1
Université de Paris, F-75006, Paris, France.
2
Université de Paris, CNRS, Institut Jacques Monod, F-75006, Paris, France.
3
Université Paris-Saclay, Institut de Biologie F. Jacob, Commissariat à l’Energie Atomique,
Fontenay aux Roses, France.
Laëtitia HERMAN-Doctorat de Génétique-2020
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Abstract
The forkead box L2 (FOXL2) and the estrogen receptor beta (ESR2) are key transcriptional regulators in ovarian granulosa cells. As little is known about the transcriptional roles of FOXL2 and its partner ESR2, we have depleted Foxl2 and Esr2in mouse primary granulosa cells to assess the molecular consequences of this intervention on their ability to bind their targets and to modulate gene expression. We show that FOXL2 is involved in a large number of regulatory actions essential for the maintenance of granulosa cells fate cell. We provide evidence showing that FOXL2 and ESR2 modulate, through a coherent feed-forward loop, an important number of common targets. Indeed, we have identified several binding sites of FOXL2 that are not recognized when ESR2 is depleted. Moreover, a large number of these binding sites are localized in the vicinity of genes involved in cell migration and adhesion. Consistently, we show that both FOXL2 and ESR2 modulate together the cell's ability to migrate and invade via their co-regulation of Phactr1 expression. Specifically, we show that granulosa cells depleted for either Foxl2 and Esr2 exhibit decreased migration velocity and cell invasion ability and this is paralleled by the depletion of Phactr1 itself. We provide also a functional link between AKT and FOXL2 on a specific pool of genes where AKT inhibits FOXL2 activity. Our results taken all together point to novel pathways regulated by FOXL2 and its partner ESR2 shedding a new light on the role of both transcription factors in granulosa cells.
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Introduction
FOXL2 is a highly conserved gene encoding a forkhead transcription factor (TF) required for the differentiation and maintenance of granulosa cells, those surrounding the oocyte in ovarian follicles (1–3). Foxl2−/− mice are devoid of mature granulosa cells (2) and depletion of Foxl2 in mature granulosa cells results in their “molecular” transdifferentiation involving an upregulation of markers of Sertoli cells, the somatic cells of the male sex cords (3). In humans, heterozygous mutations of
FOXL2 underlie the blepharophimosis/ptosis/epicanthus inversus syndrome (BPES), where an eyelid
hypoplasia can be associated or not with premature ovarian insufficiency (POI)(4). FOXL2 pathogenic variants impairing its DNA-binding and/or transcriptional activity are responsible for cases of BPES with POI (5, 6). Furthermore, a unique damaging variant of FOXL2 (FOXL2 c.402C>G, C134W at the protein level) has been identified in the vast majority of adult-type granulosa cell tumors (AGCTs), showing the important role of FOXL2 for granulosa cell fate and function (7).
Several proteins that interact with FOXL2 have been identified by ourselves and others, including DEAD box-containing protein DP103 and TFs such as the estrogen receptor ESR1 (encoding estrogen receptor alpha), the orphan nuclear receptor SF-1 and SMAD3(8). Interestingly, mice depleted for both estrogen receptors (Esr1 and Esr2) display a transdifferentiation of granulosa cells similar to that observed when Foxl2 is depleted in mature cells (9). Moreover, previous data from our group, show that FOXL2 and ESR2/Estrogen (E2) repress the expression of the testis determining factor SOX9 in ovarian follicular cells (1).Despite such progresses, the mechanisms by which FOXL2 and its partners regulate granulosa cell differentiation are not well understood. This is due, at least in part, to the difficulty in the isolation of primary granulosa cells at different maturation stages and to the fact that the deletion/depletion of Foxl2 triggers a rapid transdifferentiation of such cells into Sertoli-like cells as pointed out above (3). Moreover, cellular models, such as GCT-derived cell lines or ovaries from constitutive knocked-out mice are far from being satisfactory (because of aforementioned transdifferentiation issue) and cannot thus provide a bona fide overview of FOXL2 transcriptional targets.
In contrast to AGCTs, juvenile GCTs (J-CGTs) are less well-characterized in molecular terms. FOXL2 has been found under-expressed in such tumors (10) and more recently, we identified pathogenic variants affecting AKT1 as their main molecular hallmark. Indeed, we found that more than 60% of the tumor samples studied bore somatic in-frame tandem duplications affecting the pleckstrin homology domain (PHD) of AKT1 whereas the others carried potentially damaging point mutations at conserved residues (11). To date, no link has been made between these two different GCT types and a potential (dys)regulation of FOXL2 expression or function by AKT.
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Here, we explore target gene regulation by FOXL2 and ESR2 on genome-wide scale. Specifically, this work was performed using murine primary follicular cells through a knockdown (KD) approach coupled to RNA-sequencing (RNA-seq) and ChIP-seq. These analyses allowed us to uncover a thorough core of FOXL2 and ESR2 transcriptional targets in the somatic cells of preantral-follicles, which includes as expected many key genes of ovarian function. A ChIP-seq analysis of FOXL2 in these cells suggests that it mainly binds to regions located in intergenic regions quite far from its targets, and provides a resource of potential regulatory elements. RNA-seq allowed us to find multiple common cellular processes altered by the depletion of either Foxl2 or Esr2, notably those regarding cell migration and the regulation of extracellular matrix (ECM) organization. Moreover, we show that FOXL2 and ESR2 are required, for normal expression of Phactr1 (Phosphatase and actin regulator 1). Phactr1 is an interactor of the protein phosphatase 1 (PP1), of which it modulates the activity, and of G-actin (12, 13).The regulation of its expression by FOXL2 and ESR2, through a newly identified regulatory element, is required for efficient granulosa cell migration, invasion and adhesion. We also document an interesting functional interaction between FOXL2 and PI3K/AKT, also describe to drive cell migration and invasion, regulating the actin cytoskeleton (14, 15) and the secretion of matrix metalloproteases (16).
Taken together, our transcriptomic and genome-wide location data help understand the role of both FOXL2 and ESR2 in the biology of ovarian granulosa cells.
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