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Saadi et al.
1
Identification of a Dominant Negative Homeodomain Mutation
in Rieger Syndrome
Irfan Saadi‡, Elena V. Semina§, Brad A. Amendt¶,||, David J. Harris**, Kenneth P. Murphy‡‡, Jeffrey C.
Murray‡,§,§§, and Andrew F. Russo‡,¶,¶¶
‡Genetics Program, Departments of §Pediatrics, ‡‡Biochemistry, §§Biological Sciences, and ¶Physiology
and Biophysics, University of Iowa; Iowa City, IA 52242. **Children’s Mercy Hospital, Kansas City, MO
64108.
|| Present address: Department of Biological Sciences, The University of Tulsa, Tulsa, OK 74104
¶¶ Correspondence should be addressed to A.F.R.
e-mail: [email protected]
Tel: (319) 335-7872
Fax: (319) 335-7330
Copyright 2001 by The American Society for Biochemistry and Molecular Biology, Inc.
JBC Papers in Press. Published on April 11, 2001 as Manuscript M008592200 by guest on M
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Running title
“Dominant negative Rieger syndrome mutation”
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Abstract
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 wildtype protein, with an inability to synergise with
another transcription factor, Pit-1. However, when the K88E DNA was cotransfected with wildtype
PITX2, analogous to the patient genotype, the K88E mutant suppressed the synergism of wildtype
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
wildtype 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.
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Introduction
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 kD 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 (6,7,8). In addition, PITX2 transactivates a reporter gene with multimers
of the bicoid binding site (6). Amendt et al. (6) have shown 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 (11,12,13,14). Interestingly, a mutation found in a Rieger syndrome
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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 K50 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 synergise with the transcription factor, Pit-1. Unexpectedly, in contrast to T68P, K88E suppressed the
Pit-1 synergism of wildtype PITX2. These results support a model in which a dominant negative mutant
protein can contribute to phenotypic variation within the haploinsufficiency Rieger syndrome.
Materials and Methods
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 is 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
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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 are
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 wildtype and mutant cDNAs correspond to PITX2
isoform “a”. The K88E point mutation was created by PCR-based site-directed mutagenesis (16) using
Pfu DNA polymerase (Stratagene) and confirmed by sequencing. Protein preparations were done similar
to as described (6). The GST moiety was removed by PreScission protease (Amersham Pharmacia
Biotech). Proteins were quantitated by Bradford assays (BioRad) and visualized on SDS-polyacrylamide
gels.
The wildtype 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) reactions. One microgram of DNA
was used in TNT reactions that were carried out according to the protocol supplied with the TNT kit
(Invitrogen).
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The 32P-labeled probe for 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 protein on ice for 15
minutes. The samples were electrophoresed for 3.5 hours at 280 V on 8% polyacrylamide gels as
described (6).
Computer modeling
The structure of Antp homeodomain-DNA complex was obtained from the Protein Database1
[accession no. 1AHD] (14). The computer program SYBYL (Tripos, Inc.) was used to model the wildtype
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;
∆energy for termination = 0.05 kcal/mol), and the complex was reannealed around the residue change
(dielectric constant = 80; ∆energy for termination = 0.05 kcal/mol; reannealing ‘hot’ radius = 6 Å,
‘interesting’ radius = 12 Å) and the entire complex minimized (dielectric constant = 80; ∆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 = ΣEstr + ΣEbend + ΣEoop + ΣEtors + ΣEvdw + [ΣEele + ΣEdist_c +
ΣEang_c + ΣEtors_c + ΣErange_c + ΣEmulti + ΣE field 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 non-bonded
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interactions; ele = electrostatic interactions; dist_c = distance constraints; tors_c = torsion angle
constraints; range_c = range constraints; multi = multifit.
Cell culture and reporter gene assays
The wildtype 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 β-galactosidase (6) or an SV40 β-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 ∆8-128 (20), Pit-1 ∆POU (21), and Pit-1
W261C (22) expression plasmids were provided by S.J. Rhodes. The MEKK (MEK Kinase amino acids
380 to 672) expression vector was obtained from Stratagene.
COS7 and CHO cells for Figures 4, 5, 6 and 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 µF) or CHO (340 mV; 960 µF) cells. 1 µg of CMV-
βgal (COS7) or pSV-βgal (CHO) was also added to each sample. For Figures 8, 9, 10 and 11, 2x105
COS7, CHO, or GH3 cells were cultured in 12-well plates and transfected by lipofection in the absence of
antibiotics. FuGene 6 (Roche) was used for COS7 (9 µL) and CHO cells (3 µL), whereas, Lipofectamine
2000 (Gibco BRL) was used for GH3 cells (4 µL). Assays used 0.5 µg of each construct for CHO and
1 The atomic coordinates for the NMR structure of this protein are available in Research Collaboratory for
Structural Bioinformatics Protein Databank (http://www.rcsb.org/pdb/) under PDB # 1AHD (18).
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GH3 cells, while 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 hrs. Following incubation, the cells were scraped, lysed and assayed
using luciferase assay reagents (Promega) and β-galactosidase assay reagents (Tropix Inc.). Luciferase
activities were corrected for both transfection efficiency and protein levels (Bradford assays) (BioRad).
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
Identification of the K88E mutation
The pedigree of the affected proband and family with a de novo mutation is shown in Figure 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→G change in codon 88 (Fig. 1c) resulting in a lysine (AAG) to glutamic acid
(GAG) substitution.
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K88E is defective in DNA binding
We evaluated the DNA binding activity of the mutant PITX2 protein. Wildtype PITX2 has been shown
to bind TAATCC bicoid consensus sequence in an electrophoretic mobility shift assay (EMSA) (Fig. 2a)
(6). In contrast, the K88E mutant showed little or no binding to bicoid probe (Fig. 2a). Similar amounts of
bacterial-synthesized wildtype PITX2 and K88E mutant proteins were used (Fig. 2b). EMSA was also
done with similar amounts of wildtype and mutant PITX2 proteins obtained from in vitro transcription and
translation (TNT) reactions (Fig. 2c-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).
In order 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 spectroscopy2. 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 (Q50), 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
2 The atomic coordinates for the NMR structure of these proteins are available in Research Collaboratory
for Structural Bioinformatics Protein Databank (http://www.rcsb.org/pdb/) under PDB # 1AHD, 1APL,
1HDD (18).
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PITX2 homeodomain-DNA complex, including the substitution of Q50 to K50 and TAATGG to TAATCC,
with recalculated local annealing and energy minimization. In the case of wildtype PITX2, K50 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/mole higher than wildtype PITX2 complex. The greatest contributions to the difference were from
angle bending energy (∆Ebend = 24.5 kcal/mol) and torsional energy (∆Etor = 21.4 kcal/mol).
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.
Wildtype PITX2 transactivated a luciferase reporter gene containing the thymidine kinase promoter with
four TAATCC binding sites (TK-Bic) approximately 5-fold (Fig. 4a). Wildtype PITX2 specifically activates
this reporter and does not transactivate the parental TK-luciferase or the CMV-βgal 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). Wildtype
PITX2 activated the prolactin promoter approximately 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 wildtype and K88E mutant PITX2 proteins expressed in
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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.
K88E does not synergise 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 wildtype PITX2 yielded about 350-fold transactivation of the prolactin
reporter plasmid in CHO cells (Fig. 5a, lane 4). In contrast, the K88E mutant did not synergize with Pit-1
(Fig. 5a, lane 7). This is similar to the T68P mutant, which also failed to synergise with Pit-1 (Fig. 5a, lane
10) (6).
K88E suppresses wildtype PITX2 synergism with Pit-1
In order to explain the marked phenotype of the patient carrying the K88E mutation, we tested the
possibility that the mutant protein might interfere with the wildtype protein activity. Indeed, the presence
of one wildtype and one mutant allele in Rieger syndrome can be viewed as an autosomal dominant as
well as haploinsufficiency disorder. To simulate this situation, we co-transfected equal amounts of
wildtype PITX2 and K88E mutant DNA with either the TK-Bic (Fig. 4a) or prolactin (Fig. 4b) reporter
plasmids and found no significant effect on wildtype activity. We then repeated the same experiments in
the presence of Pit-1. Surprisingly, the K88E mutant protein suppressed the synergistic effect of wildtype
PITX2 with Pit-1 significantly (Fig. 5, lane 8). In contrast, the T68P mutant did not suppress the
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synergism of wildtype PITX2 with Pit-1 (Fig. 5, lane 11). As a control for the increased amount of
transfected DNA, transfection with additional wildtype PITX2 DNA yielded similar activity as seen with 5
µg wildtype PITX2 (Fig. 5, lane 5). As additional controls, K88E did not suppress the co-transfected CMV
β-galactosidase or an SV40 β-galactosidase reporters. Titration of the K88E mutant DNA in the co-
transfection assay indicated that approximately equimolar amounts of wildtype and K88E DNAs are
required for significant inhibition of wildtype PITX2 activity (Fig. 6). Again, titration of the T68P mutant
DNA did not result in any inhibition of wildtype synergism (Fig. 6). In order to confirm the dose-dependent
nature of the dominant-negative effect, we transfected additional wildtype PITX2 DNA to the equimolar
amounts of wildtype and K88E (Fig. 7). The additional amount of wildtype 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
titration curve in Fig. 6 (lane 3).
We wanted to know if K88E could suppress Pit-1 independent activation of the prolactin promoter.
We used a constitutively active MEK kinase (MEKK), which stimulates the MAP (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 MEKK 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.
In order to determine the dependence of this dominant-negative effect on Pit-1 DNA binding,
cotransfections of wildtype and mutant PITX2 were done in the presence of Pit-1 with TK-Bic reporter
construct which lacks the Pit-1 binding site in COS7 cells (Fig. 9). Wildtype PITX2 activated the TK-Bic
reporter approximately 5-fold (Fig. 9, lane 2) as shown in Fig. 4, while both K88E (Fig. 9, lane 5) and
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T68P (Fig. 9, lane 7) showed little or no transactivation. As expected, the presence of Pit-1 did not affect
the transactivation of wildtype PITX2 (Fig. 9, lane 4), K88E (Fig. 9, lane 6), or T68P (Fig. 9, lane 8).
Cotransfection of the wildtype 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, 12). This implies that the Pit-1 binding site is
required for the dominant negative effect.
In order to map the protein domains required for the K88E suppression of wildtype activity, we tested
several Pit-1 and PITX2 mutants (Fig. 10). PITX2 ∆C39 lacks the C-terminal 39 amino acids that have
been shown to interact with Pit-1 (6). Pit-1 ∆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 ∆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). Figure 10 shows the cotransfection of wildtype PITX2 and K88E mutant in
the presence of Pit-1 mutants with the prolactin reporter in CHO cells. Wildtype PITX2 (Fig. 10, lane 2)
and wildtype Pit-1 (Fig. 10, lane 3) show approximately 15-fold activation of prolactin promoter.
Cotransfection of both PITX2 and Pit-1 showed a synergistic response of approximately 60-fold (Fig. 10,
lane 4). The lower fold activation seen here compared to 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 wildtype PITX2 and wildtype Pit-1 (Fig. 10, lane 5) showed significantly
reduced activation (p < 0.009) as expected. The PITX2 ∆C39 mutant that 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).
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Surprisingly, in CHO cells, PITX2 ∆C39 showed a strong synergism with Pit-1 (Fig. 10, lane7) which was
suppressed by cotransfection of K88E (Fig. 10, lane 8, p < 0.001). The Pit-1 ∆8-128 mutant (Fig. 10, lane
9) that lacks the major transactivation domain showed significantly reduced activity than wildtype Pit-1
(Fig. 10, lane 3, p < 0.0004). Interestingly, this mutant which lacks the reported PITX1 interaction domain
was also able to synergize with wildtype PITX2 (Fig. 10, lane 10). This synergism was again suppressed
by K88E mutant coexpression (Fig. 10, lane 11). The PITX2 C∆39 and Pit-1 ∆8-128 results imply that, in
CHO cells, physical interaction of Pit-1 with PITX2 is not required for synergism. Both Pit-1 ∆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 ∆POU (Fig. 10, lane 13) and Pit-1 W261C (Fig. 10, lane
16) mutants with wildtype 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 wildtype 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 wildtype PITX2 and
K88E mutant resulted in lower, albeit statistically non-significant (p < 0.14) transactivation. Results from
Figures 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.
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 wildtype PITX2 (Fig. 11a, lane 2), or Pit-1
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(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 wildtype 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.
Discussion
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 which 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 (∆E = 38 kcal/mol) must be considered more qualitative
than quantitative, a ∆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
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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 et al. (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 wildtype PITX2
activity in the presence of Pit-1. In contrast, another PITX2 missense mutation, T68P, that abolishes
transactivation properties (6), does not repress wildtype PITX2 synergism with Pit-1. As 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 wildtype PITX2 and the K88E mutant proteins.
Previous studies have indicated that PITX2 can form homodimers (6,27). One mechanism for the
dominant negative inhibition would be that the K88E protein could heterodimerize with wildtype PITX2
and that the wildtype PITX2-K88E heterodimer would be unable to synergise with Pit-1. Simply
interpreted, our results imply that wildtype 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 wildtype PITX2 in CHO cells was significant only in the presence of Pit-1 DNA
binding activity. Hence, it seems likely that the wildtype 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.
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We propose that the dominant negative nature of the PITX2-K88E wildtype-mutant heterodimer is
dictated by its inability to synergise 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 ∆C39 mutant (6). We have now shown that
both PITX2 ∆C39, which lacks the Pit-1 interacting residues (6), and Pit1 ∆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
∆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.
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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
(28,29,30). 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 et al. (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, S.D. Hirsch for reagents and assistance; and S.J. Rhodes
(Indiana University-Purdue University Indianapolis, IN) for kindly providing the Pit-1 mutants. This work
was supported by National Institutes of Health grants DE13076 and EY12384.
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Figure legends
Figure 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 A
to G 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.
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Figure 2. K88E mutant protein does not bind the bicoid DNA element. a) Increasing amounts (100,
200, and 300 ng) of wildtype PITX2 and K88E mutant proteins were incubated with a probe containing the
bicoid consensus sequence (TAATCC) in an electrophoretic mobility shift assay (EMSA). The bound
complex is indicated. Competitor lanes indicate incubation of 200 ng protein with 33-fold molar excess of
unlabeled probe prior to 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 (following PreScission protease cleavage to
remove the GST moiety), with prestained molecular weight standards (Gibco BRL) indicated. The
wildtype and K88E proteins are the same size. c) 5µl of wildtype PITX2, K88E, and T68P in vitro
transcription and translation (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 wildtype PITX2,
K88E, and T68P TNT products showing comparable expression. The molecular weight markers (Gibco
BRL) are indicated. The wildtype, K88E, and T68P TNT products are the same size.
Figure 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).
Figure 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
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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 to reporter alone ± S.E.M. from 5 independent
experiments. *, significantly different from wildtype 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
4 independent experiments are shown as the mean fold-activation compared to reporter alone ± S.E.M.
*, significantly different from wildtype 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 wildtype and/or K88E PITX2 expression vector
DNAs. The prestained molecular weight standards (Gibco BRL) and PITX2 bands are indicated. The WT
PITX2 and K88E proteins are the same size.
Figure 5. K88E mutant protein prevents wildtype 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 wildtype (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 to
reporter alone ± S.E.M. from 5 independent experiments.
Figure 6. K88E mutant protein suppresses wildtype PITX2 synergism with Pit-1 in a dose-
dependent manner. CHO cells were cotransfected with prolactin-luciferase reporter (5µg), 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
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in activation relative to 5 µg of WT PITX2 alone (*, p < 0.028; **, p < 0.006). The data represent 4
independent experiments with the mean fold-activation ± S.E.M.
Figure 7. Wildtype PITX2 can rescue the dominant negative effect of the K88E mutant. CHO cells
were cotransfected with prolactin-luciferase reporter (5µg), 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 5µg 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 3 independent experiments
with the mean fold-activation ± S.E.M.
Figure 8. K88E mutant cannot suppress MEKK activation of the prolactin promoter. CHO cells
were transfected by lipofection with prolactin-luciferase reporter plasmid (0.5 µg) alone or in combination
with MEKK, and/or K88E mutant (0.5 µg each). The luciferase activity is shown as the mean fold-
activation compared to reporter alone ± S.E.M. from 4 independent experiments.
Figure 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 to reporter alone ± S.E.M. from 4 independent experiments.
Figure 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
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PITX2, PITX2 ∆C39, K88E, Pit-1, Pit-1 ∆8-128, Pit-1 ∆POU, and/or Pit-1 W261C (0.5 µg each) as
indicated. Cotransfection of equal amounts of K88E mutant and wildtype (WT) Pit-1 with WT PITX2 (0.5
µg each; lane 5; **, p < 0.009) or PITX2 ∆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 ∆C39 (lane 7), respectively. Similarly,
cotransfection of wildtype PITX2, K88E, and Pit-1 ∆8-128 (lane 11; **, p < 0.017) yielded decreased
activation than wildtype PITX2 and Pit-1 ∆8-128 (lane 10). Cotransfection of wildtype PITX2, and K88E
with Pit-1 ∆POU (lane 14; *, p < 0.04) or Pit-1 W261C (lane 17; *, p < 0.04) yielded marginally decreased
activation than wildtype PITX2 and Pit-1 ∆POU (lane 13) or PITX2 and Pit-1 W261C (lane 16),
respectively. The activity is shown as mean fold-activation compared to reporter alone ± S.E.M. from 4
independent experiments.
Figure 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 to
reporter alone ± S.E.M. 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 to reporter alone ± S.E.M. from 4 independent
experiments.
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c)
a)
b)
Figure 1
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Bound
WTPITX2 K88EC
omp
Pro
be O
nly
Com
p
30
60
40
20
WT P
ITX2
K88E
PITX2
a) b)
Bound
WT
PITX2
K88E
T68P
Probe
Onl
y
c)
WT
PITX2
K88E
T68Pd)
70
60
40PITX2
Figure 2
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Wildtype PITX2
K50
G5G6
K88E
E50
G5G6
PITX2 homeodomain
DNA
Sense: TAATC5C6
Antisense: ATTAG5G6
CarbonOxygenHydrogenNitrogenPhosphorus
Figure 3
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0
1
2
3
4
5
6
1 2 3 4
Fo
ld A
ctiv
atio
n
PITX2K88E
--
+- +
-
a)
*
++
LucTK-Bic
b)
0
2
4
6
8
10
12
14
16
18
1 2 3 4
Fo
ld A
ctiv
atio
n
PITX2K88E
--
+- +
-
LucProlactin 2.5
*
++
*
Figure 4
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60
40
30
K88E - - 55
PITX2 5 10 5-
-
-
PITX2
c)
Figure 4
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0
50
100
150
200
250
300
350
400
450
*
Fo
ld A
ctiv
atio
n
K88E - 55- 5-PITX2 5
-
-10 55
Pit-1 +
-
++ +- -
-
-
+
-
-
T68P 5
5
+
- - - - - 5
-
- - -
-
-
-
5
-
-
+
LucProlactin 2.5
1 2 3 4 5 6 7 8 119 10
Figure 5
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20
40
60
80
100
120
140
160
1 2 3 4 5 6
WT PITX2 55 55 5
WT, K88E,or T68P
1-
5
WT
K88E
Act
ivat
ion
(% c
on
tro
l)
*
**
*LucProlactin 2.5
T68P
32 4 5
Figure 6
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PITX2+
10Pit-1
K88E5
-5+
5 5
+
0
20
40
60
80
100
120
1 2 3
*
LucProlactin 2.5
Figure 7
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MEKK +K88E
+--
- +
0
1
2
3
4
5
6
7
1 2 3
LucProlactin 2.5
Figure 8
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0.0
1.0
2.0
3.0
4.0
5.0
6.0
7.0
8.0
1 2 3 4 5 6 7 8 9 10 11 12
Fo
ld A
ctiv
atio
n
K88E - -++ --PITX2 +
+
--+
Pit-1 +
-
-- +- -
-
-
+
-
-
T68P +
+
-
- - - + + -
-
- - -
+
+
-
-
+
+
+
+
+
+
-
-
LucTK-Bic
Figure 9
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0.0
20.0
40.0
60.0
80.0
100.0
120.0
140.0
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
Pit-1 + --+ --K88E -
-
-+-
PITX2 +
-
++ ++ -
-
+
-
-
-
Pit-1 ∆8-128 -
-
-
- - + + + -
-
- - -
-
-
+
-
-
+
+
-
-
+
-
+
Fo
ld A
ctiv
atio
n
Pit-1 ∆POU
Pit-1 W261C
+
+
-
-
-
-
-
-
-- - - - - +- - - + - -+
+- - - - - -- - - - + +-
LucProlactin 2.5
****
* *
-- - - - - -- - - - - --PITX2 ∆C39
+ +
- +
- --
-
-
- --
- --
- --
+ ++
**
Figure 10
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0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
1.8
1 2 3 4 5
Fo
ld A
ctiv
atio
n
b)
PITX2
++
++
Pit-1
T68P
K88E-
----
- -- -
---
-
- --
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----
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LucProlactin 2.5
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Figure 11
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Jeffrey C. Murray and Andrew F. RussoIrfan Saadi, Elena V. Semina, Brad A. Amendt, David J. Harris, Kenneth P. Murphy,
Identification of a dominant negative homeodomain mutation in Rieger syndrome
published online April 11, 2001J. Biol. Chem.
10.1074/jbc.M008592200Access the most updated version of this article at doi:
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