stimulated activation of the prolactin promoter (Fig. 8, lane 3). Thus, the dominant negative effect of K88E requires Pit-1 DNA binding or its interaction with PITX2 or both.


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Fig. 8.   K88E mutant cannot suppress MEK kinase activation of the prolactin promoter. CHO cells were transfected by lipofection with prolactin-luciferase reporter plasmid (0.5 µg) alone or in combination with MEK kinase (MEKK) and/or K88E mutant (0.5 µg each). The luciferase activity is shown as the mean fold-activation compared with reporter alone ± S.E. from four independent experiments.

To determine the dependence of this dominant-negative effect on Pit-1 DNA binding, cotransfections of wild type and mutant PITX2 were done in the presence of Pit-1 with a TK-Bic reporter construct, which lacks the Pit-1 binding site in COS7 cells (Fig. 9). Wild type PITX2 activated the TK-Bic reporter ~5-fold (Fig. 9, lane 2) as shown in Fig. 4, whereas both K88E (Fig. 9, lane 5) and T68P (Fig. 9, lane 7) showed little or no transactivation. As expected, the presence of Pit-1 did not affect the transactivation of wild type PITX2 (Fig. 9, lane 4), K88E (Fig. 9, lane 6), or T68P (Fig. 9, lane 8). Cotransfection of the wild type and the K88E mutant in the absence of Pit-1 (Fig. 9, lane 9) or in the presence of Pit-1 (Fig. 9, lane 10) did not show any dominant negative effect. Likewise, the T68P mutant was not dominant negative as expected (Fig. 9, lanes 11 and 12). This implies that the Pit-1 binding site is required for the dominant negative effect.


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Fig. 9.   Pit-1 binding site is required for the dominant negative effect of the K88E mutant. COS7 cells were transfected by lipofection with TK-Bic reporter plasmid (1.5 µg) alone or in combination with Pit-1, PITX2, K88E, and/or T68P (1.5 µg each). The luciferase activity is shown as the mean fold-activation compared with reporter alone ± S.E. from four independent experiments.

To map the protein domains required for the K88E suppression of wild type activity, we tested several Pit-1 and PITX2 mutants (Fig. 10). PITX2 Delta C39 lacks the C-terminal 39 amino acids that have been shown to interact with Pit-1 (6). Pit-1 Delta 8-128 lacks the N-terminal 120 amino acid residues that constitute the major transactivation domain (20). These N-terminal residues have been shown to interact with the closely related PITX1 (10). Pit-1 Delta POU lacks the POU-specific domain of Pit-1 required for DNA binding (21). Finally, Pit-1 W261C contains the mutation identified in the Snell dwarf mice that disables the POU DNA domain (22). Fig. 10 shows the cotransfection of wild type PITX2 and K88E mutant in the presence of Pit-1 mutants with the prolactin reporter in CHO cells. Wild type PITX2 (Fig. 10, lane 2) and wild type Pit-1 (Fig. 10, lane 3) show ~15-fold activation of prolactin promoter. Cotransfection of both PITX2 and Pit-1 showed a synergistic response of ~60-fold (Fig. 10, lane 4). The lower fold activation seen here compared with that seen in Fig. 5 (lane 4) could be due to differences in methods of transfection, electroporation for Fig. 5 versus lipofection for Fig. 10. The coexpression of K88E mutant with wild type PITX2 and wild type Pit-1 (Fig. 10, lane 5) showed significantly reduced activation (p < 0.009) as expected. The PITX2 Delta C39 mutant, which lacks the C-terminal 39 amino acids shown to interact with Pit-1, has been reported to synergize poorly with Pit-1 in COS cells (20). Surprisingly, in CHO cells, PITX2 Delta C39 showed a strong synergism with Pit-1 (Fig. 10, lane 7), which was suppressed by cotransfection of K88E (Fig. 10, lane 8, p < 0.001). The Pit-1 Delta 8-128 mutant (Fig. 10, lane 9), which lacks the major transactivation domain, showed significantly reduced activity than wild type Pit-1 (Fig. 10, lane 3, p < 0.0004). Interestingly, this mutant that lacks the reported PITX1 interaction domain was also able to synergize with wild type PITX2 (Fig. 10, lane 10). This synergism was again suppressed by K88E mutant coexpression (Fig. 10, lane 11). The PITX2 CDelta 39 and Pit-1 Delta 8-128 results imply that, in CHO cells, physical interaction of Pit-1 with PITX2 is not required for synergism. Both Pit-1 Delta POU (Fig. 10, lane 12) and Pit-1 W261C (Fig. 10, lane 15) mutants that are defective in Pit-1 DNA binding showed little or no transactivation. Cotransfection of Pit-1 Delta POU (Fig. 10, lane 13) and Pit-1 W261C (Fig. 10, lane 16) mutants with wild type PITX2 did not show synergistic activation, underscoring the need for Pit-1 DNA binding for synergism. Coexpression of K88E resulted in some suppression of the activity, although to a lesser degree (about 3-fold) than seen with wild type Pit-1 (6-fold) (Fig. 10, lanes 14 and 17, respectively). These results are consistent with the trend seen in Fig. 4b, where coexpression of wild type PITX2 and K88E mutant resulted in lower, albeit statistically nonsignificant (p < 0.14) transactivation. Results from Figs. 9 and 10 thus show that both Pit-1 binding site and Pit-1 DNA binding activity are required for a pronounced dominant negative effect. Furthermore, the K88E suppression of PITX2-Pit-1 synergism does not require the respective C-terminal or N-terminal domains of these proteins.


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Fig. 10.   Pit-1 DNA binding is required for the dominant negative effect of K88E mutant. CHO cells were transfected by lipofection with the prolactin-luciferase reporter DNA alone (0.5 µg) or with PITX2, PITX2 Delta C39, K88E, Pit-1, Pit-1 Delta 8-128, Pit-1 Delta POU, and/or Pit-1 W261C (0.5 µg each) as indicated. Cotransfection of equal amounts of K88E mutant and WT Pit-1 with WT PITX2 (0.5 µg each (lane 5); **, p < 0.009) or PITX2 Delta C39 (0.5 µg each (lane 8); **, p < 0.001) yielded decreased activation relative to WT Pit-1 and WT PITX2 (lane 4) or PITX2 Delta C39 (lane 7), respectively. Similarly, cotransfection of wild type PITX2, K88E, and Pit-1 Delta 8-128 (lane 11; **, p < 0.017) yielded decreased activation from wild type PITX2 and Pit-1 Delta 8-128 (lane 10). Cotransfection of wild type PITX2 and K88E with Pit-1 Delta POU (lane 14; *, p < 0.04) or Pit-1 W261C (lane 17; *, p < 0.04) yielded marginally decreased activation from wild type PITX2 and Pit-1 Delta POU (lane 13) or PITX2 and Pit-1 W261C (lane 16), respectively. The activity is shown as mean fold-activation compared with reporter alone ± S.E. from four independent experiments.

K88E Suppresses Prolactin Promoter Activity in GH3 Cells-- We wanted to see if K88E mutant would suppress the activity of endogenous Pitx2 and Pit-1. For this experiment, we used the GH3 pituitary cell line. GH3 cells are known to express the endogenous Pitx2 and Pit-1 genes (10). Under our conditions, overexpression of wild type PITX2 (Fig. 11a, lane 2) or Pit-1 (Fig. 11a, lane 3) did not significantly change prolactin promoter activity in GH3 cells, suggesting that the endogenous Pitx2 and Pit-1 proteins were not rate-limiting. Interestingly, overexpression of the K88E mutant (Fig. 11a, lane 4) was able to significantly suppress the prolactin promoter activity in GH3 cells (p < 0.005). In contrast, overexpression of the T68P mutant did not affect promoter activity (Fig. 11a, lane 5). As a control, we used the TK-Bic reporter (Fig. 11b), which lacks Pit-1 binding site. Neither the K88E or T68P mutants were able to suppress the activity of TK-Bic reporter in GH3 cells (Fig. 11b). This control underscores the need for the Pit-1 binding site for the dominant negative effect of K88E mutant. Hence the K88E mutant can repress wild type PITX2 activity in both heterologous cells and in cells that normally express the Pitx2 gene. Furthermore, in both situations, Pit-1 interaction with DNA is required for repression by the K88E mutant.


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Fig. 11.   K88E mutant protein suppresses prolactin promoter activity in GH3 cells. a, GH3 cells were transfected by lipofection with the prolactin-luciferase reporter DNA alone or with Pit-1, PITX2, K88E, or T68P (1 µg each) as indicated. Transfection of K88E mutant yielded decreased activation relative to WT PITX2 alone (*, p < 0.005). The activity is shown as mean fold-activation compared with reporter alone ±S.E. from 18 independent experiments. b, GH3 cells were transfected by lipofection with the TK-Bic reporter DNA alone or with Pit-1, PITX2, K88E, or T68P (1 µg each) as indicated. The activity is shown as mean fold-activation compared with reporter alone ±S.E. from four independent experiments.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In this study we have focused on a point mutation, K88E, that changes a single amino acid in the recognition helix of the homeodomain from a lysine to a glutamic acid. We show that the K88E mutant is defective in its capacity to bind DNA and transactivate synthetic and natural promoters in transfection assays. Our computer modeling suggests that the defect in DNA binding of the K88E mutant homeodomain is due to the steric constraints that require significant unfavorable bending and torsional angles in the DNA. This energetic penalty results in a very large decrease in the binding constant. Although the exact value of the energy difference (Delta E = 38 kcal/mol) must be considered more qualitative than quantitative, a Delta E of this size would result in a decrease in the binding constant by about 30 orders of magnitude. Interestingly, the only documented homeodomain proteins with a glutamic acid at position 50 are Zmhox1a and Zmhox1b maize proteins that appear to bind very divergent sequences (26). The T68P mutant protein is also defective in DNA binding. Amendt et al. (6) used bacterially expressed T68P protein in EMSA to show that it could still bind the bicoid probe but with reduced binding capacity. Kozlowski and Walter (15) used total extracts from cells transfected with T68P DNA in their EMSA to show that the mutant protein cannot bind DNA. We have used T68P protein obtained from in vitro TNT reactions in EMSA to show greatly diminished binding to the bicoid DNA probe. The quantitative differences observed are likely due to different types of protein preparations and binding conditions.

Most surprisingly, the K88E protein acts in a dominant negative manner to repress wild type PITX2 activity in the presence of Pit-1. In contrast, another PITX2 missense mutation, T68P, that abolishes transactivation properties (6), does not repress wild type PITX2 synergism with Pit-1. Because the K88E mutant protein does not appear to have any intrinsic repressor activity, the dominant negative effect is likely due to the formation of nonfunctional complexes between wild type PITX2 and the K88E mutant proteins. Previous studies indicate that PITX2 can form homodimers (6, 27). One mechanism for the dominant negative inhibition would be that the K88E protein could heterodimerize with wild type PITX2 and that the wild type PITX2-K88E heterodimer would be unable to synergize with Pit-1. Simply interpreted, our results imply that wild type PITX2-K88E mutant dimers are nonfunctional. At face value, this would explain the dominant-negative effect of K88E. However, the repression cannot be that simple since the suppression of wild type PITX2 in CHO cells was significant only in the presence of Pit-1 DNA binding activity. Hence, it seems likely that the wild type PITX2-K88E heterodimer is still able to bind DNA. Furthermore, the T68P mutant has decreased DNA binding, defective transactivation, and synergism (6, 15), yet it is not a dominant-negative mutant protein.

We propose that the dominant negative nature of the PITX2-K88E wild type-mutant heterodimer is dictated by its inability to synergize with other transcription factors, such as Pit-1. In support of this hypothesis, K88E was unable to suppress Pit-1-independent activation of the prolactin promoter by a constitutively active MEK kinase. Interestingly, the suppression by K88E requires a functional, but not physical, interaction between PITX2 and Pit-1. The C-terminal domain of PITX2 that binds Pit-1 is not required for repression by K88E. In this regard, we found that the requirement for the C-terminal 39 residues for synergism is cell-specific. It appears that these residues are not required for synergism in at least 3 cell lines, CHO, LS8, and N2A, but are required in COS7 and HeLa cells. The basis for this specificity is not known but is suggestive that other factors are involved in PITX2 transactivation, as we had previously speculated based on data from the PITX2 Delta C39 mutant (6). We have now shown that both PITX2 Delta C39, which lacks the Pit-1 interacting residues (6), and Pit1 Delta 8-128, which lacks the reported PITX1 interacting residues (10), are capable of synergism in CHO cells and that this synergism can be suppressed by K88E.

In a further test of the role that Pit-1 plays in K88E suppression, we have shown that Pit-1 DNA binding activity is required. This was shown by complementary experiments using a reporter lacking a Pit-1 binding site (TK-bicoid) and by using mutant Pit-1 proteins that lack DNA binding activity (Pit-1 Delta POU and Pit-1 W261C). Taken together, these data imply that in CHO cells, Pit-1 and PITX2 DNA binding activities may be sufficient for the dominant negative effect of K88E. Finally, we showed that K88E can suppress the prolactin promoter in the pituitary adenoma GH3 cell line, which expresses the endogenous Pit-1 and Pitx2 genes. The ability of K88E to suppress the prolactin promoter in GH3 cells supports the potential physiological relevance of K88E activity in the pituitary.

Our results describe the first molecular characterization of a dominant negative missense mutation in a homeodomain. The other examples of dominant negative mutations in homeodomain proteins have been truncations that remove the transactivation domain yet retain the DNA binding homeodomain (28-30). Hence, the K88E mutation represents a new type of homeodomain mutation in human disorders. We propose that the phenotypic variation of Rieger syndrome can be determined by a continuum of mutations in PITX2 ranging from null deletions (1) to dominant negative proteins. This proposal is in agreement with recent studies by Kozlowski and Walter (15), who showed that two mutations with mild defects in PITX2 activities were correlated with milder anterior segment aberrations (iris hypoplasia and iridogoniodysgenesis syndrome) than seen in most Rieger patients. This perspective suggests an approach to understanding genotype-phenotype correlations for other transcription factor disorders, such as Saethre-Chotzen syndrome (TWIST) (31, 32), Waardenburg syndrome (PAX3) (33, 34), aniridia, and Peter's Anomaly (PAX6) (35, 36).

    ACKNOWLEDGEMENTS

We thank the family for their cooperation and willingness to participate in the study and for providing photos and dental x-rays, L. B. Sutherland and S.D. Hirsch for reagents and assistance, and S. J. Rhodes (Indiana University-Purdue University Indianapolis, IN) for kindly providing the Pit-1 mutants.

    FOOTNOTES

* This work was supported by National Institutes of Health Grants DE13076 and EY12384.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

|| Present address: Dept. of Biological Sciences, The University of Tulsa, Tulsa, OK 74104.

¶¶ To whom correspondence should be addressed. Tel.: 319-335-7872; Fax: 319-335-7330; E-mail: andrew-russo@uiowa.edu.

Published, JBC Papers in Press, April 11, 2001, DOI 10.1074/jbc.M008592200

    ABBREVIATIONS

The abbreviations used are: TNT, transcription and translation; CMV, cytomegalovirus; MEK, mitogen-activated protein kinase; CHO, Chinese hamster ovary; EMSA, electrophoretic mobility shift assay; Antp, Antennapedia; WT, wild type.

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