Doctor of Philosophy, Universite De Paris Vii (2011)
Anne Brunet, Postdoctoral Faculty Sponsor
Aging is a complex process that affects multiple organs. Modeling aging and age-related diseases in the lab is challenging because classical vertebrate models have relatively long lifespans. Here, we develop the first platform for rapid exploration of age-dependent traits and diseases in vertebrates, using the naturally short-lived African turquoise killifish. We provide an integrative genomic and genome-editing toolkit in this organism using our de-novo-assembled genome and the CRISPR/Cas9 technology. We mutate many genes encompassing the hallmarks of aging, and for a subset, we produce stable lines within 2-3 months. As a proof of principle, we show that fish deficient for the protein subunit of telomerase exhibit the fastest onset of telomere-related pathologies among vertebrates. We further demonstrate the feasibility of creating specific genetic variants. This genome-to-phenotype platform represents a unique resource for studying vertebrate aging and disease in a high-throughput manner and for investigating candidates arising from human genome-wide studies.
View details for DOI 10.1016/j.cell.2015.01.038
View details for PubMedID 25684364
Trimethylation of histone H3 at lysine 4 (H3K4me3) is a chromatin modification known to mark the transcription start sites of active genes. Here, we show that H3K4me3 domains that spread more broadly over genes in a given cell type preferentially mark genes that are essential for the identity and function of that cell type. Using the broadest H3K4me3 domains as a discovery tool in neural progenitor cells, we identify novel regulators of these cells. Machine learning models reveal that the broadest H3K4me3 domains represent a distinct entity, characterized by increased marks of elongation. The broadest H3K4me3 domains also have more paused polymerase at their promoters, suggesting a unique transcriptional output. Indeed, genes marked by the broadest H3K4me3 domains exhibit enhanced transcriptional consistency rather than increased transcriptional levels, and perturbation of H3K4me3 breadth leads to changes in transcriptional consistency. Thus, H3K4me3 breadth contains information that could ensure transcriptional precision at key cell identity/function genes.
View details for DOI 10.1016/j.cell.2014.06.027
View details for PubMedID 25083876
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.
View details for DOI 10.1038/onc.2012.298
View details for PubMedID 22797072
Forkhead box (FOX) proteins constitute an evolutionarily conserved family of transcription factors with a central role not only during development, but also in the adult organism. Thus, the misregulation and/or mutation of FOX genes often induce human genetic diseases, promote cancer or deregulate ageing. Indeed, germinal FOX gene mutations cause diseases ranging from infertility to language and/or speech disorders and immunological defects. Moreover, because of their central role in signalling pathways and in the regulation of homeostasis, somatic misregulation and/or mutation of FOX genes are associated with cancer. FOX proteins have undergone diversification in terms of their sequence, regulation and function. In addition to dedicated roles, evidence suggests that Forkhead factors have retained some functional redundancy. Thus, combinations of slightly defective alleles might induce disease phenotypes in humans, acting as quantitative trait loci. Uncovering such variants would be a big step towards understanding the functional interdependencies of different FOX members and their implications in complex pathologies.
View details for DOI 10.1016/j.tig.2011.03.003
View details for Web of Science ID 000291567400003
View details for PubMedID 21507500
FOXL2 is a transcription factor that is essential for ovarian function and maintenance, the germline mutations of which are responsible for the Blepharophimosis Ptosis Epicanthus-inversus Syndrome (BPES), often associated with premature ovarian failure. Recent evidence has linked FOXL2 downregulation or somatic mutation (p.Cys134Trp) to cancer, although underlying molecular mechanisms remain unclear. Using a functional genomic approach, we find that FOXL2 modulates cell-cycle regulators in a way which tends to induce G1 arrest. Indeed, FOXL2 upregulation promotes cell accumulation in G1 phase and protects cells from oxidative damage, notably by promoting oxidized DNA repair and by increasing the amounts of anti-oxidant agent glutathione. In agreement with clinical observations, we find that FOXL2-mutated versions leading to BPES along with ovarian dysfunction mostly fail to transactivate cell-cycle and DNA repair targets, whereas mutations leading to isolated craniofacial defects (and normal ovarian function) activate them correctly. Interestingly, these assays revealed a mild promoter-specific hypomorphy of the tumor-associated mutation (p.Cys134Trp). Finally, the SIRT1 deacetylase suppresses FOXL2 activity on targets linked to cell-cycle and DNA repair in a dose-dependent manner. Accordingly, we find that SIRT1 inhibition by nicotinamide limits proliferation, notably by increasing endogenous FOXL2 amount/activity. The body of evidence presented here supports the idea that FOXL2 plays a key role in granulosa cell homeostasis, the failure of which is central to ovarian ageing and tumorigenesis. As granulosa cell tumors respond poorly to conventional chemotherapy, our findings on the deacetylase inhibitor nicotinamide provide an interesting option for targeted therapy.
View details for DOI 10.1093/hmg/ddr042
View details for Web of Science ID 000289311400001
View details for PubMedID 21289058
Cellular responses to environmental or physiological cues rely on transduction pathways that must ensure discrimination between different signals. These cascades 'crosstalk' and lead to a combinatorial regulation. This often results in different combinations of post-translational modifications (PTMs) on target proteins, which might act as a molecular barcode. Although appealing, the idea of the existence of such a code for transcription factors is debated. Using general arguments and recent evidence, we propose that a PTM code is not only possible but necessary in the context of transcription factors regulating multiple processes. Thus, the coding potential of PTM combinations should both provide a further layer of information integration from several transduction pathways and warrant highly specific cellular outputs.
View details for DOI 10.1016/j.tcb.2009.02.003
View details for Web of Science ID 000266623500001
View details for PubMedID 19328693
Mutations in the retinoblastoma tumor suppressor gene Rb are involved in many forms of human cancer. In this study, we investigated the early consequences of inactivating Rb in the context of cellular reprogramming. We found that Rb inactivation promotes the reprogramming of differentiated cells to a pluripotent state. Unexpectedly, this effect is cell cycle independent, and instead reflects direct binding of Rb to pluripotency genes, including Sox2 and Oct4, which leads to a repressed chromatin state. More broadly, this regulation of pluripotency networks and Sox2 in particular is critical for the initiation of tumors upon loss of Rb in mice. These studies therefore identify Rb as a global transcriptional repressor of pluripotency networks, providing a molecular basis for previous reports about its involvement in cell fate pliability, and implicate misregulation of pluripotency factors such as Sox2 in tumorigenesis related to loss of Rb function.
View details for DOI 10.1016/j.stem.2014.10.019
View details for PubMedID 25467916
How an individual's longevity is affected by the opposite sex is still largely unclear. In the nematode Caenorhabditis elegans, the presence of males accelerated aging and shortened the life span of individuals of the opposite sex (hermaphrodites), including long-lived or sterile hermaphrodites. The male-induced demise could occur without mating and required only exposure of hermaphrodites to medium in which males were once present. Such communication through pheromones or other diffusible substances points to a nonindividual autonomous mode of aging regulation. The male-induced demise also occurred in other species of nematodes, suggesting an evolutionary conserved process whereby males may induce the disposal of the opposite sex to save resources for the next generation or to prevent competition from other males.
View details for DOI 10.1126/science.1244160
View details for Web of Science ID 000330343700047
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-?2?, 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.
View details for DOI 10.1093/hmg/dds170
View details for Web of Science ID 000305822700014
View details for PubMedID 22544055
FOXL2 is a gene encoding a forkhead transcription factor. Its mutations or misregulation have been shown to cause the blepharophimosis-ptosis-epicanthus inversus (BPES) syndrome and more recently have been associated with the development of Ovarian Granulosa Cell Tumors (OGCT). BPES is a genetic disorder involving mild craniofacial abnormalities often associated with premature ovarian failure. OGCTs are endocrine malignancies, accounting for 2-5% of ovarian cancers, the treatment of which is still challenging. In this review we summarize recent data concerning FOXL2 transcriptional targets and molecular partners, its post-translational modifications, its mutations and its involvement in newly discovered pathophysiological processes. In the ovary, FOXL2 is involved in the regulation of cholesterol and steroid metabolism, apoptosis, reactive oxygen species detoxification and cell proliferation. Interestingly, one of the main roles of FOXL2 is also to preserve the identity of ovarian granulosa cells even at the adult stage and to prevent their transdifferentiation into Sertoli-like cells. All these recent advances indicate that FOXL2 is central to ovarian development and maintenance. The elucidation of the impact of FOXL2 germinal and somatic mutations will allow a better understanding of the pathogenesis of BPES and of OGCTs.
View details for DOI 10.1016/j.mce.2011.06.019
View details for Web of Science ID 000303641500008
View details for PubMedID 21763750
Here we present the Transcription Factor Encyclopedia (TFe), a new web-based compendium of mini review articles on transcription factors (TFs) that is founded on the principles of open access and collaboration. Our consortium of over 100 researchers has collectively contributed over 130 mini review articles on pertinent human, mouse and rat TFs. Notable features of the TFe website include a high-quality PDF generator and web API for programmatic data retrieval. TFe aims to rapidly educate scientists about the TFs they encounter through the delivery of succinct summaries written and vetted by experts in the field. TFe is available at http://www.cisreg.ca/tfe.
View details for DOI 10.1186/gb-2012-13-3-r24
View details for Web of Science ID 000308544200009
View details for PubMedID 22458515
A recent study by Greer et al. in the nematode C. elegans has shown transgenerational epigenetic inheritance of longevity in the descendants of worms deficient for subunits of a complex responsible for histone H3 lysine 4 trimethylation (H3K4me3). In this commentary, we discuss the implications of this epigenetic memory of longevity and the potential mechanisms underlying this phenomenon. The transgenerational inheritance of longevity could result from heritable depletion of H3K4me3 at particular aging-regulating gene loci that would only be progressively replenished. The epigenetic memory of longevity could also be explained by the transgenerational transmission of other molecules, for example other proteins or non-coding RNAs. The discovery of an epigenetic memory of longevity in worms raises the intriguing possibility that environmental cues modulating longevity in ancestors might affect subsequent generations in a non-Mendelian manner. Another remaining intriguing question is whether transgenerational inheritance of longevity also exists in other species, including mammals.
View details for DOI 10.4161/worm.19157
View details for PubMedID 24058828
Chromatin modifiers regulate lifespan in several organisms, raising the question of whether changes in chromatin states in the parental generation could be incompletely reprogrammed in the next generation and thereby affect the lifespan of descendants. The histone H3 lysine 4 trimethylation (H3K4me3) complex, composed of ASH-2, WDR-5 and the histone methyltransferase SET-2, regulates Caenorhabditis elegans lifespan. Here we show that deficiencies in the H3K4me3 chromatin modifiers ASH-2, WDR-5 or SET-2 in the parental generation extend the lifespan of descendants up until the third generation. The transgenerational inheritance of lifespan extension by members of the ASH-2 complex is dependent on the H3K4me3 demethylase RBR-2, and requires the presence of a functioning germline in the descendants. Transgenerational inheritance of lifespan is specific for the H3K4me3 methylation complex and is associated with epigenetic changes in gene expression. Thus, manipulation of specific chromatin modifiers only in parents can induce an epigenetic memory of longevity in descendants.
View details for DOI 10.1038/nature10572
View details for Web of Science ID 000297059700038
View details for PubMedID 22012258
FOXL2 is a gene encoding a forkhead transcription factor, whose germline mutations are responsible for the blepharophimosis ptosis epicanthus inversus syndrome. We have previously shown that expression levels of FOXL2 in a series of juvenile ovarian granulosa cell tumors (OGCTs) were markedly reduced. More recently, a whole-transcriptome 'next-generation' sequencing study has identified the somatic mutation p.Cys134Trp as recurring in adult OGCTs. This mutation may thus provide the tumor with either a striking proliferative potential or increased survival abilities. These studies of FOXL2 in OGCTs suggest that it may act as a tumor suppressor gene. This is in line with the fact that other forkhead transcription factors have already been involved in the etiology of cancer. Indeed, an in-depth review of existing data on FOXL2 reveals that its target genes and molecular partners can often be linked to cancer progression.
View details for DOI 10.1016/j.bbcan.2009.09.002
View details for Web of Science ID 000274665700001
View details for PubMedID 19747961
FOXL2 is a forkhead transcription factor, essential for ovarian function, whose mutations are responsible for the blepharophimosis syndrome, characterized by craniofacial defects, often associated with premature ovarian failure. Here, we show that cell stress upregulates FOXL2 expression in an ovarian granulosa cell model. Increased FOXL2 transcription might be mediated at least partly by self-activation. Moreover, using 2D-western blot, we show that the response of FOXL2 to stress correlates with a dramatic remodeling of its post-translational modification profile. Upon oxidative stress, we observe an increased recruitment of FOXL2 to several stress-response promoters, notably that of the mitochondrial manganese superoxide dismutase (MnSOD). Using several reporter systems, we show that FOXL2 transactivation is enhanced in this context. Models predict that gene upregulation in response to a signal should eventually be counterbalanced to restore the initial steady state. In line with this, we find that FOXL2 activity is repressed by the SIRT1 deacetylase. Interestingly, we demonstrate that SIRT1 transcription is, in turn, directly upregulated by FOXL2, which closes a negative-feedback loop. The regulatory relationship between FOXL2 and SIRT1 prompted us the test action of nicotinamide, an inhibitor of sirtuins, on FoxL2 expression/activity. According to our expectations, nicotinamide treatment increases FoxL2 transcription. Finally, we show that 11 disease-causing mutations in the ORF of FOXL2 induce aberrant regulation of FOXL2 and/or regulation of the FOXL2 stress-response target gene MnSOD. Taken together, our results establish that FOXL2 is an actor of the stress response and provide new insights into the pathogenic consequences of FOXL2 mutations.
View details for DOI 10.1093/hmg/ddn389
View details for Web of Science ID 000262719900004
View details for PubMedID 19010791
The Forkhead transcription factor FOXL2 plays a crucial role in ovarian development and maintenance. In humans, its mutations lead to craniofacial abnormalities, isolated or associated with ovarian dysfunction. Using a combinatorial approach, we identified and characterized a FoxL2 response element (FLRE) and showed that it is highly specific and that it diverges from that of other Forkhead transcription factors. This specificity should prevent aberrant regulation of FOXL2 targets by other members of the family and should prevent ectopic activation of the ovarian differentiation program in testes. We provide evidence that the FLRE is used in naturally occurring promoters. We show that polyAlanine expansions of FOXL2, which are the most frequent pathogenic mutations, induce a length-dependent loss of response on different artificial promoter reporters depending on the number and sequence of the FLREs that they contain. Thus, we provide clear mechanistic evidence explaining how the architecture of promoters influences their sensitivity to decreased transcription factor availability. Furthermore, we speculate that the generally absent ovarian phenotype of patients carrying the most frequent polyAlanine expansion should come from its ability to properly regulate high-affinity ovarian targets. The existence of critical high-affinity ovarian targets would be compatible with the role of FOXL2 in reproduction and ensure developmental and functional robustness. Taken together, our results give mechanistic insights on the molecular pathogenesis of FOXL2 polyAlanine expansions.
View details for DOI 10.1093/hmg/ddn209
View details for Web of Science ID 000259766800003
View details for PubMedID 18635577
The transcription factor Forkhead box L subfamily member 2 (FOXL2) is involved in craniofacial development and ovarian function. Using 2-DE and immunoblotting, we show that it is highly modified post-translationally. The most outstanding feature of its migration profile is the presence of two distinct modification "trains" and the absence of intermediates. A theoretical analysis of the modification profile of FOXL2 suggests that it undergoes parallel processive/concerted modifications. The absence of intermediates is compatible with the recruitment of poorly modified FOXL2 into a post-translational "modification factory."
View details for DOI 10.1002/pmic.200800084
View details for Web of Science ID 000258503400013
View details for PubMedID 18604817