Human Molecular Genetics, 2002, Vol. 11, No. 7 743-753
© 2002 Oxford University Press
A molecular basis for differential developmental anomalies in Axenfeld–Rieger syndrome
Department of Biological Sciences, The University of Tulsa, 600 South College Avenue, Tulsa, OK 74104-3189, USA and 1Department of Pediatrics, The University of Iowa, Iowa City, IA 52242, USA
Received November 16, 2001; Revised and Accepted February 4, 2002.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Pitx2, a bicoid-like homeodomain transcription factor and Dlx2 are two transcriptional markers observed during early tooth development. PITX2 binds to bicoid and bicoid-like elements in the Dlx2 promoter and activates this promoter 30-fold in Chinese hamster ovary cells. Mutations in PITX2 associated with Axenfeld–Rieger syndrome (ARS) provided the first link of this homeodomain transcription factor to tooth development. We are investigating the molecular basis of developmental anomalies associated with human PITX2 mutations. A phenotypically less severe ARS mutant (without tooth anomalies), PITX2 R84W, has a similar DNA binding specificity compared to wild-type PITX2 and transactivates the Dlx2 promoter. This mutation is associated with iris hypoplasia (IH); in contrast a Rieger syndrome mutation, PITX2 T68P, which presents clinically with the full spectrum of developmental anomalies (including tooth anomalies), is unable to transactivate the Dlx2 promoter. Since Dlx2 expression is required for tooth and craniofacial development the lack of tooth anomalies in the patient with IH may be due to the residual activity of this mutant in activating the Dlx2 promoter. We demonstrate that PITX2 phosphorylation increases PITX2 and PITX2 R84W DNA binding. The PITX2 T68P ARS mutation occurs at a protein kinase C phosphorylation site in the homeodomain. Surprisingly, phosphorylation of PITX2 T68P is increased compared to wild-type PITX2 but has little effect on its DNA binding activity. Altogether these data suggest a molecular mechanism for tooth development involving Dlx2 gene expression in ARS patients.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Axenfeld–Rieger syndrome (ARS) (MIM 180500) is an autosomal dominant disorder of morphogenesis. ARS is a phenotypically heterogeneous disorder characterized by malformations of the eyes, teeth and umbilicus. The Axenfeld–Rieger group of anomalies includes Rieger syndrome, Axenfeld anomaly and Rieger anomaly [or Rieger eye malformation, iris hypoplasia (IH)], which display ocular features only (for reviews see 1 and 2). Axenfeld anomaly patients have posterior embryotoxon and abnormal iris tissue crossing the angle formed between the iris and cornea (iridocorneal angle) and attaching to the trabecular meshwork. Rieger anomaly patients have all of the abnormalities seen in Axenfeld anomaly with the addition of iris changes such as hypoplastic stroma, a displaced pupil (corectopia) or extra holes in the iris (polycoria). The most important ocular feature of the Axenfeld–Rieger family of diseases is glaucoma, which develops in
50% of affected individuals (3). Rieger syndrome is a disease with ocular, facial, dental and umbilical anomalies as primary manifestations. Rieger syndrome is considered to be part of ARS, a spectrum of diseases that share many ocular characteristics (3). Rieger syndrome is the most extreme member of the family of diseases called ARS. The diseases of ARS have a wide spectrum of clinical features including tooth abnormalities, IH and glaucoma. IH is the mildest of the disorders characterized solely by ocular defects including abnormal development of the iris stroma and early-onset glaucoma (4). These related anomalies make up a group of diseases termed the anterior segment dysgenesis disorders. These anomalies may result from developmental arrest of the anterior segment structures derived from the neural crest cells that form the corneal endothelium, iris stroma and trabecular meshwork (3). The interference with corneal endothelial differentiation may result in anterior segment dysgenesis during early stages of ocular development (5). ARS has been genetically linked to loci at chromosomes 4q25, 6p25, 13q14 and very recently chromosome 11 (1,2,6–8). The most common site for chromosomal abnormalities associated with Rieger syndrome occurs in the region 4q25. This region encodes the homeodomain transcription factor PITX2, identified using a positional cloning method (6). PITX2 mutations have been associated with several patients with ARS and most mutations occur in the homeodomain (1,6,9,10). The affected locus at chromosome 6p25 encodes a forkhead-like transcription factor FOX-C1. Mutations in FOX-C1 have been found in ARS patients and Axenfeld–Rieger anomaly, which has the ocular phenotype of ARS without the syndromic features (7,11–13). A second locus for Rieger syndrome (Rieger syndrome II) was identified on chromosome 13q14, but the affected gene at this locus has not yet been identified (14). Recently, a fourth loci for Rieger syndrome has been identified on chromosome 11. This loci encodes the paired-like transcription factor PAX6 and small deletions of this gene are associated with ARS (8).
PITX2 is a member of the bicoid-like homeobox transcription factor family (6,15). The homeobox gene family members play fundamental roles in the genetic control of development, including pattern formation and determination of cell fate (for reviews see 16–18). The homeobox proteins contain a 60 amino acid homeodomain that binds DNA. PITX2 contains a lysine at position 50 in the third helix of the homeodomain that is characteristic of the Bicoid-related proteins (19–21). This lysine residue selectively recognizes the 3'-CC dinucleotide adjacent to the TAAT core (17,22). It has been shown that PITX2 can bind the DNA sequence 5'-TAATCC-3' (15), which is also recognized by Bicoid protein (23). Several PITX2 isoforms have been identified and all differ at the N-terminus (1,24–26). In this report we use the PITX2A isoform to study ARS mutations and phosphorylation.
Pitx2 is expressed very early during tooth development in the tooth bud epithelium (27–29). The expression of Pitx2 is restricted to the dental epithelium and Pitx2 transcripts can be detected as early as day 8.5 during mouse tooth morphogenesis (27,29). Pitx2 expression remains specific to the oral epithelium with a progressive restriction to the dental placodes, followed by high level expression in the dental lamina and enamel knot in embryonic tooth primordia. Postnatal expression is still detected in relatively undifferentiated epithelial tissue in the tooth germs, in the later developing second and third molar anlage. Pitx2 transcripts are found in the pre-ameloblasts, although the levels are lower, and it is absent from the fully differentiated ameloblasts (27). We have recently shown the presence of Pitx2 transcripts and protein in a cell line (LS-8) derived from neonatal mouse molar epithelium (30).
Patients with Rieger syndrome present clinically with missing teeth among other anomalies (6). Teeth anomalies occur as abnormally small teeth (microdontia) giving rise to spaces between teeth, misshapened teeth, and missing teeth (hypodontia). The clinical presentations of ARS patients with regard to tooth anomalies are varied and may include all of the aforementioned anomalies or only one. In older ARS patients it has been observed that their teeth can become brittle and they suffer tooth loss. The analysis of Rieger syndrome patients provided the first link of PITX2 involvement in tooth development. We have previously shown that some of the naturally occurring PITX2 mutations associated with Rieger syndrome are defective for either DNA binding or transcriptional activation (15). Thus, the molecular basis of tooth anomalies in Rieger syndrome appear to be the inability of PITX2 to activate genes involved in tooth morphogenesis (for a review see 1). Taken together, these data support an early role for PITX2 in tooth morphogenesis.
Outside of the pituitary only two target genes of PITX2 have been identified to date. We have shown that the PLOD (procollagen lysyl hydroxylase) and Dlx2 genes are regulated by PITX2 (30,31). Since the pituitary is largely unaffected in ARS patients the molecular basis of the developmental anomalies must reside in genes that regulate the development of the affected structures.
Dlx2, a member of the distal-less gene family, has been established as a regulator of branchial arch development (32,33). Homozygous mutants of Dlx2 have abnormal development of forebrain cells and craniofacial abnormalities in developing neural tissue, Dlx genes exhibit both sequential and overlapping expression, implying that temporospatial regulation of Dlx genes is tightly regulated (34). Within the mandibular and maxillary divisions of the first branchial arch, whose mesenchyme and epithelium eventually form the teeth, Dlx2 is expressed proximally in the mesenchyme and distally in the epithelium (33). Dlx genes are believed to play a role in tooth morphogenesis because homozygous Dlx1/Dlx2 mutants are missing maxillary molars (35). There are no obvious tooth abnormalities in either Dlx1 or Dlx2 mutant mice. However, these mice do show abnormalities in development of specific facial skeletal elements (32,36). The Dlx1 and Dlx2 double mutant mice show normal development and cyto-differentiation of the incisors and mandibular molars in newborn mice but the maxillary molar teeth were missing (35). Maxillary molar tooth development was halted at the epithelial thickening stage and no bud- or cap-stage tooth germs were identified. In Pitx–/+ mutant mice the tooth phenotypes are small, misshapen or mal-occluded teeth (37). Pitx2hd–/– mice mandibular teeth arrested as tooth buds and maxillary teeth arrested at the placode stage (38). Thus, there appear to be differences in the teeth phenotypes between the Pitx2 and Dlx1/Dlx2 mutant mice. However, it is also clear that humans with PITX2 mutations present with different phenotypes than mice.
We are investigating the molecular basis of selected PITX2 mutations causing Rieger syndrome and related diseases. In this report we provide evidence for the molecular mechanism of two phenotypically different ARS mutations using a naturally occurring promoter that is required for tooth development. Our data demonstrate that the PITX2 R84W mutation produces a protein that has identical DNA binding specificity to the wild-type protein and can activate the Dlx2 promoter at levels we speculate are high enough to allow for normal tooth development in this patient. In contrast, an ARS mutation, PITX2 T68P, which affects tooth development is unable to transactivate the Dlx2 promoter. The patient with this mutation presents with abnormal tooth development. While we initially speculated that the PITX2 T68P mutation inhibited protein kinase C (PKC) phosphorylation at this site we were surprised to find that this mutation acts to increase phosphorylation at this site but has little effect on the activity of this mutant.
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RESULTS |
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Dlx2 gene expression is differentially regulated by ARS PITX2 mutations
We have previously reported that the naturally occurring full-length Dlx2 promoter was activated by PITX2 (30). The Dlx2 promoter contains multiple bicoid and bicoid-like sites as denoted in Figure 1A. The Dlx2 promoter contains three consensus 5'-TAATCC-3' bicoid sites and five non-consensus bicoid sites that have one nucleotide substitution within the DNA element. These bicoid-like elements are 5'-TATTCC-3', 5'-TTATCC-3', 5'-TAAGCC-3', and 5'-CAATCC-3'. We have previously shown that PITX2 can bind to these elements (30,39). The Dlx2 promoter was linked to the luciferase gene (Fig. 1A) and used as the reporter plasmid. We compared the activities of the minimal Dlx2 promoter (Dlx2-200-luc) which contains only the TAATAA box and 200 bp of 5' flanking sequences to the full-length promoter (Dlx2-3276-luc) containing all of the upstream regulatory elements (Fig. 1A). Transfection of Chinese hamster ovary (CHO) cells with Dlx2-3276-luc and PITX2 resulted in 30-fold activation of this promoter, which contains multiple bicoid and bicoid-like elements (Fig. 1B). Although there do not appear to be any bicoid-like sites within the minimal promoter of Dlx2 we observe a 4-fold activation in the presence of PITX2. PITX2 does not activate the same luciferase construct containing the TK promoter, suggesting that it is not binding a site in the vector (Fig. 1B) (15). We use this construct since it is not activated by PITX2 compared to the pGL3 vectors (Promega, Madison, WI) which contain multiple bicoid sites in the vector sequence and are activated by PITX2 (data not shown). Thus, we cannot rule out that PITX2 may be interacting with other transcription factors to activate transcription from this construct or that it is binding to other DNA sites in the minimal promoter. Two ARS PITX2 mutations, PITX2 T68P and PITX2 R84W, were co-transfected with the full-length and minimal Dlx2 promoter. We have previously shown that PITX2 T68P was unable to activate an artificial TK-Bicoid-luciferase reporter (15). Here we demonstrate that PITX2 T68P is also unable to transactivate the naturally occurring Dlx2 promoter (Fig. 1B). Surprisingly, PITX2 R84W activated Dlx2-3276-luc 6-fold and did not activate the Dlx2-200-luc minimal promoter reporter (Fig. 1B). Similar results were observed with transfected HeLa cells (data not shown). Western blot analysis was performed using 10 µg transfected cell lysates to demonstrate equal expression of each PITX2 protein. We used an antibody against PITX2 (P2R10), which recognizes an epitope in the N-terminus flanking the homeodomain (28). All three proteins are expressed in similar amounts and appear to be stable in these cells (Fig. 1C).
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DNA binding specificity of the PITX2 R84W mutant protein
Since the PITX2 R84W mutation affected the transcriptional properties of PITX2 we wanted to determine whether this was due to a lack of DNA binding specificity. We have previously shown that the PITX2 T68P mutant had reduced bicoid DNA binding and a loss of DNA binding specificity (15). To examine PITX2 and PITX2 R84W DNA binding activity these proteins were expressed in bacteria as glutathione S-transferase (GST) fusion proteins and the GST moiety was cleaved from the protein. The cleaved proteins were analyzed on an SDS-polyacrylamide gel and detected by Coomassie blue staining (data not shown). All protein preparations were quantitated and 80 ng of protein were used in the electrophoretic mobility shift assays (EMSAs). Unlike the PITX2 T68P mutant, the PITX2 R84W mutant bound to the bicoid element (5'-TAATCC-3') with similar activity compared to wild-type (Fig. 2A). We next asked whether the mutation affected binding specificity. To determine the specificity of PITX2 R84W binding, competition analyses were performed using non-specific oligonucleotides and oligonucleotides matching known homeobox binding sites. Addition of 50-fold molar excess of unlabeled DNA containing sites for the Hmx, Msx and Ftz classes caused <20% competition for PITX2 binding to the bicoid probe (Fig. 2C). In contrast, >85% of the PITX2–bicoid DNA complex could be competed using unlabeled bicoid DNA for both wild-type and PITX2 R84W (Fig. 2B and C). Interestingly, the P3 paired site (5'-TAATCTGATTA-3') can also compete for both wild-type and PITX2 R84W binding to the bicoid element (Fig. 2). We also tested other binding sites and found no loss of binding specificity by PITX2 R84W (data not shown). These data indicate that the PITX2 R84W mutant protein has the same DNA binding specificity as wild-type PITX2.
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Phosphorylation of PITX2 by PKC
Analysis of the PITX2 protein revealed 10 consensus PKC sites (S/T X K/R, K/R XX S/T, K/R X S/T), located throughout the PITX2 protein (Fig. 3A). There also appears to be several casein kinase II (CKII) sites (S/T XX EX) located within the PITX2 protein. We asked if PITX2 could be phosphorylated by these two serine/threonine kinases (PKC and CKII) and a third serine/threonine kinase [protein kinase A (PKA)] all known to phosphorylate homeodomain proteins. PITX2 protein (1.0 µg) purified from bacteria as described previously (2) was incubated with CKII, PKA or PKC. We observed specific phosphorylation of PITX2 with PKC and no phosphorylation by either CKII or PKA (Fig. 3B). The phosphorylation of PITX2 was very efficient as the gel shown in Figure 3B was exposed to film for only 30 min and quantitation of the gel reveals a high level of phosphorylation by PKC.
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Two PITX2 mutant proteins associated with ARS are phosphorylated by PKC
The PITX2 R84W mutant retains normal DNA binding activity and reduced transcriptional activity while the PITX2 T68P mutant has defective binding and transcriptional activity. We have shown that PITX2 is specifically phosphorylated by PKC. The molecular mechanism of the PITX2 T68P mutant appears to be due to reduced DNA binding (15). However, abnormal phosphorylation of PITX2 and PITX2 mutants could affect their activities during development.
A possible mechanism for the loss of transcriptional activity by PITX2 mutations associated with ARS could be due to inefficient phosphorylation. To examine this possibility we determined the phosphorylation efficiency of the PITX2 T68P and PITX2 R84W mutant proteins. The PKC site located in the homeodomain is changed by the PITX2 T68P mutation, which places a proline residue between the serine and arginine residues of the S/T X K/R PKC site (Fig. 3A). While this change is not predicted to disrupt this site we asked if it had any influence on its activity. Quantitation of the phosphorylated proteins revealed an increase in PITX2 T68P phosphorylation by PKC compared to wild-type (Fig. 4A and B). Phosphorylation of PITX2 R84W was identical to wild-type as expected since this mutation does not appear to affect a PKC site in the protein. To further analyze this effect we expressed and purified from bacteria the wild-type homeodomain peptide, PITX2 HD, and the PITX2 T68P peptide homeodomain, PITX2 T68P HD. These peptides were then phosphorylated by PKC and analyzed on a denaturing polyacrylamide gel and quantitated using the Molecular Dynamics STORM PhosphoImager (Molecular Dynamics, Amersham Pharmacia Biotech, Piscataway, NJ). The wild-type homeodomain is efficiently phosphorylated but unexpectedly we observed a 1.5–2.0-fold increase in phosphorylation of the PITX2 T68P homeodomain peptide compared to PITX2 HD (Fig. 5). Interestingly, the T68P mutation appears to increase the efficiency of phosphorylation at this site. Furthermore, this result suggests that phosphorylation occurs through a serine residue at this site, since the threonine was replaced with a proline. We have shown through PITX2 mutational studies that serine residues are phosphorylated in the consensus PKC sites (L.Sutherland, H.Espinoza, K.Chappell, J.Hall, P.Green, T.Hjalt and B.Amendt, submitted for publication). The naturally occurring ARS PITX2 mutation in the HD supports our mutational studies demonstrating phosphorylation of serine residues.
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PITX2 transcriptional activity is stimulated by phorbol 12-myristate 13-acetate (PMA)
Since PITX2 was phosphorylated by PKC in vitro we next asked if PKC activated phosphorylation by PMA would increase PITX2 and PITX2 mutant transcriptional activity. Addition of PMA to PITX2 transfected CHO cells increased the activation of the Dlx2-3276-luc reporter from 30-fold activation to 65-fold (Fig. 6). PMA alone (without PITX2 expression) activated the Dlx2-3276-luc promoter 3-fold compared to controls without PMA (Fig. 6). We observed a small activation of the minimal Dlx2-200-luc reporter with PITX2 and addition of PMA from 4-fold activation to 8-fold (Fig. 6). This is not unexpected since phosphorylation of PITX2 stimulates its transcriptional activity as seen with the full-length Dlx2 promoter. Thus, these data could suggest that PITX2 phosphorylation acts to facilitate protein interactions. Addition of PMA to PITX2 R84W transfected CHO cells increased the activation of Dlx2-3276 reporter from 6-fold to 13-fold while PITX2 T68P transcriptional activity was not affected (Fig. 6). Thus, we have shown that activated PKC by PMA results in increased PITX2 and PITX2 R84W transcriptional activity but not PITX2 T68P.
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Phosphorylation of PITX2 and PITX2 R84W acts to increase its DNA binding activity
We have shown that PITX2 is phosphorylated by PKC and that activation of PKC causes an increase in PITX2 transcriptional activity. But we did not understand the mechanism of this increased transcriptional activity. One possible explanation would be increased DNA binding activity. To examine this possibility we performed EMSAs with phosphorylated PITX2 proteins to determine whether phosphorylation affected DNA binding. Two concentrations of purified PITX2 and mutant proteins were analyzed on native polyacrylamide gels. We observed increased DNA binding for both the PITX2 and PITX2 R84W phosphorylated proteins. Interestingly, phosphorylation acts to facilitate dimer formation of these proteins (Fig. 7). Quantitation of the EMSA gels demonstrated a 2-fold increase in binding by both PITX2 and PITX2 R84W proteins. In contrast, the phosphorylated PITX2 T68P mutant protein demonstrated only a slight increase in binding to the bicoid probe (Fig. 7). The PITX2 T68P mutant protein has an overall reduced DNA binding activity for the Dlx2 bicoid labeled oligonucleotide (Fig. 7). This is somewhat different from what we reported for PITX2 T68P binding to our original artificial bicoid probe (15). Using the naturally occurring Dlx2 bicoid probe we observe significantly less binding, demonstrating that sequences flanking the bicoid site can influence PITX2 binding. Therefore, our data indicate that the PITX2 R84W mutation causes a less severe phenotype because the protein can bind DNA similar to the way in which wild-type is activated by PKC phosphorylation and retains some transcriptional activity. However, the PITX2 T68P mutation produces a protein with increased phosphorylation activity but reduced DNA binding activity and defective transcriptional activity.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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In this report we provide direct evidence using a natural PITX2 target gene for the molecular basis of the phenotypic variation associated with ARS. We have focused on two PITX2 mutations associated with ARS, which present clinically with very different phenotypes. The PITX2 T68P mutation was identified in an individual with Rieger syndrome, the most severe of the ARS disorders, and clinically presents with the full spectrum of development abnormalities (6,15) These defects include ocular anterior chamber anomalies causing glaucoma, dental hypoplasia, mild craniofacial dysmorphism and umbilical stump abnormalities. The PITX2 R84W mutation was identified in 15 patients of a five-generation pedigree with IH (4,40). This mutation represents the least severe phenotype associated with the ARS disorders, affecting mainly eye development. However, one patient did present with tooth anomalies but this may be due to other causes. Both of these disorders are characterized by ocular defects and the lack of other anomalies in IH suggests that these mutations have distinct molecular effects during development.
Different DNA binding and transcriptional properties of two ARS mutant proteins
We have previously shown that the PITX2 T68P mutant protein has reduced DNA binding activity, loss of binding specificity and defective transcriptional activation (1,15). We have compared the DNA binding and transcriptional activity of PITX2 T68P to the phenotypically less severe mutation PITX2 R84W. In contrast to the PITX2 T68P mutant, the PITX2 R84W mutant retained normal DNA binding and specificity compared to wild-type protein. It was recently reported that the PITX2 R84W mutation caused a reduction in binding activity, but binding specificity was not addressed by those investigators (9). A possible explanation for the differences may be due to their use of proteins expressed in nuclear extracts with a 4 kDa flag epitope tag and inability to quantitate the amount of PITX2 proteins used in their experiments. However, both of our PITX2 T68P mutant proteins demonstrated reduced DNA binding activity. A possible explanation for the lack of or reduced transcriptional activity by these mutant proteins could be a change in their binding specificity. This would allow binding to different DNA elements which might not be present in the natural targets of PITX2. To address this we asked if these mutant proteins could bind more efficiently to other known homeodomain binding sites. We have shown that the PITX2 T68P mutation produces a protein with altered DNA binding specificity, which is in contrast to the PITX2 R84W mutant protein reported in this study.
The transcriptional properties between these two mutant proteins are also quite different. We have recently identified the Dlx2 promoter as a natural target of PITX2 (30). Dlx2, a member of the distal-less gene family, has been established as a regulator of branchial arch development (32,33). Within the mandibular and maxillary divisions of the first branchial arch, whose mesenchyme and epithelium eventually form the teeth, Dlx2 is expressed proximally in the mesenchyme and distally in the epithelium (33). Pitx2 has been identified as the first transcriptional marker of tooth development and precedes Dlx2 expression. PITX2 binds to both consensus bicoid elements (5'-TAATCC-3') within the Dlx2 promoter and non-consensus bicoid elements such as 5'-TATTCC-3' and 5'-TTATCC-3'. However, PITX2 binds to the consensus bicoid element as a dimer, which we speculate facilitates transcriptional activation (30). We use the natural Dlx2 promoter to characterize the transcriptional properties of these two mutant proteins. Our data demonstrate a molecular basis for the lack of dental anomalies associated with the PITX2 R84W mutation since this mutant can activate the Dlx2 promoter 6-fold. PITX2 R84W, while able to bind DNA as a monomer similar to wild-type, does not form dimers similar to wild-type. Thus, one explanation for the lack of transcriptional activation may be its reduced ability to form homodimers. We have recently shown that PITX2 homodimers occur naturally in a cell line endogenously expressing PITX2 (30). However, the more phenotypically severe mutation, PITX T68P, is unable to activate the Dlx2 promoter. We speculate that the 6-fold activation of the Dlx2 promoter by PITX2 R84W would allow for normal tooth development in patients with this mutation. A dosage response model has been proposed to explain the differential organ development seen in Pitx2 heterozygous (+/–) and homozygous (–/–) mice (37). Recently, investigators have shown that thresholds of the PITX2C isoform dictate development of asymmetric organ morphogenesis (41). These models would explain the normal development of teeth in patients with the PITX2 R84W mutation as this protein would allow for some expression of the Dlx2 gene. ARS has been postulated to arise from haploinsufficiency and the PITX2 R84W mutant activity combined with the levels of PITX2 expressed from the normal allele would provide an increased Dlx2 dosage response compared to the PITX2 T68P mutant.
The role of PITX2 phosphorylation
We have shown that PITX2 is phosphorylated by PKC and that phosphorylation increases DNA binding activity. Here we demonstrate that two ARS mutant proteins are phosphorylated by PKC and the PITX2 T68P mutation acts to increase phosphorylation at that site. Since phosphorylation of wild-type protein increased its activity we expected that hyperphosphorylation of PITX2 T68P would produce a gain-of-function mutant. However, increased PITX2 T68P phosphorylation only had a minimal effect on DNA binding. Thus, the proline mutation probably imparts a conformational change in the protein that generally inhibits its DNA binding activity. Interestingly, the PITX2 T68P mutation suggests that the serine residue is phosphorylated by PKC, since removal of the threonine residue does not inhibit phosphorylation. Furthermore, phosphorylation of both PITX2 and PITX2 R84W acts to increase dimer formation. We speculate that PITX2 phosphorylation may also act to facilitate protein–protein interactions. We have previously shown that protein interactions can occur through the C-terminal tail of PITX2 and phosphorylation may regulate these interactions. Altogether, these results suggest that PITX2 DNA binding and transcriptional activation can be modulated by PKC.
We have provided evidence of how PITX2 mutations give rise to two ARS phenotypes and specifically a molecular basis for normal tooth development in one patient and dental anomalies in another. In ARS the PITX2 T68P point mutation lies in helix 2 at position 30 of the homeodomain, whereas the R84W point mutation is in helix 3 at position 46 of the homeodomain. We have found no other proteins with a proline at position 30 and it appears that this position can accommodate some changes in amino acid identity without a complete loss of DNA binding activity. Interestingly, our previous report using the PITX2 T68P mutant protein revealed only a 2-fold reduction in DNA binding activity using an artificial bicoid oligonucleotide (15). In this report we use the naturally occurring Dlx2 consensus bicoid element and flanking sequences and find that it binds poorly to the natural element. In the PITX2 R84W mutant the R residue at position 84 may be similar to the K residue of En, which contacts the sugar residue of the DNA backbone. However, our data indicate that this position can accommodate amino acid changes without adverse effects.
The molecular basis of ARS appears to result from reduced activation of PITX2 arising from the various missense mutations found in the homeodomain and C-terminal tail. However, we recently reported a dominant negative effect from co-expression of a PITX2 K88E mutation with the wild-type gene. These proteins may interact to abolish the interaction of wild-type with other PITX2 interacting proteins such as Pit-1 (42). Furthermore, a new study has identified two PITX2 mutations associated with ARS (10). Interestingly, one mutation appears to yield a mutant PITX2 protein with reduced DNA binding activity compared to wild-type but results in an increase in transcriptional activation. Our results indicate that PITX2 may also stimulate Dlx2 transcription without binding to a bicoid site as seen with the Dlx2 minimal promoter and TK promoter. However, we cannot rule out that it may be binding to a non-bicoid-like sequence in the Dlx2 minimal promoter. Thus, it appears that a combination of phosphorylation, inactivity, dominant negative and dominant positive mutations will all contribute to the variable ARS phenotypes.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Expression and reporter constructs
Expression plasmids containing the cytomegalovirus (CMV) promoter linked to the PITX2, PITX2 T68P and PITX2 truncated DNA were constructed in pcDNA 3.1 MycHisC (Invitrogen) (15,39). To make plasmid PITX2 R84W, which has a point mutation at position 84, an arginine was changed to a tryptophan by a two-step megaprimer PCR technique, described previously (15). The first PCR reaction to generate the megaprimer used a BamHI sense primer (5'-CGGGATCCCGGGGAAATGGAGACCAACTGCCGC-3') and an antisense primer to the PITX2 homeodomain sequence (5'-GAACCAAACCCAGACTCGGGCTTCCGT-3') where the point mutation is underlined. A HindIII antisense primer (5'-GGCCCAAGCTTCACGGGCCGGTCCAC-3') and the megaprimer were used to generate the mutant PITX2 cDNA. All PCR reactions were performed as described previously (15). Construction of the Dlx2 promoter plasmids were described previously (30). All constructs were confirmed by DNA sequencing. A CMV ß-galactosidase reporter plasmid (Clontech) was co-transfected in all experiments as a control for transfection efficiency.
Approximately 10 µg of transfected CHO cell lysates were analyzed in western blots. Following SDS gel electrophoresis, the proteins were transferred to PVDF filters (Millipore), immunoblotted and detected using PITX2 antibody P2R10 (28) and enhanced chemiluminescence reagents from Amersham Pharmacia Biotech.
Cell culture, transient transfections, luciferase and ß-galactosidase assays
CHO and HeLa cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 5% fetal bovine serum (FBS) and penicillin/streptomycin in 60 mm dishes and transfected by electroporation. CHO and HeLa cells were mixed with 2.5 µg of expression plasmids, 5 µg of reporter plasmid and 0.5 µg of CMV ß-galactosidase plasmid plated in 60 mm culture dishes and fed with 5% FBS and DMEM. Electroporation of CHO cells was at 360 V and 950 µF (Bio-Rad), cells were fed 24 h prior to transfection. HeLa cells were transfected by electroporation at 220 V and 950 µF, cells were fed 24 h prior to transfection. PMA activation of PKC was performed after transfection by adding 100 ng/ml to the culture medium. Transfected cells were incubated for 24 h then lysed and assayed for reporter activities and protein content by Bradford assay (Bio-Rad). Luciferase was measured using reagents from Promega. ß-Galactosidase was measured using the Galacto-Light Plus reagents (Tropix Inc.). All luciferase activities were normalized to ß-galactosidase activity. Expression of transiently expressed PITX2 proteins was demonstrated using our PITX2 antibody (28).
Expression and purification of GST–PITX2 fusion proteins
The human PITX2, PITX2 mutations and deletion constructs were PCR amplified from a cDNA clone as described previously (15). PITX2 T68P homeodomain was amplified using 100 ng PITX2 T68P DNA template (15), sense primer 5'-CCGGGATCCCAAAGGCGGCAGCGGACT-3' containing a BamHI site and antisense primer 5'-GTACTGCAGATGCGGCCGCCTCCCTCTTTCTCCATTTG-3' containing a NotI restriction site. The PITX2 PCR products were cloned into the pGex6P2 GST vector (Amersham Pharmacia Biotech) as described previously (15,39). The plasmids were transformed into BL21 cells. Protein was isolated as described previously (15). PITX2 proteins were cleaved from the GST moiety using 80 U PreScission Protease (Amersham Pharmacia Biotech) per ml of glutathione Sepharose. Purified proteins used in the binding assays have been previously described (39) or reported in this manuscript. The cleaved proteins were analyzed on SDS-polyacrylamide gels and quantitated by the Bradford protein assay (Bio-Rad).
Electrophoretic mobility shift assay
Complementary oligonucleotides containing a Dlx2 bicoid site with flanking partial BamHI ends were annealed and filled with Klenow polymerase to generate 32P-labeled probes for EMSAs, as described previously (43). The sequence of the sense oligonucleotide for probe Dlx TAATCC was 5'-gatccGCTCATGCCTGTAATCCCAGCACTCAGGg-3' and antisense 5'-gatccCCTGAGTGCTGGGATTACAGGCATGAGCg-3', leaving the four base overhangs (lower case) which were end filled and labeled. The Dlx2 bicoid site is underlined.
Standard binding assays were performed as described previously (39). Either 80 or 160 ng of the bacterial expressed and purified PITX2 proteins was used in the assays. The samples were electrophoresed, visualized and quantitated as described previously, except quantitation of dried gels was performed on the Molecular Dynamics STORM PhosphoImager (15).
Kinase assays
PITX2 and PITX2 mutant proteins (1 µg) were incubated in kinase buffer (50 mM Tris–Cl pH 8.0, 100 mM NaCl, 5 mM MgCl2, 4 mM DTT), kinase (PKA, Boehringer Mannheim, Indianapolis, IN), (CKII or PKC; Promega) and [
-32P]ATP (Amersham Pharmacia Biotech) for 30 min at 30°C. Proteins were then separated by electrophoresis on 12.5% polyacrylamide gels. Gels were exposed to imaging film (Kodak X-OMAT AR) and to a Phosphor screen for quantitation on the Storm 860 Phosphorimager system (Molecular Dynamics). Kinase assays were also performed with 5 µM bisindolylmaleimide (BIM, Sigma, St Louis, MO), a known inhibitor of PKC.
Phosphorylated proteins for gel shifts
Proteins were incubated with kinase buffer and 2.5 mM ATP in the presence or absence of PKC at 30°C for 30 min representing phosphorylated and unphosphorylated protein, respectively.
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ACKNOWLEDGEMENTS |
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We thank Kimberly Chappell, Lisa Morton, John Hall and Crystal (Zoe) Hansen for excellent technical assistance and Drs Jeffrey C.Murray and Andrew F.Russo (University of Iowa) for reagents and helpful discussions. We also thank Drs Paul Sharpe and Bethan Thomas (King’s College, University of London) for the Dlx2 promoter plasmid. Support for this research was provided from grants 1 RO1 DE13941 from the National Institute of Dental and Craniofacial Research and American Heart Association 9960299Z to Brad A.Amendt.
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FOOTNOTES |
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+ To whom correspondence should be addressed. Tel: +1 918 631 3328; Fax: +1 918 631 2762; Email: brad-amendt@utulsa.edu
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REFERENCES |
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