[Show abstract][Hide abstract] ABSTRACT: Gonadotropin-releasing hormone (GnRH) plays a key role in the vertebrate reproductive system by stimulating biosynthesis and secretion of pituitary gonadotropins. However, the potential involvement of microRNAs (miRNAs) on this activation has still to be explored. In this study, we investigated the role of miRNA-132 and miRNA-212, two tandemly expressed miRNAs that target the same transcripts, on GnRH-induced follicle-stimulating hormone (FSH) expression. We first showed that GnRH stimulation of FSH secretion was reduced and Fshb mRNA abolished by blocking miR-132/212 action in rat pituitary cells. In mouse LβT2 gonadotrope cells, the GnRH stimulation of Fshb mRNA was also demonstrated to be dependent on miR-132/212 and reproduced by overexpressing one or both miRNAs. We then showed that the miR-132/212-mediated action of GnRH involved a post-transcriptional decrease of Sirtuin 1 (SIRT1) deacetylase. The lower level of SIRT1 deacetylase correlated with an increase in the acetylated form of Forkhead Box O 1 (FOXO1), a transcriptional repressor of Fshb. Interestingly, we show that the acetylated mimicking mutant of FOXO1 was localized outside the nucleus, thus alleviating its repressive effect on Fshb transcription. Overall, we demonstrate that the GnRH stimulation of Fshb expression is dependent on miR-132/212 and involves a SIRT1-FOXO1 pathway. This is the first demonstration of an obligatory microRNA pathway in the GnRH-regulated expression of a gonadotropin gene.
Full-text · Article · Jan 2015 · Molecular Endocrinology
[Show abstract][Hide abstract] ABSTRACT: eLife digest
In female mammals, granulosa cells in the ovaries help egg cells to grow and develop by secreting nutrients and estrogens—the female sex hormones. A protein called FOXL2 helps granulosa cells to develop and functions by binding to the DNA of the cells to switch certain genes either on or off.
In humans, mutations in the gene that codes for the FOXL2 protein are associated with granulosa cell tumors and with a loss of female fertility in early adulthood. In addition, if the amount of FOXL2 is artificially reduced in granulosa cells in female mice, the cells take on many of the characteristics of supporting cells found in the testes of males.
To investigate in more detail how FOXL2 works, Georges et al. grew mouse granulosa cells in the laboratory to identify the DNA sequences where FOXL2 will bind, and to uncover how this binding affects gene expression. Georges et al. conclude that FOXL2 orchestrates a network involving many different proteins that allows estrogen to be produced and used by granulosa cells; and in doing so these cells maintain their identity as ovarian cells. FOXL2 was also shown to work closely with the receptor proteins that detect the sex hormones, and which help to control whether particular sex-specific genes are switched on or off.
One particularly important role of FOXL2 in granulosa cells is that it represses a gene called Sox9. By repressing Sox9, the granulosa cells do not transform into their counterparts normally found in testes. Although FOXL2 was previously reported to directly regulate the Sox9 gene, Georges et al. find that it also acts through other molecules, and that there are alternative ways in which it can do so.
Although Georges et al. have established some of the ways that FOXL2 functions, this protein can work via other pathways; these will require further investigation to be fully understood.
[Show abstract][Hide abstract] ABSTRACT: Aberrant ovarian granulosa cell proliferation and apoptosis may lead to granulosa cell tumors (GCT), the pathogenesis of which involves transcription factors GATA4, FOXL2, and SMAD3. FOXL2 gene harbors a point mutation (C134W) in a vast majority of GCTs. GATA4 is abundantly expressed in GCTs and its expression correlates with poor prognosis. The TGF-β mediator SMAD3 promotes GCT cell survival through NF-κB activation, and interacts with FOXL2. Here, we find that the expression patterns of these factors overlap in the normal human ovary and 90 GCTs, and positively correlate with each other and with their mutual target gene CCND2, which is a key factor for granulosa cell proliferation. We have explored the molecular interactions of FOXL2, GATA4, and SMAD3 and their roles in the regulation of CCND2 using co-immunoprecipitation, promoter transactivation, and cell viability assays in human GCT cells. We found that not only SMAD3, but also GATA4 physically interact with both wild type and C134W-mutated FOXL2. GATA4 and SMAD3 synergistically induce a 8-fold increase in CCND2 promoter transactivation, which is 50% reduced by both FOXL2 types. We confirmed that wild type FOXL2 significantly decreases cell viability. Interestingly, GATA4 and SMAD3 caused a marked reduction of GCT cell apoptosis induced by wild type FOXL2. Thus, the effects of GATA4 and SMAD3 on both cell viability and apoptosis are distinct from those of wild type FOXL2; a perturbation of this balance due to the oncogenic FOXL2 mutation is likely to contribute to GCT pathogenesis.
[Show abstract][Hide abstract] ABSTRACT: Ovarian granulosa cell tumors (OGCT) are the most frequent kind of sex cord-stromal tumors, and represent ∼2-5% of all ovarian malignancies. OGCTs exist as two entities, juvenile and adult types, with specific clinical and pathological characteristics. The molecular pathogenesis of these tumors has just begun to be unraveled. Indeed, recent studies have indicated that mutation and/or misregulation of the key ovarian transcription factor FOXL2 has a role in OGCT formation, although the mechanisms remain unclear. To better understand the molecular characteristics of OGCT, we studied the transcriptomic profiles of ten human adult-type OGCT samples, as well as ethnically matched granulosa cell (GC) controls. We find that the OGCT samples analyzed herein exhibit several hallmarks of cancer, including increased expression of genes linked to cell proliferation, but decreased expression of those conferring sensitivity to cell death. Moreover, genes differentially expressed in OGCTs are significantly enriched for known FOXL2 target genes, consistently with the prevalence of FOXL2 somatic mutation in these tumors. Expression of these targets is altered in a way expected to promote malignant transformation, for instance, through induction of genes associated with faster cell cycling and downregulation of genes associated with cell death. Over time, such defects may be responsible at least partly for the malignant transformation of healthy GCs into OGCT. These insights into the molecular pathogenesis of OGCTs may open the way to new efforts in the development of more targeted therapeutic strategies for OGCT patients.Oncogene advance online publication, 16 July 2012; doi:10.1038/onc.2012.298.
[Show abstract][Hide abstract] ABSTRACT: 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.
No preview · Article · Apr 2012 · Human Molecular Genetics
[Show abstract][Hide abstract] ABSTRACT: In a previous work, using an interspecific recombinant congenic mouse model, we reported a genomic region of 23 Mb on mouse chromosome 11 implicated in testis weight decrease and moderate teratozoospermia (∼20-30%), a Quantitative Trait Locus (QTL) called Ltw1. The objective of the present study is to identify the gene underlying this phenotype.
In the present study, we refined the QTL position to a 5 Mb fragment encompassing only 11 genes. We showed that the low testis weight phenotype was due to kinetic alterations occurring during the first wave of the spermatogenesis where we could point out to an abnormal lengthening of spermatocyte prophase. We identify Fidgetin-like 1 (Fignl1) as the gene underlying the phenotype, since if fulfilled both the physiological and molecular characteristics required. Indeed, amongst the 11 positional candidates it is the only gene that is expressed during meiosis at the spermatocyte stage, and that presents with non-synonymous coding variations differentiating the two mouse strains at the origin of the cross.
This work prompted us to propose Fignl1 as a novel actor in mammal's male meiosis dynamics which has fundamental interest. Besides, this gene is a new potential candidate for human infertilities caused by teratozoospermia and blockades of spermatogenesis. In addition this study demonstrates that interspecific models may be useful for understanding complex quantitative traits.
[Show abstract][Hide abstract] ABSTRACT: Histological sections of epididymal duct at 28 and 35 days post partum in 97 C and B6 mice. At 35 DPP, we observed the presence of spermatozoa in the epididymal duct lumen of B6 mice whereas only abnormal/apoptotic round cells are visible in 97 C epididymis. These cellular elements are normally present in the epididymal duct lumen at 28 DPP consequent to the setting up of the first wave of spermatogenesis.
[Show abstract][Hide abstract] ABSTRACT: Expressional data of Pms1 during the first wave of spermatogenesis. RT-PCR amplification of Pms1 ORF from testis cDNA of B6 mice at 10, 16, 20, 22, 24 and 30 days post partum. The specific amplification product is undetectable at 10, 16, and 20 DPP when germ cells are only represented by spermatogonies and spermatocytes in the tubules. It became observable from 22 DPP, concomitantly with the apparition of spermatids which begun to differentiate in the tubules.
[Show abstract][Hide abstract] ABSTRACT: Phylogenetic conservation of Fignl1 across vertebrate species. In blue are represented amino-acids of the B6 type, while in yellow are represented amino-acids of the spretus type. The amino-acids located at positions 379, 397 and 599 are apparently strictly specific of spretus SEG/Pas. It is interesting to notice that S99C is generally specific of non mammal species (except the monotremata).
[Show abstract][Hide abstract] ABSTRACT: SUMO-FOXL2 Bodies are not enriched in SC-35, Pol2, RNA Helicase A or TMG-capped RNA. COS-7 cells were transfected with FOXL2-GFP and SUMO1. A representative cell with SUMO-FOXL2 Bodies is shown. SC-35 (i), RNA HA (ii), Pol2 (iii) and TMG-capped RNAs (iv) localization were detected by immunofluorescence, and DNA by Hoechst 33342 staining, pointing to an absence of colocalisation with SUMO-FOXL2 Bodies. This excludes the hypothesis that SUMO-FOXL2 Bodies are splicing factories, nucleoli, Cajal Bodies, gems or transcription factories.
[Show abstract][Hide abstract] ABSTRACT: Spectrum attribution of the chymotryptic peptide TLDPACEDMFEKGNY in its unmodified (upper panel) and acetylated form (lower panel). Most intense peaks of the y-series (in blue) and b-series (in red) are well conserved between the two spectra but do not allow for a very precise localisation of the modification. A more intense acetylated precursor could help obtaining more detailed fragmentation spectra and confirm the localisation of the modification.
[Show abstract][Hide abstract] ABSTRACT: Spectrum attribution of the tryptic peptide KGWQNSIR in its unmodified (upper panel) and acetylated form (lower panel). The monocharged y-series of ions (in blue) is well conserved between spectra and goes from y1 to y7, all peaks being unshifted, which localize the 42 Da mass shift to the first amino acid of the sequence, which indeed is a lysine. The b-series of ions (in red) are much less intense, but the most intense peaks of the first spectrum (b2, b6, b7) are found in the second spectrum shifted by 42 Da, confirming the presence of an acetyl group on the N-terminal lysine of this peptide.
[Show abstract][Hide abstract] ABSTRACT: FOXL2 is a transcription factor essential for ovarian development and maintenance. It is mutated in the genetic condition called Blepharophimosis Ptosis Epicantus inversus Syndrome (BPES) and in cases of isolated premature ovarian failure. We and others have previously shown that FOXL2 undergoes several post-translational modifications.
Here, using cells in culture, we show that interference with FOXL2 SUMOylation leads to a robust inhibition of its transactivation ability, which correlates with a decreased stability. Interestingly, FOXL2 SUMOylation promotes its transient recruitment to subnuclear structures that we demonstrate to be PML (Promyelocytic Leukemia) Nuclear Bodies. Since PML bodies are known to be sites where post-translational modifications of nuclear factors take place, we used tandem mass spectrometry to identify new post-translational modifications of FOXL2. Specifically, we detected four phosphorylated, one sulfated and three acetylated sites.
By analogy with other transcription factors, we propose that PML Nuclear Bodies might transiently recruit FOXL2 to the vicinity of locally concentrated enzymes that could be involved in the post-translational maturation of FOXL2. FOXL2 acetylation, sulfation, phosphorylation as well as other modifications yet to be discovered might alter the transactivation capacity of FOXL2 and/or its stability, thus modulating its global intracellular activity.
[Show abstract][Hide abstract] ABSTRACT: Spectrum attribution of the chymotryptic peptide ASYPEPEDAAGAL in its unmodified (upper panel) and sulfated form (lower panel). b-series of ions (in red) are well conserved between the two spectrum, with a 80 Da shift in the second spectrum. The doubly charged ions of the b-series are however much more intense in the second spectrum, compared to the monocharged ions. For example the intensity ratio of b122+ over b12+ is close to 3 in the first spectrum, and close to 30 in the second one. Other ions or other spectra are coherent with this observation. The y-series of ions is only represented by two proeminent peaks (in blue) in the first spectrum, corresponding to fragmentations before the two prolines of the peptide. This suggests that one of the peptide positive charges is solvated N-terminally of prolines, favoring fragmentation at these points, which is coherent with the higher pKa of proline amino function, compared to other amino acids. These two peaks completely disappear in the second spectrum, suggesting that the modification sequesters the positive charge, presumably through hydrogen bonding between the sulphate oxygens and carbonyl groups of the primary chain. This is also coherent with the prevalence of doubly-charged fragments in the second spectrum. One additional peak, attributed to y7+-NH3 by MASCOT appears in the second spectrum. Comparison with other spectra shows that this attribution is erroneous and that this fragment bears two positive charges. It may be due to an unusual c-ion, corresponding to the neutral loss of the last amino-acid.
[Show abstract][Hide abstract] ABSTRACT: Spectrum attribution of the chymotryptic peptide DHDSKTGALHSRLDQGQF in its unmodified (upper panel) and acetylated form (lower panel). The monocharged y-series of ions (in blue) allows to localize the 42 Da modification in the Asp-His-Asp-Ser-Lys subfragment as peaks corresponding to y9 to y13 fragment are well defined and unshifted between spectra. A more intense acetylated precursor could help obtaining more detailed fragmentation spectra and confirm the localisation of the modification.on lysine 366.
[Show abstract][Hide abstract] ABSTRACT: Spectrum attribution of the tryptic peptide EPEPPPSPGK in its unmodified (upper panel) and phosphorylated form (lower panel). Proeminent neutral loss fragments corresponding to loss of H3P04, H3P04+H2O, H3P04+NH3 or H3P04+H2O+NH3 are present in the second spectrum (in green). Fragments attributed to the y-series of ions in the first spectrum (in blue) are conserved in the second spectrum (with the exception of y5) and bear the 80 Da modification, making it possible to localize it in the Pro-Pro-Ser sugfragment (y3 does not bear the modification as y6 does). Fragments attributed to the b-series of ions in the first spectrum (in red), most of them affected by an H2O neutral loss are also well conserved and confirm the localisation of the modification. Indeed b2 to b6 fragments are unaffected in size, whereas b8 is found with a −18 Da weight due to loss of an H2O group along with the phosphate (+80 Da–98 Da). This again localises the modification in the Pro-Ser subfragment and confirms the attribution.
[Show abstract][Hide abstract] ABSTRACT: Spectrum attribution of the chymotryptic peptide AAAAAAAAAAAAAAGPGSPGAAAVVKGL in its unmodified (upper panel) and phosphorylated form (lower panel). Neutral loss fragments characteristic of th phosphorylation are present but with pour abundance (in green). Spectra are extremely complex due to the size of the peptide but many peaks are attributed giving these peptides very high scores (XCorr = 6.97 for the upper spectrum, XCorr = 7.16 for the lower one) and little doubt about their attribution. Most peaks of the y-series (in blue) bearing the modification are present and well conserved between spectra, which allows to localize the modification in the Pro-Gly-Ser subfragment (y10 does not bear the modification, y13 does), further confirming the attribution.
[Show abstract][Hide abstract] ABSTRACT: Spectrum attribution of the chymotryptic peptide SPASPATAAPPAPAPTSAPGLQF in its unmodified (upper panel) and phosphorylated form (lower panel). Some neutral loss fragment (in green) characteristic of phosphorylation may be observed, but are once again of very low abundance. The fragmentation pattern is overall rather poor, but the main peaks are well conserved between spectra, notably y14 and y15 (in blue) and b9 and b14 (in red). This allows to localize the modification in the Ser-Pro-Ala-Ser-Pro-Ala-Thr-Ala-Ala subfragment, which still contains three possible sites for phosphorylation. Indeed both b9 and b14 are detected with both a +80 Da and a −18 Da shift in the second spectrum, whereas y14 and y15 are unshifted. A small peak attributed to y19 and unshifted between the spectra suggests that the modification is localised in the Ser-Pro-Ala-Ser subfragment, eliminating Thr329 as a phosphorylated residue, but this peak has a too low intensity to strictly conclude. According to the GPS 2.1 phospohrylation prediction tool, Ser326 is the residue most likely to be phosphorylated.
[Show abstract][Hide abstract] ABSTRACT: Spectrum attribution of the chymotryptic peptide LNNSWPLPQPPSPMPY in its unmodified (upper panel) and phosphorylated form (lower panel). Neutral loss fragments are detected (in green) but are very minor. Peaks of the y-series (in blue) y7, y9, y11 and y12 are well conserved and bear the 80 Da modification in the second spectrum, allowing to localize it in the Pro-Pro-Ser-Pro-Met-Pro-Tyr subfragment. The b-series of ions are well conserved between spectra but give little informations about the modification position. Only small peaks attributed to y2 and b14 that are conserved between spectra suggest that the serine may more likely bear the modification (indeed y2 appears unshifted whereas b14 is shifted by 80 Da). However the intensity of this peaks does not allow to strictly conclude that serine 211 is the modified residue in this case.