Foxc1 and Foxc2 function in osteochondral progenitors for the progression through chondrocyte hypertrophy and mineralization of the primary ossification center.

The forkhead box transcription factor genes Foxc1 and Foxc2 are expressed in the condensing mesenchyme of the developing skeleton prior to the onset of chondrocyte differentiation. To determine the roles of these transcription factors in limb development we deleted both Foxc1 and Foxc2 in lateral plate mesoderm using the Prx1-cre mouse line. Resulting compound homozygous mice died shortly after birth with exencephaly, and malformations to this sternum and limb skeleton. Notably distal limb structures were preferentially affected, with the autopods displaying reduced or absent mineralization. The radius and tibia bowed and the ulna and fibula were reduced to an unmineralized rudimentary structure. Molecular analysis revealed reduced expression of Ihh leading to reduced proliferation and delayed chondrocyte hypertrophy at E14.5. At later ages, Prx1-cre;Foxc1Δ/ Δ;Foxc2 Δ / Δ embryos exhibited restored Ihh expression and an expanded COLX-positive hypertrophic chondrocyte region, indicating a delayed exit and impaired remodeling of the hypertrophic chondrocytes. Osteoblast differentiation and mineralization were disrupted at the osteochondral junction and in the primary ossification center (POC). Levels of OSTEOPONTIN were elevated in the POC of compound homozygous mutants, while expression of Phex was reduced, indicating that impaired OPN processing by PHEX may underlie the mineralization defect we observe. Together our findings suggest that Foxc1 and Foxc2 act at different stages of endochondral ossification. Initially these genes act during the onset of chondrogenesis leading to the formation of hypertrophic chondrocytes. At later stages Foxc1 and Foxc2 are required for remodeling of HC and for Phex expression required for mineralization of the POC.

Assessment of Growth Plate Chondrocytes Proliferative Activity in Embryonic Endochondral Ossification via Ki-67 Immunofluorescence.

Cell proliferation is one of the key events that regulates organism development. In the limb, chondrocytes differentiate into a multi-layered cellular template called the growth plate. Chondrocyte proliferation is essential to provide the necessary cells that allow growth of a bone. Deregulated cell proliferation will lead to truncated bone elements. Immunofluorescence is a biological technique that uses specific antibodies to detect the subcellular localization of a proliferative marker within cellular or tissue context. In this chapter, we illustrate how to perform immunofluorescence to detect the localization of Ki-67 (a marker of actively growing/proliferating chondrocytes) in order to assess the growth fraction of the columnar chondrocytes in the growth plate in paraffin-embedded mouse tissue limb.

Loss of Foxc1 and Foxc2 function in chondroprogenitor cells disrupts endochondral ossification.

Endochondral ossification initiates the growth of the majority of the mammalian skeleton and is tightly controlled through gene regulatory networks. The forkhead box transcription factors Foxc1 and Foxc2 regulate aspects of osteoblast function in the formation of the skeleton, but their roles in chondrocytes to control endochondral ossification are less clear. Here, we demonstrate that Foxc1 expression is directly regulated by the activity of SRY (sex-determining region Y)-box 9, one of the earliest transcription factors to specify the chondrocyte lineage. Moreover, we demonstrate that elevated expression of Foxc1 promotes chondrocyte differentiation in mouse embryonic stem cells and loss of Foxc1 function inhibits chondrogenesis in vitro. Using chondrocyte-targeted deletion of Foxc1 and Foxc2 in mice, we reveal a role for these factors in chondrocyte differentiation in vivo. Loss of both Foxc1 and Foxc2 caused a general skeletal dysplasia predominantly affecting the vertebral column. The long bones of the limbs were smaller, mineralization was reduced, and organization of the growth plate was disrupted; in particular, the stacked columnar organization of the proliferative chondrocyte layer was reduced in size and cell proliferation was decreased. Differential gene expression analysis indicated disrupted expression patterns of chondrogenesis and ossification genes throughout the entire process of endochondral ossification in chondrocyte-specific Foxc1/Foxc2 KO embryos. Our results suggest that Foxc1 and Foxc2 are required for normal chondrocyte differentiation and function, as loss of both genes results in disorganization of the growth plate, reduced chondrocyte proliferation, and delays in chondrocyte hypertrophy that prevents ossification of the skeleton.

A Novel Locus Predicts Spermatogenic Recovery among Childhood Cancer Survivors Exposed to Alkylating Agents.

Exposure to high doses of alkylating agents is associated with increased risk of impaired spermatogenesis among nonirradiated male survivors of childhood cancer, but there is substantial variation in this risk. Here we conducted a genetic study for impaired spermatogenesis utilizing whole-genome sequencing data from 167 nonirradiated male childhood cancer survivors of European ancestry from the St. Jude Lifetime Cohort treated with cyclophosphamide equivalent dose (CED) ≥4,000 mg/m2. Sperm concentration from semen analysis was assessed as the primary outcome. Common variants (MAF > 0.05) were adjusted for age at cancer diagnosis, CED, and top principal components. Rare/low-frequency variants (MAF ≤ 0.05) were evaluated jointly by various functional annotations and 4-kb sliding windows. A novel locus at 7q21.3 containing TAC1/ASNS was associated with decreased sperm concentration (rs7784118: P = 3.5 × 10-8). This association was replicated in two independent samples of SJLIFE survivors of European ancestry, including 34 nonirradiated male survivors treated with 0 < CED < 4,000 mg/m2 (P = 3.1 × 10-4) and 24 male survivors treated with CED ≥4,000 mg/m2 and radiotherapy <40 Gray (P = 0.012). No association was observed among survivors not exposed to alkylating agents included in the CED (P > 0.29). rs7784118 conferred 3.48- and 9.73-fold increases in risk for clinically defined oligospermia and azoospermia and improved prediction of normospermic, oligospermic, and azoospermic states by 13.7%, 5.3%, and 21.7%. rs7784118 was associated with decreased testosterone level, increased levels of follicle stimulating and luteinizing hormones, and 8.52-fold increased risk of Leydig cell failure. Additional research is warranted to determine how this SNP influences spermatogenesis and to assess its clinical utility in characterizing high-risk survivors and guiding intervention strategies. SIGNIFICANCE: The identified genetic markers harbor potential clinical utility in characterizing high-risk survivors and guiding intervention strategies including pretreatment patient counseling and use of fertility preservation services.

FOXC1 negatively regulates BMP-SMAD activity and Id1 expression during osteoblast differentiation.

Bone morphogenetic proteins regulate a diverse range of biological processes through their activation of SMAD1, SMAD5, or SMAD8 proteins that, in turn, regulate gene expression. These SMAD transcription factors achieve a layer of functional specificity in different cell types largely through actions with additional transcriptional regulatory molecules. In this study, we demonstrate that the forkhead box C1 (FOXC1) transcription factor can modulate bone morphogenetic protein (BMP) signaling to impair the expression of BMP4-responsive genes and prevent the efficient osteoblast differentiation. We demonstrate that repression occurs downstream of BMP signaling and impacts the ability SMAD1 or SMAD5 to activate gene expression. Repression of SMAD activity requires FOXC1 DNA-binding capacity and the transcriptional inhibitory domain of FOXC1. We report that FOXC1 inhibits BMP4 induction of Id1 expression and identify a motif in the regulatory region of mouse Id1 gene that FOXC1 binds. We determine that this inhibition by FOXC1 binding does not affect SMAD1, SMAD5, or SMAD8 binding to its target sequence in the Id1 gene. Finally, we determine that the elevated expression of FOXC1 can reduces expression osteogenic differentiation genes in mouse embryonic stems directed to the osteoblast lineage through BMP4 treatment. Together, these findings indicate that FOXC1 can negatively regulate certain aspects of BMP4 signaling required for osteoblast differentiation. We propose that FOXC1 acts to attenuate the initial BMP-activated pathways that establish osteoblast differentiation and allow for terminal osteoblast differentiation to conclude.

Molecular analysis of NPAS3 functional domains and variants.

BACKGROUND: NPAS3 encodes a transcription factor which has been associated with multiple human psychiatric and neurodevelopmental disorders. In mice, deletion of Npas3 was found to cause alterations in neurodevelopment, as well as a marked reduction in neurogenesis in the adult mouse hippocampus. This neurogenic deficit, alongside the reduction in cortical interneuron number, likely contributes to the behavioral and cognitive alterations observed in Npas3 knockout mice. Although loss of Npas3 has been found to affect proliferation and apoptosis, the molecular function of NPAS3 is largely uncharacterized outside of predictions based on its high homology to bHLH-PAS transcription factors. Here we set out to characterize NPAS3 as a transcription factor, and to confirm whether NPAS3 acts as predicted for a Class 1 bHLH-PAS family member. RESULTS: Through these studies we have experimentally demonstrated that NPAS3 behaves as a true transcription factor, capable of gene regulation through direct association with DNA. NPAS3 and ARNT are confirmed to directly interact in human cells through both bHLH and PAS dimerization domains. The C-terminus of NPAS3 was found to contain a functional transactivation domain. Further, the NPAS3::ARNT heterodimer was shown to directly regulate the expression of VGF and TXNIP through binding of their proximal promoters. Finally, we assessed the effects of three human variants of NPAS3 on gene regulatory function and do not observe significant deficits. CONCLUSIONS: NPAS3 is a true transcription factor capable of regulating expression of target genes through their promoters by directly cooperating with ARNT. The tested human variants of NPAS3 require further characterization to identify their effects on NPAS3 expression and function in the individuals that carry them. These data enhance our understanding of the molecular function of NPAS3 and the mechanism by which it contributes to normal and abnormal neurodevelopment and neural function.

FOXC1 Regulates FGFR1 Isoform Switching to Promote Invasion Following TGFß-Induced EMT.

Epithelial-to-mesenchymal transition (EMT) is an important physiologic process that drives tissue formation during development, but also contributes to disease pathogenesis, including fibrosis and cancer metastasis. Elevated expression of the FOXC1 transcription factor has been detected in several metastatic cancers that have undergone EMT. Therefore, mechanistic insight into the role of FOXC1 in the initiation of the EMT process was sought. It was determined that although Foxc1 transcript expression was elevated following TGFß1-induced EMT of NMuMG cells, FOXC1 was not required for this induction. RNA sequencing revealed that the mRNA levels of FGF receptor 1-isoform IIIc (Fgfr1-IIIc), normally activated upon TGFß1 treatment, were reduced in Foxc1 knockdown cells, and overexpression of Foxc1 was sufficient to induce Fgfr1-IIIc expression, but not EMT. Chromatin immunoprecipitation experiments demonstrated that FOXC1 binds to an Fgfr1 upstream regulatory region and that FOXC1 activates an Fgfr1 promoter element. Furthermore, elevated expression of Foxc1 led to increased Fgfr1-IIIc transcript. Foxc1 knockdown impaired the FGF2-mediated three-dimensional migratory ability of NMuMG cells, which was rescued by expression of FGFR1. In addition, elevated expression of FOXC1 and FGFR1 was also observed in migratory mesenchymal MDA-MB-231 breast cancer cells. Together, these results define a role for FOXC1 in specifying an invasive mesenchymal cell type by promoting FGFR1 isoform switching following induction of TGFß1-mediated EMT. Mol Cancer Res; 15(10); 1341-53. ©2017 AACR.

Muscle dysfunction caused by loss of Magel2 in a mouse model of Prader-Willi and Schaaf-Yang syndromes.

Prader-Willi syndrome is characterized by severe hypotonia in infancy, with decreased lean mass and increased fat mass in childhood followed by severe hyperphagia and consequent obesity. Scoliosis and other orthopaedic manifestations of hypotonia are common in children with Prader-Willi syndrome and cause significant morbidity. The relationships among hypotonia, reduced muscle mass and scoliosis have been difficult to establish. Inactivating mutations in one Prader-Willi syndrome candidate gene, MAGEL2, cause a Prader-Willi-like syndrome called Schaaf-Yang syndrome, highlighting the importance of loss of MAGEL2 in Prader-Willi syndrome phenotypes. Gene-targeted mice lacking Magel2 have excess fat and decreased muscle, recapitulating altered body composition in Prader-Willi syndrome. We now demonstrate that Magel2 is expressed in the developing musculoskeletal system, and that loss of Magel2 causes muscle-related phenotypes in mice consistent with atrophy caused by altered autophagy. Magel2-null mice serve as a preclinical model for therapies targeting muscle structure and function in children lacking MAGEL2 diagnosed with Prader-Willi or Schaaf-Yang syndrome.

Contribution of growth differentiation factor 6-dependent cell survival to early-onset retinal dystrophies.

Retinal dystrophies are predominantly caused by mutations affecting the visual phototransduction system and cilia, with few genes identified that function to maintain photoreceptor survival. We reasoned that growth factors involved with early embryonic retinal development would represent excellent candidates for such diseases. Here we show that mutations in the transforming growth factor-ß (TGF-ß) ligand Growth Differentiation Factor 6, which specifies the dorso-ventral retinal axis, contribute to Leber congenital amaurosis. Furthermore, deficiency of gdf6 results in photoreceptor degeneration, so demonstrating a connection between Gdf6 signaling and photoreceptor survival. In addition, in both murine and zebrafish mutant models, we observe retinal apoptosis, a characteristic feature of human retinal dystrophies. Treatment of gdf6-deficient zebrafish embryos with a novel aminopropyl carbazole, P7C3, rescued the retinal apoptosis without evidence of toxicity. These findings implicate for the first time perturbed TGF-ß signaling in the genesis of retinal dystrophies, support the study of related morphogenetic genes for comparable roles in retinal disease and may offer additional therapeutic opportunities for genetically heterogeneous disorders presently only treatable with gene therapy.

Initiation of early osteoblast differentiation events through the direct transcriptional regulation of Msx2 by FOXC1.

Hierarchal transcriptional regulatory networks function to control the correct spatiotemporal patterning of the mammalian skeletal system. One such factor, the forkhead box transcription factor FOXC1 is necessary for the correct formation of the axial and craniofacial skeleton. Previous studies have demonstrated that the frontal and parietal bones of the skull fail to develop in mice deficient for Foxc1. Furthermore expression of the Msx2 homeobox gene, an essential regulator of calvarial bone development is absent in the skull mesenchymal progenitors of Foxc1 mutant mice. Thus we sought to determine whether Msx2 was a direct target of FOXC1 transcriptional regulation. Here, we demonstrate that elevated expression of FOXC1 can increase endogenous Msx2 mRNA levels. Chromatin immunoprecipitation experiments reveal that FOXC1 occupies a conserved element in the MSX2 promoter. Using a luciferase reporter assay, we demonstrate that FOXC1 can stimulate the activity of the both human and mouse MSX2 promoters. We also report that reducing FOXC1 levels by RNA interference leads to a decrease in MSX2 expression. Finally, we demonstrate that heterologous expression of Foxc1 in C2C12 cells results in elevated alkaline phosphatase activity and increased expression of Runx2 and Msx2. These data indicate that Foxc1 expression leads to a similar enhanced osteogenic differentiation phenotype as observed with Msx2 overexpression. Together these findings suggest that a Foxc1->Msx2 regulatory network functions in the initial stages of osteoblast differentiation.

PITX2 is involved in stress response in cultured human trabecular meshwork cells through regulation of SLC13A3.

PURPOSE: Mutations of the PITX2 gene cause Axenfeld-Rieger syndrome (ARS) and glaucoma. In this study, the authors investigated genes directly regulated by the PITX2 transcription factor to gain insight into the mechanisms underlying these disorders. METHODS: RNA from nonpigmented ciliary epithelium cells transfected with hormone-inducible PITX2 and activated by mifepristone was subjected to microarray analyses. Data were analyzed using dCHIP algorithms to detect significant differences in expression. Genes with significantly altered expression in multiple microarray experiments in the presence of activated PITX2 were subjected to in silico and biochemical analyses to validate them as direct regulatory targets. One target gene was further characterized by studying the effect of its knockdown in a cell model of oxidative stress, and its expression in zebrafish embryos was analyzed by in situ hybridization. RESULTS: Solute carrier family 13 sodium-dependent dicarboxylate transporter member 3 (SLC13A3) was identified as 1 of 47 potential PITX2 target genes in ocular cells. PITX2 directly regulates SLC13A3 expression, as demonstrated by luciferase reporter and chromatin immunoprecipitation assays. Reduction of PITX2 or SLC13A3 levels by small interfering RNA (siRNA)-mediated knockdown augmented the death of transformed human trabecular meshwork cells exposed to hydrogen peroxide. Zebrafish slc13a3 is expressed in anterior ocular regions in a pattern similar to that of pitx2. CONCLUSIONS: The results indicate that SLC13A3 is a direct downstream target of PITX2 transcriptional regulation and that levels of PITX2 and SLC13A3 modulate responses to oxidative stress in ocular cells.

Severe molecular defects of a novel FOXC1 W152G mutation result in aniridia.

PURPOSE: FOXC1 mutations result in Axenfeld-Rieger syndrome, a disorder characterized by a broad spectrum of malformations of the anterior segment of the eye and an elevated risk for glaucoma. A novel FOXC1 W152G mutation was identified in a patient with aniridia. Molecular analysis was conducted to determine the functional consequences of the FOXC1 W152G mutation. METHODS: Site-directed mutagenesis was used to introduce the W152G mutation into the FOXC1 complementary DNA. The levels of W152G protein expression and the functional abilities of the mutant protein were determined. RESULTS: After screening for mutations in PAX6, CYP1B1, and FOXC1, a novel FOXC1 W152G mutation was identified in a newborn boy with aniridia and congenital glaucoma. Molecular analysis of the W152G mutation revealed that the mutant protein has severe molecular consequences in FOXC1, including defects in phosphorylation, protein folding, DNA-binding ability, inability to transactivate a reporter gene, and nuclear localization. Although W152G has molecular defects similar to those of the previously studied FOXC1 L130F mutation, W152G causes a more severe phenotype than L130F. Both the W152G and the L130F mutations result in the formation of protein aggregates in the cytoplasm. However, unlike the L130F aggregates, the W152G aggregates do not form microtubule-dependent inclusion bodies, known as aggresomes. CONCLUSIONS: Severe molecular consequences, including the inability of the W152G protein aggregates to form protective aggresomes, may underlie the aniridia phenotype that results from the FOXC1 W152G mutation.

Human p32 is a novel FOXC1-interacting protein that regulates FOXC1 transcriptional activity in ocular cells.

PURPOSE: Mutations in the human forkhead box C1 gene (FOXC1) cause Axenfeld-Rieger (AR) malformations, often leading to glaucoma. Understanding the function of FOXC1 necessitates characterizing the proteins that interact with FOXC1. This study was undertaken to isolate FOXC1-interacting proteins and determine their effects on FOXC1. METHODS: To identify FOXC1-interacting proteins, a human trabecular meshwork (HTM) yeast two-hybrid (Y2H) cDNA library was screened. The interaction and colocalization between FOXC1 and its putative protein partner were confirmed by Ni(2+) pull-down assays, immunoprecipitation, and immunofluorescence, respectively. The electrophoretic mobility shift assay (EMSA) was used to study the effect of the interacting protein on FOXC1 DNA-binding ability. Dual luciferase assays using FOXC1 reporter plasmids in HTM cells were performed to determine the effect of the interaction on FOXC1 transcription activity. RESULTS: The human p32 protein was isolated as a putative FOXC1-interacting protein from a Y2H screen. The interaction of FOXC1 with p32 was confirmed by Ni-pull-down assays and immunoprecipitation. Although p32 is predominantly cytoplasmic, the portion of p32 that is within the nucleus colocalizes with FOXC1. The FOXC1 forkhead domain (FHD) was identified as the p32 interaction domain. p32 significantly inhibited FOXC1-mediated transcription activation in a dose-dependent manner but did not affect FOXC1 DNA-binding ability. Of interest, a FOXC1 mutation F112S displayed an impaired interaction with p32. CONCLUSIONS: In the study, the human p32 protein as a novel regulator of FOXC1-mediated transcription activation. Failure of p32 to interact with FOXC1 containing the disease-causing F112S mutation indicates that impaired protein interaction may be a disease mechanism for AR malformations.

FOXC1 is required for cell viability and resistance to oxidative stress in the eye through the transcriptional regulation of FOXO1A.

Mutations in the human FOXC1 transcription factor gene underlie Axenfeld-Rieger (AR) syndrome, a disorder characterized by anterior segment malformations in the eye and glaucoma. Through the use of an inducible FOXC1 protein, along with an intermediate protein synthesis blocker, we have determined direct targets of FOXC1 transcriptional regulation. FOXC1 regulates the expression of FOXO1A and binds to a conserved element in the FOXO1A promoter in vivo. The zebrafish foxO1a orthologs exhibit a robust expression pattern in the periocular mesenchyme. Furthermore, FOXO1A expression is reduced in cultured human trabecular meshwork (TM) cells and in the zebrafish developing eye when FOXC1 expression is knocked down by siRNAs and morpholino antisense oliognucleotides, respectively. We also demonstrate that reduced FOXC1 expression increases cell death in cultured TM cells in response to oxidative stress, and increases cell death in the developing zebrafish eye. These studies have uncovered a novel role for FOXC1 as an essential mediator of cellular homeostasis in the eye and indicate that a decreased resistance to oxidative stress may underlie AR-glaucoma pathogenesis. Given that FOXO1A influences cellular homeostasis when positively or negatively regulated; the dysregulation of FOXO1A activities in the eye through FOXC1 loss of function mutations and FOXC1 gene duplications provides an explanation into how seemingly similar human disorders can arise from both increases and decreases in FOXC1 gene dose.

FGF19 is a target for FOXC1 regulation in ciliary body-derived cells.

The forkhead C1 (FOXC1) transcription factor is involved in the development and regulation of several organs, including the eye, where FOXC1 alterations cause iris, trabecular meshwork and corneal anomalies. Using nickel agarose chromatin enrichment with human anterior segment cells, we previously identified the fibroblast growth factor 19 (FGF19) locus as a gene potentially regulated by FOXC1. Here, we demonstrate that FGF19 is a direct target of FOXC1 in the eye. FOXC1 positively regulates FGF19 expression in corneal and periocular mesenchymal cells in cell culture and in zebrafish embryos. Through the FGFR4 tyrosine kinase, FGF19 promotes MAPK phosphorylation in the developing and mature cornea. During development, loss of either FOXC1 or FGF19 results in complementary, but distinct, anterior segment dysgeneses. This study reveals an important role for FOXC1 in the direct regulation of the FGF19-FGFR4-MAPK pathway to promote both the development and maintenance of anterior segment structures within the eye.

Regulation of FOXC1 stability and transcriptional activity by an epidermal growth factor-activated mitogen-activated protein kinase signaling cascade.

Mutations in the FOXC1 transcription factor gene result in Axenfeld Rieger malformations, a disorder that affects the anterior segment of the eye, the teeth, and craniofacial structures. Individuals with this disorder possess an elevated risk for developing glaucoma. Previous work in our laboratory has indicated that FOXC1 transcriptional activity may be regulated by phosphorylation. We report here that FOXC1 is a short-lived protein (t 1/2< 30 min), and serine 272 is a critical residue in maintaining proper stability of FOXC1. Furthermore, we have demonstrated that activation of the ERK1/2 mitogen-activated protein kinase through epidermal growth factor stimulation is required for maximal FOXC1 transcriptional activation and stability. Finally, we have demonstrated that FOXC1 is targeted to the ubiquitin 26 S proteasomal degradation pathway and that amino acid residues 367-553, which include the C-terminal transactivation domain of FOXC1, are essential for ubiquitin incorporation and proteolysis. These results indicate that FOXC1 protein levels and activity are tightly regulated by post-translational modifications.

Functional interactions between FOXC1 and PITX2 underlie the sensitivity to FOXC1 gene dose in Axenfeld-Rieger syndrome and anterior segment dysgenesis.

Axenfeld-Rieger ocular dysgenesis is associated with mutations of the human PITX2 and FOXC1 genes, which encode transcription factors of the homeodomain and forkhead types, respectively. We have identified a functional link between FOXC1 and PITX2 which we propose underpins the similar Axenfeld-Rieger phenotype caused by mutations of these genes. FOXC1 and PITX2A physically interact, and this interaction requires crucial functional domains on both proteins: the C-terminal activation domain of FOXC1 and the homeodomain of PITX2. Immunofluorescence further shows PITX2A and FOXC1 to be colocalized within a common nuclear subcompartment. Furthermore, PITX2A can function as a negative regulator of FOXC1 transactivity. This work ties both proteins into a common pathway and offers an explanation of why increased FOXC1 gene dosage produces a phenotype resembling that of PITX2 deletions and mutations. Ocular phenotypes arise despite the deregulated expression of FOXC1-target genes through mutations in FOXC1 or PITX2. Ultimately, PITX2 loss of function mutations have a compound effect: the reduced expression of PITX2-target genes coupled with the extensive activation of FOXC1-regulated targets. Our findings indicate that the functional interaction between FOXC1 and PITX2A underlies the sensitivity to FOXC1 gene dosage in Axenfeld-Rieger syndrome and related anterior segment dysgeneses.

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.

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.

FOXC1 transcriptional regulatory activity is impaired by PBX1 in a filamin A-mediated manner.

FOXC1 mutations underlie Axenfeld-Rieger syndrome, an autosomal dominant disorder that is characterized by a spectrum of ocular and nonocular phenotypes and results in an increased susceptibility to glaucoma. Proteins interacting with FOXC1 were identified in human nonpigmented ciliary epithelial cells. Here we demonstrate that FOXC1 interacts with the actin-binding protein filamin A (FLNA). In A7 melanoma cells possessing elevated levels of nuclear FLNA, FOXC1 is unable to activate transcription and is partitioned to an HP1alpha, heterochromatin-rich region of the nucleus. This inhibition is mediated through an interaction between FOXC1 and the homeodomain protein PBX1a. In addition, we demonstrate that efficient nuclear and subnuclear localization of PBX1 is mediated by FLNA. Together, these data reveal a mechanism by which structural proteins such as FLNA can influence the activity of a developmentally and pathologically important transcription factor such as FOXC1. Given the resemblance of the skeletal phenotypes caused by FOXC1 loss-of-function mutations and FLNA gain-of-function mutations, this inhibitory activity of FLNA on FOXC1 may contribute to the pathogenesis of FLNA-linked skeletal disorders.

Structural and functional analyses of disease-causing missense mutations in the forkhead domain of FOXC1.

Five missense mutations (P79L, P79T, I91S, I91T and R127H) within the forkhead DNA-binding domain of the FOXC1 transcription factor, identified in patients with Axenfeld-Rieger (AR) malformations, were studied to identify the effects of these mutations on FOXC1 structure and function. Molecular modeling and threading analyses predict that the I91S and T mutations may generate local disruptions to the structure of the forkhead domain while the R127H mutation alters the electrostatic charge of the DNA binding surface of the forkhead domain. The P79L and T mutations are not predicted to grossly perturb the structure of the forkhead domain. Biological analyses indicate that all of these missense mutations cause a range of FOXC1 perturbations, including nuclear localization defects, reduced or abolished DNA binding capacity, and a reduction in the transactivation capacity of FOXC1. These experiments extend our previous hypothesis that reduced transactivation of appropriate target genes by FOXC1, underlie AR malformations mapping to human chromosome 6p25. Importantly, these results can also be applied to predict the consequences of the molecular effects of mutations of other FOX genes that have analogous missense mutations, including FOXP2, FOXE3 and FOXC2.