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The Heterokonta or Stramenopile phylum comprises clades of unicellular photosynthetic species, which are promising for a broad range of biotechnological applications, based on their capacity to capture atmospheric CO2 via photosynthesis and produce biomolecules of interest. These molecules include triacylglycerol (TAG) loaded inside specific cytosolic bodies, called the lipid droplets (LDs). Understanding TAG production and LD biogenesis and function in photosynthetic stramenopiles is therefore essential, and is mostly based on the study of a few emerging models, such as the pennate diatom Phaeodactylum tricornutum and eustigmatophytes, such as Nannochloropsis and Microchloropsis species. The biogenesis of cytosolic LD usually occurs at the level of the endoplasmic reticulum. However, stramenopile cells contain a complex plastid deriving from a secondary endosymbiosis, limited by four membranes, the outermost one being connected to the endomembrane system. Recent cell imaging and proteomic studies suggest that at least some cytosolic LDs might be associated to the surface of the complex plastid, via still uncharacterized contact sites. The carbon length and number of double bonds of the acyl groups contained in the TAG molecules depend on their origin. De novo synthesis produces long-chain saturated or monounsaturated fatty acids (SFA, MUFA), whereas subsequent maturation processes lead to very long-chain polyunsaturated FA (VLC-PUFA). TAG composition in SFA, MUFA, and VLC-PUFA reflects therefore the metabolic context that gave rise to the formation of the LD, either via an early partitioning of carbon following FA de novo synthesis and/or a recycling of FA from membrane lipids, e.g., plastid galactolipids or endomembrane phosphor- or betaine lipids. In this review, we address the relationship between cytosolic LDs and the complex membrane compartmentalization within stramenopile cells, the metabolic routes leading to TAG accumulation, and the physiological conditions that trigger LD production, in response to various environmental factors.

Transit amplification (TA), a state of combined, rapid proliferative expansion and differentiation of stem cell-descendants, remains poorly defined at the molecular level. The Polycomb Repressive Complex 1 (PRC1) protein BMI1 has been localized to TA compartments, yet its exact role in TA is unclear. PRC1 proteins control gene expression, cell proliferation and DNA-damage repair. Coordination of such DNA-templated activities during TA is predicted to be crucial to support DNA replication and differentiation-associated transcriptional programming. We here examined whether chondrogenesis provides a relevant biological context for synchronized coordination of these chromatin-based tasks by BMI1. Taking advantage of a prominently featuring TA-phase during chondrogenesis in vitro and in vivo, we here report that TA is completely dependent on intact PRC1 function. BMI1-depleted chondrogenic progenitors rapidly accumulate double strand DNA breaks during DNA replication, present massive non-H3K27me3-directed transcriptional deregulation and fail to undergo chondrogenic TA. Genome-wide accumulation of Topoisomerase 2α and Geminin suggests a model in which PRC1 synchronizes replication and transcription during rapid chondrogenic progenitor expansion. Our combined data reveals for the first time a vital cell-autonomous role for PRC1 during chondrogenesis. We provide evidence that chondrocyte hyper-replication and hypertrophy represent a unique example of programmed senescence in vivo. These findings provide new perspectives on PRC1 function in development and disease., The authors received financial support from the European Molecular Biology Organization (Germany; www.embl.de) ASTF5-2009 (F.S.) and the René Vogels foundation (F.S.); grant BFU2010-18146 (MINECO, MV) and Oncocycle program S2010/BMD2470 (Comunidad de Madrid, MV); the Dutch Science Organization (ZonMW-NWO; www.NWO.nl) VIDI grant 864.12.007 (H.M.); VIDI grant 016.046.362 (J.W.V.); Netherlands Reuma foundation (The Netherlands; www.ruemafonds.nl) grant LLP14 (J.W.V., L. van Rhijn, Maastricht, The Netherlands) and a transnational University of Limburg grant (J.W.V., B.W.G.).

Chez les mammiferes, le genome de l’embryon nouvellement feconde est d’abord transcriptionnellement inactif. Le developpement embryonnaire depend alors strictement de l’heritage maternel en ARN et en proteines presents dans l’ovocyte et accumules avant l’ovulation. La mise en route de l’expression des genes embryonnaires se fait ulterieurement lors de la phase appelee « activation du genome embryonnaire (EGA) », a un moment de la periode preimplantatoire variable selon les especes. Cette phase d’activation est dependante de la presence des composants de la machinerie transcriptionnelle de base mais aussi de l’evolution de l’organisation des genomes parentaux apres la fecondation. En effet, au cours des premiers cycles embryonnaires, les noyaux subissent une reorganisation intense qui constituerait un element cle dans la regulation du developpement embryonnaire.

Polycomb-group (PcG) and trithorax-group (trxG) genes encode important regulators of homeotic genes, repressors and activators, respectively. They act through epigenetic mechanisms that maintain chromatin structure. The corto gene of Drosophila melanogaster encodes a co-factor of these regulators belonging to the Enhancer of Trithorax and Polycomb class. We have previously shown that Corto maintains the silencing of the homeotic gene Abdominal-B in the embryo and that it interacts with a cyclin, Cyclin G, suggesting that it could be a major actor in the connection between Polycomb/Trithorax function and the cell cycle. We show here that inactivation of Cyclin G by RNA interference leads to rotated genitalia and cuticle defects in the posterior abdomen of pupae and that corto genetically interacts with Cyclin G for generating these phenotypes. Examination of these pupae shows that development of the dorsal histoblast nests that will give rise to the adult epithelium is impaired in the posterior segments which identity is specified by Abdominal-B. Using a line that expresses LacZ in the Abdominal-B domain, we show that corto maintains Abdominal-B repression in the pupal epithelium whereas Cyclin G maintains its activation. These results prompt us to propose that the interaction between the Enhancer of Trithorax and Polycomb Corto and Cyclin G is involved in regulating the balance between cell proliferation and cell differentiation during abdominal epithelium development.

International audience; Although the MK3 gene was originally found deleted in some cancers, it is highly expressed in others. The relevance of MK3 for oncogenesis is currently not clear. We recently reported that MK3 controls ERK activity via a negative feedback mechanism. This prompted us to investigate a potential role for MK3 in cell proliferation. We here show that overexpression of MK3 induces a proliferative arrest in normal diploid human fibroblasts, characterized by enhanced expression of replication stress-and senescence-associated markers. Surprisingly, MK3 depletion evokes similar senescence characteristics in the fibroblast model. We previously identified MK3 as a binding partner of Polycomb Repressive Complex 1 (PRC1) proteins. In the current study we show that MK3 overexpression results in reduced cellular EZH2 levels and concomitant loss of epigenetic H3K27me3-marking and PRC1/chromatin-occupation at the CDKN2A/INK4A locus. In agreement with this, the PRC1 oncoprotein BMI1, but not the PCR2 protein EZH2, bypasses MK3-induced senescence in fibroblasts and suppresses P16$^{INK4A}$ expression. In contrast, BMI1 does not rescue the MK3 loss-of-function phenotype, suggesting the involvement of multiple different checkpoints in gain and loss of MK3 function. Notably, MK3 ablation enhances proliferation in two different cancer cells. Finally , the fibroblast model was used to evaluate the effect of potential tumorigenic MK3 driver-mutations on cell proliferation and M/SAPK signaling imbalance. Taken together, our findings support a role for MK3 in control of proliferation and replicative lifespan , in part through concerted action with BMI1, and suggest that the effect of MK3 modulation or mutation on M/ SAPK signaling and, ultimately, proliferation, is cell context-dependent.

Background In the mouse zygote, DNA methylation patterns are heavily modified, and differ between the maternal and paternal pronucleus. Demethylation of the paternal genome has been described as an active and replication-independent process, although the mechanisms responsible for it remain elusive. Recently, 5-hydroxymethylcytosine has been suggested as an intermediate in this demethylation. Methodology/Principal Findings In this study, we quantified DNA methylation and hydroxymethylation in both pronuclei of the mouse zygote during the replication period and we examined their patterns on the pericentric heterochromatin using 3D immuno-FISH. Our results demonstrate that 5-methylcytosine and 5-hydroxymethylcytosine localizations on the pericentric sequences are not complementary; indeed we observe no enrichment of either marks on some regions and an enrichment of both on others. In addition, we show that DNA demethylation continues during DNA replication, and is inhibited by aphidicolin. Finally, we observe notable differences in the kinetics of demethylation and hydroxymethylation; in particular, a peak of 5-hydroxymethylcytosine, unrelated to any change in 5-methylcytosine level, is observed after completion of replication. Conclusions/Significance Together our results support the already proposed hypothesis that 5-hydroxymethylcytosine is not a simple intermediate in an active demethylation process and could play a role of its own during early development.

Remerciements : INRA, plate-forme MIMA2 (Microscopie et Imagerie des Micro-organismes, Animaux et Aliments), Centre de Jouy en Josas; Somatic cell nuclear transfer (SCNT) is the injection of a donor nucleus into an enucleated egg. Despite the use of this technology for many years in research, it is still quite inefficient. One of the causes for this is thought to be incorrect or incomplete genome reprogramming. Embryos produced by nuclear transfer (cloned embryos) very often present abnormal epigenetic signatures and irregular chromatin reorganization. Of these two issues, the issue of chromatin rearrangements within the nuclei after transfer is the least studied. It is known that cloned embryos often present pericentromeric heterochromatin clumps very similar to the chromocenters structures present in the donor nuclei. Therefore, it is believed that the somatic nuclear configuration of donor nuclei, especially that of the chromocenters, is not completely lost after nuclear transfer, in other words, not well reprogrammed. To further investigate pericentromeric heterochromatin reorganization after nuclear transfer, we decided to study its rearrangements in cumulus-derived clones using several related epigenetic markers such as H3S10P, H3K9me3, and the double marker H3K9me3S10P. We observed that two of these markers, H3S10P and H3K9me3S10P, are the ones found on the part of the pericentromeric heterochromatin that is remodeled correctly, resembling exactly the embryonic heterochromatin configuration of naturally fertilized embryos. Conversely, H3K9me3 and heterochromatin protein 1 beta (HP1β)-associated protein were also detected in the perinuclear clumps of heterochromatin, making obvious the maintenance of the somatic epigenetic signature within these nuclear regions. Our results demonstrate that H3S10P and H3K9me3S10P could be good candidates for evaluating heterochromatin reorganization following nuclear reprogramming.

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In mammals, totipotent embryos are formed by fusion of highly differentiated gametes. Acquisition of totipotency concurs with chromatin remodeling of parental genomes, changes in the maternal transcriptome and proteome, and zygotic genome activation (ZGA). The inefficiency of reprogramming somatic nuclei in reproductive cloning suggests that intergenerational inheritance of germline chromatin contributes to developmental proficiency after natural conception. Here we show that Ring1 and Rnf2, components of Polycomb-repressive complex 1 (PRC1), serve redundant transcriptional functions during oogenesis that are essential for proper ZGA, replication and cell cycle progression in early embryos, and development beyond the two-cell stage. Exchange of chromosomes between control and Ring1/Rnf2-deficient metaphase II oocytes reveal cytoplasmic and chromosome-based contributions by PRC1 to embryonic development. Our results strongly support a model in which Polycomb acts in the female germline to establish developmental competence for the following generation by silencing differentiation-inducing genes and defining appropriate chromatin states.

After fertilization in mammals, the genome of the newly formed embryo is first transcriptionally inactive. Development is then strictly dependent on the maternally inherited RNA and proteins present in the oocyte that were accumulated before ovulation during oocyte growth and maturation. The onset of transcription specific to the embryo, referred to as "embryonic genome activation (EGA)", is initiated later during development at various preimplantation stages according to species. Transcriptional activity can be underlined thanks to several approaches such as precursors incorporation in newly synthesized RNA and expression of reporter genes. These studies show that EGA is established in two phases: a "minor" one, first with reduced transcriptional activity and that does not require any specific transcription factor; second, a "major" phase with rapidly increasing transcription. Upon major activation, newly synthesized RNA/proteins are essential for further embryonic development. EGA is dependent on the availability and activity of the basal transcriptional machinery components but also on the structural modifications of the nuclei after fertilization. Indeed, during the first embryonic cycles, the maternal and paternal genome undergo intense chromatin remodeling that could be a key regulator of embryonic transcription.

International audience; BACKGROUND: Polycomb (PcG) and trithorax (trxG) genes encode proteins involved in the maintenance of gene expression patterns, notably Hox genes, throughout development. PcG proteins are required for long-term gene repression whereas TrxG proteins are positive regulators that counteract PcG action. PcG and TrxG proteins form large complexes that bind chromatin at overlapping sites called Polycomb and Trithorax Response Elements (PRE/TRE). A third class of proteins, so-called "Enhancers of Trithorax and Polycomb" (ETP), interacts with either complexes, behaving sometimes as repressors and sometimes as activators. The role of ETP proteins is largely unknown. METHODOLOGY/PRINCIPAL FINDINGS: In a two-hybrid screen, we identified Cyclin G (CycG) as a partner of the Drosophila ETP Corto. Inactivation of CycG by RNA interference highlights its essential role during development. We show here that Corto and CycG directly interact and bind to each other in embryos and S2 cells. Moreover, CycG is targeted to polytene chromosomes where it co-localizes at multiple sites with Corto and with the PcG factor Polyhomeotic (PH). We observed that corto is involved in maintaining Abd-B repression outside its normal expression domain in embryos. This could be achieved by association between Corto and CycG since both proteins bind the regulatory element iab-7 PRE and the promoter of the Abd-B gene. CONCLUSIONS/SIGNIFICANCE: Our results suggest that CycG could regulate the activity of Corto at chromatin and thus be involved in changing Corto from an Enhancer of TrxG into an Enhancer of PcG.

International audience; Mitogen-activated protein kinase (MAPK) cascades are evolutionary conserved transduction pathways involved in many cellular processes. Kinase modules are associated with scaffold proteins that regulate signaling by providing critical spatial and temporal specificities. Some of these scaffold proteins have been shown to be conserved, both in sequence and function. In mouse, the scaffold MP1 (MEK Partner 1) forms a signaling complex with MEK1 and ERK1. In this work, we focus on Drosophila MP1 (dMP1). We show that dMP1 is expressed ubiquitously during embryonic and larval development. By in vitro and in vivo experiments, we show that dMP1 is located in the cytoplasm and the nuclei, and that it interacts with MEK and ERK. Genetic studies with transgenic Drosophila lines allowing either dMP1 over-expression or dMP1 down-regulation by RNA interference highlight dMP1 function in the control of cell differentiation during development of the Drosophila wing.

Background Polycomb-group genes (PcG) encode proteins that maintain homeotic (Hox) gene repression throughout development. Conversely, trithorax-group (trxG) genes encode positive factors required for maintenance of long term Hox gene activation. Both kinds of factors bind chromatin regions called maintenance elements (ME). Our previous work has shown that corto, which codes for a chromodomain protein, and dsp1, which codes for an HMGB protein, belong to a class of genes called the Enhancers of trithorax and Polycomb (ETP) that interact with both PcG and trxG. Moreover, dsp1 interacts with the Hox gene Scr, the DSP1 protein is present on a Scr ME in S2 cells but not in embryos. To understand better the role of ETP, we addressed genetic and molecular interactions between corto and dsp1. Results We show that Corto and DSP1 proteins co-localize at 91 sites on polytene chromosomes and co-immunoprecipitate in embryos. They interact directly through the DSP1 HMG-boxes and the amino-part of Corto, which contains a chromodomain. In order to search for a common target, we performed a genetic interaction analysis. We observed that corto mutants suppressed dsp1 1 sex comb phenotypes and enhanced Antp Scx phenotypes, suggesting that corto and dsp1 are simultaneously involved in the regulation of Scr. Using chromatin immunoprecipitation of the Scr ME, we found that Corto was present on this ME both in Drosophila S2 cells and in embryos, whereas DSP1 was present only in S2 cells. Conclusion Our results reveal that the proteins Corto and DSP1 are differently recruited to a Scr ME depending on whether the ME is active, as seen in S2 cells, or inactive, as in most embryonic cells. The presence of a given combination of ETPs on an ME would control the recruitment of either PcG or TrxG complexes, propagating the silenced or active state.

International audience; In $Drosophila$, PcG complexes provide heritable transcriptional silencing of target genes. Among them, the ESC/E(Z) complex is thought to play a role in the initiation of silencing whereas other complexes such as the PRC1 complex are thought to maintain it. PcG complexes are thought to be recruited to DNA through interaction with DNA binding proteins such as the GAGA factor, but no direct interactions between the constituents of PcG complexes and the GAGA factor have been reported so far. The Drosophila corto gene interacts with $E(z)$ as well as with genes encoding members of maintenance complexes, suggesting that it could play a role in the transition between the initiation and maintenance of PcG silencing. Moreover, corto also interacts genetically with $Trl$, which encodes the GAGA factor, suggesting that it may serve as a mediator in recruiting PcG complexes. Here, we show that Corto bears a chromo domain and we provide evidence for $in\ vivo$ association of Corto with ESC and with PC in embryos. Moreover, we show by GST pull-down and two-hybrid experiments that Corto binds to E(Z), ESC, PH, SCM and GAGA and co-localizes with these proteins on a few sites on polytene chromosomes. These results reinforce the idea that Corto plays a role in PcG silencing, perhaps by con-fering target specificity.