Project description:FOXL2 is a lineage determining transcription factor in the ovary, but its direct targets and modes of action are not fully characterized. Interaction of FOXL2 with several members of the nuclear receptor familly of transcription factors has been described, and many nuclear receptors play a key role in ovarian biology. Here, we explore the targets of FOXL2 and five nuclear receptors in murine primary follicular cells. siRNA targeting FOXL2 or control siRNA were used in conjunction with siRNAs targeting five different nuclear receptors or control siRNAs (2x6=12 conditions). Each condition was analysed in completely independent biological duplicates (i.e. cell from different animals grown independently)
Project description:FOXL2 is a lineage determining transcription factor in the ovary, but its direct targets and modes of action are not fully characterized. Interaction of FOXL2 with several members of the nuclear receptor familly of transcription factors has been described, and many nuclear receptors play a key role in ovarian biology. Here, we explore the targets of FOXL2 and five nuclear receptors in murine primary follicular cells.
Project description:FOXL2 is a transcription factor essential for female fertility, expressed in somatic cells of the ovary, notably granulosa cells. In the mouse, Foxl2 deletion leads to partial sex reversal postnatally. However, deletion of the gene in 8-week-old females leads to granulosa to Sertoli cell transdifferentiation. We hypothesised that different outcomes of Foxl2 deletion in embryonic versus adult ovary may depend on a different role played across ovarian development. Therefore, we characterised the dynamics of gene expression and chromatin accessibility changes in purified murine granulosa cells across key developmental stages (E14.5, 1 and 8 weeks). We then performed genome-wide identification of FOXL2 target genes and on-chromatin interacting partners by ChIP-SICAP. We found that FOXL2 regulates more genes at postnatal stages, through the interaction with factors regulating primordial follicle activation (PFA), such as NR5A2, and others regulating steroidogenesis including AR and ESR2. As a proof of principle experiment, we chose one FOXL2 interactor, Ubiquitin specific protease 7 (USP7) and showed that deletion of this gene in granulosa cells leads to a blockage of PFA, impaired ovary development and sterility. Our study constitutes a comprehensive resource for exploration of the molecular mechanisms of ovarian development and causes of female infertility.
Project description:The Foxl2 transcription factor is required for ovarian function during follicular development. Our approach to begin to understand Foxl2 function is through the identification of Foxl2 regulated genes in the ovary. Transiently transfected KK1 mouse granulosa cells were used to identify genes that are potentially regulated by Foxl2. KK1 cells were transfected in three groups (mock, activated, and repressed) and twenty-four hours later RNA was isolated and submitted for Affymetrix microarray analysis.
Project description:The Foxl2 transcription factor is required for ovarian function during follicular development. Our approach to begin to understand Foxl2 function is through the identification of Foxl2 regulated genes in the ovary. Transiently transfected KK1 mouse granulosa cells were used to identify genes that are potentially regulated by Foxl2. KK1 cells were transfected in three groups (mock, activated, and repressed) and twenty-four hours later RNA was isolated and submitted for Affymetrix microarray analysis. Experiment Overall Design: To increase the potential of Foxl2 to alter gene expression levels of putative target genes, two fusions were constructed consisting of Foxl2 fused to the activation domain of the Herpes simplex virus VP16 transcription factor (Foxl2-VP16) and Foxl2 fused to the repression domain of the murine MAD transcription factor (Foxl2-MAD). Levels of gene expression were compared between mock transfected cells and those that were transfected with Foxl2-VP16 and Foxl2-MAD, respectively.
Project description:The identity of the gonads is determined by which fate, ovarian granulosa cell or testicular Sertoli cell, the bipotential somatic cell precursors choose to follow. In most vertebrates, the fate of granulosa cells is controlled by a conserved regulator FOXL2. To understand how FOXL2 elicits its fate-determining action, we performed genome-wide analysis of FOXL2 chromatin occupancy in fetal ovaries. Combining genome-wide analysis of FOXL2 binding in the fetal ovary with transcriptomic analyses of Foxl2 gain-of-function and Foxl2 loss-of-function models, we identified potential pathways responsible for the feminizing action of FOXL2. Finally, comparison of FOXL2 genome-wide occupancy in the fetal ovary with testis-determining factor SOX9 genome-wide occupancy in the fetal testis revealed extensive overlaps, implying that antagonistic signals between FOXL2 and SOX9 occur at the chromatin level.
Project description:Sex determination of the gonads begins with fate specification of gonadal supporting cells into either ovarian granulosa cells or testicular Sertoli cells. This process of fate specification hinges on a balance of transcriptional control. We discovered that the transcription factor RUNX1 is enriched in the fetal ovary in rainbow trout, turtle, mouse, and human. In the mouse, RUNX1 marks the supporting cell lineage and becomes granulosa cell-specific as the gonads differentiate. RUNX1 plays complementary/redundant roles with FOXL2 to maintain fetal granulosa cell identity, and combined loss of RUNX1 and FOXL2 results in masculinization of the fetal ovaries. To determine whether interplay between RUNX1 and FOXL2 occurs at the chromatin level, we performed genome-wide analysis of RUNX1 chromatin occupancy in E14.5 ovaries. The top de novo motif identified in RUNX1 ChIP-seq matched the RUNX motif. We found that RUNX1 chromatin occupancy was partially overlapping with FOXL2 chromatin occupancy in fetal ovaries.
Project description:It has previously been shown that FOXL2 and ESR1 cooperate to repress the testis-determining gene Sox9 in murine granulosa cells, and suggested that FOXL2/ESR1 cooperation may be central to granulosa cell differentiation (Uhlenhaut et al., 2009). However, no study has so far compared the DNA-binding of FOXL2 and ESR1 at the genomic level or analyzed the impact of FOXL2 on ESR1 binding to its regulatory elements. Here, we have analyzed and compared the genomic locations recognized by ESR1 and FOXL2 in E2-treated primary murine granulosa cells. Input DNA, FOXL2 and ESR1 ChIP
Project description:We have generated a humanized Foxl2 mouse model by genome editing to evaluate in vivo the role of mutated FOXL2 in the development of murine Adult Granulosa Cell Tumors