| forkhead box L2 | OKDB#: 1109 |
| Symbols: | FOXL2 | Species: | human | ||
| Synonyms: | BPES, PFRK, POF3, BPES1, PINTO | Locus: | 3q22.3 in Homo sapiens |
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| General Comment |
The forkhead domain is a monomeric DNA binding motif that defines a rapidly growing family of eukaryotic transcriptional regulators. Genetic and biochemical data suggest a central role in embryonic development for genes encoding forkhead proteins.
There are 2 forms of the blepharophimosis/ptosis/epicanthus inversus syndrome (BPES). In one form, called
type I, eyelid abnormalities are associated with ovarian failure. Crisponi et al. (2001) refined the physical mapping of the BPES critical
region and positionally cloned a novel, putative winged helix/forkhead transcription factor gene, FOXL2, in which they
found mutations to produce truncated proteins in type I families and larger proteins in type II. Consistent with an
involvement in those tissues, FOXL2 was selectively expressed in the mesenchyme of developing mouse eyelids and in
adult ovarian follicles; in adult humans, it appeared predominantly in the ovary.
FOXL2: a central transcription factor of the ovary. Georges A 2013 et al.
FOXL2 is a gene encoding a forkhead transcription factor preferentially expressed in the ovary, the eyelids and the pituitary. Its germline mutations are responsible for the blepharophimosis-ptosis-epicanthus inversus syndrome (BPES), which associates eyelid and mild craniofacial defects with primary ovarian insufficiency. Recent studies have shown the involvement of FOXL2 in virtually all stages of ovarian development and function, as well as in granulosa cell-related pathologies. A central role of FOXL2 is the lifetime maintenance of granulosa cell identity through the repression of testis-specific genes. Recently, a highly recurrent somatic FOXL2 mutation leading to the p.C134W subtitution has been linked to the development of Granulosa Cell Tumors in the adult (GCTs), which account for up to 5 % of ovarian malignancies. In this review, we summarize data on FOXL2 modulators, targets, partners and post-translational modifications. Despite the progresses made thus far, a better understanding of the impact of FOXL2 mutations and of the molecular aspects of its function are required to rationalise its implication in various pathophysiological processes.
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Foxl2 and Its Relatives Are Evolutionary Conserved Players in Gonadal Sex Differentiation. Bertho S et al. (2016) Foxl2 is a member of the large family of Forkhead Box (Fox) domain transcription factors. It emerged during the last 15 years as a key player in ovarian differentiation and oogenesis in vertebrates and especially mammals. This review focuses on Foxl2 genes in light of recent findings on their evolution, expression, and implication in sex differentiation in animals in general. Homologs of Foxl2 and its paralog Foxl3 are found in all metazoans, but their gene evolution is complex, with multiple gains and losses following successive whole genome duplication events in vertebrates. This review aims to decipher the evolutionary forces that drove Foxl2/3 gene specialization through sub- and neo-functionalization during evolution. Expression data in metazoans suggests that Foxl2/3 progressively acquired a role in both somatic and germ cell gonad differentiation and that a certain degree of sub-functionalization occurred after its duplication in vertebrates. This generated a scenario where Foxl2 is predominantly expressed in ovarian somatic cells and Foxl3 in male germ cells. To support this hypothesis, we provide original results showing that in the pea aphid (insects) foxl2/3 is predominantly expressed in sexual females and showing that in bovine ovaries FOXL2 is specifically expressed in granulosa cells. Overall, current results suggest that Foxl2 and Foxl3 are evolutionarily conserved players involved in somatic and germinal differentiation of gonadal sex.//////////////////Temporal Differences in Granulosa Cell Specification in the Ovary Reflect Distinct Follicle Fates in Mice. Mork L et al. The embryonic origins of ovarian granulosa cells have been a subject of debate for decades. By tamoxifen-induced lineage tracing of Foxl2-expressing cells, we show that descendants of the bipotential supporting cell precursors in the early gonad contribute granulosa cells to a specific population of follicles in the medulla of the ovary that begin to grow immediately after birth. These precursor cells arise from the proliferative ovarian surface epithelium and enter mitotic arrest prior to upregulating Foxl2. Granulosa cells that populate the cortical primordial follicles activated in adult life derive from the surface epithelium perinatally and enter mitotic arrest at that stage. Ingression from the surface epithelium dropped to undetectable levels by Postnatal Day 7, when most surviving oocytes were individually encapsulated by granulosa cells. These findings add complexity to the standard model of sex determination in which the Sertoli and granulosa cells of the adult testis and ovary directly stem from the supporting cell precursors of the bipotential gonad.
NCBI Summary: This gene encodes a forkhead transcription factor. The protein contains a fork-head DNA-binding domain and may play a role in ovarian development and function. Expansion of a polyalanine repeat region and other mutations in this gene are a cause of blepharophimosis syndrome and premature ovarian failure 3. [provided by RefSeq, Jul 2016] |
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| General function | Nucleic acid binding, DNA binding, Transcription factor | ||||
| Comment | Deletion of FOXL2 by CRISPR promotes cell cycle G0/G1 restriction in KGN cells. Tang B et al. (2018) Forkhead box L2 (FOXL2), a member of the forkhead family of transcription factors, is important in eyelid and ovary differentiation. Although the function of FOXL2 in organogenesis has been investigated, the detailed mechanisms by which FOXL2 mediates cellular process remain to be fully elucidated. Few FOXL2‑knockout cell lines have been reported, which has limited molecular mechanism investigations. CRISPR is a novel gene editing technique that has been widely used in human genetic diseases. In the present study, FOXL2 was disrupted using clustered regularly interspaced short palindromic repeats (CRISPR), and screening of a stable knockout cell line was performed in human ovarian granulosa KGN cells. Three sites (F404, F425 and F446) around the ATG start codon on the FOXL2 DNA sequence were constructed in a guide RNA lentivirus. Targeting F425 was most efficient, and western blot analysis and DNA sequencing of the resulting cells suggested that both FOXL2 alleles were fully disrupted. In addition, flow cytometry results indicated that the knockout of FOXL2 restricted cell cycle progression at the G0/G1 phase. In addition, the expression levels of cell cycle mediators cyclin D1 and cyclin‑dependent kinase 4 were reduced. These results confirmed that FOXL2 disruption in KGN cells is associated with the cell cycle attenuation.////////////////// FOXL2 posttranslational modifications mediated by GSK3?determine the growth of granulosa cell tumours. Kim JH 2014 et al. Approximately 97% of patients with ovarian granulosa cell tumours (GCTs) bear the C134W mutation in FOXL2; however, the pathophysiological mechanism of this mutation is unknown. Here we report how this mutation affects GCT development. Sequential posttranslational modifications of the C134W mutant occur where hyperphosphorylation at serine 33 (S33) by GSK3?induces MDM2-mediated ubiquitination and proteasomal degradation. In contrast, S33 of wild-type FOXL2 is underphosphorylated, leading to its SUMOylation and stabilization. This prominent hyperphosphorylation is also observed at S33 of FOXL2 in GCT patients bearing the C134W mutation. In xenograft mice, the S33 phosphorylation status correlates with the oncogenicity of FOXL2, and the inhibition of GSK3?efficiently represses GCT growth. These findings reveal a previously unidentified regulatory mechanism that determines the oncogenic attributes of the C134W mutation via differential posttranslational modifications of FOXL2 in GCT development. ///////////////////////// Differential Apoptotic and Proliferative Activities of Wild-type FOXL2 and Blepharophimosis-ptosis-epicanthus Inversus Syndrome (BPES)-associated Mutant FOXL2 Proteins. Kim JH 2013 et al. FOXL2 is an essential transcription factor that is required for proper development of the ovary and eyelid. Mutations in FOXL2 cause an autosomal dominant genetic disorder, blepharophimosis-ptosis-epicanthus inversus syndrome (BPES). BPES type I patients have eyelid malformation and premature ovarian failure leading to infertility, whereas women with type II BPES are fertile or subfertile. In the present study, we evaluated and compared apoptotic and antiproliferative activities of wild-type (WT) and mutant FOXL2 proteins found in BPES type I and II in human granulosa cell tumor-derived KGN cells. Ectopic expression of WT FOXL2 induced apoptosis and inhibited cell cycle progression in human granulosa cells. In contrast, mutated FOXL2s found in BPES type I significantly reduced these activities, whereas mutated FOXL2s in BPES type II showed intermediate activities. Furthermore, mutant FOX L2 proteins were defective in activating transcription of target genes including Caspase 8, TNF-R1, FAS, p21, and BMP4, which regulate apoptosis, proliferation, and differentiation of granulosa cells. Thus, decreased apoptotic and antiproliferative activities caused by mutant forms of FOXL2 found in BPES patients may at least partially contribute to the pathophysiology of ovarian dysfunction. ///////////////////////// Granulosa cell tumor mutant FOXL2(C134W) suppresses GDF-9 and activin A-induced follistatin transcription in primary granulosa cells. McTavish KJ et al. A single somatic FOXL2 mutation (FOXL2(C134W)) was identified in almost all granulosa cell tumor (GCT) patients. In the pituitary, FOXL2 and Smad3 coordinately regulate activin stimulation of follistatin transcription. We explored whether a similar regulation occurs in the ovary, and whether FOXL2(C134W) has altered activity. We show that in primary granulosa cells, GDF-9 and activin increase Smad3-mediated follistatin transcription. In contrast to findings in the pituitary, FOXL2 negatively regulates GDF-9 and activin-stimulated follistatin transcription in the ovary. Knockdown of endogenous FOXL2 confirmed this inhibitory role. FOXL2(C134W) displayed enhanced inhibitory activity, completely ablating GDF-9 and activin-induced follistatin transcription. GDF-9 and activin activity was lost when either the smad binding element or the forkhead binding element were mutated, indicating that both sites are required for Smad3 actions. This study highlights that FOXL2 negatively regulates follistatin expression within the ovary, and that the pathogenesis of FOXL2(C134W) may involve an altered interaction with Smad3. Temporal Differences in Granulosa Cell Specification in the Ovary Reflect Distinct Follicle Fates in Mice. Mork L et al. The embryonic origins of ovarian granulosa cells have been a subject of debate for decades. By tamoxifen-induced lineage tracing of Foxl2-expressing cells, we show that descendants of the bipotential supporting cell precursors in the early gonad contribute granulosa cells to a specific population of follicles in the medulla of the ovary that begin to grow immediately after birth. These precursor cells arise from the proliferative ovarian surface epithelium and enter mitotic arrest prior to upregulating Foxl2. Granulosa cells that populate the cortical primordial follicles activated in adult life derive from the surface epithelium perinatally and enter mitotic arrest at that stage. Ingression from the surface epithelium dropped to undetectable levels by Postnatal Day 7, when most surviving oocytes were individually encapsulated by granulosa cells. These findings add complexity to the standard model of sex determination in which the Sertoli and granulosa cells of the adult testis and ovary directly stem from the supporting cell precursors of the bipotential gonad. Mutant Forkhead L2 (FOXL2) Proteins Associated with Premature Ovarian Failure (POF) Dimerize with Wild-Type FOXL2, Leading to Altered Regulation of Genes Associated with Granulosa Cell Differentiation. Kuo FT et al. Premature ovarian failure in the autosomal dominant disorder blepharophimosis-ptosis-epicanthus inversus is due to mutations in the gene encoding Forkhead L2 (FOXL2), producing putative truncated proteins. We previously demonstrated that FOXL2 is a transcriptional repressor of the steroidogenic acute regulatory (StAR), P450SCC (CYP11A), P450aromatase (CYP19), and cyclin D2 (CCND2) genes, markers of ovarian follicle proliferation and differentiation. Furthermore, we found that mutations of FOXL2 may regulate wild-type FOXL2, leading to loss of transcriptional repression of CYP19, similar to StAR. However, the regulatory mechanisms underlying these premature ovarian failure-associated mutations remain largely unknown. Therefore, we examined the effects of a FOXL2 mutant protein on the transcriptional repression of the CYP19 promoter by the full-length protein. We found that mutant FOXL2 exerts a dominant-negative effect on the repression of CYP19 by wild-type FOXL2. Both wild-type and mutant FOXL2 and can form homo- and heterodimers. We identified a minimal -57-bp human CYP19 promoter containing two potential FOXL2-binding regions and found that both wild-type and mutant FOXL2 can bind to either of these regions. Mutational analysis revealed that either site is sufficient for transcriptional repression by wild-type FOXL2, and the dominant-negative effect of mutant FOXL2, but these are eliminated when both sites are mutated. These findings confirm that mutant FOXL2 exerts a dominant-negative effect on wild-type FOXL2's activity as a transcriptional repressor of key genes in ovarian follicle differentiation and suggest that this is likely due to heterodimer formation and possibly also competition for DNA binding. Impaired FSH{beta} Expression in the Pituitaries of Foxl2 Mutant Animals. Justice NJ et al. Forkhead box L2 (FoxL2) is required for ovarian development and differentiation. FoxL2 is also expressed in the pituitary where it has been implicated in the development and regulation of gonadotropes, which secrete LH and FSH, the endocrine signals that regulate folliculogenesis in the ovary and spermatogenesis in the testis. Here, we show that FoxL2 is not required for the specification of gonadotropes; the pituitaries of Foxl2 mutant mice contain normal numbers of gonadotropes that express glycoprotein a subunit and LH? Whereas the specification of gonadotropes and all other hormonal cell types is normal in the pituitaries of Foxl2 mutant animals, FSH?levels are severely impaired in both male and female animals, suggesting that FoxL2 is required for normal Fshb expression. The size of the pituitary is reduced in proportion to the smaller body size of Foxl2 mutants, with a concomitant increase in the pituitary cellular density. In primary pituitary cultures, activin induces FSH secretion and Fshb mRNA expression in cells from wild-type mice. In cells from Foxl2 mutant mice, however, FSH secretion is not detected, and activin is unable to drive Fshb expression, suggesting that the mechanism of activin-dependent activation of Fshb transcription is impaired. However, a small number of gonadotropes in the ventromedial region of the pituitaries from Foxl2 mutant mice maintain FSH?expression, suggesting that a FoxL2- and activin-independent mechanism can drive Fshb transcription. These data indicate that, in addition to its role in the ovary, FoxL2 function in the pituitary is required for normal expression of FSH. Sumoylation of Forkhead L2 by Ubc9 is required for its activity as a transcriptional repressor of the Steroidogenic Acute Regulatory gene. Kuo FT et al. Forkhead L2 (FOXL2) is a member of the forkhead/hepatocyte nuclear factor 3 (FKH/HNF3) gene family of transcription factors and acts as a transcriptional repressor of the Steroidogenic Acute Regulatory (StAR) gene, a marker of granulosa cell differentiation. FOXL2 may play a role in ovarian follicle maturation and prevent premature follicle depletion leading to premature ovarian failure. In this study, we found that FOXL2 interacts with Ubc9, an E2-conjugating enzyme that mediates sumoylation, a key mechanism in transcriptional regulation. FOXL2 and Ubc9 are co-expressed in granulosa cells of small and medium ovarian follicles. FOXL2 is sumoylated by Ubc9, and this Ubc9-mediated sumoylation is essential to the transcription activity of FOXL2 on the StAR promoter. As FOXL2 is endogenous to granulosa cells, we generated a stable cell line expressing FOXL2 and found that activity of the StAR promoter in this cell line is greatly decreased in the presence of Ubc9. The sumoylation site was identified at lysine 25 of FOXL2. Mutation of lysine 25 to arginine leads to loss of transcriptional repressor activity of FOXL2. Taken together, we propose that Ubc9-mediated sumoylation at lysine 25 of FOXL2 is required for transcriptional repression of the StAR gene and may be responsible for controlling the development of ovarian follicles. An evolutionary and functional analysis of FoxL2 in rainbow trout gonad differentiation Baron D, et al . FOXL2 is a forkhead transcription factor involved in ovarian development and function. Here, we have studied the evolution and pattern of expression of the FOXL2 gene and its paralogs in fish. We found well conserved FoxL2 sequences (FoxL2a) and divergent genes, whose forkhead domains belonged to the class L2 and were shown to be paralogs of the FoxL2a sequences (named FoxL2b). In the rainbow trout, FoxL2a and FoxL2b were specifically expressed in the ovary, but displayed different temporal patterns of expression. FoxL2a expression correlated with the level of aromatase, the key enzyme in estrogen production, and an estrogen treatment used to feminize genetically male individuals elicited the up-regulation of both paralogs. Conversely, androgens or an aromatase inhibitor down-regulated FoxL2a and FoxL2b in females. We speculate that there is a direct link between estrogens and FoxL2 expression in fish, at least during the period where the identity of the gonad is sensitive to hormonal treatments. Foxl2, a Forkhead Transcription Factor, Modulates Nonclassical Activity of the Estrogen Receptor-{alpha} Kim SY et al. Foxl2 is a forkhead transcription factor required for ovary development and ovarian follicle maturation. In this report, we identified and characterized a functional relationship between Foxl2 expression and estrogen receptor (ER)-alpha signaling. We show that Foxl2 has no effect on classical ERalpha-mediated transcription, which occurs through canonical estrogen response elements. However, Foxl2 suppresses ERalpha signaling through nonclassical tethered transcriptional pathways. Specifically, the selective ER modulator tamoxifen stimulates activator protein-1 (AP1)-dependent transcription via the ERalpha, and this enhancement is blocked by Foxl2. Two lines of evidence suggest that Foxl2 suppression is mediated by physical interactions with ERalpha rather than direct action at AP1 binding sites. First, ERalpha is coimmunoprecipitated with Foxl2. Second, activation of a upstream activating sequence (UAS) reporter by Gal4-cJun in the presence of ERalpha and tamoxifen was blocked by Foxl2, demonstrating suppression in the absence of an AP1 site. Cyclooxygenase-2 (COX2), which is required for ovulation, was identified through expression profiling as a candidate physiological target for nonclassical ERalpha signaling and thus modulation by ERalpha/Foxl2 interactions. This possibility was confirmed by two sets of experiments. COX2 protein levels were induced by ERalpha in the presence of tamoxifen, and protein expression was suppressed by Foxl2. In addition, ERalpha stimulation of the COX2 promoter was repressed by Foxl2. We conclude that ERalpha and Foxl2 interact and that Foxl2 selectively suppresses ERalpha-mediated transcription of AP1-regulated genes. These data provide a potential point of convergence for ERalpha and Foxl2 to regulate ovarian development and function. The forkhead transcription factor foxl2 is sumoylated in both human and mouse: sumoylation affects its stability, localization, and activity. Marongiu M et al. The FOXL2 forkhead transcription factor is expressed in ovarian granulosa cells, and mutated FOXL2 causes the blepharophimosis, ptosis and epicanthus inversus syndrome (BPES) and predisposes to premature ovarian failure. Inactivation of Foxl2 in mice demonstrated its indispensability for female gonadal sex determination and ovary development and revealed its antagonism of Sox9, the effector of male testis development. To help to define the regulatory activities of FOXL2, we looked for interacting proteins. Based on yeast two-hybrid screening, we found that FOXL2 interacts with PIAS1 and UBC9, both parts of the sumoylation machinery. We showed that human FOXL2 is sumoylated in transfected cell lines, and that endogenous mouse Foxl2 is comparably sumoylated. This modification changes its cellular localization, stability and transcriptional activity. It is intriguing that similar sumoylation and regulatory consequences have also been reported for SOX9, the male counterpart of FOXL2 in somatic gonadal tissues. | ||||
| Cellular localization | Cytoplasmic, Nuclear | ||||
| Comment | Conventional and unconventional interactions of the transcription factor FOXL2 uncovered by a proteome-wide analysis. Penrad-Mobayed M et al. (2020) Beyond the study of its transcriptional target genes, the identification of the various interactors of a transcription factor (TF) is crucial to understand its diverse cellular roles. We focused on FOXL2, a winged-helix forkhead TF important for ovarian development and maintenance. FOXL2 has been implicated in diverse cellular processes, including apoptosis, the control of cell cycle or the regulation of steroid hormone synthesis. To reliably identify partners of endogenous FOXL2, we performed a proteome-wide analysis using co-immunoprecipitation in the murine granulosa cell-derived AT29c and the pituitary-derived alpha-T3 cell lines, using three antibodies targeting different parts of the protein. Following a stringent selection of mass spectrometry data on the basis of identification reliability and protein enrichment, we identified a core set of 255 partners common to both cell lines. Their analysis showed that we could co-precipitate several complexes involved in mRNA processing, chromatin remodeling and DNA replication and repair. We further validated (direct and/or indirect) interactions with selected partners, suggesting an unexpected role for FOXL2 in those processes. Overall, this comprehensive analysis of the endogenous FOXL2 interactome sheds light on its numerous and diverse interactors and unconventional cellular roles.////////////////// FOXL2C134W-induced CYP19 expression via cooperation with SMAD3 in HGrC1 cells. Belli M et al. (2018) Germline knockout studies in female mice demonstrated an essential role for FOXL2 in early follicle development, while an inducible granulosa cell (GC) specific deletion of Foxl2 in adults has shown ovary-to-testis somatic sex reprogramming. In women, over 120 different germline mutations in the FOXL2 gene have been shown to cause blepharophimosis/ptosis/epicantus inversus syndrome associated with or without primary ovarian insufficiency. By contrast, a single somatic mutation (FOXL2C134W) accounts for almost all adult-type GC tumors (aGCTs). To test the hypothesis that FOXL2C134W differentially regulates the expression of aGCT markers, we investigated the effect of FOXL2C134W on Inhibin B and P450 aromatase expression using a recently established human GC line (HGrC1), which we now show to bear two normal alleles of FOXL2. Neither FOXL2wt nor FOXL2C134W regulate INHBB mRNA expression. However, FOXL2C134W selectively displays a 50-fold induction of CYP19 mRNA expression dependent upon activin A. Mechanistically, the CYP19 promoter is activated in a similar way by FOXL2C134W interaction with SMAD3, but not by FOXL2wt. SMAD2 had no effect. Moreover, FOXL2C134W interactions with SMAD3 and with the FOX binding element located at -199 bp upstream of the ATG initiation codon of CYP19 are more sustainable than FOXL2wt. Thus, FOXL2C134W potentiates CYP19 expression in HGrC1 cells via enhanced recruitment of SMAD3 to a proximal FOX binding element. These findings may explain the pathophysiology of estrogen excess in patients with aGCT.////////////////// The CpG Island in the Murine Foxl2 Proximal Promoter Is Differentially Methylated in Primary and Immortalized Cells. Tran S 2013 et al. Forkhead box L2 (Foxl2), a member of the forkhead transcription factor family, plays important roles in pituitary follicle-stimulating hormone synthesis and in ovarian maintenance and function. Mutations in the human FOXL2 gene cause eyelid malformations and premature ovarian failure. FOXL2/Foxl2 is expressed in pituitary gonadotrope and thyrotrope cells, the perioptic mesenchyme of the developing eyelid, and ovarian granulosa cells. The mechanisms governing this cell-restricted expression have not been described. We mapped the Foxl2 transcriptional start site in immortalized murine gonadotrope-like cells, L?2, by 5' rapid amplification of cDNA ends and then PCR amplified approximately 1 kb of 5' flanking sequence from murine genomic DNA. When ligated into a reporter plasmid, the proximal promoter conferred luciferase activity in both homologous (L?2) and, unexpectedly, heterologous (NIH3T3) cells. In silico analyses identified a CpG island in the proximal promoter and 5' untranslated region, suggesting that Foxl2 transcription might be regulated epigenetically. Indeed, pyrosequencing and quantitative analysis of DNA?methylation?using real-time PCR revealed Foxl2 proximal promoter hypomethylation in homologous compared to some, though not all, heterologous cell lines. The promoter was also hypomethylated in purified murine gonadotropes. In vitro promoter methylation completely silenced reporter activity in heterologous and homologous cells. Collectively, the data suggest that differential proximal promoter DNA methylation may contribute to cell-specific Foxl2 expression in some cellular contexts. However, gonadotrope-specific expression of the gene cannot be explained by promoter hypomethylation alone. ///////////////////////// The Transcriptional Targets of Mutant FOXL2 in Granulosa Cell Tumours. Rosario R et al. Despite their distinct biology, granulosa cell tumours (GCTs) are treated the same as other ovarian tumours. Intriguingly, a recurring somatic mutation in the transcription factor Forkhead Box L2 (FOXL2) 402C>G has been found in nearly all GCTs examined. This investigation aims to identify the pathogenicity of mutant FOXL2 by studying its altered transcriptional targets. Discovery of novel protein partners of the transcription factor FOXL2 provides insights into its physiopathological roles. L'h?D et al. FOXL2 transcription factor is responsible for the BPES Syndrome, a genetic disease involving craniofacial malformations often associated with ovarian failure. Recently, a somatic FOXL2 mutation (p.C134W) has been reported in more than 95% of adult-type granulosa cell tumors (GCTs). Here, we have identified ten 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-t2a, which acted as a repressor on most promoters, increased wild-type 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 wild-type 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 wild-type 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. Deletions in the polyAlanine-containing transcription factor FOXL2 lead to intranuclear aggregationMoumne L, et al . Mutations of FOXL2, a gene encoding a forkhead transcription factor, have been shown to cause the blepharophimosis-ptosis-epicanthus inversus syndrome. This genetic disorder is characterized by eyelid and craniofacial abnormalities associated or not with premature ovarian failure. We have previously shown that mutant FOXL2 with an expanded polyAlanine (polyAla) tract forms large aggregates both in the nucleus and in the cytoplasm of transfected cells, whereas the wild-type protein localizes in the nucleus in a rather diffuse manner. Premature stop codons in FOXL2 have been considered so far as null alleles. However, we demonstrate here that such nonsense mutations may lead to the production of N-terminally truncated proteins by re-initiation of translation downstream of the stop codon. Surprisingly, the truncated proteins strongly aggregate in the nucleus, partially localize in the cytoplasm and retain a fraction of the wild-type protein. We also show that a complete deletion of the polyAla tract of FOXL2 induces a significant intranuclear aggregation. Our results enlarge the spectrum of mutations inducing FOXL2 aggregation. Essential but differential role of FOXL2(wt) and FOXL2(C134W) in GDF-9 stimulation of follistatin transcription in co-operation with Smad3 in the human granulosa cell line COV434. Nonis D et al. The FOXL2(C134W) mutation has been identified in virtually all adult granulosa cell tumors (GCTs). Here we show that the exogenous FOXL2 expression is necessary for GDF-9 stimulation of follistatin transcription in the human GCT cell line, COV434 that lacks endogenous FOXL2 expression. Interestingly, in the presence of Smad3 co-expression, FOXL2(C134W) negated GDF-9 stimulation of follistatin transcription. However, mutation of the Smad binding element (SBE) located in the intronic enhancer elements in the follistatin gene restored normal FOXL2 activity to FOXL2(C134W), thus the altered activity of FOXL2(C134W) is dependent on the ability of Smad3 to directly bind the SBE. Mutation of the FOXL2 binding element (FBE) or the FBE and SBE completely prevented GDF-9 activity, suggesting that the FBE is essential for GDF-9 stimulation in COV434. Overall, our study supports the view that altered interaction of FOXL2(C134W) with co-factors may underlie the pathogenesis of this mutation in GCTs. | ||||
| Ovarian function | Follicle development, Initiation of primordial follicle growth, Follicle atresia, Steroid metabolism, Germ cell development, Early embryo development | ||||
| Comment | Restoration of the destroyed crown of multirooted teeth]. [Kopeĭkin VN et al. (1988)////////////////// Oocytes suppress FOXL2 expression in cumulus cells in mice. Emori C et al. (2020) Cumulus cells and mural granulosa cells (MGCs) play distinct roles during follicular development, and normal development of these cell lineages is critical for the female fertility. Transcriptomic diversification between the two cell-lineages is obviously a critical mechanism for their functional diversification; however, the transcriptional regulators responsible for this event have not been fully defined. In this study, we sought to identify key transcriptional regulators responsible for the differential gene expression between the two cell-lineages. In silico analysis of transcriptomic comparison between cumulus cells and MGCs identified several candidate regulators responsible for the diversification of the two cell-lineages. Among them, we herein focused on Forkhead box L2 (FOXL2) and showed that expression of FOXL2 as well as its target transcripts were differentially regulated between cumulus cells and MGCs. The lower expression of FOXL2 in cumulus cells seemed to be due to the suppression by oocyte-derived paracrine signals. These results suggest that FOXL2 is one of the critical transcription factors that determine cumulus cell and MGC lineages under the control of oocytes.////////////////// FOXL2 Is an Essential Activator of SF-1-Induced Transcriptional Regulation of Anti-Müllerian Hormone in Human Granulosa Cells. Jin H et al. (2016) Anti-Müllerian hormone (AMH) is required for proper sexual differentiation by regulating the regression of the Müllerian ducts in males. Recent studies indicate that AMH could be an important factor for maintaining the ovarian reserve. However, the mechanisms of AMH regulation in the ovary are largely unknown. Here, we provide evidence that AMH is an ovarian target gene of steroidogenic factor-1 (SF-1), an orphan nuclear receptor required for proper follicle development. FOXL2 is an evolutionally conserved transcription factor, and its mutations cause blepharophimosis, ptosis, and epicanthus inversus syndrome (BPES), wherein affected females display eyelid defects and premature ovarian failure (POF). Notably, we found that functional FOXL2 is essential for SF-1-induced AMH regulation, via protein-protein interactions between FOXL2 and SF-1. A BPES-inducing mutant of FOXL2 (290-291delCA) was unable to interact with SF-1 and failed to mediate the association between SF-1 and the AMH promoter. Therefore, this study identified a novel regulatory circuit for ovarian AMH production; specifically, through the coordinated interplay between FOXL2 and SF-1 that could control ovarian follicle development.////////////////// WNT/β-catenin and p27/FOXL2 differentially regulate supporting cell proliferation in the developing ovary. Gustin SE et al. (2016) Sexual development is initiated through differentiation of testicular Sertoli cells or ovarian granulosa cells. Although these supporting cells are considered to develop from common bipotential precursors, recent evidence suggests that distinct supporting cell populations are present in the ovary, with one providing granulosa cells of the medullary follicles and the other providing granulosa cells of the cortical follicles, the latter of which support lifelong fertility. Here, we demonstrate that XX fetal gonads contain GATA4 expressing supporting cells that either enter mitotic arrest, or remain proliferative. Blocking WNT signalling reduces XX supporting cell proliferation, while stabilising β-catenin signalling promotes proliferation, indicating that the renewal of pre-granulosa cells is dependent on WNT/β-catenin signalling in the proliferative supporting cell population. In contrast, XX supporting cells express p27 and FOXL2 and are maintained in mitotic arrest. Although FOXL2 is required for maintaining high levels of p27 expression, it is dispensable for entry and maintenance of mitotic arrest in XX supporting cells. Combined our data suggest that both medullary and cortical precursors arise from a common GATA4 expressing cell type. In addition, this work indicates that a balance between supporting cell self-renewal and differentiation is maintained in the developing ovary by relative WNT/β-catenin and p27/FOXL2 activities. This study provides significant new insights into the origin and formation of ovarian follicles and evidence supporting a common fetal origin of medullary and cortical granulosa cells.////////////////// The transcription factor FOXL2 mobilizes estrogen signaling to maintain the identity of ovarian granulosa cells. Georges A et al. (2014) FOXL2 is a lineage determining transcription factor in the ovary, but its direct targets and modes of action are not fully characterized. In this study, we explore the targets of FOXL2 and five nuclear receptors in murine primary follicular cells. We found that FOXL2 is required for normal gene regulation by steroid receptors, and we show that estrogen receptor beta (ESR2) is the main vector of estradiol signaling in these cells. Moreover, we found that FOXL2 directly modulates Esr2 expression through a newly identified intronic element. Interestingly, we found that FOXL2 repressed the testis-determining gene Sox9 both independently of estrogen signaling and through the activation of ESR2 expression. Altogether, we show that FOXL2 mobilizes estrogen signaling to establish a coherent feed-forward loop repressing Sox9. This sheds a new light on the role of FOXL2 in ovarian maintenance and function.////////////////// Mouse Forkhead L2 (FOXL2) maintains repression of FSH-dependent genes in the granulosa cell. Kuo FT et al. The forkhead transcription factor FOXL2 (Forkhead box L2) is expressed in granulosa cells of small and medium follicles in the mouse ovary. Foxl2 female knockout mice exhibit primordial follicle depletion and primary ovarian failure, but evidence from adult female conditional Foxl2 knockout mice suggests FOXL2 may also play a significant role in maintenance of ovarian differentiation at stages beyond the primordial follicle and initial wave of folliculogenesis. We previously showed that human FOXL2 functions as a transcriptional repressor of several key genes involved in granulosa cell proliferation and differentiation, including StAR, P450aromatase (CYP19), P450scc (CYP11A) and cyclinD2 (CCND2). To elucidate the role of mouse FOXL2, we determined its role in transcriptional regulation in CHO cells, and then confirmed our findings in mouse granulosa cells. We found that mouse FOXL2 represses the activities of the mouse Star, Cyp19, Cyp11a promoters in CHO cells, but may not repress the Ccnd2 promoter, and identified the minimal mouse Star, Cyp19 and Cyp11a promoter regions responsive to FOXL2 regulation. We then knocked down Foxl2 in mouse granulose cells using siRNA, which resulted in significantly increased expression levels of mouse Star, Cyp19 and Cyp11a, but not Ccnd2. To increase Foxl2 expression levels, we generated a mouse Foxl2 lentiviral construct and used it to infect mouse granulosa cells. Following lentiviral infection, the expression levels of mouse Star, Cyp19 and Cyp11a, but not Ccnd2, decreased significantly. These data confirm that mouse FOXL2 functions as a transcriptional repressor of key granulosa cell genes that influence ovarian development. Transcription factor FOXL2 protects granulosa cells from stress and delays cell cycle: role of its regulation by the SIRT1 deacetylase. Benayoun BA et al. FOXL2 is a transcription factor essential for ovarian function and maintenance, whose germline mutations are responsible for the Blepharophimosis syndrome (BPES), often associated with premature ovarian failure/ageing. Recent evidence has linked FOXL2 downregulation or somatic mutation (p.Cys134Trp) to cancer, though underlying molecular mechanisms remain unclear. Using a functional genomics approach, we find that FOXL2 modulates cell cycle regulators in a way which should lead to G1 arrest. Indeed, FOXL2 upregulation promotes cell accumulation in the 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. Since granulosa cell tumors respond poorly to conventional chemotherapy, our findings on the deacetylase inhibitor nicotinamide provide an interesting option for targeted therapy. Aromatase Is a Direct Target of FOXL2: C134W in Granulosa Cell Tumors via a Single Highly Conserved Binding Site in the Ovarian Specific Promoter. Fleming NI et al. Granulosa cell tumors (GCT) of the ovary often express aromatase and synthesize estrogen, which in turn may influence their progression. Recently a specific point mutation (C134W) in the FOXL2 protein was identified in >94% of adult-type GCT and it is likely to contribute to their development. A number of genes are known to be regulated by FOXL2, including aromatase/CYP19A1, but it is unclear which are direct targets and whether the C134W mutation alters their regulation. Recently, it has been reported that FOXL2 forms a complex with steroidogenic factor 1 (SF-1) which is a known regulator of aromatase in granulosa cells. Differential apoptotic activities of wild-type FOXL2 and the adult-type granulosa cell tumor-associated mutant FOXL2 (C134W). Kim JH et al. Some mutations in FOXL2 result in premature ovarian failure accompanied by blepharophimosis, ptosis, epicanthus inversus syndrome type I disease, and FOXL2-null mice exhibit developmental defects in granulosa cells. Recently, FOXL2 c.402C>G, a new somatic mutation that leads to a p.C134W change, was found in the majority of adult-type ovarian granulosa cell tumors (GCTs). In this study, we investigated the possible mechanisms by which the C134W mutation contributes to the development of GCTs. Wild-type (WT) and mutant FOXL2 displayed differential apoptotic activities. Specifically, WT FOXL2 induced significant granulosa cell death, but the mutant exhibited minimal cell death. The FOXL2-induced apoptotic response was greatly dependent on caspase 8, BID and BAK because the depletion of any of these three proteins inhibited FOXL2 from eliciting the full apoptotic response. Activation of caspase 8 and subsequent increased production of truncated BID, and oligomerization of BAK, and release of cytochrome c were all associated with the apoptosis induced by WT FOXL2 expression. In contrast, the mutant FOXL2 was unable to elicit the full array of apoptotic signaling responses. In addition, we found differential TNF-R1 (tumor necrosis factor-receptor 1) and Fas (CD95/APO-1) upregulation between the WT and the mutant, and the silencing of TNF-R1 or Fas and the blockage of the death signaling mediated by TNF-R1 or Fas using TNF-Fc or Fas-Fc, respectively, resulted in significant attenuations of FOXL2-induced apoptosis. Moreover, granulosa cells that expressed either WT FOXL2 or mutant exhibited distinct cell death sensitivities on activation of death receptors and deprivation of serum. Thus, the differential activities of FOXL2 and its mutant may partially account for the pathophysiology of GCT development.Oncogene advance online publication, 29 November 2010; doi:10.1038/onc.2010.541. FOXL2 Interacts with Steroidogenic Factor-1 (SF-1) and Represses SF-1-Induced CYP17 Transcription in Granulosa Cells. Park M et al. Mutations in FOXL2 are responsible for blepharophimosis-ptosis-epicanthus inversus syndrome (BPES) type I, in which affected women exhibit premature ovarian failure. FOXL2-null mice showed defects in granulosa cell development during folliculogenesis. We screened a rat ovarian yeast two-hybrid cDNA library to identify FOXL2-interacting proteins and found steroidogenic factor-1 (SF-1). Here, we show that human FOXL2 and SF-1 proteins interact in human granulosa cells and that FOXL2 negatively regulates the transcriptional activation of a steroidogenic enzyme, CYP17, by SF-1. Furthermore, FOXL2 mutants found in blepharophimosis-ptosis-epicanthus inversus syndrome type I patients lost the ability to repress CYP17 induction mediated by SF-1. Chromatin immunoprecipitation and EMSA results further revealed that FOXL2 inhibited the binding of SF-1 to the CYP17 promoter, whereas the FOXL2 mutants failed to block this interaction. Therefore, this study identifies a novel regulatory role for FOXL2 on a key steroidogenic enzyme and provides a possible mechanism by which mutations in FOXL2 disrupt normal ovarian follicle development. Human forkhead L2 represses key genes in granulosa cell differentiation including aromatase, P450scc, and cyclin D2. Bentsi-Barnes IK et al. FOXL2 is expressed in granulosa cells (GC) of small and medium ovarian follicles, functions as a repressor of the human steroidogenic acute regulatory gene, a marker of a GC differentiation, and its mutation is associated with premature ovarian failure (POF) in women with blepharophimosis-ptosis-epicanthus inversus syndrome (BPES), type I. We now report that FOXL2 also represses the transcription of aromatase, P450scc, and cyclin D2, three other key genes involved in GC proliferation, differentiation, and steroidogenesis, and that a FOXL2 mutation found in patients with BPES type I, also fails to repress aromatase transcription, further supporting a role for FOXL2 in follicle maturation. Foxl2 functions in sex determination and histogenesis throughout mouse ovary development. Garcia-Ortiz JE et al. ABSTRACT: BACKGROUND: Partial loss of function of the transcription factor FOXL2 leads to premature ovarian failure in women. In animal models, Foxl2 is required for maintenance, and possibly induction, of female sex determination independently of other critical genes, e.g., Rspo1. Here we report expression profiling of mouse ovaries that lack Foxl2 alone or in combination with Wnt4 or Kit/c-Kit. RESULTS: Following Foxl2 loss, early testis genes (including Inhbb, Dhh, and Sox9) and several novel ovarian genes were consistently dysregulated during embryonic development. In the absence of Foxl2, expression changes affecting a large fraction of pathways were opposite those observed in Wnt4-null ovaries, reinforcing the notion that these genes have complementary actions in ovary development. Loss of one copy of Foxl2 revealed strong gene dosage sensitivity, with molecular anomalies that were milder but resembled ovaries lacking both Foxl2 alleles. Furthermore, a Foxl2 transgene disrupted embryonic testis differentiation and increased the levels of key female markers. CONCLUSIONS: The results, including a comprehensive principal component analysis, 1) support the proposal of dose-dependent Foxl2 function and anti-testis action throughout ovary differentiation; and 2) identify candidate genes for roles in sex determination independent of FOXL2 (e.g., the transcription factors IRX3 and ZBTB7C) and in the generation of the ovarian reserve downstream of FOXL2 (e.g., the cadherin-domain protein CLSTN2 and the sphingomyelin synthase SGMS2). The gene inventory is a first step toward the identification of the full range of pathways with partly autonomous roles in ovary development, and thus provides a framework to analyze the genetic bases of female fertility. To better understand the role of FOXL2 in the etiology of ovarian failure in BPES, Loffler et al et al examined its expression in embryonic ovaries of mice, chickens, and red-eared slider turtles, representatives of three phylogenetically distant vertebrate groups that have different mechanisms of sex determination. Expression of Foxl2 was detected in early ovaries of all three species around the time of sex determination and was associated with both somatic and germ cell populations in mice. Expression was sexually dimorphic in all cases. Sequence analysis of turtle and chicken FoxL2 orthologues indicated an unusually high degree of structural conservation during evolution. FoxL2 was found to be autosomal in chickens, and therefore unlikely to represent the dominant ovarian-determining gene that has been postulated to exist as a possible explanation for female heterogamety in birds. BPES may result from early abnormalities in regulating the development of the fetal ovary, rather than premature degeneration of the postnatal or adult ovary. Further, our results suggest that FOXL2 is a highly conserved early regulator of vertebrate ovarian development. Foxl2 is required for commitment to ovary differentiation Ottolenghi C, et al . Genetic control of female sex differentiation from a bipotential gonad in mammals is poorly understood. We find that mouse XX gonads lacking the forkhead transcription factor Foxl2 form meiotic prophase oocytes but then activate the genetic program for somatic testis determination. Pivotal Foxl2 action thus represses the male gene pathway at several stages of female gonadal differentiation. This suggests the possible continued involvement of sex-determining genes in maintaining ovarian function throughout female reproductive life. Potential targets of FOXL2, a transcription factor involved in craniofacial and follicular development, identified by transcriptomics. Batista F et al. FOXL2 is a gene encoding a forkhead transcription factor, whose mutations are responsible for the blepharophimosis-ptosis-epicanthus inversus syndrome that often involves 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. We have recently shown that the aromatase gene is a target of FOXL2, and only three other targets have been reported so far. To detect potential transcriptional targets of FOXL2, we used DNA chips and quantitative PCR to compare the transcriptomes of granulosa-like cells overexpressing, or not, FOXL2. This analysis showed that mediators of inflammation, apoptotic and transcriptional regulators, genes involved in cholesterol metabolism, and genes encoding enzymes and transcription factors involved in reactive oxygen species detoxification were up-regulated. On the other hand, FOXL2 down-regulated the transcription of several genes involved in proteolysis and signal transduction and in transcription regulation. A bioinformatic analysis was conducted to discriminate between potential target promoters activated and repressed by FOXL2. In addition, the promoters of strongly activated genes were enriched in forkhead recognition sites, suggesting that these genes might be direct FOXL2 targets. Altogether, these results provide insight into the activity of FOXL2 and may help in understanding the mechanisms of pathogenesis of FOXL2 mutations if the targets prove to be the same in the ovary. Loss of Wnt4 and Foxl2 leads to Female-To-Male Sex Reversal Extending to Germ Cells. Ottolenghi C et al. The discovery that the SRY gene induces male sex in humans and other mammals led to speculation about a possible equivalent for female sex. However, only partial effects have been reported for candidate genes experimentally tested so far. Here we demonstrate that inactivation of two ovarian somatic factors, Wnt4 and Foxl2, produces testis differentiation in XX mice, resulting in the formation of testis tubules and spermatogonia. These genes are thus required to initiate or maintain all major aspects of female sex determination in mammals. The two genes are independently expressed and show complementary roles in ovary morphogenesis. In addition, forced expression of Foxl2 impairs testis tubule differentiation in XY transgenic mice, and germ cell-depleted XX mice lacking Foxl2 and harboring a kit mutation undergo partial female-to-male sex reversal. The results are all consistent with an anti-testis role for Foxl2. The data suggest that the relative autonomy of the action of Foxl2, Wnt4 and additional ovarian factor(s) in the mouse should facilitate the dissection of their respective contributions to female sex determination. Positive and negative feedback regulates the transcription factor FOXL2 in response to cell stress: evidence for a regulatory imbalance induced by disease-causing mutations. Benayoun BA et al. 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 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. | ||||
| Expression regulated by | Steroids, AMH, beta-catenin | ||||
| Comment | Beta-catenin directs the transformation of testis Sertoli cells to ovarian granulosa-like cells by inducing Foxl2 expression. Li Y et al. (2017) Sertoli and granulosa cells are two major types of somatic cells in the male and female gonads,respectively. Previous studies have shown that Sertoli and granulosa cells are derived from common progenitor cells and that differentiation of these two cell types is regulated by sex differentiation genes. The signaling pathway including the adhesion and transcription factor Ctnnb1 (cadherin-associated protein, beta 1, also known as β-catenin) regulates differentiation of granulosa cells in the absence of the transcription factor Sry, and over-activation of β-catenin in the presence of Sry leads to granulosa prior to sex determination. Surprisingly, our previous study found that β-catenin over-activation in Sertoli cells after sex determination can also cause disruption of the testicular cord and aberrant testis development. However, the underlying molecular mechanism was unclear. In this study, we found that constitutive activation of Ctnnb1 in Sertoli cells led to ectopic expression of the granulosa cell-specific marker FOXL2 in testes. Co-staining experiments revealed that FOXL2-positive cells were derived from Sertoli cells, and Sertoli cells were transformed into granulosa-like cells after Ctnnb1 over-activation. Further studies demonstrated that CTNNB1 induced Foxl2 expression by directly binding to transcription factor Tcf/Lef binding sites in the FOXL2 promoter region. We also found that directly over-expression of Foxl2 indecreased the expression of Sertoli cell-specific genes in primary Sertoli cells. Taken together, these results demonstrate that repression of β-catenin (CTNNB1) signaling is required for lineage maintenance of Sertoli cells. Our study provides a new mechanism for Sertoli cell lineage maintenance during gonad development.////////////////// The anti-Müllerian hormone (AMH) induces forkhead box L2 (FOXL2) expression in primary culture of human granulosa cells in vitro. Sacchi S et al. (2017) Anti-Müllerian hormone (AMH) and forkhead box L2 (FOXL2) are two pivotal genes expressed in human granulosa cells (hGCs) where both genes share similar inhibitory functions on activation and follicular growth in order to preserve the ovarian follicle reserve. Furthermore, AMH and FOXL2 contribute to inhibit steroidogenesis, decreasing or preventing the activation of gonadotrophin-dependent aromatase CYP19A1 cytochrome P450 family 19 subfamily A member 1 (CYP19A1). The purpose of this study is to evaluate the role of AMH in regulating the expression of FOXL2. Primary cultures of hGCs were treated with increasing concentrations of recombinant human AMH (rhAMH; range 10-100 ng/ml) for 3 h. Negative controls were performed using corresponding amounts of AMH vehicle. Total RNA or proteins were purified and quantified by spectrophotometry. FOXL2 and CYP19A1 gene expression, normalized by reference gene ribosomal protein S7 (RpS7), was evaluated by RT-qPCR. Each reaction was repeated in triplicate. Statistical analysis was performed. Extracted proteins were analyzed by immunoblot using anti-FOXL2 and anti-β-actin as primary antibodies. rhAMH treatments tested did not modulate the basal expression of aromatase CYP19A1 gene. rhAMH (50 ng/ml) was able to increase FOXL2 gene expression and its intracellular content. This study demonstrated the existence of an AMH-FOXL2 relationship in hGCs. AMH is capable of increasing both gene and protein expression of FOXL2. Because FOXL2 induces AMH transcription, these ovarian factors could be finely regulated by a positive feedback loop mechanism to preserve the ovarian follicle reserve.////////////////// The Emerging Role of FOXL2 in Regulating the Transcriptional Activation Function of Estrogen Receptor β: An Insight Into Ovarian Folliculogenesis. Hirano M et al. (2016) Germline mutations of the fork-head transcriptional factor forkhead box L2 (FOXL2) predispose embryos to autosomal-dominant blepharophimosis-ptosis-epicanthus inversus syndrome with primary ovarian insufficiency in female patients, but the mechanisms of FOXL2 in ovarian follicular development remain elusive. Estrogens produced by ovarian granulosa cells and estrogen receptor (ER) α and ERβ play fundamental roles in ovarian pathophysiology, and a previous study revealed that ERα and ERβ physically interact with FOXL2. However, the underlying functions of these interactions have not been investigated. Herein, we report an ERβ-specific repressive function of FOXL2. Histological examination demonstrated that FOXL2 expression tends to be intense during early follicular development. Immunoprecipitation revealed that ERβ and FOXL2 interact in a ligand-independent manner. In vitro pull-down assays revealed a direct interaction between FOXL2 and the activation function (AF)-1/2 domain of ERβ. The expression of FOXL2 represses the ligand-dependent transcriptional activation of ERβ, but FOXL2 does not influence the ligand-dependent transcriptional activation of ERα. Consistent with these results, RNA interference-mediated depletion of FOXL2 stimulates the expression of the ERβ-downstream gene p450 aromatase. The convergence between FOXL2 functions and ERβ-mediated transcription in the ovary suggests the putative mechanism of FOXL2 in early-phase follicular development, which may be partially attributed to the regulation of ERβ-dependent gene expression.////////////////// Sense and antisense Foxl2 transcripts in mouse Cocquet J, et al . FOXL2 is a forkhead transcription factor involved in eyelid development and in the development and adult function of the ovary in mammals. In mouse, we have previously suggested the existence of two mRNA isoforms of Foxl2 that result from an alternative polyadenylation. In this study, we characterize in depth the structure and expression of these two variants. We also describe an antisense transcript that overlaps the whole Foxl2 transcription unit. This antisense transcript, called Foxl2OS (for opposite strand), yields several isoforms resulting from alternative splicing. No significant coding region was found in the Foxl2OS sequence. Foxl2OS displays a pattern of expression very similar to that of Foxl2 in the gonads during development and at the adult age. RNA FISH experiments show that both transcripts are expressed in the same cells at the same time. We suggest that Foxl2OS is a noncoding antisense RNA that may be involved in the regulation of Foxl2. All in all our results provide new insights about the organization of the murine Foxl2 locus. This might help us understand its regulation and function. Aromatase inhibition reduces expression of FOXL2 in the embryonic chicken ovary Dev Dyn. 2005 . Synergistic Activation of the Mc2r Promoter by FOXL2 and NR5A1 in Mice. Yang WH et al. FOXL2 is the earliest ovarian marker and plays an important role in regulation of cholesterol and steroid metabolism, inflammation, apoptosis, and ovarian development and function. Mutations and deficiencies of the human FOXL2 gene have been shown to cause blepharophimosis-ptosis-epicanthus inversus syndrome (BPES) as well as premature ovarian failure. Although Foxl2 interacts with Nr5a1 and up-regulates cyp19a1a gene transcription in fish, FOXL2 represses the transcriptional activity of the gene that codes for steroidogenic acute regulatory protein (Star) in mice. Most of the recent studies have heavily focused on the FOXL2 target genes (Star and Cyp19a1) in the ovaries. Hence, it is of importance to search for other downstream targets of FOXL2 and for the possibility of FOXL2 expression in non-ovary tissues. Herein, we demonstrate that the interplay between FOXL2 and NR5A1 regulates Star and melanocortin 2 receptor (Mc2r) gene expression in mammalian systems. Both FOXL2 and NR5A1 are expressed in ovary and adrenal gland tissues. As expected, FOXL2 represses and NR5A1 enhances the promoter activity of Star. Notably, the promoter activity of Mc2r is activated by FOXL2 in a dose-dependent manner. Surprisingly, we found that FOXL2 and NR5A1 synergistically up-regulate the transcriptional activity of Mc2r. By mapping the Mc2r promoter, we provide evidence that distal NR5A1 response elements (-1410 and -975) are required for synergistic activation by FOXL2 and NR5A1. These results suggest that the interplay between FOXL2 and NR5A1 on the Mc2r promoter functions as a novel mechanism for regulating MC2R-mediated cell signaling as well as steroidogenesis in adrenal glands. | ||||
| Ovarian localization | Cumulus, Granulosa, Theca, Stromal cells, Ovarian tumor | ||||
| Comment | Pisarska MD, et al reported that Forkhead L2 Is Expressed in the Ovary and Represses the Promoter Activity of the Steroidogenic Acute Regulatory Gene. The forkhead transcription factor FOXL2 is expressed in somatic cells of the human ovary prior to follicle formation. Duffin K et al. Interactions between germ cells and surrounding somatic cells are central to ovarian development as well as later function. Disruption of these interactions arising from abnormalities in either cell type can lead to premature ovarian failure (POF). The forkhead transcription factor FOXL2 is a candidate POF factor, and mutations in the FOXL2 gene are associated with syndromic and non-syndromic ovarian failure. Foxl2-deficient mice display major defects in primordial follicle activation with consequent follicle loss, and earlier roles in gonadal development and sex determination have also been suggested. However, despite its importance no data presently exist on its expression in the developing human ovary. Expression of FOXL2 mRNA was demonstrated in the human fetal ovary between 8 weeks and 19 weeks gestation, thus from soon after sex determination to primordial follicle development. Expression in the ovary was higher after 14 weeks than at earlier gestation weeks and was very low in the fetal testis at all ages examined. Immunolocalisation revealed FOXL2 expression to be confined to somatic cells, both adjacent to germ cells and those located in the developing ovarian stroma. These cells are the site of action of oocyte-derived activin signalling, but in vitro treatment of human fetal ovaries with activin failed to reveal any regulation of FOXL2 transcription by this pathway. In summary, the expression of FOXL2 in somatic cells of the developing human ovary before and during follicle formation supports a conserved and continuing role for this factor in somatic/germ cell interactions from the earliest stages of human ovarian development. Premature ovarian failure in a subgroup of women with Blepharophimosis-Ptosis-Epicanthus Inversus type 1 syndrome has been associated with nonsense mutations in the gene encoding a Forkhead transcription factor, Forkhead L2 (FOXL2). However, the exact function of FOXL2 in the ovary is unclear. We investigated the expression of FOXL2 in the mouse ovary during follicular development and maturation by RT-PCR and in situ hybridization. The FOXL2 mRNA is expressed in ovaries throughout development and adulthood, and is localized to the undifferentiated granulosa cells in small and medium follicles, as well as cumulus cells of preovulatory follicles. FOXL2 belongs to a group of transcription factors capable of interacting with specific DNA sequences in diverse gene promoters. With the presence of multiple putative forkhead DNA consensus sites, the promoter of the human Steroidogenic Acute Regulatory (StAR) gene was used to test for regulation by FOXL2. Co-transfection studies revealed that wild-type FOXL2 represses the activity of the StAR promoter, and the first 95 bp upstream of the transcriptional start site of the StAR gene is sufficient for FOXL2 repression. Electrophoretic mobility shift assays confirmed that FOXL2 interacts directly with this region. Analyses using FOXL2 mutants also demonstrated the importance of the entire alanine/proline-rich carboxyl terminus of FOXL2 for transcriptional repression. Furthermore, these mutations produce a protein with a dominant negative effect that disables the transcriptional repressor activity of wild-type FOXL2. Dominant negative mutations of FOXL2 could increase expression of StAR and other follicle differentiation genes in small and medium follicles to accelerate follicle development, resulting in increased initial recruitment of dormant follicles and thus the premature ovarian failure phenotype. Isolation of chicken homolog of the FOXL2 gene and comparison of its expression patterns with those of aromatase during ovarian development. Govoroun MS, et al . Mutations in the forkhead transcription factor gene FOXL2 are involved in ovarian failure, which occurs in human BPES syndrome. This syndrome presents a sexually dimorphic expression, specific to the ovary in several vertebrates. We cloned the open reading frame of chicken FOXL2 (cFoxL2) and studied cFoxL2 expression in developing gonads and during adulthood to examine the role of FOXL2 in ovarian differentiation and function in birds. The spatial and temporal dynamics of cFoxL2 and aromatase expression were analyzed in parallel by using real-time quantitative reverse transcriptase-polymerase chain reaction and immunohistochemistry in attempt to investigate the possible role of cFoxL2 in the regulation of aromatase. The expression patterns of cFoxL2 and aromatase transcripts were highly correlated during the sex-differentiation period (4.7-12.7 days of incubation). Aromatase and cFoxL2 proteins were colocalized in the medullar part of female gonads on embryonic day 14. Fourteen days after hatching, cFoxL2 protein was mainly detected in granulosa cells of developing follicles. In adult ovary follicular envelopes, apart from granulosa cells, cFoxL2 transcript and protein were detected at lower levels in theca cells where aromatase was present. A high level of cFoxL2 transcription was also observed in maturing and ovulated oocytes. Our results confirm that FoxL2 is an early regulator of ovarian development in birds and may be involved in aromatase transcription regulation. Developmental Dynamics, 2004. Molecular cloning and analysis of gonadal expression of Foxl2 in the medaka, Oryzias latipes. Nakamoto M et al. Foxl2 is a member of the winged helix/forkhead family of transcription factors and is known to be involved in ovarian development in some vertebrates. To address the role of Foxl2 in ovarian differentiation in medaka, we isolated Foxl2 cDNA and analyzed its expression patterns during sex differentiation. Expression of Foxl2 started in somatic cells surrounding germ cells in XX gonads, just after initiation of ovarian differentiation, and was maintained in granulosa cells throughout ovarian development. In the adult ovary, Foxl2 was expressed in previtellogenic and vitellogenic follicles, but expression ceased in postvitellogenic follicles. In contrast, Foxl2 mRNA could not be detected in testes. In addition, Foxl2 and aromatase mRNAs were co-localized in some somatic cells located on the ventral side of developing XX gonads. Our results suggested that Foxl2 was not involved in ovarian determination, but was involved in differentiation of granulosa cells in medaka. Extinction of FOXL2 expression in aggressive ovarian granulosa cell tumors in children. Kalfa N et al. OBJECTIVE: In the female gonad, FOXL2 is a key factor for proper differentiation of granulosa cells (GC) during folliculogenesis and its expression persists in the ovary after birth. The aim of this multicentric nationwide study was to determine whether FOXL2 expression varies during tumoral proliferation of GC cells in juvenile ovarian GC tumors (OGCT). DESIGN: Nationwide retrospective study. SETTING: University Hospital of Montpellier, Department of Hormonology. PATIENT(S): Between 1994 and 2004, 26 patients with juvenile OGCT were reported in the TGM95 database of the French Society for Childhood Cancer (SFCE) and from eight pediatric endocrinology centers. Immunohistochemistry was performed using an anti-FOXL2 antibody. INTERVENTION(S): Immunohistochemistry studies of FOXL2 on OGCT slides. MAIN OUTCOME MEASURE(S): Level of FOXL2 expression within the tumor, International Federation of Gynecology and Obstetrics classification, and tumor recurrences. RESULT(S): FOXL2 expression was absent in the GC of 10 patients and was markedly reduced in the cells of 4 patients. Precocious pseudopuberty was more frequently the revealing symptom in the children with conserved FOXL2 expression. Patients with no or reduced expression of FOXL2 more frequently exhibited associated hemorrhagic ascites, higher mitotic activity in the tumor, and significantly more advanced oncologic staging. All patients requiring complementary treatment (n = 7; chemotherapy or complementary surgery) had reduced expression of FOXL2 in the tumor. All recurring OGCT exhibited a complete extinction of FOXL2 expression (n = 3). CONCLUSION(S): These results show that FOXL2 is not expressed or is underexpressed in juvenile OGCT with an aggressive pattern of progression, and it thus may be a prognostic factor for these tumors. Mutational analysis of FOXL2 codon 134 in granulosa cell tumour of ovary and other human cancers. Kim MS et al. A missense somatic mutation in the FOXL2 gene affecting codon 134 has recently been reported in granulosa cell tumour (GCT) and thecoma of the ovary. Such a recurrent nature of the mutation strongly suggests that the FOXL2 mutation may play an important role in the development of ovarian sex cord-stromal tumours. The aim of this study was to characterize the FOXL2 mutation in human tumour tissues. We analysed 1353 tumour tissues from various origins, including ovarian tumours and other common cancers, by single-strand conformation polymorphism analysis. We found the FOXL2 codon 134 missense mutation in 53 of 56 adult GCTs (94.6%) and two of the 16 thecomas (12.5%), but none in other tumours. Histologically, FOXL2 mutation-negative adult GCT showed that GCT cells were admixed with fibrothecomatous cells, and FOXL2 mutation-positive thecomas showed that luteinized theca cells were predominant. However, immunostaining of either inhibin alpha or FOXL2 did not differentiate the FOXL2 mutation status of adult GCTs and thecomas. There was no FOXL1 mutation and no common oncogenic mutation in the adult GCTs and thecomas. Our data indicate that the FOXL2 codon 134 mutation occurs exclusively in GCT and thecoma, and suggest the possibility that the development of most GCTs and a fraction of thecomas may be dependent on this mutation. Our data also suggest that the FOXL2 mutation status, as well as some histological features, may be important in the diagnosis of ovarian sex cord-stromal tumours. Copyright (c) 2010 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. | ||||
| Follicle stages | Secondary, Antral, Preovulatory, microRNA | ||||
| Comment | Single nucleotide polymorphisms at miR-146a/196a2 and their primary ovarian insufficiency-related target gene regulation in granulosa cells. Cho SH et al. (2017) MicroRNAs post-transcriptionally regulate gene expression in animals and plants. The aim of this study was to identify new target genes for microRNA polymorphisms (miR-146aC>G and miR-196a2T>C) in primary ovarian insufficiency (POI). We cloned and transfected miR-146aC>G and miR-196a2T>C into human granulosa cells and used microarrays and qPCR-arrays to examine the changes in the messenger RNA expression profile. We show miR-146aC>G and miR-196a2T>C change the mRNA expression patterns in granulosa cell. In each case, mRNAs were up or down-regulated after treatments with miR-146a C or G and miR-196a2 T or C. We found that miR-146a led to a significantly altered regulation of the mRNA levels of FOXO3, FOXL2 and CCND2 compared to controls. We also found that the polymorphisms of miR-146a led to a significantly altered regulation of CCND2 and FOXO3. Our results suggest that miR-146aC>G and miR-196a2T>C can regulate the levels of many of their target transcripts. In addition, specific target genes of miR-146aC>G polymorphisms may be involved in granulosa cell regulation.////////////////// Molecular cloning and gene expression of Foxl2 in the Nile tilapia, Oreochromis niloticus. Wang et al . A Foxl2 cDNA was cloned from the Nile tilapia ovary by RT-PCR and subsequent RACE. Alignment of known Foxl2 sequences from vertebrates confirmed the conservation of the Foxl2 open reading frame and protein sequences, especially the forkhead domain and C-terminal region, while some homopolymeric runs of amino acids are found only in mammals but not in non-mammalian vertebrates. RT-PCR revealed that Foxl2 is expressed in the tilapia brain (B), pituitary (P), gill, and gonads (G), with the highest level of expression in the ovary, reflecting the involvement of Foxl2 in B-P-G axis. Northern blotting and in situ hybridization also revealed an evident sexual dimorphic expression pattern in the gonads. Foxl2 mRNA was mainly detected in the granulosa cells surrounding the oocytes. The ovarian expression of Foxl2 in tilapia begins early during the differentiation of the gonads and persists until adulthood, implying the involvement of Foxl2 in fish gonad differentiation and the maintenance of ovarian function. FOXL2 Is a Sensitive and Specific Marker for Sex Cord-Stromal Tumors of the Ovary. Al-Agha OM et al. Sex cord-stromal tumors (SCSTs) of the ovary are relatively uncommon tumors. Diagnosis of SCST rests primarily on the histomorphology of these tumors, and tumors with an atypical or unconventional appearance can pose diagnostic challenges. Previously, we had identified FOXL2 (402C?G) mutation as being characteristic of adult granulosa cell tumors (aGCTs). However, molecular screening for this mutation is not always possible and adds time and cost to the diagnostic process. In this study, we investigated the potential diagnostic use of immunostaining for FOXL2 on formalin-fixed paraffin-embedded tissue sections. Using a commercially available polyclonal antiserum against FOXL2 protein, immunoexpression of FOXL2 was tested in 501 ovarian tumor samples, including 119 SCSTs, using whole tissue sections and tissue microarrays. Staining was correlated with FOXL2 mutation status. In addition, we compared FOXL2 immunoexpression with that of a-inhibin and calretinin, the 2 traditional immunomarkers of SCST, in a subset of 89 SCSTs. FOXL2 immunostaining was present in 95 of 119 (80%) SCSTs, including >95% of aGCTs, juvenile granulosa cell tumors, fibromas, and sclerosing stromal tumors. Only 50% of Sertoli-Leydig cell tumors (N=40) expressed FOXL2. One of 11 steroid cell tumors and 3 of 3 female adnexal tumors of probable Wolffian origin showed FOXL2 immunoreactivity, whereas all other non-SCSTs tested (N=368) were negative for FOXL2 expression. Thus, the sensitivity and specificity of FOXL2 immunoreactivity for SCST are 80% and 99%, respectively. The FOXL2 (402C?G) mutation was confirmed to be both a sensitive and relatively specific indicator of aGCT. Forty-five of 119 SCSTs were mutation positive. These cases were 39 of 42 (93%) aGCTs, 3 of 40 Sertoli-Leydig cell tumors, 2 of 5 thecomas, and 1 of 4 (25%) SCSTs of unclassified type. SCSTs harboring a FOXL2 mutation consistently immunoexpressed FOXL2 (44 of 45, 98%), but FOXL2 immunostaining was also seen in many SCSTs that lacked a mutation (49 of 73, 67%). FOXL2 immunostaining showed higher sensitivity for the diagnosis of SCST, compared with a-inhibin and calretinin, and FOXL2 staining was typically more intense in positive cases compared with either a-inhibin or calretinin. In the SCSTs that were negative for FOXL2 expression, a-inhibin and/or calretinin immunostaining yielded positive results. In conclusion, FOXL2 is a relatively sensitive and highly specific marker for SCST. FOXL2 staining is present in almost all SCSTs with a FOXL2 mutation, and also in a majority of SCSTs without a mutation. FOXL2, together with a-inhibin and calretinin, forms an immunomarker panel that will result in positive staining with 1 or more markers in essentially all cases of SCST. | ||||
| Phenotypes |
POF (premature ovarian failure) |
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| Mutations |
18 mutations
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| Genomic Region | show genomic region | ||||
| Phenotypes and GWAS | show phenotypes and GWAS | ||||
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| created: | Feb. 16, 2001, 4:40 p.m. | by: |
hsueh email:
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| last update: | Dec. 4, 2020, 10:36 p.m. | by: | hsueh email: |
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