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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: Mutations of the transcription factor FOXL2, involved in cranio-facial and ovarian development, lead to the Blepharophimosis Syndrome. Here, we have systematically replaced the amino acids of the helices of the forkhead domain (FHD) of FOXL2 by glycine residues to assess the impact of such substitutions. A number of mutations lead to protein mislocalization, aggregation and to partial or complete loss of transactivation ability on a series of luciferase reporter systems. To rationalize the results of this glycine mutation scan, we have modeled the structure of the FHD by comparison with crystallographic data available for other FHDs. We failed to detect a clear-cut correlation between protein mislocalization or aggregation and the position of the mutation. However, we found that the localization of the side chain of each amino acid strongly correlated with the impact of its mutation on FOXL2 transactivation capacity. Indeed, when the side chains of the amino acids involved in the helices of the forkhead are supposed to point towards the hydrophobic core formed by the three main helices, a loss of function was observed. On the contrary, if the side chains point outward the hydrophobic core, protein function was preserved. The extension of this analysis to natural mutants shows that a similar correlation can be found for BPES mutations associated or not with ovarian dysfunction. Our findings reveal new insights into the molecular effects of FOXL2 mutations affecting the FHD, which represent two-thirds of intragenic mutations, and provide the first predictive tool of their effects.
Human Molecular Genetics 06/2011; 20(17):3376-85. · 7.64 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: In a previous work, using an interspecific recombinant congenic mouse model, we reported a genomic region of 23 Mb on mouse chromosome 11 implicated in testis weight decrease and moderate teratozoospermia (∼20-30%), a Quantitative Trait Locus (QTL) called Ltw1. The objective of the present study is to identify the gene underlying this phenotype.
In the present study, we refined the QTL position to a 5 Mb fragment encompassing only 11 genes. We showed that the low testis weight phenotype was due to kinetic alterations occurring during the first wave of the spermatogenesis where we could point out to an abnormal lengthening of spermatocyte prophase. We identify Fidgetin-like 1 (Fignl1) as the gene underlying the phenotype, since if fulfilled both the physiological and molecular characteristics required. Indeed, amongst the 11 positional candidates it is the only gene that is expressed during meiosis at the spermatocyte stage, and that presents with non-synonymous coding variations differentiating the two mouse strains at the origin of the cross.
This work prompted us to propose Fignl1 as a novel actor in mammal's male meiosis dynamics which has fundamental interest. Besides, this gene is a new potential candidate for human infertilities caused by teratozoospermia and blockades of spermatogenesis. In addition this study demonstrates that interspecific models may be useful for understanding complex quantitative traits.
PLoS ONE 01/2011; 6(11):e27582. · 4.09 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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ABSTRACT: Positional cloning of the quantitative trait locus (QTL) still encounters numerous difficulties, which explains why thousands of QTL have been mapped, while only a few have been identified at the molecular level. Here, we focus on a specific mapping tool that exists in plant and animal model species: interspecific recombinant congenic strains (IRCSs) or interspecific nearly isogenic lines (NILs). Such panels exhibit a much higher sequence diversity than intraspecific sets, thus enhancing the contrasts between phenotypes. In animals, it allows statistical significance to be reached even when using a limited number of individuals. Therefore, we argue that interspecific resources may constitute a major genetic tool for positional cloning and for understanding some bases of speciation mechanisms.
BioEssays 02/2010; 32(2):132-42. · 4.95 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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Paul Laissue,
Gaétan Burgio, David l'Hôte,
Gilles Renault,
Carmen Marchiol-Fournigault,
Didier Fradelizi,
Marc Fellous,
Catherine Serres,
Xavier Montagutelli,
Philippe Monget,
Daniel Vaiman
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ABSTRACT: Recurrent Spontaneous Abortion (RSA) is a frequent pathology affecting 1 to 5% of couples. In approximately 50 % of cases, the aetiology is unknown suggesting a subtle interaction between genetic and environmental factors. Previous attempts to describe genetic factors using the candidate gene approach have been relatively unsuccessful due to the physiological, cellular and genetic complexity of mammalian reproduction. Indeed, fertility can be considered as a quantitative feature resulting from the interaction of genetic, epigenetic and environmental factors. Herein, we identified Quantitative Trait Loci (QTL) associated with diverse embryonic lethality phenotypes and the subsequent embryonic resorption in 39 inter-specific recombinant congenic mice strains, using in vivo ultrasound bio-microscopy. The short chromosomal intervals related to the phenotypes will facilitate the study of a restricted number of candidate genes which are potentially dysregulated in patients affected by RSA.
The International journal of developmental biology 02/2009; 53(4):623-9. · 2.16 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: In mammals, male fertility is a quantitative feature determined by numerous genes. Until now, several wide chromosomal regions involved in fertility have been defined by genetic mapping approaches; unfortunately, the underlying genes are very difficult to identify. Here, 53 interspecific recombinant congenic mouse strains (IRCSs) bearing 1-2% SEG/Pas (Mus spretus) genomic fragments disseminated in a C57Bl/6J (Mus domesticus) background were used to systematically analyze male fertility parameters. One of the most prominent advantages of this model is the possibility of analyzing stable phenotypes in living animals. Here, we demonstrate the possibility in one-step fine mapping for several fertility traits. Focusing on strains harboring a unique spretus fragment, we could unambiguously localize two testis and one prostate weight-regulating QTL (Ltw1, Ltw2, and Lpw1), four QTL controlling the sperm nucleus shape (Sh1, Sh2, Sh3, and Sh4), and one QTL influencing sperm survival (Dss1). In several cases, the spretus DNA fragment was small enough to propose sound candidates. For instance, Spata1, Capza, and Tuba7 are very strong candidates for influencing the shape of the sperm head. Identifying new genes implied in mammalian fertility pathways is a necessary prerequisite for clarifying their molecular grounds and for proposing diagnostic tools for masculine infertilities.
Genetics 08/2007; 176(3):1907-21. · 4.01 Impact Factor
