From the Genetics Program, Departments of
§ Pediatrics,
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
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ABSTRACT |
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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.
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.
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; 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
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- 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 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).
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 ( 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- 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).
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
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.
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.
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 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.
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 ( 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 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 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).
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
energy for termination = 0.05 kcal/mol), 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 was 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 =
Estr +
Ebend +
Eoop +
Etors +
Evdw + (
Eele +
Edist c +
Eang c +
Etors c +
Erange c +
Emulti +
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.
-galactosidase (6) or an
SV40
-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 MEK kinase (amino acids 380-672) expression
vector was obtained from Stratagene.
-galactosidase (COS7) or
pSV-
-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
-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
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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.
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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.
Ebend = 24.5 kcal/mol) and torsional energy (
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).
-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.
View larger version (14K):
[in a new window]
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.
-galactosidase or SV40
-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).
View larger version (16K):
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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.
View larger version (10K):
[in a new window]
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.
View larger version (8K):
[in a new window]
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.
View larger version (15K):
[in a new window]
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.
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). 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
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
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
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 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 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.
View larger version (18K):
[in a new window]
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 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 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 wild type PITX2,
K88E, and Pit-1
8-128 (lane 11; **,
p < 0.017) yielded decreased activation from wild type
PITX2 and Pit-1
8-128 (lane 10).
Cotransfection of wild type 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 from
wild type 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 with reporter alone ± S.E. from four independent
experiments.
View larger version (14K):
[in a new window]
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
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 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.
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.
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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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.
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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
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ABBREVIATIONS |
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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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