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ABSTRACT: FOXL2 transcription factor is responsible for the Blepharophimosis Ptosis Epicantus inversus Syndrome (BPES), a genetic disease involving craniofacial malformations often associated with ovarian failure. Recently, a somatic FOXL2 mutation (p.C134W) has been reported in >95% of adult-type granulosa cell tumors. Here, we have identified 10 novel FOXL2 partners by yeast-two-hybrid screening and co-immunoprecipitation. Most BPES-inducing mutated FOXL2 proteins display aggregation in cultured cells. Here, we show that two of the partners (NR2C1 and GMEB1) can be sequestered in such aggregates. This co-aggregation can contribute to the pathogenesis of FOXL2 mutations. We have also measured the effects of FOXL2 interactants on the transcriptional regulation of a series of target promoters. Some of the partners (CXXC4, CXXC5, BANF1) were able to repress FOXL2 activity indistinctively of the promoter. Interestingly, CREM-τ2α, which acted as a repressor on most promoters, increased wild-type (WT) FOXL2 activity on two promoters (PTGS2 and CYP19A1), but was unable to increase the activity of the oncogenic mutant p.C134W. Conversely, GMEB1, which also acted as a repressor on most promoters and increased WT FOXL2 activity on the Per2 promoter, increased to a greater extent the activity of the p.C134W variant. Interestingly, partners with intrinsic pro-apoptotic effect were able to increase apoptosis induction by WT FOXL2, but not by the p.C134W mutant, whereas partners with an anti-apoptotic effect decreased apoptosis induction by both FOXL2 versions. Altogether, these results suggest that the p.C134W mutated form fails to integrate signals through protein-protein interactions to regulate target promoter subsets and in particular to induce cell death.
Human Molecular Genetics 04/2012; 21(14):3264-74. · 7.64 Impact Factor
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ABSTRACT: FOXL2 is a gene encoding a forkhead transcription factor. Its mutations or misregulation have been shown to cause the blepharophimosis-ptosis-epicanthus inversus (BPES) syndrome and more recently have been associated with the development of Ovarian Granulosa Cell Tumors (OGCT). BPES is a genetic disorder involving mild craniofacial abnormalities often associated with premature ovarian failure. OGCTs are endocrine malignancies, accounting for 2-5% of ovarian cancers, the treatment of which is still challenging. In this review we summarize recent data concerning FOXL2 transcriptional targets and molecular partners, its post-translational modifications, its mutations and its involvement in newly discovered pathophysiological processes. In the ovary, FOXL2 is involved in the regulation of cholesterol and steroid metabolism, apoptosis, reactive oxygen species detoxification and cell proliferation. Interestingly, one of the main roles of FOXL2 is also to preserve the identity of ovarian granulosa cells even at the adult stage and to prevent their transdifferentiation into Sertoli-like cells. All these recent advances indicate that FOXL2 is central to ovarian development and maintenance. The elucidation of the impact of FOXL2 germinal and somatic mutations will allow a better understanding of the pathogenesis of BPES and of OGCTs.
Molecular and Cellular Endocrinology 07/2011; 356(1-2):55-64. · 4.19 Impact Factor
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ABSTRACT: Foxn4, a member of the N-family forkhead transcription factors, controls fate decision in mouse retina and spinal cord as well as in zebrafish heart. Analysis of Foxn4 amino acid sequence revealed the presence of a region homologous to the activation domain of its close relative Foxn1 in between C-terminal amino acids 402 and 455 of Foxn4 protein. The requirement of Foxn4 putative activation domain remains to be elucidated. Using a gain-of function approach in rat and chick retinal explants, we report that deletion of Foxn4 putative activation domain results in a complete loss of its activity during retinogenesis. Target promoter transcription assay indicates that this domain is critical for Foxn4 transcriptional regulatory properties in vitro. Accordingly, in chick retinal explants, this domain is required for proper regulation of target retinogenic factors expression by Foxn4. Thus, our study demonstrates that the domain between amino acids 402 and 455 is necessary for Foxn4 transcriptional activity both in vitro and in the retina.
Journal of Molecular Neuroscience 06/2011; 46(2):315-23. · 2.50 Impact Factor
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ABSTRACT: Forkhead box (FOX) proteins constitute an evolutionarily conserved family of transcription factors with a central role not only during development, but also in the adult organism. Thus, the misregulation and/or mutation of FOX genes often induce human genetic diseases, promote cancer or deregulate ageing. Indeed, germinal FOX gene mutations cause diseases ranging from infertility to language and/or speech disorders and immunological defects. Moreover, because of their central role in signalling pathways and in the regulation of homeostasis, somatic misregulation and/or mutation of FOX genes are associated with cancer. FOX proteins have undergone diversification in terms of their sequence, regulation and function. In addition to dedicated roles, evidence suggests that Forkhead factors have retained some functional redundancy. Thus, combinations of slightly defective alleles might induce disease phenotypes in humans, acting as quantitative trait loci. Uncovering such variants would be a big step towards understanding the functional interdependencies of different FOX members and their implications in complex pathologies.
Trends in Genetics 06/2011; 27(6):224-32. · 10.06 Impact Factor
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ABSTRACT: FOXL2 is a transcription factor that is essential for ovarian function and maintenance, the germline mutations of which are responsible for the Blepharophimosis Ptosis Epicanthus-inversus Syndrome (BPES), often associated with premature ovarian failure. Recent evidence has linked FOXL2 downregulation or somatic mutation (p.Cys134Trp) to cancer, although underlying molecular mechanisms remain unclear. Using a functional genomic approach, we find that FOXL2 modulates cell-cycle regulators in a way which tends to induce G1 arrest. Indeed, FOXL2 upregulation promotes cell accumulation in G1 phase and protects cells from oxidative damage, notably by promoting oxidized DNA repair and by increasing the amounts of anti-oxidant agent glutathione. In agreement with clinical observations, we find that FOXL2-mutated versions leading to BPES along with ovarian dysfunction mostly fail to transactivate cell-cycle and DNA repair targets, whereas mutations leading to isolated craniofacial defects (and normal ovarian function) activate them correctly. Interestingly, these assays revealed a mild promoter-specific hypomorphy of the tumor-associated mutation (p.Cys134Trp). Finally, the SIRT1 deacetylase suppresses FOXL2 activity on targets linked to cell-cycle and DNA repair in a dose-dependent manner. Accordingly, we find that SIRT1 inhibition by nicotinamide limits proliferation, notably by increasing endogenous FOXL2 amount/activity. The body of evidence presented here supports the idea that FOXL2 plays a key role in granulosa cell homeostasis, the failure of which is central to ovarian ageing and tumorigenesis. As granulosa cell tumors respond poorly to conventional chemotherapy, our findings on the deacetylase inhibitor nicotinamide provide an interesting option for targeted therapy.
Human Molecular Genetics 02/2011; 20(9):1673-86. · 7.64 Impact Factor
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ABSTRACT: FOXL2 is a transcription factor essential for ovarian development and maintenance. It is mutated in the genetic condition called Blepharophimosis Ptosis Epicantus inversus Syndrome (BPES) and in cases of isolated premature ovarian failure. We and others have previously shown that FOXL2 undergoes several post-translational modifications.
Here, using cells in culture, we show that interference with FOXL2 SUMOylation leads to a robust inhibition of its transactivation ability, which correlates with a decreased stability. Interestingly, FOXL2 SUMOylation promotes its transient recruitment to subnuclear structures that we demonstrate to be PML (Promyelocytic Leukemia) Nuclear Bodies. Since PML bodies are known to be sites where post-translational modifications of nuclear factors take place, we used tandem mass spectrometry to identify new post-translational modifications of FOXL2. Specifically, we detected four phosphorylated, one sulfated and three acetylated sites.
By analogy with other transcription factors, we propose that PML Nuclear Bodies might transiently recruit FOXL2 to the vicinity of locally concentrated enzymes that could be involved in the post-translational maturation of FOXL2. FOXL2 acetylation, sulfation, phosphorylation as well as other modifications yet to be discovered might alter the transactivation capacity of FOXL2 and/or its stability, thus modulating its global intracellular activity.
PLoS ONE 01/2011; 6(10):e25463. · 4.09 Impact Factor
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Bérénice A Benayoun,
Sandrine Caburet,
Aurélie Dipietromaria,
Adrien Georges,
Barbara D'Haene,
P J Eswari Pandaranayaka,
David L'Hôte,
Anne-Laure Todeschini,
Sankaran Krishnaswamy,
Marc Fellous,
Elfride De Baere,
Reiner A Veitia
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ABSTRACT: The somatic mutation in the FOXL2 gene c.402C>G (p.Cys134Trp) has recently been identified in the vast majority of adult ovarian granulosa cell tumors (OGCTs) studied. In addition, this mutation seems to be specific to adult OGCTs and is likely to be a driver of malignant transformation. However, its pathogenic mechanisms remain elusive.
We have sequenced the FOXL2 open reading frame in a panel of tumor cell lines (NCI-60, colorectal carcinoma cell lines, JEG-3, and KGN cells). We found the FOXL2 c.402C>G mutation in the adult OGCT-derived KGN cell line. All other cell lines analyzed were negative for the mutation. In order to gain insights into the pathogenic mechanism of the p.Cys134Trp mutation, the subcellular localization and mobility of the mutant protein were studied and found to be no different from those of the wild type (WT). Furthermore, its transactivation ability was in most cases similar to that of the WT protein, including in conditions of oxidative stress. A notable exception was an artificial promoter known to be coregulated by FOXL2 and Smad3, suggesting a potential modification of their interaction. We generated a 3D structural model of the p.Cys134Trp variant and our analysis suggests that homodimer formation might also be disturbed by the mutation.
Here, we confirm the specificity of the FOXL2 c.402C>G mutation in adult OGCTs and begin the exploration of its molecular significance. This is the first study demonstrating that the p.Cys134Trp mutant does not have a strong impact on FOXL2 localization, solubility, and transactivation abilities on a panel of proven target promoters, behaving neither as a dominant-negative nor as a loss-of-function mutation. Further studies are required to understand the specific molecular effects of this outstanding FOXL2 mutation.
PLoS ONE 01/2010; 5(1):e8789. · 4.09 Impact Factor
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ABSTRACT: A transcription reaction relies on the specific recognition of cis-regulatory regions containing short DNA motifs. Such sequences are bound by transcription factors (TFs) involved in the recruitment, direct or not, of the transcriptional machinery. A eukaryotic genome can contain tens of genes encoding TFs that recognize very similar consensus DNA target sequences. In this review, we explore in a simple way how TFs coexpressed in the same cells and recognizing generic consensus sites with generic DNA-binding domains can achieve a specific modulation of target gene expression. We dissect the strategy followed by eukaryotes, which involves the formation of complex nucleoprotein structures involving many TFs and their cognate binding sites. This multiplicity of actors increases the effective length of the target DNA recognized by the TFs and might help paralogous TFs establish specific interactions. From this perspective, eukaryotic gene regulation implies the cooperation of several TFs, which is also the basis of information integration. Such cooperative TFs are likely to form a combinatorial partner code whose ultimate molecular hallmark is the assembly of enhanceosome-like structures ensuring the formation of an activation surface that is complementary to other coactivators and to the transcriptional machinery itself.
The FASEB Journal 09/2009; 24(2):346-56. · 5.71 Impact Factor
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ABSTRACT: Mutations of FOXL2 are responsible for the Blepharophimosis-Ptotsis-Epicantus-inversus Syndrome (BPES), involving complex eyelid malformations often associated with premature ovarian failure (POF). Loss-of-function mutations are expected to lead to BPES associated with POF, whereas hypomorphic mutations would lead to BPES without ovarian dysfunction. However, multiple exceptions to the genotype-phenotype correlation have been described and missense mutations in the forkhead domain can lead to either type of BPES. This renders almost impossible the prediction of a POF condition from a given genotype. Moreover, no clear-cut correlation between nuclear and/or cytoplasmic aggregation or cytoplasmic retention of mutant FOXL2 forms and the BPES type has been established thus far. Here, we dissect the molecular and functional effects of 10 FOXL2 mutants, known to induce BPES associated with POF or not. We found a correlation between the transcriptional activity of FOXL2 variants on two different reporter promoters and the type of BPES. We used this functional classification framework to explore the behavior of 18 missense mutations leading to BPES of unknown type. The reporters used enabled us to assess the risk of POF associated with these mutations. Moreover, we document a previously overlooked correlation between subcellular mislocalization and aggregation of mutant FOXL2 and the type of BPES, known or predicted using our reporter assays. Thus, intranuclear aggregation and cytoplasmic mislocalization of mutant FOXL2 may be considered as loose predictors of ovarian dysfunction. The functional classification tool described here is a first step towards circumventing the lack of a clear-cut genotype-phenotype correlation in BPES.
Human Molecular Genetics 07/2009; 18(17):3324-33. · 7.64 Impact Factor
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P Laissue,
B Lakhal, B A Benayoun,
A Dipietromaria,
R Braham,
H Elghezal,
P Philibert,
A Saâd,
C Sultan,
M Fellous,
R A Veitia
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ABSTRACT: FOXL2 encodes a forkhead transcription factor whose mutations are responsible for the blepharophimosis-ptosis-epicanthus inversus syndrome (BPES), involving craniofacial/palpebral abnormalities often associated with premature ovarian failure (POF).
We describe a FOXL2 variant (p.Gly187Asp) in a case of POF without BPES. The subcellular localisation of FOXL2-G187D was normal but its transactivation capacity tested on two reporter promoters, one of which should be relevant to the ovary, was significantly lower than that of normal FOXL2. However, FOXL2-G187D was able to activate strongly a reporter construct driven by the promoter of Osr2 (odd-skipped related 2 transcription factor), which we have suggested to be a crucial target of FOXL2 in the craniofacial region. This is compatible with the absence of BPES in our patient.
Our data provide evidence in favour of the implication of FOXL2 variants in non-syndromic POF and confirm the regulatory interaction between FOXL2 and OSR2 whose perturbation might contribute to the palpebral abnormalities observed in BPES patients.
Journal of Medical Genetics 06/2009; 46(7):455-7. · 6.36 Impact Factor
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ABSTRACT: Cellular responses to environmental or physiological cues rely on transduction pathways that must ensure discrimination between different signals. These cascades 'crosstalk' and lead to a combinatorial regulation. This often results in different combinations of post-translational modifications (PTMs) on target proteins, which might act as a molecular barcode. Although appealing, the idea of the existence of such a code for transcription factors is debated. Using general arguments and recent evidence, we propose that a PTM code is not only possible but necessary in the context of transcription factors regulating multiple processes. Thus, the coding potential of PTM combinations should both provide a further layer of information integration from several transduction pathways and warrant highly specific cellular outputs.
Trends in cell biology 04/2009; 19(5):189-97. · 12.12 Impact Factor
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ABSTRACT: The gene FOXL2 encodes a forkhead transcription factor whose mutations are responsible for the blepharophimosis ptosis epicanthus-inversus syndrome. This genetic disorder is characterized by eyelid and mild craniofacial abnormalities often in association with premature ovarian failure. FOXL2 orthologs are found throughout the animal phylum and its sequence is highly conserved in vertebrates. FOXL2 is one of the earliest ovarian markers and it offers, alongwith its targets, a model to study ovarian development and function. In this chapter, we review recent data concemingits mutations, targets, regulation and functions. Studies of the cellular consequences of FOXL2 mutations seem to indicate that aggregation is a common pathogenic mechanism. However, no reliable genotype/phenotype correlation has been established to predict the exact impact of point mutations in the coding region of FOXL2. FOXL2 has been suggested to be involved in the regulation of cholesterol homeostasis, steroid metabolism, apoptosis, reactive oxygen species detoxification and inflammation processes. Interestingly, all these processes are not equally affected by FOXL2 mutations. The elucidation of the impact of the FOXL2 function in the ovary will allow a better understanding of normal ovarian development and function as well as the pathogenic mechanisms underlying BPES.
Advances in experimental medicine and biology 01/2009; 665:207-26. · 1.09 Impact Factor
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ABSTRACT: FOXL2 is a forkhead transcription factor, essential for ovarian function, whose mutations are responsible for the blepharophimosis syndrome, characterized by craniofacial defects, often associated with premature ovarian failure. Here, we show that cell stress upregulates FOXL2 expression in an ovarian granulosa cell model. Increased FOXL2 transcription might be mediated at least partly by self-activation. Moreover, using 2D-western blot, we show that the response of FOXL2 to stress correlates with a dramatic remodeling of its post-translational modification profile. Upon oxidative stress, we observe an increased recruitment of FOXL2 to several stress-response promoters, notably that of the mitochondrial manganese superoxide dismutase (MnSOD). Using several reporter systems, we show that FOXL2 transactivation is enhanced in this context. Models predict that gene upregulation in response to a signal should eventually be counterbalanced to restore the initial steady state. In line with this, we find that FOXL2 activity is repressed by the SIRT1 deacetylase. Interestingly, we demonstrate that SIRT1 transcription is, in turn, directly upregulated by FOXL2, which closes a negative-feedback loop. The regulatory relationship between FOXL2 and SIRT1 prompted us the test action of nicotinamide, an inhibitor of sirtuins, on FoxL2 expression/activity. According to our expectations, nicotinamide treatment increases FoxL2 transcription. Finally, we show that 11 disease-causing mutations in the ORF of FOXL2 induce aberrant regulation of FOXL2 and/or regulation of the FOXL2 stress-response target gene MnSOD. Taken together, our results establish that FOXL2 is an actor of the stress response and provide new insights into the pathogenic consequences of FOXL2 mutations.
Human Molecular Genetics 12/2008; 18(4):632-44. · 7.64 Impact Factor
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ABSTRACT: 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.
Human Molecular Genetics 10/2008; 17(20):3118-27. · 7.64 Impact Factor
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ABSTRACT: The transcription factor Forkhead box L subfamily member 2 (FOXL2) is involved in craniofacial development and ovarian function. Using 2-DE and immunoblotting, we show that it is highly modified post-translationally. The most outstanding feature of its migration profile is the presence of two distinct modification "trains" and the absence of intermediates. A theoretical analysis of the modification profile of FOXL2 suggests that it undergoes parallel processive/concerted modifications. The absence of intermediates is compatible with the recruitment of poorly modified FOXL2 into a post-translational "modification factory."
Proteomics 08/2008; 8(15):3118-23. · 4.43 Impact Factor
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Jeyabalan Nallathambi,
Paul Laissue,
Frank Batista, Bérénice A Benayoun,
Corinne Lesaffre,
Lara Moumné,
Pj Eswari Pandaranayaka,
Kim Usha,
Sankaran Krishnaswamy,
Periasamy Sundaresan,
Reiner A Veitia
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ABSTRACT: 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. (c) 2008 Wiley-Liss, Inc.
Human Mutation 05/2008; 29(8):E123-31. · 5.69 Impact Factor
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ABSTRACT: FOXL2 is a gene encoding a forkhead transcription factor, whose germline mutations are responsible for the blepharophimosis ptosis epicanthus inversus syndrome. We have previously shown that expression levels of FOXL2 in a series of juvenile ovarian granulosa cell tumors (OGCTs) were markedly reduced. More recently, a whole-transcriptome ‘next-generation’ sequencing study has identified the somatic mutation p.Cys134Trp as recurring in adult OGCTs. This mutation may thus provide the tumor with either a striking proliferative potential or increased survival abilities. These studies of FOXL2 in OGCTs suggest that it may act as a tumor suppressor gene. This is in line with the fact that other forkhead transcription factors have already been involved in the etiology of cancer. Indeed, an in-depth review of existing data on FOXL2 reveals that its target genes and molecular partners can often be linked to cancer progression.
Biochimica et Biophysica Acta (BBA) - Reviews on Cancer.
