A cellular model provides insights into the pathogenicity of the oncogenic FOXL2 somatic variant p.Cys134Trp.

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.

The forkhead DNA-binding domain binds specific G2-rich RNA sequences.

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.

The Oncogenic FOXL2 C134W Mutation Is a Key Driver of Granulosa Cell Tumors.

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.

Recurrent missense variants in clonal hematopoiesis-related genes present in the general population.

Clonal hematopoiesis (CH) consists in an abnormal expansion of a hematopoietic stem cell bearing an advantageous somatic variant. A survey of known recurrent somatic missense variants in DNMT3A, SF3B1, SRSF2, and TP53, some of the most prominent genes underlying CH of indeterminate potential (CHIP), in gnomAD noncancer database shows the presence of 73 variants. Many of them reach frequencies higher than 0.01% in various populations and, in many cases, are enriched in specific populations. Consistent with a potential involvement in CHIP, we found that the age distribution of the carriers is shifted towards old ages. Moreover, the variant allele frequencies are on average lower than 50%, expected for germline heterozygous variants. The pervasive presence of some of such variants in blood DNA from elder individuals is compatible with CHIP of somatic origin. On practical grounds, CHIP can lead to misclassification of somatic variants in cancer-predisposition genes as inherited, which bear consequences for the affected individuals and their families.

Pathogenic "germline" variants associated with myeloproliferative disorders in apparently normal individuals: Inherited or acquired genetic alterations?

Myeloproliferative syndromes (MPS) are hematologic malignancies due to the expansion of an abnormal hematopoietic stem cell. They include chronic myeloid leukemia (CML) and non-CML MPS such as polycythemia vera, essential thrombocythemia and primary myelofibrosis. The latter are distinguished by somatic pathogenic variants affecting JAK2, CALR, or MPL genes. Apparent germline pathogenic variants have been reported in the general population. Here, we found that two gnomAD data-sets report more homozygotes than expected for the JAK2 c.1849G > T(Val617Phe) variant. We propose that somatic gene conversion can explain the presence of those unexpected homozygotes in normal populations. Consistently, homozygous individuals are older than 65 years. We also found a lower-than-expected frequency of the JAK2 variant in younger individuals suggesting that somatic mutation can underlie its presence in (at least some) heterozygotes. Regarding pathogenic variants in MPL and CALR, they are also present in the gnomAD data-sets explored. However, we cannot conclude that such seemingly germline variants are in fact somatic alterations. These results suggest that apparently normal individuals bearing MPS-related variants can be subclinical/undiagnosed MPS cases of somatic origin. It would be interesting to assess the hematologic phenotype of such individuals and the presence of the relevant variants in other tissues.

Gene-dosage issues: a recurrent theme in whole genome duplication events.

Two recent studies have addressed the long-term consequences of whole genome duplications (WGD). Specifically, they analyzed transcriptomes of the plant Arabidopsis thaliana and of four salmonids to assess the impact of WGD on gene expression. These studies point to commonalities in gene expression adjustments after polyploidization that we outline and discuss below.

One Hundred Years of Gene Balance: How Stoichiometric Issues Affect Gene Expression, Genome Evolution, and Quantitative Traits.

A century ago experiments with the flowering plant Datura stramonium and the fruit fly Drosophila melanogaster revealed that adding an extra chromosome to a karyotype was much more detrimental than adding a whole set of chromosomes. This phenomenon was referred to as gene balance and has been recapitulated across eukaryotic species. Here, we retrace some developments in this field. Molecular studies suggest that the basis of balance involves stoichiometric relationships of multi-component interactions. This concept has implication for the mechanisms controlling gene expression, genome evolution, sex chromosome evolution/dosage compensation, speciation mechanisms, and the underlying genetics of quantitative traits.

FOXL2 in adult-type granulosa cell tumour of the ovary: oncogene or tumour suppressor gene?

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.

Genomic exploration of the targets of FOXL2 and ESR2 unveils their implication in cell migration, invasion, and adhesion.

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.

Forkhead Transcription Factors in Health and Disease.

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.

A missense in HSF2BP causing primary ovarian insufficiency affects meiotic recombination by its novel interactor C19ORF57/BRME1.

Primary Ovarian Insufficiency (POI) is a major cause of infertility, but its etiology remains poorly understood. Using whole-exome sequencing in a family with three cases of POI, we identified the candidate missense variant S167L in HSF2BP, an essential meiotic gene. Functional analysis of the HSF2BP-S167L variant in mouse showed that it behaves as a hypomorphic allele compared to a new loss-of-function (knock-out) mouse model. Hsf2bpS167L/S167L females show reduced fertility with smaller litter sizes. To obtain mechanistic insights, we identified C19ORF57/BRME1 as a strong interactor and stabilizer of HSF2BP and showed that the BRME1/HSF2BP protein complex co-immunoprecipitates with BRCA2, RAD51, RPA and PALB2. Meiocytes bearing the HSF2BP-S167L variant showed a strongly decreased staining of both HSF2BP and BRME1 at the recombination nodules and a reduced number of the foci formed by the recombinases RAD51/DMC1, thus leading to a lower frequency of crossovers. Our results provide insights into the molecular mechanism of HSF2BP-S167L in human ovarian insufficiency and sub(in)fertility.

DHH pathogenic variants involved in 46,XY disorders of sex development differentially impact protein self-cleavage and structural conformation.

In humans, pathogenic variants in the DHH gene underlie cases of 46,XY gonadal dysgenesis. DHH is part of the Hedgehog family of proteins, which require extensive processing, including self-cleavage of the precursor for efficient signalling. In our work, we have assessed the effect of several human DHH pathogenic variants involved in recessive complete or partial gonadal dysgenesis, on protein processing and sub-cellular localization. We found that a subset of variants was unable to perform self-cleavage, which correlated albeit not perfectly with an altered subcellular localization of the resulting proteins. For the processing-proficient variants, we used structural modelling tools and molecular dynamic (MD) simulations to predict the potential impact of the variants on protein conformation and/or interaction with partners. Our study contributes to a better understanding of the molecular mechanisms involved in DHH dysfunction leading to 46,XY disorders of sex development.

The Gene Balance Hypothesis: Epigenetics and Dosage Effects in Plants.

Dosage effects in plants are caused by changes in the copy number of chromosomes, segments of chromosomes, or multiples of individual genes. Genes often exhibit a dosage effect in which the amount of product is closely correlated with the number of copies present. However, when larger segments of chromosomes are varied, there are trans-acting effects across the genome that are unleashed that modulate gene expression in cascading effects. These appear to be mediated by the stoichiometric relationship of gene regulatory machineries. There are both positive and negative modulations of target gene expression, but the latter is the plurality effect. When this inverse effect is combined with a dosage effect, compensation for a gene can occur in which its expression is similar to the normal diploid regardless of the change in chromosomal dosage. In contrast, changing the whole genome in a polyploidy series has fewer relative effects as the stoichiometric relationship is not disrupted. Together, these observations suggest that the stoichiometry of gene regulation is important as a reflection of the mode of assembly of the individual subunits involved in the effective regulatory macromolecular complexes. This principle has implications for gene expression mechanisms, quantitative trait genetics, and the evolution of genes depending on the mode of duplication, either segmentally or via whole-genome duplication.

Causes and effects of haploinsufficiency.

Haploinsufficiency is a form of genetic dominance and is the underlying mechanism of numerous human inherited conditions in which the causal genes are sensitive to altered dosage. This review examines the poorly understood relationships between haploinsufficiency, dosage sensitivity and genetic dominance, whose common theme is the existence of nonlinear relationships between genotype and phenotype. We present an up-to-date account of the bases of haploinsufficiency from the perspective of theoretical and experimental models. We also discuss human conditions caused by haploinsufficiency, including developmental syndromes and cancer. Connections between the understanding of these conditions' genetic mechanisms and advances in treatments are also described.

Genomic Balance and Speciation.

The role of genomic balance in accumulating species hybrid incompatibilities is discussed. Aneuploidy has been shown to produce more global modulations than polyploidy with the responsible genes being transcription factors and signaling components involved in molecular complexes, illustrating a stoichiometric component to gene expression. Genomic imbalance is usually detrimental to the organism and in many cases results in lethality. Here, it is proposed that once gene flow is prevented between or within populations by various speciation initiating processes, the stoichiometric relationship of members of macromolecular complexes can change via compensatory drift with the eventual result of newly established functional balances. However, when these new relationships are brought together in interspecific hybrids, detrimental consequences will occur. We suggest that these detrimental interactions contribute to hybrid incompatibilities.

High-throughput Exploration of the Network Dependent on AKT1 in Mouse Ovarian Granulosa Cells.

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.

A truncating MEIOB mutation responsible for familial primary ovarian insufficiency abolishes its interaction with its partner SPATA22 and their recruitment to DNA double-strand breaks.

BACKGROUND: Primary Ovarian Insufficiency (POI), a major cause of infertility, affects about 1-3% of women under forty years of age. Although there is a growing list of causal genetic alterations, POI remains mostly idiopathic. METHODS: We performed exome sequencing (WES) of two sisters affected with POI, one unaffected sister and their mother from a consanguineous family. We assessed the impact of the identified MEIOB variant with a minigene assay and by sequencing illegitimate transcripts from the proband's leukocytes. We studied its functional impact on the interaction between MEIOB with its partner SPATA22 and their localization to DNA double-strand breaks (DSB). FINDINGS: We identified a homozygous variant in the last base of exon 12 of MEIOB, which encodes a factor essential for meiotic recombination. This variant was predicted to strongly affect MEIOB pre-mRNA splicing. Consistently, a minigene assay showed that the variant induced exon 12 skipping, which was confirmed in vivo in the proband's leukocytes. Aberrant splicing leads to the production of a C-terminally truncated protein that cannot interact with SPATA22, abolishing their recruitment to DSBs. INTERPRETATION: This truncating MEIOB variant is expected to provoke meiotic defects and a depleted follicular stock, as in Meiob-/- mice. This is the first molecular defect reported in a meiosis-specific single-stranded DNA-binding protein (SSB) responsible for POI. We hypothesise that alterations in other SSB proteins could explain cases of syndromic or isolated ovarian insufficiency. FUND: Université Paris Diderot, Fondation pour la Recherche Médicale, Fondation ARC contre le cancer, Commissariat à l'Energie Atomique and Institut Universitaire de France.

Advances in the Molecular Pathophysiology, Genetics, and Treatment of Primary Ovarian Insufficiency.

Primary ovarian insufficiency (POI) affects ∼1% of women before 40 years of age. The recent leap in genetic knowledge obtained by next generation sequencing (NGS) together with animal models has further elucidated its molecular pathogenesis, identifying novel genes/pathways. Mutations of >60 genes emphasize high genetic heterogeneity. Genome-wide association studies have revealed a shared genetic background between POI and reproductive aging. NGS will provide a genetic diagnosis leading to genetic/therapeutic counseling: first, defects in meiosis or DNA repair genes may predispose to tumors; and second, specific gene defects may predict the risk of rapid loss of a persistent ovarian reserve, an important determinant in fertility preservation. Indeed, a recent innovative treatment of POI by in vitro activation of dormant follicles proved to be successful.

Gene Expression Dominance in Allopolyploids: Hypotheses and Models.

The classical example of nonadditive contributions of the two parents to allopolyploids is nucleolar dominance, which entails silencing of one parental set of ribosomal RNA genes. This has been observed for many other loci. The prevailing explanation for this genome-wide expression disparity is that the two merged genomes differ in their transposable element (TE) complement and in their level of TE-mediated repression of gene expression. Alternatively, and not exclusively, gene expression dominance may arise from mismatches between trans effectors and their targets. Here, we explore quantitative models of regulatory mismatches leading to gene expression dominance. We also suggest that, when pairs of merged genomes are similar from one allopolyploidization event to another, gene-level and genome dominance patterns should also be similar.