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International audience; Lipid droplets (LDs) are found in all lineages of living organisms, whether prokaryotes or eukaryotes. Their structure is rather simple with a core of hydrophobic molecules, mostly Triacylglycerols and Sterol Esters, surrounded by a monolayer of polar lipids with associated proteins. They are constitutively present in storage organs, such as seeds in plants, or adipose tissue in mammals, while they can form in response to various abiotic or biotic stresses in other plant organs and in microalgae. Their biogenesis has been mostly studied in yeast and animals, yet remains poorly understood in plants. In particular, the central role of the photosynthetic plastid in lipid metabolism raises questions about its role in LD biogenesis. Over the years, many functions of LDs have emerged in addition to the obvious role in carbon and energy storage. While some functions appear broadly distributed among organisms and organs, other appear specific to certain tissues and/or lineages. The present review aims at highlighting specific aspects of LD biogenesis and functions in all photosynthetic organisms, from the historic model plant Arabidopsis thaliana to emerging secondary endosymbiont models such as the diatom Phaeodactylum tricornutum.

Data Paper; International audience; Diatoms are one of the largest groups in phytoplankton biodiversity. Understanding their response to nitrogen variations, present from micromolar to near-zero levels in oceans and fresh waters, is essential to comprehend their ecological success. Nitrogen starvation is used in biotechnological processes, to trigger the remodeling of carbon metabolism in the direction of fatty acids and triacylglycerol synthesis. We evaluated whole proteome changes in Phaeodactylum tricornutum after 7 days of cultivation with 5.5-mM nitrate (+N) or without any nitrogen source (-N). On a total of 3768 proteins detected in biological replicates, our analysis pointed to 384 differentially abundant proteins (DAP). Analysis of proteins of lower abundance in -N revealed an arrest of amino acid and protein syntheses, a remodeling of nitrogen metabolism, and a decrease of the proteasome abundance suggesting a decline in unselective whole-proteome decay. Analysis of proteins of higher abundance revealed the setting up of a general nitrogen scavenging system dependent on deaminases. The increase of a plastid palmitoyl-ACP desaturase appeared as a hallmark of carbon metabolism rewiring in the direction of fatty acid and triacylglycerol synthesis. This dataset is also valuable to select gene candidates for improved biotechnological properties.

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.

International audience; Diatoms are a major phylum of phytoplankton biodiversity and a resource considered for biotechnological developments, as feedstock for biofuels and applications ranging from food, human health or green chemistry. They contain a secondary plastid limited by four membranes, the outermost one being connected with the endoplasmic reticulum (ER). Upon nitrogen stress, diatoms reallocate carbon to triacylglycerol storage inside lipid droplets (LDs). The comprehensive glycerolipid and sterol composition and the architecture of diatom LDs are unknown. In Phaeodactylum tricornutum, LDs are in contact with plastid, mitochondria and uncharacterized endomembranes. We purified LDs from nitrogen-starved P. tricornutum cells to high purity level (99 mol% triacylglycerol of total glycerolipids). We used the Stramenopile Lipid Droplet Protein (StLDP) as a previously validated marker for the identity of P. tricornutum LD. Amphipathic lipids surrounding LDs consist of a betaine lipid, diacylglycerylhydroxymethyltrimethyl-beta-alanine (0.4 mol%); sulfoquinovosyldiacylglycerol (0.35 mol%); phosphatidylcholine (0.15 mol%) and one sterol, brassicasterol. By contrast with whole cell extracts, the betaine lipid from LDs only contains eicosapentaenoic acid paired with palmitoleic or palmitolenic acids. This polar lipid composition suggests a budding of LDs from the cytosolic leaflet of the plastid outermost membrane. LD pigments reveal a specific accumulation of beta-carotene. The LD proteome obtained from three independent biological replicates, based on stringent filtering of extracted data, and following subtraction of proteins downregulated by nitrogen starvation, highlights a core proteome of 86 proteins, including StLDP. LD-associated proteins suggest connections with vesicular trafficking (coatomer, clathrin), cytoskeleton, plastid and mitochondria. Unsuspected LD-associated function includes protein synthesis (ribosomes), folding (chaperones), posttranslational modifications and quality control (ubiquitination and ERAD pathway), possibly preparing translation of specific mRNAs. The detection of histone proteins, as previously demonstrated in drosophila embryo LDs, also suggests the storage of nucleosome components, preparing cell division and chromatin packaging, when cells are not stressed anymore.

International audience; Immunostaining is widely used in cell biology for the in situ detection of proteins in fixed cells. The method is based on the specificity of antibodies for recognizing and binding to a selected target, combined with immunolabeling techniques for microscopic imaging. Antibodies with high specificities for modified nucleotides have also been widely developed, and among those, antibodies that recognize modified cytosine: 5-methylcytosine (5mC), and more recently, its derivates 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC). To allow for their detection, primary antibody signals can be amplified using secondary antibodies coupled to fluorophores for immunofluorescence, or other molecules for immunocytochemistry.Immunostaining can be used to gain information on the spatial distribution and levels of DNA methylation states within the nucleus. Although the resolution remains quite low in genomic terms, advanced microscopy techniques and image analysis can obtain detailed spatial information content from immunostained sites. The technique complements genomic approaches that permit the assessment of DNA methylation on specific sequences, but that cannot provide global nuclear spatial context. Immunostaining is an accessible method of great benefit in several cases: when working with limited material (such as embryos or primary cells), to quickly assess at the level of individual cells the effect of siRNA, drugs, or biological processes that promote or inhibit DNA methylation or demethylation, or to study the 3D nuclear organization of regions with high DNA methylation, such as constitutive heterochromatin.Here, we review and outline protocols for the fluorescent and enzymatic immunodetection of DNA methylation in the nuclei of cells, tissue sections, and mammalian embryos.

Immunostaining is widely used in cell biology for the in situ detection of proteins in fixed cells. The method is based on the specificity of antibodies for recognizing and binding to a selected target, combined with immunolabeling techniques for microscopic imaging. Antibodies with high specificities for modified nucleotides have also been widely developed, and among those, antibodies that recognize modified cytosine: 5-methylcytosine (5mC), and more recently, its derivates 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC). To allow for their detection, primary antibody signals can be amplified using secondary antibodies coupled to fluorophores for immunofluorescence, or other molecules for immunocytochemistry.Immunostaining can be used to gain information on the spatial distribution and levels of DNA methylation states within the nucleus. Although the resolution remains quite low in genomic terms, advanced microscopy techniques and image analysis can obtain detailed spatial information content from immunostained sites. The technique complements genomic approaches that permit the assessment of DNA methylation on specific sequences, but that cannot provide global nuclear spatial context. Immunostaining is an accessible method of great benefit in several cases: when working with limited material (such as embryos or primary cells), to quickly assess at the level of individual cells the effect of siRNA, drugs, or biological processes that promote or inhibit DNA methylation or demethylation, or to study the 3D nuclear organization of regions with high DNA methylation, such as constitutive heterochromatin.Here, we review and outline protocols for the fluorescent and enzymatic immunodetection of DNA methylation in the nuclei of cells, tissue sections, and mammalian embryos.

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; Despite the progressive increase in the use of Assisted Reproductive Technologies (ART) (2.9 % of births in France in 2012), the safety of ART associated procedures remains a matter of concern. Especially, an increase in the prevalence of imprinting disorders has been reported among ART issued children, pointing to potential epigenetic defaults induced by such procedures. To eliminate confounding factors and precisely isolate the impact of embryo culture conditions on embryonic epigenome, we chose the rabbit embryo as a model since its embryonic genome activation (EGA) and associated epigenetic modifications are close to the human embryo. We compared DNA methylation and hydroxymethylation in in vivo developed embryos to those of in vitro cultured embryos using either a single medium (Global (Life global)) or a sequential medium (G1/G2 (Vitrolife)). Both of these media are currently used in human ART, and the choice between single or sequential media for embryo long-culture remains a puzzling question. In vivo, DNA methylation progressively decreases from the early 4-cell stage until the late 8-cell stage, while hydroxymethylation decreases during the 4 and 8-cell stages, then transiently increases at the 16-32-cell stage. Interestingly, our results evidenced culture condition-specific alterations in both demethylation and hydroxymethylation kinetics. We thus wondered whether the expression of genes encoding DNMT and TET enzymes, responsible for DNA methylation and hydroxymethylation respectively, were affected by in vitro culture conditions. TET1 and TET2 were underexpressed in in vitro cultured embryos after EGA. Whether these alterations in enzyme expression and in DNA demethylation/hydroxymethylation impact embryonic gene expression and fetal phenotype is currently under investigation.

Session 1: DNA methylation, pluripotency and gene expression; DNA methylation is deeply reprogrammed in early mammalian embryos. After an initial phase of demethylation, that is thought to be linked with the embryonic genome activation (EGA) and the reprogramming to totipotency, remethylation at the blastocyst stage goes along with the first differentiation steps. The discovery of 5-hydroxymethylcytosine as an intermediate of active DNA demethylation but also as a potential player of gene activation and of pluripotency has added a new layer of complexity to methylation reprogramming in early embryos. While mouse is the most widely studied model of mammalian development, it has a very early EGA (2-cell stage) that sets it apart from other mammalian species and makes it difficult to separate reprogramming events linked with fertilization from those linked to EGA. We have thus decided to take advantage of the rabbit model which exhibits a later EGA (8/16-cell stage), and have recently reported that demethylation in rabbit embryos is delayed as compared to mouse and that methylation reaches its lowest point at the time of EGA. In addition, our study has shown that the kinetics of demethylation in in vitro cultured embryos differs from that of in vivo developed ones. In vitro culture is widely used for human Assisted Reproductive Technologies (ART), and it is of major importance to determine the impact it can have on epigenetic reprogramming. Indeed, in some cases consequences of inappropriate reprogramming during early development can be observed only much later in life, which makes them difficult to observe and evaluate. Moreover, different kinds of culture media are currently in use, and there is so far very little evidence to help deciding which is the most appropriate one. In this work, we have compared the dynamics of methylation and hydroxymethylation during preimplantation development of in vivo fertilized rabbit embryos that were collected at the 1-cell stage and subsequently cultured in vitro in 2 different media commonly used in human ART. Our first results indicate that there is indeed an effect of the culture medium used on DNA hydroxymethylation.

ln mammals, the embryonic genome is first transcriptionnally inactive alter fertilization. The onset of embryonic gene expression is initiated later during the preimplantation developmental period, during the so-called "embryonic genome activation (EGA)". EGA is dependent on the presence of the basal transcriptional machinery components but also on parental genomes reprogramming. Among the mechanisms of this reprogramming, it appears that modifications of epigenetic marks are essential. One of the main reprogrammed elements is pericentric heterochromatin and the related epigenetic mark H3K9me3 (Martin et al., 2006). Remarkably, it has been shown that interference with pericentric heterochromatin reprogramming allers development (Probst et al., 2010; Santenard et al., 2010). To further investigate pericentric heterochromatin reorganization, we decided to study three other epigenetic markers, H3S10P 1 H3K9me3S10P and 5-hydroxymethylcytosine (5-hMeC) in early mouse embryos. Our results indicate that H3K9me3S1OP followed similar dynamics but only labeled the maternai pericentric heterochromatin, even more specifically than H3K9me3. On the other hand, we found that H3S1OP overlaps with the pericentric sequences at the 1-cell and 2-cell stages on both parental genomes. We then looked at 5-hMeC that is believed to complement 5-MeC during paternal genome demethylation at 1-cell. We demonstrate thal 5-MeC is mainly enriched on the paternal pericentric heterochromatin whil5-hMeC overlaps only with a subset of the maternai pericentric sequences. Moreover, their kinetics do not exactly complement each other; in particular, a peak of 5-hMeC is observed at the end of the 1-cell stage in bath pronuclei. Based on these findings, we used these epigenetic modifications as markers of pericentric heterochromatin reprogramming in embryos derived by nuclear transfer (NT). NT refers to the injection of a donor nucleus into an enucleated egg. However only a very small proportion of NT embryos can develop to term alter transfer into recipient uteri and it is believed that one of the causes for this is incomplete genome reprogramming. ln mouse, we indeed showed that remodeling into an embryonic-like nuclear organization occurs quite rapidly alter NT, within two-cell cycles. However, abnormal nuclear remodeling is frequently observed: NT embryos olten present irregular chromatin reorganization with pericentromeric heterochromatin clumps like in the donor nuclei, suggesting thal the somalie nuclear configuration is not completely lost alter NT. lnterestingly, the percentage of such abnormalities is correlated with developmental efficiency and seems to depend on the differentiation state of the starting material. When we studied epigenetic markers in these embryos, we observed thal two of these markers, H3S10P and H3K9me3S10P, are the ones found on the part of the pericentromeric heterochromatin which is correctly remodeled, resembling exactly the embryonic heterochromatin configuration of naturally fertilized embryos. Conversely, H3K9me3 and HP1associated protein were also detected in the perinuclear clumps of heterochromatin, making obvious the maintenance of the somalie epigenetic signature within these nuclear regions. Finally, we also observed that transient treatment with histone deacetylase inhibitors (trichostatin A or scriptaid) improves nuclear remodeling in NT embryos as weil as embryonic development at subsequent stages. Together, the results suggest that proper organization of constitutive heterochromatin in embryos is involved in the initial reprogramming steps and might have long term consequences. We also

DNA methylation of cytosines (5-mC) plays a crucial role in the epigenetic regulation of gene expression throughout development. The pattern of 5-mC is accurately established by de nova DNA methyltransferases (DNMT3a-3b) and inherited through mitosis by a maintenance DNA methyltransferase (DNMT1). Recently, another DNA modification, 5- hydroxymethylcytosine (5-hmC), was reported. 5-hmC is generated from 5-mC by TET proteins, and couId be an intermediate in demethylation pathways. Given the potential role of 5-hmC in nuclear reprogramming, it is crucial to obtain data in species with longer developmental lime courses than the mouse. This study aims to determine the tissue-, cell­ and nuclear content and distribution of 5-mC and 5-hmC in different bovine lineages during development. Bovine foetal (brain, liver, heart) and extraembryonic (allantois, amnion, chorion and cotyledon) tissues were collected at day 60 of gestation from 3 lemales and 3 males. Genomic DNA was prepared and digested by EcoRI, a methylation-non sensitive enzyme, to homogenize ils size. The 5-hmC or 5-mC content was measured using specifie 5-hmC or 5-mC immunoassays (Epigentek) and compared with mouse tissues (brain, muscle and liver from 3 females and 3 males at day 15 of gestation). ln arder to determine the distribution of 5-mC and 5-hmC in tissues/cells/nuclei, we performed immunostaining experiments with 5-mC and 5-hmC antibodies (Eurogentec and Active Motif). Image acquisitions were performed using the high-resolution Apotome microscope (Zeiss, MIMA2 facility, INRA, Jouy en Josas). Finally, the expression of the genes encoding DNMT3a, 3b, DNMT1 and TET was analysed in the different bovine tissues by quantitative PCR. Our data provide the first overview of the tissue/cell/nuclear distribution of 5-mC and 5-hmC during the development in bovine with a locus on the lineage origin, and will be a useful reference to determine the epigenetic alterations induced by situations affecting nuclear reprogramming, such as nuclear transfer

In the mouse zygote, DNA methylation patterns undergo important changes and differ in 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 are not yet fully understood. Recently, 5-hydroxymethylcytosine has been suggested as an intermediate in active demethylation. To investigate this question, we have quantified DNA methylation and hydroxymethylation in both pronuclei of the mouse zygote with a focus on the replication period and 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. Together our results support the 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.

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.

1047 m/s n° 12, vol. 28, decembre 2012 DOI : 10.1051/medsci/20122812009 6. Palop JJ, Chin J, Roberson ED, et al. Aberrant excitatory neuronal activity and compensatory remodeling of inhibitory hippocampal circuits in mouse models of Alzheimer’s disease. Neuron 2007 ; 55 : 697-711. 7. Palop JJ, Mucke L. Epilepsy and cognitive impairments in Alzheimer disease. Arch Neurol 2009 ; 66 : 435-40. 8. Verret L, Mann EO, Hang GB, et al. Inhibitory interneuron deficit links altered network activity and cognitive dysfunction in Alzheimer model. Cell 2012 ; 149 : 708-21. 9. Herrmann CS, Demiralp T. Human EEG gamma oscillations in neuropsychiatric disorders. Clin Neurophysiol 2005 ; 116 : 2719-33. 10. Cardin JA, Carlen M, Meletis K, et al. Driving fastspiking cells induces gamma rhythm and controls sensory responses. Nature 2009 ; 459 : 663-7. 11. Catterall WA, Kalume F, Oakley JC. NaV1.1 channels and epilepsy. J Physiol 2010 ; 588 : 1849-59. REFERENCES

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.

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In mammals, the embryonic genome is first transcriptionally inactive after fertilization. Embryonic development is then strictly dependent on the maternally inherited RNA and proteins accumulated before ovulation and present in the oocyte cytoplasm. The onset of embryonic gene expression is initiated later during development, i.e. during the "embryonic genome activation (EGA)". EGA takes place at various preimplantation stages according to species and is dependent on the presence of the basal transcriptional machinery components but also on parental genomes reorganizations after fertilization. Indeed, during the first embryonic cycles, nuclei undergo intense remodeling that could be a key regulator of embryonic development.

International audience; The organization of centromeric and pericentric heterochromatin in 1-cell fertilized mouse embryos is completely different from the organization observed in somatic cells. This unique organization is also observed in 60% of embryos obtained by nuclear transfer using embryonic stem cell (ESNT). Here, we show that remodelling into a zygotic-like organization also occurs after somatic cell nuclear transfer (SCNT), supporting the hypothesis that reorganization of constitutive heterochromatin can occur regardless of the source and differentiation state of the starting material. However, remodelling efficiency in SCNT embryos appears lower than in ESNT embryos and is accompanied by a lower developmental potential. Interestingly, treatment of SCNT embryos with the deacetylase inhibitor trichostatin A (TSA)improves nuclear remodelling at the first cell stage as well as development to term, but not to blastocyst. Together, the results suggest that proper organization of constitutive heterochromatin in early embryos has long-term effects, especially in cloning procedures.

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National audience

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.

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.

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.

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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.

This review was written for the COST Action FA1201 Epiconcept: Epigenetics and Periconception Environment; International audience; A classical model of epigenetic reprogramming of methyl-cytosine-phosphate-guanine (CpG) dinucleotides within the genome of the early embryo involves a process of active demethylation of the paternally derived genome immediately following fertilisation, creating marked asymmetry in global cytosine methylation levels in male and female pronuclei, followed by passive demethylation of the maternally derived genome over subsequent cell cycles. This model has dominated thinking in developmental epigenetics over recent decades. Recent re-analyses of the model show that demethylation of the paternally derived genome is more modest than formerly thought and results in overall similar levels of methylation of the paternal and maternal pronuclei in presyngamal zygotes, although there is little evidence for a pervasive process of passive demethylation during the cleavage stage of development. In contrast, the inner cell mass of the blastocyst shows some loss of methylation within specific classes of loci. Improved methods of chemical analysis now allow global base-level analysis of modifications to CpG dinucleotides within the cells of the early embryo, yet the low cost and convenience of the immunolocalisation techniques mean that they still have a valuable place in the analysis of the epigenetics of embryo development. In this review we consider the key strengths and weaknesses of this methodology and some factors required for its valid use and interpretation.

DNA methylation of cytosines (5-mC) plays a crucial role in the epigenetic regulation of gene expression throughout development. The pattern of 5-mC is accurately established by de novo DNA methyltransferases (DNMT3a-3b) and inherited through mitosis by a maintenance DNA methyltransferase (DNMT1). Recently, another DNA modification, 5-hydroxymethylcytosine (5-hmC), was reported. 5-hmC is generated from 5-mC by TET proteins, and could be an intermediate in demethylation pathways. This study aims to determine the tissue- and cell- content of the 5-mC and 5-hmC amount by different approaches in mouse and in bovine species. Given the potential role of 5-hmC in nuclear reprogramming, it is crucial to obtain data in species with longer developmental time courses than the mouse.

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International audience; The reprogramming of DNA methylation in early embryos has been considered to be essential for the reprogramming to totipotency, the embryonic genome activation (EGA) and subsequent development. However, its degree appears to differ as a function of species and it may be altered by the in vitro enviromnent. While the rabbit is a pertinent mode!for species with a delayed EGA because both in vivo and in vitro developed embryos are easily available, the status of DNA methylation levels in both parental genomes after fertilization remains controversial. In order to generate precise data on the DNA methylation status in rabbit zygotes, we first of ali defined five pronuclear (PN) stages during the first cell cycle, which we subsequently used to classizy in vivo and in vitro developed rabbit zygotes. This allowed us to precisely quantizy the methylated DNA during the one cell stage, as weil as the total DNA content, whlch was used for normalization. We thus showed that both pronuclei display distinct patterns of DNA methylation reprogramming. In the maternai pronucleus (MP) the methylation leve!remains constant throughout the one cell stage, thanks to maintenance methylation during the S-phase. Conversely, in the paternal pronucleus (PP) partial demethylation occurs bcforc replication, probably as a result of active DNA demethylation, while maintenance methylation subsequent!y talœs place during the S-phase. Interestingly, we showed that PP DNA methylation reprogramming is partially altered by the in vitro enviromnent. Taken together, our approach evidences that rabbit is one of the species displaying partial DNA demethylation in the PP, and for the first time demonstrates maintenance methylation activity in both pronuclei during the first S phase.