RESUMO
Transcription factors contain a DNA-binding domain ensuring specific recognition of DNA target sequences. The family of forkhead (FOX) transcription factors is composed of dozens of paralogs in mammals. The forkhead domain (FHD) is a segment of about 100 amino acids that binds an A-rich DNA sequence. Using DNA and RNA PCR-SELEX, we show that recombinant FOXL2 proteins, either wild-type or carrying the oncogenic variant C134W, recognize similar DNA-binding sites. This suggests that the oncogenic variant does not alter the intrinsic sequence-specificity of FOXL2. Most importantly, we show that FOXL2 binds G2-rich RNA sequences whereas it virtually fails to bind similar sequences in DNA chemistry. Interestingly, a statistically significant subset of genes responding to the knock-down of FOXL2/Foxl2 harbor such G2-rich sequences and are involved in crucial signaling pathways and cellular processes. In addition, we show that FOXA1, FOXO3a and chimeric FOXL2 proteins containing the FHD of the former are also able to interact with some of the preferred FOXL2-binding sequences. Our results point to an unexpected and novel characteristic of the forkhead domain, the biological relevance of which remains to be explored.
Assuntos
DNA , Fatores de Transcrição Forkhead , Animais , Fatores de Transcrição Forkhead/metabolismo , Sequência de Bases , Domínios Proteicos , Sítios de Ligação/genética , DNA/genética , Mamíferos/genéticaRESUMO
Forkhead box (FOX) proteins belong to an evolutionarily conserved family of transcription factors that has evolved by gene/genome duplication. FOX family members have undergone sequence and regulatory diversification. However, they have retained some degree of functional redundancy, in addition to playing specific roles, both during development and in the adult. Genetic alterations or misregulation of FOX genes underlie human genetic diseases, cancer, and/or aging. In this review, we provide an updated overview of the main characteristics of the members of this family, in terms of breadth of expression, protein domain composition, evolution, and function.
Assuntos
Evolução Molecular , Fatores de Transcrição Forkhead/metabolismo , Neoplasias/genética , Envelhecimento/fisiologia , Animais , Fatores de Transcrição Forkhead/química , Fatores de Transcrição Forkhead/genética , Regulação da Expressão Gênica , Doenças Genéticas Inatas/genética , Humanos , Camundongos , Família MultigênicaRESUMO
BACKGROUND: FOXL2 is a transcription factor expressed in ovarian granulosa cells. A somatic variant of FOXL2 (c.402 C > G, p.Cys134Trp) is the hallmark of adult-type granulosa cell tumours. METHODS: We generated KGN cell clones either heterozygous for this variant (MUT) or homozygous for the wild-type (WT) allele by CRISPR/Cas9 editing. They underwent RNA-Seq and bioinformatics analyses to uncover pathways impacted by deregulated genes. Cell morphology and migration were studied. RESULTS: The differentially expressed genes (DEGs) between WT/MUT and WT/WT KGN cells (DEGs-WT/MUT), pointed to several dysregulated pathways, like TGF-beta pathway, cell adhesion and migration. Consistently, WT/MUT cells were rounder than WT/WT cells and displayed a different distribution of stress fibres and paxillin staining. A comparison of the DEGs-WT/MUT with those found when FOXL2 was knocked down (KD) in WT/WT KGN cells showed that most DEGs-WT/MUT cells were not so in the KD experiment, supporting a gain-of-function (GOF) scenario. MUT-FOXL2 also displayed a stronger interaction with SMAD3. CONCLUSIONS: Our work, aiming at better understanding the GOF scenario, shows that the dysregulated genes and pathways are consistent with this idea. Besides, we propose that GOF might result from an enhanced interaction with SMAD3 that could underlie an ectopic capacity of mutated FOXL2 to bind SMAD4.
Assuntos
Proteína Forkhead Box L2 , Tumor de Células da Granulosa , Proteína Forkhead Box L2/genética , Proteína Forkhead Box L2/metabolismo , Humanos , Feminino , Tumor de Células da Granulosa/genética , Tumor de Células da Granulosa/patologia , Linhagem Celular Tumoral , Movimento Celular/genética , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/patologia , Neoplasias Ovarianas/metabolismo , Proteína Smad3/genética , Proteína Smad3/metabolismo , Sistemas CRISPR-Cas , Regulação Neoplásica da Expressão GênicaRESUMO
FOXL2 and ESR2 are key transcriptional regulators in ovarian granulosa cells. To explore their transcriptional roles and their interplay, we have depleted Foxl2 and Esr2 in mouse primary granulosa cells to assess their ability to bind their targets and/or to modulate gene expression and cellular functions. We show that FOXL2 is involved in a large number of regulatory actions essential for the maintenance of granulosa cell fate. A parallel ChIP-seq analysis showed that FOXL2 mainly binds to sites located in intergenic regions quite far from its targets. A bioinformatic analysis demonstrated that FOXL2-activated genes were enriched in peaks associated with the H3K27ac mark, whereas FOXL2-repressed genes were not, suggesting that FOXL2 can activate transcription through binding to enhancer sites. We also identified about 500 deregulated genes upon Esr2 silencing, of which one third are also targets of FOXL2. We provide evidence showing that both factors modulate, through a coherent feed-forward loop, a number of common targets. Many of the FOXL2/ESR2 targets are involved in cell motility and, consistently, granulosa cells depleted for either Foxl2 or Esr2 exhibit decreased migration, invasion and adhesion. This effect is paralleled by the depletion of their target Phactr1, involved in actin cytoskeleton dynamics. Our analysis expands the number of direct and indirect transcriptional targets of both FOXL2 and ESR2, which deserve investigation in the context of adult-type granulosa cell tumors whose molecular diagnostic hallmark is the presence of the C134W FOXL2 pathogenic variant.
Assuntos
Receptor beta de Estrogênio/metabolismo , Proteína Forkhead Box L2/metabolismo , Células da Granulosa/fisiologia , Animais , Adesão Celular , Linhagem Celular Tumoral , Movimento Celular , Receptor beta de Estrogênio/genética , Feminino , Proteína Forkhead Box L2/genética , Edição de Genes , CamundongosRESUMO
A recurrent mutation in FOXL2 (c.402C>G; p.C134W) is present in over 95% of adult-type granulosa cell tumours (AGCTs). In contrast, various loss-of-function mutations in FOXL2 lead to the development of blepharophimosis, ptosis, and epicanthus inversus syndrome (BPES). BPES is characterised by an eyelid malformation often accompanied with primary ovarian insufficiency. Two recent studies suggest that FOXL2 C402G is a gain- or change-of-function mutation with altered DNA-binding specificity. Another study proposes that FOXL2 C402G is selectively targeted for degradation, inducing somatic haploinsufficiency, suggesting its role as a tumour suppressor. The latter study relies on data indicative of an FOXL2 allelic imbalance in AGCTs. Here we present RNA-seq data as genetic evidence that no real allelic imbalance is observed at the transcriptomic level in AGCTs. Additionally, there is no loss of protein expression in tumours harbouring the mutated allele. These data and other features of this mutation compared to other oncogenes and tumour suppressor genes argue strongly against FOXL2 being a tumour suppressor in this context. Given the likelihood that FOXL2 C402G is oncogenic, targeting the variant protein or its downstream consequences is the most viable path forward to identifying an effective treatment for this cancer. © 2021 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
Assuntos
Proteína Forkhead Box L2/genética , Tumor de Células da Granulosa/genética , Oncogenes/genética , Feminino , Humanos , MutaçãoRESUMO
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.
Assuntos
Proteína Forkhead Box L2/metabolismo , Células da Granulosa/metabolismo , Hipófise/metabolismo , Mapas de Interação de Proteínas , Proteoma/metabolismo , Animais , Células Cultivadas , Feminino , Células da Granulosa/citologia , Camundongos , Hipófise/citologia , Proteoma/análiseRESUMO
The PI3K/AKT signaling pathway is known to regulate a broad range of cellular processes, and it is often altered in several types of cancers. Recently, somatic AKT1 mutations leading to a strong activation of this kinase have been reported in juvenile granulosa cell tumors. However, the molecular role of AKT1 in the supporting cell lineage of the ovary is still poorly understood. To get insights into its function in such cells, we depleted Akt1 in murine primary granulosa cells and assessed the molecular consequences at both the transcript and protein levels. We were able to corroborate the involvement of AKT1 in the regulation of metabolism, apoptosis, cell cycle, or cytoskeleton dynamics in this ovarian cell type. Consistently, we showed in established granulosa cells that depletion of Akt1 provoked altered directional persistent migration and increased its velocity. This study also allowed us to put forward new direct and indirect targets of the kinase. Indeed, a series of proteins involved in intracellular transport and mitochondrial physiology were significantly affected by Akt1 depletion. Using in silico analyses, we also propose a set of kinases and transcription factors that can mediate the action of AKT1 on the deregulated transcripts and proteins. Taken altogether, our results provide a resource of direct and indirect AKT1 targets in granulosa cells and may help understand its roles in this ovarian cell type.
Assuntos
Células da Granulosa/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Animais , Linhagem Celular , Movimento Celular , Feminino , Regulação da Expressão Gênica , Genoma , Camundongos , Peptídeos/metabolismo , Fosforilação , Processamento de Proteína Pós-Traducional , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fatores de Transcrição/metabolismoRESUMO
Primary ovarian insufficiency (POI) implies the cessation of menstruation for several months in women before the age of 40 years and is a major cause of infertility. The study of the contribution of genetic factors to POI has been fueled by the use of whole exome sequencing (WES). Here, to uncover novel causative pathogenic variants and risk alleles, WES has been performed in 12 patients with familial POI (eight unrelated index cases and two pairs of sisters) and six women with early menopause and family history of POI (four index cases and one pair of sisters). Likely causative variants in NR5A1 and MCM9 genes were identified as well as a variant in INHA that requires further investigation. Moreover, we have identified more than one candidate variant in 3 out of 15 familial cases. Taken together, our results highlight the genetic heterogeneity of POI and early menopause and support the hypothesis of an oligogenic inheritance of such conditions, in addition to monogenic inheritance.
Assuntos
Inibinas/genética , Proteínas de Manutenção de Minicromossomo/genética , Insuficiência Ovariana Primária/genética , Fator Esteroidogênico 1/genética , Adolescente , Adulto , Alelos , Exoma/genética , Feminino , Predisposição Genética para Doença , Humanos , Insuficiência Ovariana Primária/patologia , Fatores de Risco , Sequenciamento do Exoma , Adulto JovemRESUMO
Juvenile granulosa cell tumors (JGCTs) of the ovary are pediatric neoplasms representing 5% of all granulosa cell tumors (GCTs). Most GCTs are of adult type (AGCTs) and bear a mutation in the FOXL2 gene. The molecular basis of JGCTs is poorly understood, although mutations in the GNAS gene have been reported. We have detected in-frame duplications within the oncogene AKT1 in >60% of the JGCTs studied. Here, to evaluate the functional impact of these duplications and the existence of potential co-driver alterations, we have sequenced the transcriptome of four JGCTs and compared them with control transcriptomes. A search for gene variants detected only private alterations probably unrelated with tumorigenesis, suggesting that tandem duplications are the best candidates to underlie tumor formation in the absence of GNAS alterations. We previously showed that the duplications were specific to JGCTs. However, the screening of eight AGCTs samples without FOXL2 mutation showed the existence of an AKT1 duplication in one case, also having a stromal luteoma. The analysis of RNA-Seq data pinpointed a series of differentially expressed genes, involved in cytokine and hormone signaling and cell division-related processes. Further analyses pointed to the existence of a possible dedifferentiation process and suggested that most of the transcriptomic dysregulation might be mediated by a limited set of transcription factors perturbed by AKT1 activation. Finally, we show that commercially available AKT inhibitors can modulate the in vitro activity of various mutated forms. These results shed light on the pathogenesis of JGCTs and provide therapeutic leads for a targeted treatment.
Assuntos
Tumor de Células da Granulosa/genética , Mutação , Neoplasias Ovarianas/genética , Proteínas Proto-Oncogênicas c-akt/genética , Adolescente , Divisão Celular/genética , Criança , Pré-Escolar , Citocinas , Análise Mutacional de DNA , Feminino , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Tumor de Células da Granulosa/metabolismo , Hormônios , Humanos , Lactente , Recém-Nascido , Neoplasias Ovarianas/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Transdução de Sinais/genéticaRESUMO
Specific recognition of cis-regulatory regions is essential for correct gene regulation in response to developmental and environmental signals. Such DNA sequences are recognized by transcription factors (TFs) that recruit the transcriptional machinery. Achievement of specific sequence recognition is not a trivial problem; many TFs recognize similar consensus DNA-binding sites and a genome can harbor thousands of consensus or near-consensus sequences, both functional and nonfunctional. Although genomic technologies have provided large-scale snapshots of TF binding, a full understanding of the mechanistic and quantitative details of specific recognition in the context of gene regulation is lacking. Here, we explore the various ways in which TFs recognizing similar consensus sites distinguish their own targets from a large number of other sequences to ensure specific cellular responses.
Assuntos
Regulação da Expressão Gênica , Fatores de Transcrição/metabolismo , Animais , Sítios de Ligação , Cromatina/genética , Cromatina/metabolismo , Humanos , Ligação Proteica , Sequências Reguladoras de Ácido Nucleico , Transdução de Sinais , Transcrição GênicaRESUMO
PURPOSE: We aimed to identify the genetic cause in a cohort of 11 unrelated cases and two sisters with 46,XX SRY-negative (ovo)testicular disorders of sex development (DSD). METHODS: Whole-exome sequencing (n = 9), targeted resequencing (n = 4), and haplotyping were performed. Immunohistochemistry of sex-specific markers was performed on patients' gonads. The consequences of mutation were investigated using luciferase assays, localization studies, and RNA-seq. RESULTS: We identified a novel heterozygous NR5A1 mutation, c.274C>T p.(Arg92Trp), in three unrelated patients. The Arg92 residue is highly conserved and located in the Ftz-F1 region, probably involved in DNA-binding specificity and stability. There were no consistent changes in transcriptional activation or subcellular localization. Transcriptomics in patient-derived lymphocytes showed upregulation of MAMLD1, a direct NR5A1 target previously associated with 46,XY DSD. In gonads of affected individuals, ovarian FOXL2 and testicular SRY-independent SOX9 expression observed. CONCLUSIONS: We propose NR5A1, previously associated with 46,XY DSD and 46,XX primary ovarian insufficiency, as a novel gene for 46,XX (ovo)testicular DSD. We hypothesize that p.(Arg92Trp) results in decreased inhibition of the male developmental pathway through downregulation of female antitestis genes, thereby tipping the balance toward testicular differentiation in 46,XX individuals. In conclusion, our study supports a role for NR5A1 in testis differentiation in the XX gonad.Genet Med 19 4, 367-376.
Assuntos
Proteínas de Ligação a DNA/genética , Sequenciamento do Exoma/métodos , Perfilação da Expressão Gênica/métodos , Proteínas Nucleares/genética , Transtornos Ovotesticulares do Desenvolvimento Sexual/genética , Análise de Sequência de RNA/métodos , Fator Esteroidogênico 1/genética , Fatores de Transcrição/genética , Feminino , Predisposição Genética para Doença , Haplótipos , Humanos , Masculino , Modelos Moleculares , Mutação de Sentido Incorreto , Ovário/metabolismo , Transtornos Ovotesticulares do Desenvolvimento Sexual/metabolismo , Linhagem , Polimorfismo de Nucleotídeo Único , Fator Esteroidogênico 1/química , Fator Esteroidogênico 1/metabolismo , Testículo/metabolismo , Regulação para Cima , Adulto JovemRESUMO
Leber congenital amaurosis (LCA) is a severe autosomal-recessive retinal dystrophy leading to congenital blindness. A recently identified LCA gene is NMNAT1, located in the LCA9 locus. Although most mutations in blindness genes are coding variations, there is accumulating evidence for hidden noncoding defects or structural variations (SVs). The starting point of this study was an LCA9-associated consanguineous family in which no coding mutations were found in the LCA9 region. Exploring the untranslated regions of NMNAT1 revealed a novel homozygous 5'UTR variant, c.-70A>T. Moreover, an adjacent 5'UTR variant, c.-69C>T, was identified in a second consanguineous family displaying a similar phenotype. Both 5'UTR variants resulted in decreased NMNAT1 mRNA abundance in patients' lymphocytes, and caused decreased luciferase activity in human retinal pigment epithelial RPE-1 cells. Second, we unraveled pseudohomozygosity of a coding NMNAT1 mutation in two unrelated LCA patients by the identification of two distinct heterozygous partial NMNAT1 deletions. Molecular characterization of the breakpoint junctions revealed a complex Alu-rich genomic architecture. Our study uncovered hidden genetic variation in NMNAT1-associated LCA and emphasized a shift from coding to noncoding regulatory mutations and repeat-mediated SVs in the molecular pathogenesis of heterogeneous recessive disorders such as hereditary blindness.
Assuntos
Regiões 5' não Traduzidas , Variações do Número de Cópias de DNA , Amaurose Congênita de Leber/genética , Mutação , Nicotinamida-Nucleotídeo Adenililtransferase/genética , Alelos , Elementos Alu , Criança , Pontos de Quebra do Cromossomo , Mapeamento Cromossômico , Biologia Computacional/métodos , Consanguinidade , Éxons , Feminino , Expressão Gênica , Estudos de Associação Genética , Genótipo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Amaurose Congênita de Leber/diagnóstico , Masculino , Linhagem , Fenótipo , RNA Mensageiro/genética , Análise de Sequência de DNA , Adulto JovemRESUMO
BACKGROUND: Ovarian granulosa cell tumors (GCTs) are the most frequent sex cord-stromal tumors. Several studies have shown that a somatic mutation leading to a C134W substitution in the transcription factor FOXL2 appears in more than 95% of adult-type GCTs. Its pervasive presence suggests that FOXL2 is the main cancer driver gene. However, other mutations and genomic changes might also contribute to tumor formation and/or progression. METHODS: We have performed a combined comparative genomic hybridization and transcriptomic analyses of 10 adult-type GCTs to obtain a picture of the genomic landscape of this cancer type and to identify new candidate co-driver genes. RESULTS: Our results, along with a review of previous molecular studies, show the existence of highly recurrent chromosomal imbalances (especially, trisomy 14 and monosomy 22) and preferential co-occurrences (i.e. trisomy 14/monosomy 22 and trisomy 7/monosomy 16q). In-depth analyses showed the presence of recurrently broken, amplified/duplicated or deleted genes. Many of these genes, such as AKT1, RUNX1 and LIMA1, are known to be involved in cancer and related processes. Further genomic explorations suggest that they are functionally related. CONCLUSIONS: Our combined analysis identifies potential candidate genes, whose alterations might contribute to adult-type GCT formation/progression together with the recurrent FOXL2 somatic mutation.
Assuntos
Fatores de Transcrição Forkhead/genética , Perfilação da Expressão Gênica , Tumor de Células da Granulosa/genética , Proteínas de Neoplasias/biossíntese , Hibridização Genômica Comparativa , Subunidade alfa 2 de Fator de Ligação ao Core/biossíntese , Subunidade alfa 2 de Fator de Ligação ao Core/genética , Proteínas do Citoesqueleto/biossíntese , Proteínas do Citoesqueleto/genética , Variações do Número de Cópias de DNA/genética , Feminino , Proteína Forkhead Box L2 , Regulação Neoplásica da Expressão Gênica , Estudos de Associação Genética , Genômica , Tumor de Células da Granulosa/patologia , Humanos , Proteínas de Neoplasias/genética , Mutação Puntual , Proteínas Proto-Oncogênicas c-akt/biossíntese , Proteínas Proto-Oncogênicas c-akt/genéticaRESUMO
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.
Assuntos
Blefarofimose/metabolismo , Fatores de Transcrição Forkhead/metabolismo , Anormalidades da Pele/metabolismo , Animais , Apoptose , Blefarofimose/genética , Blefarofimose/fisiopatologia , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Feminino , Proteína Forkhead Box L2 , Fatores de Transcrição Forkhead/genética , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mutação de Sentido Incorreto , Folículo Ovariano/metabolismo , Regiões Promotoras Genéticas , Ligação Proteica , Transporte Proteico , Anormalidades da Pele/genética , Anormalidades da Pele/fisiopatologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Ativação Transcricional , Anormalidades UrogenitaisRESUMO
Transposable elements play a fundamental role in genome evolution. It is proposed that their mobility, activated under stress, induces mutations that could confer advantages to the host organism. Transcription of the Ty1 LTR-retrotransposon of Saccharomyces cerevisiae is activated in response to a severe deficiency in adenylic nucleotides. Here, we show that Ty2 and Ty3 are also stimulated under these stress conditions, revealing the simultaneous activation of three active Ty retrotransposon families. We demonstrate that Ty1 activation in response to adenylic nucleotide depletion requires the DNA-binding transcription factor Tye7. Ty1 is transcribed in both sense and antisense directions. We identify three Tye7 potential binding sites in the region of Ty1 DNA sequence where antisense transcription starts. We show that Tye7 binds to Ty1 DNA and regulates Ty1 antisense transcription. Altogether, our data suggest that, in response to adenylic nucleotide reduction, TYE7 is induced and activates Ty1 mRNA transcription, possibly by controlling Ty1 antisense transcription. We also provide the first evidence that Ty1 antisense transcription can be regulated by environmental stress conditions, pointing to a new level of control of Ty1 activity by stress, as Ty1 antisense RNAs play an important role in regulating Ty1 mobility at both the transcriptional and post-transcriptional stages.
Assuntos
Adenina/metabolismo , Regulação Fúngica da Expressão Gênica , RNA Antissenso/biossíntese , Retroelementos , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Transativadores/metabolismo , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Deleção de Genes , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Estresse Fisiológico/genética , Transativadores/genética , Ativação Transcricional , TranscriptomaRESUMO
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.
Assuntos
Ciclo Celular , Regulação para Baixo , Fatores de Transcrição Forkhead/metabolismo , Células da Granulosa/citologia , Estresse Oxidativo , Sirtuína 1/metabolismo , Linhagem Celular , Reparo do DNA , Feminino , Proteína Forkhead Box L2 , Fatores de Transcrição Forkhead/genética , Regulação da Expressão Gênica , Células da Granulosa/enzimologia , Células da Granulosa/metabolismo , Humanos , Sirtuína 1/genéticaRESUMO
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.
Assuntos
Fatores de Transcrição Forkhead/metabolismo , Animais , Células COS , Linhagem Celular , Chlorocebus aethiops , Proteína Forkhead Box L2 , Fatores de Transcrição Forkhead/química , Fatores de Transcrição Forkhead/genética , Humanos , Mutação , Estrutura Terciária de Proteína , Relação Estrutura-AtividadeRESUMO
Adult-type granulosa cell tumors (AGCT) are the most common type of malignant ovarian sex cord-stromal tumors. Most AGCTs carry the somatic variant c.402C>G (p.C134W) affecting the transcription factor FOXL2. Germline dominant variants in FOXL2 are responsible for blepharophimosis syndrome, which is characterized by underdevelopment of the eyelid. In this work, we generated a mouse model harboring the C134W variant of FOXL2 to evaluate in vivo the poorly understood oncogenic role of FOXL2. The mutation was dominant regarding eyelid hypoplasia, reminiscent of blepharophimosis syndrome. Interestingly, Foxl2+/C134W female mice had reduced fertility and developed AGCTs through a progression from abnormal ovaries with aberrant granulosa cells to ovaries with stromal hyperplasia and atypia and on to tumors in adut mice. The genes dysregulated in mouse AGCTs exhibited the hallmarks of cancer and were consistent with a gain-of-function of the mutated allele affecting TGFß signaling. A comparison of these data with previous results on human AGCTs indicated similar deregulated pathways. Finally, a mutational analysis of mouse AGCT transcriptomic data suggested the absence of additional driver mutations apart from FOXL2-C134W. These results provide a clear in vivo example in which a single mutational hit triggers tumor development associated with profound transcriptomic alterations. SIGNIFICANCE: A newly generated mouse model carrying a FOXL2 mutation characteristic of adult-type granulosa cell tumors shows that FOXL2 C134W shifts the transcriptome towards a signature of granulosa cell cancer and drives tumorigenesis.
Assuntos
Tumor de Células da Granulosa , Neoplasias Ovarianas , Anormalidades da Pele , Adulto , Feminino , Humanos , Animais , Camundongos , Tumor de Células da Granulosa/genética , Tumor de Células da Granulosa/metabolismo , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/metabolismo , Neoplasias Ovarianas/genética , Mutação , Proteína Forkhead Box L2/genéticaRESUMO
Ovarian causes of precocious pseudo-puberty (PPP) include McCune-Albright syndrome (MAS) and juvenile granulosa cell tumour (JGCT). We describe a case of PPP in which bilateral ovarian enlargement with multiple cysts progressed to unilateral JGCT. A girl aged 2.17 years presented with three months of breast development, and rapid growth. Examination showed tall stature, height +2.6 standard deviations, Tanner stage B3P2A1. A single café au lait patch was noted. Bone age was advanced at 5 years. Pelvic ultrasound showed bilaterally enlarged ovaries (estimated volumes 76 mL on the left, 139 mL on the right), each containing multiple cysts. Luteinizing hormone (LH) and follicle stimulating hormone (FSH) values before/after gonadotrophin administration were 0.43/0.18 and <0.1/<0.1 mUI/mL, serum estradiol 130 pg/mL, (prepubertal range <20 pg/mL). PPP of ovarian origin was diagnosed, and tamoxifen 20 mg daily started. However, after only seven weeks height velocity escalated and breast development increased to B3-4 with menorrhagia. Basal/stimulated LH and FSH were still suppressed at 0.13/0.25 and <0.1/<0.1 mUI/mL and, serum estradiol 184 pg/mL. Repeat imaging now showed normal right ovary (volume 1.8 mL) and a large left-sided vascular solid/cystic ovarian tumour which was excised (weight 850 g). Histology showed JGCT, International Federation of Gynecology and Obstetrics stage IA. DNA from tumour tissue showed no mutation in GNAS, exon 3 of AKT1 (which contains a mutational hotspot) or FOXL2. The observation that bilateral ovarian activity progressed to unilateral development of JGCT in this patient is novel. This case highlights current uncertainties in the ontology of JGCT, and its possible relationship with MAS.