Lecture  |   April 2003
PITs and FOXes in Ocular Genetics The Cogan Lecture
Author Affiliations
  • Michael A. Walter
    From the Departments of Ophthalmology and Medical Genetics, University of Alberta, Edmonton, Alberta, Canada.
Investigative Ophthalmology & Visual Science April 2003, Vol.44, 1402-1405. doi:
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      Michael A. Walter; PITs and FOXes in Ocular Genetics The Cogan Lecture. Invest. Ophthalmol. Vis. Sci. 2003;44(4):1402-1405. doi:

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      © ARVO (1962-2015); The Authors (2016-present)

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The overall goal of the research in my laboratory is to gain better understanding of the molecular biology of the anterior segment and, in particular, to identify genes that regulate the development and function of this tissue. We have concentrated on discovering and characterizing the genes that, when mutated, cause anterior segment dysgenesis (ASD). ASD encompasses a spectrum of inherited autosomal-dominant diseases that result from maldevelopment of the anterior segment of the eye, with the most serious clinical consequence being that patients have an elevated risk of development of secondary glaucoma. Within the ASD disease spectrum, iris hypoplasia (IH) falls at the mild end, iridogoniodysgenesis (IGD; iris hypoplasia plus goniodysgenesis) is more severe, and Axenfeld-Rieger (AR) malformations (iris hypoplasia and goniodysgenesis plus corectopia, a prominent and displaced Schwalbe’s line, and adhesions between the iris and the cornea) falls at the most severe end of the spectrum. All conditions can occur with nonocular findings that may include dental, jaw, and umbilical anomalies. Cardiac and pituitary abnormalities have also been reported in patients with anterior segment findings. Glaucoma, associated with elevated IOP has been reported in approximately 50% of patients with AR malformations, and indications are that patients with IGD have greater than a 75% risk of development of glaucoma. In this article I will concentrate on our efforts to improve our understanding of the role in the anterior segment of two genes, PITX2 and FOXC1 (the PIT and FOX of the article’s title). 
PITX2, located on chromosome 4 at q25, was cloned in 1996. 1 It contains a homeobox, a 60-amino-acid domain originally described in fruit fly homeotic or embryonic patterning genes, that is responsible for binding to DNA, localizing PITX2 to the cell nucleus, and for protein-protein interactions. PITX2 is expressed in the developing eye, jaw, and umbilicus, as well as the heart and limb buds. PITX2 mutations have been found in patients with a range of anterior segment dysgenesis phenotypes, including IH, IGD, or AR malformations. 1 2 3 4 5 Members of my laboratory have assayed the affect on PITX2 function of different PITX2 mutations found in patients. 5 6 We found that PITX2 mutations can alter PITX2 nuclear localization, DNA binding, and transactivation and that the differences in amounts of functional PITX2 protein may be the basis of the spectrum of IH, IGD, and AR malformation phenotypes. DNA-binding ability, reporter gene transactivation, and nuclear localization of recombinant PITX2 proteins containing missense mutations of the PITX2 homeodomain identified in patients with IH, IGD, and AR malformations were assayed. 5 6 Missense mutations of the PITX2 homeodomain identified in patients with the IH (R46W), IGD (R31H), or AR malformation (L16G; T30P; R53P) were introduced into recombinant PITX2 cDNA by site-directed mutagenesis. PITX2 mutant proteins expressed in COS-7 cells were determined to be stable and to localize to the nucleus; however, the R53P AR mutant also displayed cytoplasmic staining. Analysis of the five mutant PITX2 proteins by electrophoretic mobility shift and transactivation assays demonstrated reduced activity of the IH and IGDS mutant PITX2 proteins, with the IH mutant retaining the most activity in both studies, although the mutant PITX2 proteins from patients with AR malformations were shown to have no DNA-binding or transactivation abilities. Thus, the wide range of phenotypic consequences due to mutations of PITX2 appears to correlate with the amount of residual PITX2 activity (Fig. 1 , top). 
Of interest, we also found that a PITX2 mutation that resulted in increased PITX2 activity was associated with disease. 5 A valine 45-to-leucine missense mutation of PITX2 (V45L) found in a patient with AR malformation resulted in a mutant PITX2 protein with an approximately 200% increase in activity compared with the normal PITX2 protein. This information allows us to suggest that there is both a lower and upper threshold of PITX2 activity that is necessary for normal anterior segment development. Taking into account that patients with a PITX2 mutant allele also still have a normal PITX2 allele, our data indicate that PITX2 mutations that result in tissues that have less than 70% of normal PITX2 activity or more than 150% of normal PITX2 activity result in human disease. It also remains a possibility that one or more of the PITX2 missense mutations may have an unrecognized gain-of-function effect in the developing organism, although the observation that frameshift and stop codon PITX2 mutations also result in AR malformations is consistent with the “threshold” model of PITX2 effect. These results clearly demonstrate that even subtle alteration of PITX2 activity causes human disease. 
FOXC1, located at chromosome 6 at p25, was shown to cause ASD in 1998. 7 8 It contains a forkhead domain (FKD), a 110-amino-acid domain that we have determined binds to DNA, localizes FOXC1 to the cell nucleus, and is likely to be involved in protein-protein interaction. FOXC1 is widely expressed in fetal and adult tissues. FOXC1 mutations underlie human ASD mapping to 6p25, and the mouse congenital hydrocephalus phenotype. 
We molecularly dissected FOXC1 to determine the portions that are necessary to localize FOXC1 to the cell nucleus and for FOXC1 activity. 9 The two nuclear localization signals (NLSs) in FOX family members, FOXA2 and FOXF2, are found in their FHD. 10 11 Of these, the FHD N-terminal NLSs do not match any typical NLS motif. To test whether such information resides in the FOXC1 FHD, mutations were created in the FOXC1 protein that either deleted the entire FHD or regions at the N- and C-terminal boundaries of the FHD. The cellular localization of these proteins was visualized by indirect immunofluorescence. The full-length FOXC1 protein was localized exclusively to the nucleus, whereas a FOXC1 molecule without the entire FHD displayed an increased cytoplasmic localization, indicating that the FHD contains information necessary for nuclear localization of FOXC1. Analysis of the amino acid sequence of the FOXC1 FHD revealed a span of basic amino acids at positions 168-176 that may serve as a potential NLS. Deletion of these amino acids from FOXC1 (Δ168-176) resulted in localization to both the cytoplasm and nucleus. Deletion of residues 78-93 displayed a mixture of cytoplasmic and nuclear localization, whereas FOXC1 Δ78-93+Δ168-176 resulted in a predominately mixed cytoplasmic and nuclear localization similar to that observed when the entire FHD was removed. Therefore, two regions in the FHD are necessary for correct FOXC1 nuclear localization. The two putative FOXC1 NLS domains were fused to green fluorescent protein (GFP) to determine whether these signals were sufficient for nuclear localization. 9 FOXC1 fused to GFP resulted in a localization of the GFP signal to the cell nucleus. Expression of GFP+(77-93) produced a signal that was localized to both the cytoplasm and the nucleus, indicating that this portion of FOXC1 is not sufficient for nuclear localization and therefore does not contain the FOXC1 NLS. Expression of GFP+(168-176), however, led to the accumulation of GFP exclusively in the nucleus. These data indicate that only residues 168-176 were sufficient to localize GFP to the nucleus and may constitute the FOXC1 NLS domain. Taken together, the findings in our studies indicate that sequences within the FOXC1 FHD are involved in nuclear localization of FOXC1. 
Multiple Regulatory Domains within FOXC1 Regulate FOXC1 Activity
We identified functional regions in FOXC1 required for transcriptional activity to understand better how mutations of FOXC1 result in AR malformations. 9 A series of N- and C-terminal deletions flanking the FOXC1 FHD were created and tested for the ability to activate expression. At present, there are no known transcriptional targets of FOXC1, therefore a luciferase reporter containing six copies of the in vitro-selected FOXC1 binding site (5′-GTAAATAAA-3′ 12 ) was used to monitor transcriptional activation. Transfection of cells with the full-length FOXC1 cDNA resulted in an approximate 10-fold induction of luciferase activity compared with the empty expression vector alone. When N-terminal amino acids 1-29 or 1-51 were deleted from FOXC1, activation of the luciferase reporter was reduced by 50%, indicating that residues in the N terminus are necessary for full activation. In addition, removal of C-terminal residues also impaired activation as FOXC1 1-435 reduced the luciferase reporter by 58%. These results indicate that FOXC1 contains segments from amino acids 1-51 in the N terminus, and from 435-553 in the C-terminal domain that are both required for efficient activation of the FOXC1 reporter. Expression of a FOXC1 protein that has neither the both N- nor the C-terminal regions (FOXC1 51-221) was incapable of activating luciferase expression above levels observed with the empty expression vector. Of note, FOXC1 1-366 activated luciferase expression to levels nearly equivalent to full length FOXC1, whereas FOXC1 1-215 activated luciferase to levels exceeding those observed for full-length FOXC1. These results suggest that further deletion of the C terminus removed an inhibitory domain located between residues 215 and 365 that led to an enhanced activation of the reporter gene by the N-terminal trans-activation domain. 
To determine whether the FOXC1 domains identified in our analyses were transferable, the FOXC1 N- and C-terminal activation domains were fused in-frame to the GAL4 DNA-binding domain (DB), and their ability to activate transcription was tested. 9 Full-length FOXC1, fused to the GAL4 DB, activated the GAL4 luciferase reporter by 10-fold compared with the GAL4 DB alone. Expression of N-terminal FOXC1 residues 1-30 and 1-65 activated luciferase expression to levels equivalent to full-length FOXC1, suggesting that the N-terminal regions are sufficient to activate transcription. GAL4 DB+435-553 was more than 10 times more active than full-length FOXC1 and 100-fold more active than baseline luciferase activity, indicating that the principal C-terminal activation domain lies in these residues. The activation mediated by GAL4+366-553 and GAL4+215-553 was considerably lower than that of GAL4+435-553, indicating that residues between 215-434 may constrain the activation domain present in residues 435-553. These results indicate that FOXC1 is a potent transcriptional activator under complex regulatory control. 
Phosphorylation of transcription factors is a common means of regulating their activity. When extracts of COS-7 cells transfected with the FOXC1 cDNA were analyzed for protein expression, we noticed that FOXC1 migrated as multiple immunoreactive bands on an SDS-polyacrylamide gel. 9 The amounts of the higher-molecular-weight proteins were reduced when extracts were incubated with calf intestinal alkaline phosphatase (CIP), suggesting that the multiple FOXC1 bands were a result of phosphorylation. Inhibition of CIP activity by sodium vanadate (VO3) and EDTA retained the higher-mobility FOXC1 bands. The addition of CIP rendered FOXC1 more sensitive to a limited trypsin digestion than untreated FOXC1 or FOXC1 treated with CIP and VO3, as indicated by the disappearance of the full-length FOXC1 band and the appearance of smaller tryptic fragments. These data suggest that dephosphorylation of FOXC1 alters FOXC1 conformation, making it more accessible to trypsin digest. The phosphorylated regions of FOXC1 were mapped by Western analysis of FOXC1 deletion constructs. FOXC1 residues 1-215 produced only a single immunoreactive band, whereas FOXC1 1-366 was detected as multiple FOXC1 bands. The migration of these additional bands was reduced by incubation with CIP. Additional FOXC1-immunoreactive bands were also observed for FOXC1 1-466 and 1-475. 
Based on these expression patterns, we conclude that the phosphorylation of FOXC1 resulting in altered migration occurs between residues 215 and 366. When these residues were removed (FOXC1 Δ215-366), the protein migrated as a single immunoreactive band, insensitive to CIP treatment. In addition, FOXC1 Δ215-366 activated the FOXC1 reporter to approximately two times higher levels than full-length FOXC1. Amino acids 215-366 of FOXC1 contain phosphorylated residues and may induce negative modulation of transcriptional activation by FOXC1. We next investigated whether residues 215-366 contained repressor activity and could impair other trans-activation domains. When residues 215-366 fused to GAL4 DB were transfected with a GAL4-luciferase reporter under the control of the constitutively active SV40 promoter, luciferase levels were equivalent to those observed with the GAL4 DB alone. The GAL4 activator domain (AD) fused to the GAL4 DB led to a robust activation of the GAL4-SV40-luc reporter. However, when GAL4+215-366 was fused to GAL4 AD, luciferase levels were markedly reduced. Therefore residues 215-366 of FOXC1 do not contain intrinsic transcriptional repressor activity; rather, these residues may serve to inhibit the ability of adjacent activation domains. These results are consistent with the hypothesis that FOXC1 activity is regulated by phosphorylation. 
Similar to our observations for PITX2, our recent results indicate that FOXC1 mutations can alter FOXC1 protein levels, DNA binding, and transactivation. 13 Five missense mutations of the winged helix FOXC1 transcription factor, found in patients with AR, were investigated for their effects on FOXC1 structure and function. 13 Molecular modeling of the FOXC1 FHD predicted that the missense mutations would not alter FOXC1 structure. Biochemical analyses indicated that although all mutant proteins correctly localize to the cell nucleus, the I87M mutation reduce FOXC1 protein stability. DNA-binding experiments revealed that whereas the S82T and S131L mutations decrease DNA binding, the F112S and I126M mutations do not. However, the F112S and I126M mutations decrease the transactivation ability of FOXC1. All these FOXC1 mutations had the net effect of reducing FOXC1 transactivation ability. Recently, several reports 14 15 16 have indicated that patients with duplications within 6p25 that include FOXC1 also have ASD. We have recently shown that IGD in our families originally linked to chromosome 6 at p25 17 also appears to result from duplication of an interval that contains FOXC1 (Lines M, Walter M, unpublished results, 2002). It seems that patients with null, frameshift, or missense mutations of FOXC1 fall within the AR malformation end of the ASD spectrum, whereas those with FOXC1 duplications (presumably resulting in increased FOXC1 activity) tend to have the milder IGD phenotype. Most interesting, patients with FOXC1 duplications may have elevated risk of development of high IOP and/or glaucomatous field defects compared with those with the AR malformation with mutations within FOXC1 (>75% in IGD vs. ∼50% in AR malformation). Additional genotype-phenotype analyses are needed to test this hypothesis. 
Thus, similar to the PITX2 finding, FOXC1 mutations resulting in less than 80% of normal FOXC1 activity in tissues (activity from the S82T FOXC1 allele plus that of the normal FOXC1 allele) or more than 150% of normal FOXC1 activity (duplicated FOXC1 plus normal FOXC1 allele) cause human disease. However, the wide range of phenotypic consequences due to mutations of FOXC1 does not appear to correlate with the amount of residual FOXC1 activity, unlike PITX2 mutations (Fig. 1) . For example, both the I87M with less than 5% of normal FOXC1 levels, and the S82T, with 56% of normal FOXC1 activity, were found in patients with AR malformations. Therefore, although mutations of both PITX2 and FOXC1 result in ASD, the developmental mechanisms and steps that are interrupted by these mutations may be different for PITX2 and FOXC1
Consequences of PITX2 and FOXC1 Mutations on Glaucoma Progression and Treatment
In very preliminary studies, we have initiated an examination of the glaucoma-related clinical presentation of individuals with PITX2 or FOXC1 mutations, to determine whether we can draw any useful phenotype-genotype comparisons. In four AR families studied, 11 (50%) of 22 patients with FOXC1 mutations had increased IOP and/or glaucomatous field defects. These data are consistent with the previous suggestions 18 19 that glaucoma develops in approximately 50% of patients with AR. Very preliminary data suggest that the glaucoma in patients with nonsense-null FOXC1 mutations is more difficult to treat medically than that in patients with missense FOXC1 mutations. As indicated earlier, families with duplications of FOXC1 appear to have IGD malformations and to have an increased incidence of elevated IOP and glaucoma (>75%). Patients with mutations in either PITX2 or FOXC1 had a similar propensity toward development of glaucoma and early age of onset (<20 years). 
Our anecdotal evidence suggests that the IOP and glaucoma in patients with nonsense-null mutations of either PITX2 or FOXC1 are more difficult to treat with medication. The observation that medications that act to reduce aqueous production to lower IOP appear not to be effective, suggests that the glaucoma in these patients may arise due to a progressive impairment of outflow facility, aqueous production refractive to medication, particularly pressure-sensitive retinal ganglion cells, or a combination of these factors. Our preliminary evidence from the investigation of the incidence of glaucoma and its progression in patients with different PITX2 or FOXC1 mutations may suggest that the different mutations differently impair eye function, leading to glaucoma, or alternatively, that partially functional mutant alleles can delay or partially protect the eye from the development of glaucoma. However, additional studies with a much larger sample of patients will be required to determine whether any of these preliminary findings can be substantiated. Ultimately, comparisons of the treatment outcomes with the underlying genetic defects in additional patients with PITX2 or FOXC1 mutations could result in improved glaucoma treatment and the identification of patients in whom existing medication does not work. 
The anterior segment phenotypes resulting from mutations of PITX2 or FOXC1 are quite variable and are overlapping. Mutations of these genes cause autosomal dominant diseases. A single normal copy is not sufficient for normal eye development. For PITX2 and FOXC1, having less than 50% or more than 150% of normal activity causes human disease. As a result, we can conclude that anterior segment formation is under extremely exquisite control. 
However, although the amounts of residual PITX2 activity correlate directly with anterior segment dysgenesis phenotypes, the amounts of FOXC1 activity do not. This indicates that, despite the finding that mutations of either PITX2 or FOXC1 result in very similar anterior segment dysgenesis phenotypes, PITX2 and FOXC1 must act in different ways in eye development. 
Finally, mutations of either PITX2 or FOXC1 account for no more than 35% of cases of ASD (Mirzayans F, Lines M, Walter M, unpublished observations, 2002). As a result, there are probably additional genes with significant, but as yet uncharacterized, roles in anterior segment development and glaucoma. Complete understanding of the molecular biology of ASD necessitates the unraveling of the functions of multiple genes with potentially multiple roles in eye development and maintenance. It is an exciting time, and I look forward to continuing to play a part in contributing to this fascinating area of research. 
Figure 1.
Comparison of the anterior segment dysgenesis phenotypes in patients with PITX2 or FOXC1 mutations with the amounts of residual PITX2 or FOXC1 activity. Top: mutations resulting in less than 70% of normal PITX2 activity (R46W) or more than 150% of normal PITX2 activity (V45L) result in human disease. The wide range of phenotypic consequences due to mutations of PITX2 appears to correlate with the amount of residual PITX2 activity. Bottom: mutations resulting in less than 80% of normal FOXC1 activity (S82T) or more than 150% of normal FOXC1 activity (FOXC1 duplication) result in anterior segment dysgenesis. The wide range of phenotypic consequences due to missense mutations in FOXC1 does not appear to correlate with the amount of residual FOXC1 activity, unlike PITX2 mutations.
Figure 1.
Comparison of the anterior segment dysgenesis phenotypes in patients with PITX2 or FOXC1 mutations with the amounts of residual PITX2 or FOXC1 activity. Top: mutations resulting in less than 70% of normal PITX2 activity (R46W) or more than 150% of normal PITX2 activity (V45L) result in human disease. The wide range of phenotypic consequences due to mutations of PITX2 appears to correlate with the amount of residual PITX2 activity. Bottom: mutations resulting in less than 80% of normal FOXC1 activity (S82T) or more than 150% of normal FOXC1 activity (FOXC1 duplication) result in anterior segment dysgenesis. The wide range of phenotypic consequences due to missense mutations in FOXC1 does not appear to correlate with the amount of residual FOXC1 activity, unlike PITX2 mutations.
I would like to thank the past and current members of Ocular Genetics Laboratory at the University of Alberta, a truly amazing group of scientists with whom I have had the great pleasure to work. In particular, I would like to mention Kathy Kozlowski, Ramsey Saleem, and Fred Berry for the PITX2 and FOXC1 studies I have discussed in this lecture. I have also had the privilege of working with a large group of collaborators from all over the world without whom we would not have been able to conduct the studies that I have described. Of these, Robert Ritch, Elise Héon, and Vincent Raymond deserve special acknowledgment for their patients, patience, and helpful discussions over the years. Finally, I’d like to acknowledge the loving support of my spouse, Kirsten. 
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