Mutational probing of the forkhead domain of the transcription factor FOXL2 provides insights into the pathogenicity of naturally occurring mutations (original) (raw)
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Human Molecular Genetics, 2008
Mutations of the FOXL2 gene have been shown to cause blepharophimosis syndrome (BPES), characterized by an eyelid malformation associated with premature ovarian failure or not. Recently, polyalanine expansions and truncating FOXL2 mutations have been shown to lead to protein mislocalization, aggregation and altered transactivation. Here, we study the molecular consequences of 17 naturally occurring FOXL2 missense mutations. Most of them map to the conserved DNA-binding forkhead domain (FHD). The subcellular localization and aggregation pattern of the mutant FOXL2 proteins in COS-7 cells was variable and ranged from a diffuse nuclear distribution like the wild-type to extensive nuclear aggregation often in combination with cytoplasmic mislocalization and aggregation. We also studied the transactivation capacity of the mutants in FOXL2 expressing granulosa-like cells (KGN). Several mutants led to a loss-of-function, while others are suspected to induce a dominant negative effect. Interestingly, one mutant that is located outside the FHD (S217F), appeared to be hypermorphic and had no effect on intracellular protein distribution. This mutation gives rise to a mild BPES phenotype. In general, missense mutations located in the FHD lead to classical BPES and cannot be correlated with expression of the ovarian phenotype. However, a potential predictive value of localization and transactivation assays in the making of genotype -phenotype correlations is proposed. This is the first study to demonstrate that a significant number of missense mutations in the FHD of FOXL2 lead to mislocalization, protein aggregation and altered transactivation, and to provide insights into the pathogenesis associated with missense mutations of FOXL2 in human disease.
The mutations and potential targets of the forkhead transcription factor FOXL2
Molecular and Cellular Endocrinology, 2008
Mutations of FOXL2, a gene encoding a forkhead transcription factor, have been shown to cause the blepharophimosis-ptosis-epicanthus inversus (BPES) syndrome. This genetic disorder is characterized by eyelid and mild craniofacial abnormalities that can appear associated with premature ovarian failure. FOXL2 is one of the earliest ovarian markers and it offers, along with its targets, an excellent model to study ovarian development and function in normal and pathological conditions. In this review we summarize recent data concerning FOXL2, its mutations and its potential targets. Indeed, many mutations have been described in the coding sequence of FOXL2. Among them, polyAlanine expansions and premature nonsense mutations have been shown to induce protein aggregation. In the context of the ovary, FOXL2 has been suggested to be involved in the regulation of cholesterol and steroid metabolism, apoptosis, reactive oxygen species detoxification and inflammation processes. The elucidation of the impact of FOXL2 mutations on its function will allow a better understanding of the pathogenic mechanisms underlying the BPES phenotype.
Mutations of the transcription factor FOXL2, involved in cranio-facial and ovarian development lead to the Blepharophimosis-Ptosis-Epicanthus Inversus Syndrome (BPES) in human. Here, we describe nine mutations in the open reading frame of FOXL2. Six of them are novel: c.292T>A (p.Trp98Arg), c.323T>C (p.Leu108Pro), c.650C>G (p.Ser217Cys) and three frameshifts). We have performed localization and functional studies for three of them. We have observed a strong cytoplasmic mislocalization induced by the missense mutation p.Leu108Pro located in the forkhead (FKH) domain of FOXL2. In line with this, transcriptional activity assays confirmed the loss-of-function induced by this variant. Interestingly, the novel mutation p.Ser217Cys, mapping between the FKH and the polyalanine domain of FOXL2 and producing a mild eyelid phenotype, led to normal localization and transactivation. We have also modeled the structure of the FKH domain to explore the potential structural impact of the mutations reported here and other previously reported ones. This analysis shows that mutants can be sorted into two classes: those that potentially alter protein-protein interactions and those that might disrupt the interactions with DNA. Our findings reveal new insights into the molecular effects of FOXL2 mutations, especially those affecting the FKH binding domain.
Human Molecular Genetics, 2008
The Forkhead transcription factor FOXL2 plays a crucial role in ovarian development and maintenance. In humans, its mutations lead to craniofacial abnormalities, isolated or associated with ovarian dysfunction. Using a combinatorial approach, we identified and characterized a FoxL2 response element (FLRE) and showed that it is highly specific and that it diverges from that of other Forkhead transcription factors. This specificity should prevent aberrant regulation of FOXL2 targets by other members of the family and should prevent ectopic activation of the ovarian differentiation program in testes. We provide evidence that the FLRE is used in naturally occurring promoters. We show that polyAlanine expansions of FOXL2, which are the most frequent pathogenic mutations, induce a length-dependent loss of response on different artificial promoter reporters depending on the number and sequence of the FLREs that they contain. Thus, we provide clear mechanistic evidence explaining how the architecture of promoters influences their sensitivity to decreased transcription factor availability. Furthermore, we speculate that the generally absent ovarian phenotype of patients carrying the most frequent polyAlanine expansion should come from its ability to properly regulate high-affinity ovarian targets. The existence of critical high-affinity ovarian targets would be compatible with the role of FOXL2 in reproduction and ensure developmental and functional robustness. Taken together, our results give mechanistic insights on the molecular pathogenesis of FOXL2 polyAlanine expansions.
Three-Dimensional Domain Swapping Changes the Folding Mechanism of the Forkhead Domain of FoxP1
Biophysical journal, 2016
The forkhead family of transcription factors (Fox) controls gene transcription during key processes such as regulation of metabolism, embryogenesis, and immunity. Structurally, Fox proteins feature a conserved DNA-binding domain known as forkhead. Interestingly, solved forkhead structures of members from the P subfamily (FoxP) show that they can oligomerize by three-dimensional domain swapping, whereby structural elements are exchanged between adjacent subunits, leading to an intertwined dimer. Recent evidence has largely stressed the biological relevance of domain swapping in FoxP, as several disease-causing mutations have been related to impairment of this process. Here, we explore the equilibrium folding and binding mechanism of the forkhead domain of wild-type FoxP1, and of two mutants that hinder DNA-binding (R53H) and domain swapping (A39P), using size-exclusion chromatography, circular dichroism, and hydrogen-deuterium exchange mass spectrometry. Our results show that domain ...
Nature Genetics, 2001
In type I blepharophimosis/ptosis/epicanthus inversus syndrome (BPES), eyelid abnormalities are associated with ovarian failure. Type II BPES shows only the eyelid defects, but both types map to chromosome 3q23. We have positionally cloned a novel, putative winged helix/forkhead transcription factor gene, FOXL2, that is mutated to produce truncated proteins in type I families and larger proteins in type II. Consistent with an involvement in those tissues, FOXL2 is selectively expressed in the mesenchyme of developing mouse eyelids and in adult ovarian follicles; in adult humans, it appears predominantly in the ovary. FOXL2 represents a candidate gene for the polled/intersex syndrome XX sex-reversal goat.
Human Molecular Genetics, 2005
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.