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Human Molecular Genetics Advance Access originally published online on August 4, 2005
Human Molecular Genetics 2005 14(18):2619-2627; doi:10.1093/hmg/ddi295
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© The Author 2005. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oupjournals.org

The establishment of a predictive mutational model of the forkhead domain through the analyses of FOXC2 missense mutations identified in patients with hereditary lymphedema with distichiasis

Fred B. Berry1,*, Yahya Tamimi2, Michelle V. Carle1, Ordan J. Lehmann1,2 and Michael A. Walter1,2

1Department of Ophthalmology and 2Department of Medical Genetics, University of Alberta, Edmonton, Alberta, Canada T6G 2H7

* To whom correspondence should be addressed at: 832 Medical Sciences Building, University of Alberta, Edmonton, Alberta, Canada T6G 2H7. Tel: +1 7804923028; Fax: +1 7804926934; Email: fberry{at}ualberta.ca

Received June 8, 2005; Revised July 28, 2005; Accepted July 28, 2005


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The FOX family of transcription factor genes is an evolutionary conserved, yet functionally diverse class of transcription factors that are important for regulation of energy homeostasis, development and oncogenesis. The proteins encoded by FOX genes are characterized by a conserved DNA-binding domain known as the forkhead domain (FHD). To date, disease-causing mutations have been identified in eight human FOX genes. Many of these mutations result in single amino acid substitutions in the FHD. We analyzed the molecular consequences of two disease-causing missense mutations (R121H and S125L) occurring in the FHD of the FOXC2 gene that were identified in patients with hereditary lymphedema with distichiasis (LD) to test the predictive capacity of a FHD structure/function model. On the basis of the FOXC2 solution structure, both FOXC2 missense mutations are located on the DNA-recognition helix of the FHD. A mutation model based on the parologous FOXC1 protein predicts that these FOXC2 missense mutations will impair the DNA-binding and transcriptional activation ability of the FOXC2 protein. When these mutations were analyzed biochemically, we found that both mutations did indeed reduce the DNA binding and transcriptional capacity. In addition, the R121H mutation affected nuclear localization of FOXC2. Together, these data indicate that these FOXC2 missense mutations are functional nulls and that FOXC2 haploinsufficiency underlies hereditary LD and validates the predictive ability of the FOXC1-based FHD mutational model.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The forkhead domain (FHD) is a conserved 100 amino acid DNA-binding motif that is present in eukaryotic organisms ranging from yeast to humans (1Go–4Go). The structure of the FHD from several forkhead box (FOX) proteins has been solved by X-ray crystallography and NMR spectroscopy (5Go–7Go). These data reveal that the FHD is a variant of the helix–turn–helix motif; consisting of three major {alpha}-helices, a smaller fourth {alpha}-helix and three anti-parallel ß-sheets. The third {alpha}-helix aligns along the major groove of DNA and residues in this region make a number of contacts with the DNA molecule. Thus, Helix 3 is referred to as the DNA-recognition helix. The region between the two ß-sheets, which lies C-terminal to the helical bundle, form two loops that wrap around the DNA molecule and stabilize the protein–DNA interaction. It is the presence of these loop regions that gives the FHD domain its characteristic winged-like appearance and lends the FHD its winged-helix moniker.

To date, at least 43 FOX transcription factor genes have been identified in humans (4Go). Many of these proteins regulate diverse cellular processes, including cell growth and differentiation, oncogenesis and metabolic homeostasis (2Go,3Go,8Go). Furthermore, disease-causing mutations have been identified in eight human FOX genes (2Go,3Go). Mutations in FOXC1 result in ocular malformations and glaucoma associated with Axenfeld–Rieger syndrome (9Go–11Go). Hereditary lymphedema with distichiasis (LD) is attributed to FOXC2 mutations (12Go–15Go). FOXE1 mutations underlie thyroid agenesis (16Go). Mutations in FOXE3 cause anterior segment ocular dysgenesis and cataracts (17Go). Blepharophimosis–ptosis–epicanthus inversus syndrome and premature ovarian failure are caused by FOXL2 mutations (18Go). Mutations in the FOXN1 gene result in T-cell immunodeficiency, congenital alopecia and nail dystrophy (19Go). Speech acquisition disorders have been attributed to FOXP2 mutations (20Go). The X-linked syndrome of immunodysregulation, polyendocrinopathy and enteropathy is caused by mutations in FOXP3 (21Go,22Go). A summary of disease-causing mutations in FOX genes is presented in Table 1.


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  		Table 1. Summary of disease-causing mutations in FOX genes
 
LD is a rare developmental disorder that affects the formation of the lymphatic vasculature system (23Go,24Go). Affected individuals typically exhibit distichiasis, an accessary row of eyelashes. These superfluous lashes often abrade the cornea resulting in corneal epithelial defects and opacification. Additional features of FOXC2 mutations may include cardiac defects, cleft palate and extradural cysts (12Go). The reported mutations primarily comprise of nonsense mutations, insertions or deletions that introduce a premature termination codon and are predicted to form a truncated FOXC2 protein (12Go–15Go,25Go,26Go). Three nucleotide missense mutations that cause amino acid substitutions (W116R, R121H and S125L) in the FOXC2 coding region have been reported in LD patients (26Go,34Go). However, the molecular consequences of these FOXC2 mutations have not been assessed.

The analyses of disease-causing missense mutations provide valuable information regarding structure–function relationships. To date, the molecular consequence of 13 disease-causing mutations in the FOXC1 FHD have been analyzed in our laboratory (27Go–30Go). Mutations in residues that lie N-terminal to and in Helix 1 reduce DNA binding, impede nuclear localization and impair transactivation. Helix 2 amino acid mutations can impair transcriptional activation; however, do not affect nuclear localization or DNA binding. Mutations to residues in Helix 3 affect nuclear localization and grossly impair DNA binding and specificity. Mutations to residues in Wing 2 impair DNA binding and transactivation. These analyses provide a framework for a predictive model for the functional consequence of disease-causing missense mutations in the FHD of other FOX genes. In this report, we test the predictive ability of our FHD mutational model through the molecular analyses of the two known missense mutations in the FOXC2 FHD identified in patients with LD (26Go). Two FOXC2 mutations, R121H and S125L, occur at parologous residues to FOXC1. On the basis of the location of these residues in Helix 3 of the FHD, we predict that both of the FOXC2 missense mutations would impede DNA binding and subsequent transcriptional activation. As an initial assessment of our predictive FHD mutational model, we analyzed the molecular consequences of these FOXC2 missense mutations.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The FHD of FOXC1 and FOXC2 share 98% sequence identity differing at only two positions. FOXC2 contains glutamic acid residues at positions 90 and 110, whereas FOXC1 contains aspartic acid residues at the equivalent position in the FHD (Fig. 1A). Outside of the FHD, FOXC1 and FOXC2 share only about a 36% amino acid sequence identity. Similar disease-causing mutations to R121H and S125L occur at parologous positions in FOXC1 (Fig. 1A) and lie in the third {alpha}-helix of the FHD (Fig. 1B). Because this helix is involved in contacts with DNA and previous molecular analysis of mutations in Helix 3 is critical for DNA binding and transcriptional activation (27Go,28Go,31Go), we predicted that the FOXC2 missense mutations, R121H and S125L, would exhibit similar molecular defects in their function.



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  		Figure 1. Comparison of FOXC1 and FOXC2 primary amino acid sequence. (A) The amino acid sequences for FOXC1 and FOXC2 were aligned with the ClustW program. The amino acid sequence within the forkhead domain differs by only two conservative substitutions of acidic residues. The locations of mutated residues that were analyzed in this study are indicated in bold. Outside of the FHD, there is little conservation of amino acid sequence. Asterisk denotes identical amino acid substitutions. ‘Colon’ indicates conservative substitutions. ‘Dot’ represents similar amino acid residues. (B) The location of the missense mutations analyses in this study is depicted on a molecular model of the FHD from FOXA3 complexed with DNA. Both the R121H and S125L mutations lie within Helix 3 (the recognition Helix) and are predicted to face toward the surface of the DNA backbone.

 
The two FOXC2 missense mutations were individually generated by site-directed mutagenesis to analyze the molecular deficit of the FOXC2 mutations and to test the predictive capacity of our FOXC1-based FHD mutational model. Wild-type (WT) and mutant FOXC2 cDNA were subcloned into the pcDNA4 HIS MAX expression vector in-frame to the amino terminal Xpress epitope tag. When transfected into COS-7 cells and detected by immunoblotting with an anti-Xpress antibody, FOXC2 was expressed as an approximate of 70 kDa protein (Fig. 2A). FOXC2 migrated at a lower molecular weight than FOXC1, consistent with the lower predicted molecular weight of FOXC2 compared with FOXC1. Both the R121H and S125L proteins were expressed at equal levels as WT FOXC2 indicating that these missense mutations do not affect protein stability. Multiple immunoreactive bands were detected in WT and mutant FOXC2 transfected COS-7 extracts, suggesting FOXC2 is subject to post-translational modifications, such as protein phosphorylation. Interestingly, the R121H displayed a faster migration than either WT FOXC2 or S125L suggesting that R121H may not be modified to the full extent as that of WT FOXC2 or S125L.



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  		Figure 2. Expression analysis of WT and mutant FOXC2 cDNAs transfected into COS-7 cells. (A) FOXC1, WT and mutant FOXC2 recombinant proteins were detected by immunoblotting with an {alpha}-Xpress antibody. (B) FOXC2 is a phosphoprotein. COS-7 extracts transfected with WT FOXC1, WT FOXC2, R121H or S125L were incubated with CIP and the phosphatase inhibitor sodium vanadate (VO3) as indicated. FOXC1 and FOXC2 proteins were detected by immunoblotting with an {alpha}-Xpress antibody.

 
To test whether protein phosphorylation contributed to the post-translational modifications of FOXC2, COS-7 extracts transfected with WT FOXC1, WT FOXC2, R121H or S125L were incubated with calf intestinal alkaline phosphatase (CIP). As indicated in Figure 2B, the addition of CIP increased the mobility of WT FOXC1 which is consistent with previous observations that FOXC1 is phosphorylated (32Go). The mobility of WT FOXC2, R121H and S125L was also increased when cell extracts were incubated with CIP. Remarkably, CIP-treated R121H extracts exhibited an equal mobility as that of WT FOXC2 and S125L suggesting that an incomplete phosphorylation pattern accounts for the reduced mobility of R121H observed in untreated extracts.

Next, the nuclear localization of FOXC2 missense mutations was examined. COS-7 cells were cotransfected with Xpress-tagged FOXC2 cDNAs along with FOXC1-EGFP. As indicated in Figure 3, WT FOXC2 was localized exclusively to the nucleus and displayed a similar subnuclear distribution as FOXC1-EGFP. In contrast, R121H displays a gross defect in nuclear localization. Much of the R121H immunofluorescence signal was detected in the cytoplasm. FOXC1-EGFP was still localized to the nucleus, indicating that the deficit in R121H nuclear localization was not the result of a disruption in the nuclear integrity of the cells transfected with the R121H expression vector. Immunoreactivity for S125L was localized mainly to the nucleus; however, a small proportion of signal could be detected in the cytoplasm. These results indicate that impaired nuclear localization accounts for a portion of the molecular deficit exhibited by these FOXC2 mutations and indicate a role for residues in Helix 3 in nuclear localization.



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  		Figure 3. The R121H mutation disrupts efficient nuclear localization of FOXC2. COS-7 cells were cotransfected with Xpress-tagged FOXC2 (WT or mutant) along with FOXC1-EGFP. The localization of FOXC2 was detected by indirect immunofluorescence with anti-Xpress antibodies. The cells were scored for the presence of WT FOXC2, R121H or S125L in the nucleus only (N), in the nucleus and cytoplasm (N+C) or in the cytoplasm only (C). More than 200 cells from two independent immunofluorescence experiments were scored.

 
To assess the impact of the FOXC2 mutations on the DNA-binding capacity of the protein, electrophoretic mobility shift assays (EMSAs) were performed. Whole cell COS-7 extracts transfected with WT FOXC1, WT FOXC2 or mutant FOXC2 expression vectors were incubated with an in vitro derived FOXC1 consensus site (5'-GTAAATAAA-3') to test for DNA binding (33Go). As indicated in Figure 4, both WT FOXC1 and FOXC2 bound to the FOXC1 target site. The enhanced DNA binding observed with FOXC2 when compared with FOXC1 is likely due to increased FOXC2 expression levels (Fig. 2). Minimal binding to the DNA probe was observed when the mutations R121H or S125L were introduced into FOXC2. Increasing the levels of protein extract failed to improve the binding of either R121H or S125L. These results indicate that these mutations to the Helix 3 grossly impaired FOXC2 binding to its DNA-target sequence.



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  		Figure 4. FOXC2 missense mutations abolish DNA-binding activity. Whole cell extracts from COS-7 cells transfected with FOXC1 or FOXC2 expression vectors were incubated with the in vitro derived FOXC1/2 consensus oligonucleotide probe (5'-GTAAATAAA-3'). FOXC1 or FOXC2 DNA–protein complexes are indicated with a square bracket. A non-specific DNA–protein complex was observed and indicated with an asterisk. Both R121H and S125L failed to bind to this sequence even when a 3-fold excess of protein was tested.

 
Finally, the ability of WT and mutant FOXC2 to activate a luciferase reporter containing six tandem copies of the FOXC1 consensus site was examined in HeLa cells. WT FOXC2 activated this luciferase reporter by at least 40-fold compared with the empty expression vector, indicating that FOXC2 is a potent transcriptional activator (Fig. 5). Neither R121H nor S125L was able to activate the luciferase reporter above background levels, indicating that these mutations rendered FOXC2 transcriptionally inactive.



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  		Figure 5. Impaired transcriptional activation by R121H and S125L mutations. Transactivation assays were performed in HeLa cells transfected with a 6x FOXC1/2 BS-Luc reporter along with FOXC1 or FOXC2 cDNAs. Luciferase values were normalized to Renilla luciferase. Mean luciferase values from a representative experiment transfected in triplicate are presented. Error bars correspond to the standard error of the mean.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
Analyses of the FOXC2 disease-causing missense mutations
In this report, we demonstrate that the disease-causing FOXC2 mutations in the FHD grossly impair its DNA-binding ability and subsequent transcriptional activation of a luciferase reporter gene. Furthermore, our findings indicate that the R121H mutation largely disrupts the proper nuclear localization of FOXC2. On the basis of these observations of greatly reduced functional activity, these mutations are essentially null mutations. In addition to these missense mutations, LD can result from FOXC2 nonsense mutations, as well as small insertions and deletions, all of which are predicted to produce a truncated FOXC2 protein (12Go–15Go,34Go). The expression of such truncated proteins does indeed reduce FOXC2 transcriptional activation (24Go). Thus, the hypothesis that FOXC2 haploinsufficiency underlies LD (12Go) is completely supported by the lack of functional activity exhibited by FOXC2 R121H and S125L missense mutations.

Although the FOXC2 missense mutations behaved similar to the parologous FOXC1 mutations, these studies revealed a difference between FOXC2 and FOXC1 in their genetic dosage requirements. Both FOXC2 missense mutations resulted in a complete loss of FOXC2 protein function. This is in contrast to FOXC1 disease-causing missense mutations in the FHD that retain residual DNA-binding and transcriptional regulatory activity (27Go–29Go). In this situation, the underlying genetic defect is not simply FOXC1 haploinsufficiency per se, rather, a threshold of FOXC1 activity must be achieved for the proper development of the anterior segment of the eye (27Go).

The targeted disruption of a single murine Foxc1 or Foxc2 allele can result in anterior segment dysgenesis, including iris hypoplasia, small or absent Schlemm's canal, aberrantly developed trabecular meshwork, eccentric pupils and displaced Schawlbe's line reminiscent of Axenfeld–Rieger malformations caused by FOXC1 mutations in humans (35Go). LD patients with FOXC2 mutations do not typically present with severe anterior segment findings and glaucoma; however, the FOXC2 missense mutations analyzed in this report or mutations that introduce a stop codon in the FHD are associated with milder anterior segment abnormalities (36Go). These findings suggest that an intact FOXC2 FHD is required for the development of the anterior segment of the eye. FOXC2 mutations that truncate the protein after the FHD exhibit a loss of transcriptional regulatory activity (24Go). In the eye, however, these truncating mutations could exhibit sufficient transcriptional activity for normal development to proceed. Alternatively, there may be a novel functional requirement for an intact FHD. This hypothesis suggests that a similar, although lower, minimal threshold for FOXC2 activity may be required in the development of the anterior segment of the eye as is required for FOXC1.

The examination of FOXC2 missense mutations revealed that both FOXC2 missense mutations exhibited a markedly impaired DNA-binding activity. The solution structure of FOXC2 indicates that the two affected residues lie within Helix 3 and their side chains are orientated away from this helix and toward the surface of DNA (Fig. 2B) (6Go). As the arginine and serine residues at positions 121 and 125 are conserved in all known 43 human FOX genes (Fig. 6) (4Go), it suggests a fundamental role for these residues in FHD function. X-ray crystallographic analysis of the related FOXA3 FHD reveals that the FOXA3 serine residue that is equivalent to FOXC2 serine 125 makes a phosphate contact with DNA backbone (5Go) indicating a similar role for FOXC2 serine 125 consistent with our observations that the S125L mutation has serious ramifications for DNA-binding activity. Although the arginine residue at position 121 in FOXC2 is not predicted to make a DNA contact, mutation of the equivalent arginine residues in other FOX proteins is predicted to eliminate the positively charged electrostatic surface potential of Helix 3 that is thought to be crucial for binding specificity of the FHD (28Go,31Go). In addition, this arginine residue is adjacent to a histidine residue that contacts the nitrogen base of the DNA backbone in the FOXA3 crystal structure (5Go). This mutation of a lysine to a histidine at position 121 may alter the protein conformation and impair this base contact. Thus, the FOXC2 missense mutations in Helix 3 impair DNA-binding ability which ultimately reduces FOXC2 transcriptional activity.



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  		Figure 6. (A) Assignment of functional subdomains to regions of the FHD based on the molecular consequences of mutations present in the FHD of FOXC1. This figure was adapted from Saleem et al. (31Go). (B) The comparison of known missense mutations in FOX genes. A multiple sequence alignment of the FHD from human FOX genes that contain known disease-causing missense mutations as well as naturally occurring or chemically induced missense mutations of murine Fox genes. Lowercase letters denote the individual amino acid residue that is affected by missense mutation in the corresponding FOX gene. Asterisks indicate mutations that occur in CpG dinucleotides.

 
These analyses also reveal the requirement for R121 in correct nuclear localization of FOXC2. This function may be conserved among other FOX proteins because the parologous R127H mutation in FOXC1 also displays impaired nuclear localization (28Go). Although the nuclear localization signal (NLS) for FOXC2 has not been identified, work from our laboratory defined the NLS of FOXC1 to two regions in the FHD: residues 78–93 in the N-terminus of the FHD and a stretch of basic amino acid residues at positions 168–176 (32Go). As these two regions are identical to both FOXC1 and FOXC2, it is likely that these residues also serve as the NLS for FOXC2. However, these proposed NLSs in FOXC2 do not encompass the arginine residue at position 121 of FOXC2 which is invariant among all known human FOX genes (4Go). Because the basic amino acid residues lysine and arginine are typically associated with NLSs, it is also possible that basic amino acid residues in Helix 3 possess auxiliary nuclear localization activity. Alternatively, mutation of this residue may alter the overall conformation of FOXC2 and prevent its interaction with proteins necessary for correct nuclear import. We consistently observed an altered mobility of R121H compared with WT FOXC2 and S125L by SDS–poly acrylamide electrophoresis, indicative of an altered phosphorylation state. Arginine residues are themselves not phosphorylated, therefore we propose that an altered topology of the R121H mutation impedes the interaction of FOXC2 with protein kinases. Because the nuclear import of many transcription factors can be regulated by their phosphorylation status (37Go,38Go), it is possible that nuclear import of FOXC2 may also be regulated by protein phosphorylation.

Extension of a FOXC1-based FHD mutation model and conservation of FHD functional subdomains
The analysis of disease-causing missense mutations in FOX genes can provide vital insights into structure–function relationships for this class of transcription factors. Our laboratory has extensively analyzed the molecular consequence of 13 missense mutations found in the FOXC1 FHD to establish a model that predicts the outcome of missense mutations occurring in FHD of any FOX gene (27Go–31Go). Such analyses permit assignment of functional subdomains to regions of the FHD (Fig. 6A) and indicate that the FHD possesses conserved activities that extend beyond DNA binding. The fact that the known FOXC2 FHD mutations behave in a similar manner to parologous FOXC1 mutations supports our assignment of these functional subdomains to the FHD and is an important initial step in verifying the predictive value of our FHD mutational model.

Disease-causing mutations have been identified in at least eight human FOX genes, and missense mutations occurring in the FHD have been identified in six FOX genes (9Go,10Go,17Go,20Go–22Go,39Go,40Go). Naturally occurring and chemically induced mutations occur in the FHD of two mouse Fox genes (41Go,42Go). These missense mutations are distributed along the entire length of the FHD and indicate regions of functional importance (Fig. 6B). In addition, missense mutations are detected at common amino acid residues in many FOX genes and several residues harbor mutations that are present in at least three different genes. It is of particular interest that three of the FHD mutation clusters illustrated in Figure 6B result in C->T or G->A transversions at CpG dinucleotides indicating that numerous mutational hotspots are present in FOX genes.

Although the two FOXC2 missense mutations analyzed in this study grossly disrupted protein function, all FHD missense mutations may not result in a null allele, as illustrated by many FOXC1 missense mutations retaining residual DNA-binding and transcriptional activation activity (27Go–31Go). Mutations to the FHD can have profound effects on FOX protein function, altering protein stability, nuclear localization, DNA binding and transcriptional activation (27Go–30Go). Moreover, in this study, individual missense mutations in the FHD can exert multiple functional consequences (27Go,28Go). Therefore, an optimal, predictive mutational model of the FHD will draw from both structural and functional information. To ascertain the functional consequence of an individual amino acid substitution, one must consider its localization in the three-dimensional structure of the FHD and its localization to a defined functional subdomain. It is this comparative analysis that will expand the predictive nature of the FHD mutation model to allow better genotype–phenotype correlations and to improve the prediction of the genetic mechanisms underlying diseases attributed to FOX genes. Our analyses of FOXC1 and FOXC2 missense mutations are the first step towards the development of truly predictive FHD mutational models.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Plasmids and mutagenesis
Because the human FOXC2 gene is encoded by a single exon, the full-length FOXC2 cDNA was PCR-amplified from genomic DNA using GC-Rich PCR kit (Roche). The primer pairs used for amplification are as follows: FOXC2F 5'-GGATCCATGCAGGCGCGCTACTCC-3'; FOXC2R 5'-TCTAGATCAGTATTTCGTGCAGTG-3'. The PCR amplification generated a BamH1 and XbaI sites into the 5' and 3' end of the FOXC2 cDNA, respectively. The FOXC2 cDNA was subcloned into a pcDNA4C-HIS MAX expression vector (Invitrogen) in which the ApaI site in the multiple cloning site was abolished, to create Xpress-FOXC2. For mutagenesis reactions, a portion of the FOXC2 cDNA containing the FHD and flanking the ApaI and Bsu36I restriction sites was amplified and subcloned into pGEMT-Easy to create a mutagenesis template. Site-directed mutagenesis was performed on this template as described previously (27Go). The mutagenic primers used were as follows: R121H 5'-CAGAACAGCATCCACCACAACCTCTCG-3'; S125L 5'-CGCCACAACCTCTTGCTCAACGAGTGC-3'.

The mutated FHD regions were subcloned back into the Xpress-FOXC2 expression vector. All expression constructs were sequenced to verify that no additional mutations were introduced.

Cell culture and transfections
COS-7 and HeLa cells were cultured in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum. For transient transfections, cells were plated at a density of 5x105 cells in a 60 mm plate. The next day, cells were transfected with 2 µg of either Xpress-FOXC1 or Xpress FOXC2 using 6 µl of FUGENE6 reagent. Cells were harvested 48 h following transfection and processed for immunoblotting as described previously (5Go).

Phosphatase treatment of cell extracts
Whole cell extracts were treated with CIP as described previously (32Go). Cell extracts were incubated in NEB Restriction Enzyme buffer 3 (New England Biolabs) along with 15 U of CIP (New England Biolabs). NaVO3 (11 µM) was added to inhibit CIP activity in some reactions. Extracts were incubated for 1 h at 37°C. An equal volume of 2x SDS–PAGE loading buffer was added and reactions were loaded onto a 10% polyacrylamide gel.

Immunofluorescence
COS-7 cells were plated onto sterile cover slips at a density of 105 cells per cover slip. Cells were transfected with either WT, R121H or S125L Xpress-FOXC2 (500 ng) along with FOXC1-EGFP (500 ng) using FUGENE6. Eighteen hours after transfection, cells were fixed with 2% paraformaldehyde for 15 min and processed for indirect immunofluorescence. Briefly, cells were permeablized with phosphate-buffered saline containing 0.1% (v/v) Triton X-100 (PBS-X), blocked with 5% (w/v) bovine serum albumin in PBS-X and incubated with {alpha}-Xpress antibodies (1 : 500) for 1 h at room temperature. After extensive washes in PBS-X, the cells were incubated with a Cy3-conjugated {alpha}-mouse IgG secondary antibody at dilution of 1 : 500 (Jackson Immunolabs). Cells were stained with DAPI and mounted onto slides with Prolong mounting medium (Molecular Probes).

Electrophoretic mobility shift assays
EMSAs were performed using COS-7 whole cell extract transfected with either Xpress-FOXC1 or Xpress-FOXC2 (WT or mutant) as described previously (27Go). Cell extracts were equalized for expression of the transfected expression vectors.

Dual luciferase assays
FOXC1 luciferase reporter assays were performed in 24-well plates as described previously (43Go). All transfections were performed in triplicate and each experiment was performed three times.

ACKNOWLEDGEMENTS
We thank Ms M. Yu for tissue culture expertise. This work was supported by grants to M.A.W. from the Canadian Institute for Health Research (CIHR) and a Bogue Research Fellowship and a Wellcome Trust Travel Award to O.J.L. M.A.W. is an Alberta Heritage Foundation for Medical Research Senior Scholar and a CIHR Investigator. O.J.L. is a recipient of a Canada Research Chair in Glaucoma Genetics and is an AHFMR Clinical Investigator.

Conflict of Interest statement. None declared.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

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