Transcription profiling of mouse KK1 granulosa cells transfected with Foxl2-VP16 and Foxl2-MAD
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ABSTRACT: 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:In order to identify novel FOXL2 targets, we have transfected KGN cells using an expression vector containing the coding sequence of FOXL2 or, as a reference, the empty vector (mock transfection). The transcriptome perturbation induced by FOXL2 overexpression was then followed by DNA chips (i.e. FOXL2- vs. mock-transfected cells) using the platform developed by NimbleGen Systems, Inc.
Project description: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. The expression of mutant FOXL2 was reduced in the GCT cell line KGN, and wildtype and mutant FOXL2 were overexpressed in the GCT cell line COV434. Comparisons were made between the transcriptomes of control cells and cells altered by FOXL2 knockdown and overexpression, to detect potential transcriptional targets of mutant FOXL2. Comparisons were made between the transcriptomes of control cells and cells altered by FOXL2 knockdown and overexpression, to detect potential transcriptional targets of mutant FOXL2. Mutant FOXL2 was silenced in KGN cells with the use of siRNA and a non-targetting siRNA control. Wildtype and mutant FOXL2 were overexpressed in COV434 cells with the use of overexpression plasmids and an empty plasmid control. Each experiment was performed in triplicate. Total RNA was harvested at 24h post transfection and hybridised to Affymetrix U133 plus 2.0 arrays.
Project description:This SuperSeries is composed of the following subset Series: GSE12905: Foxl2 functions in sex determination and histogenesis throughout mouse ovary development, analyzed by Affymetrix arrays GSE12942: Foxl2 functions in sex determination and histogenesis throughout mouse ovary development, analyzed by Agilent arrays Refer to individual Series
Project description: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, i.e., Rspo1 and Wnt4. Here we report expression profiling of mouse ovaries that lack Foxl2 alone or in combination with Wnt4 or Kit/c-Kit, to identify ovarian targets of Foxl2 that, along with some testis genes, were dysregulated during embryonic 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. The results, including a comprehensive principal component analysis of published microarray datasets 1) support the proposal of dose-dependent Foxl2 function and anti-testis action throughout ovary differentiation; and 2) identify candidate genes for a role in sex determination independent of FOXL2 (notably, the transcription factor, ZBTB7C) and in the generation of the ovarian reserve downstream of it (e.g., the cadherin-domain protein CLSTN2, or the sphingomyelin synthase, SGMS2). The gene inventory provides a framework to analyze the genetic bases of ovarian development and female fertility. Keywords: reference design Comparison of Foxl2+/+,+/- and -/- whole ovaries at 2 timepoints delineating follicle formation
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:FOXL2 is a lineage determining transcription factor in the ovary, but its direct targets and modes of action are not fully characterized. Here, we explore the genomic targets of FOXL2. We found in particular that FOXL2 directly modulates Esr2 expression through a newly identified intronic element. Input DNA and FOXL2 ChIP
Project description:Foxl2 is a forkhead transcription factor essential for proper reproductive function in females. It is expressed in the somatic cell population of the gonad (granulosa cells) which forms the follicles of the ovary, the structures responsible for embedding and nurturing the oocytes during their development. FOXL2 directly regulate the aromatase that synthesizes estrogens CYP19A1, thus promoting female differentiation, as well as acting as a repressor of the male factors SOX9 and DMRT1.Expression is also found in the eyelids, pituitary gland and uterus. In the goat, frog and many fish species FOXL2 is a sex-determining gene which, when deleted, leads to female-to-male sex reversal.
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: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:FOXL2 is a transcription factor that plays a key role in sex determination, ovary development and maintenance. Mutations related to this gene have been described in syndromes involving premature ovarian failure and granulosa cell tumors. This kind of rare cancer (less than 5% of diagnosed ovarian cancers) has been causally associated with the FOXL2 c.402C>G, p.C134W mutation in 97% of the adult cases (AGCTs). In this study, we have used CRISPR technology to specifically eliminate the FOXL2 c.402C>G mutation in granulosa tumor cells. Our results indicate that this Cas9-mediated strategy allows the specific elimination of the mutation with no activity on the wild type allele. Granulosa cells depleted on FOXL2 c.402C>G show a reduced malignant phenotype. Specifically, we detect changes in cell proliferation, invasion, and cell death levels. In addition, we show that granulosa tumor cells become more susceptible to Dasatinib and Ketoconazole treatments when FOXL2 c.402C>G allele is eliminated. Our transcriptomic and proteomic analyses indicate that CRISPR-modified granulosa tumor cells significantly change their expression signature towards a wild type like phenotype. Finally, this expression signature has led us to discover new compounds with antiproliferative and proapoptotic effects on granulosa cell tumor cells. Our results demonstrate the potential of CRISPR for specifically targeting and eliminating a granulosa cell tumor-causing mutation, as well as its therapeutic potential for the treatment of this rare ovarian cancer.