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1. Lipid Droplets in Unicellular Photosynthetic Stramenopiles

Author

Nolwenn Guéguen, Damien Le Moigne, Alberto Amato, Juliette Salvaing, and Eric Maréchal

Subjects
heterokont, stramenopile, Nannochloropsis, Microchloropsis, Phaeodactylum, lipid droplet (LD), Plant culture, SB1-1110
Abstract

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.

Published
2021
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2. PUB11-Dependent Ubiquitination of the Phospholipid Flippase ALA10 Modifies ALA10 Localization and Affects the Pool of Linolenic Phosphatidylcholine

Author

Juliette Salvaing, César Botella, Catherine Albrieux, Valérie Gros, Maryse A. Block, and Juliette Jouhet

Subjects
glycerolipid, flippase, ubiquitination, membrane domain, lipid trafficking, Plant culture, SB1-1110
Abstract

Biogenesis of photosynthetic membranes depends on galactolipid synthesis, which relies on several cell compartments, notably the endoplasmic reticulum (ER) and the chloroplast envelope. Galactolipid synthesis involves lipid trafficking between both membrane compartments. In Arabidopsis, ALA10, a phospholipid flippase of the P4 type-ATPase family, counteracts the limitation of monogalactosyldiacylglycerol (MGDG) production and has a positive effect on leaf development. ALA10 locates in distinct domains of the ER depending on the ALIS (ALA interacting subunit) subunit it interacts with: close to the plasma membrane with ALIS1, or next to chloroplasts with ALIS5. It interacts with FAD2 (Fatty acid desaturase 2) and prevents accumulation of linolenic (18:3) containing phosphatidylcholine (PC) stimulating an increase of MGDG synthesis. Here we report that ALA10 interacts with PUB11 (plant U-box type 11), an E3 protein ubiquitin ligase, in vitro and in vivo. ALA10 is however ubiquitinated and degraded by the 26S proteasome in a PUB11-independent process. In pub11 null mutant, the proteasome-dependent degradation of ALA10 is retained and ALA10 is still subject to ubiquitination although its ubiquitination profile appears different. In the absence of PUB11, ALA10 is constrained to the ER close to chloroplasts, which is the usual location when ALA10 is overexpressed. Additionally, in this condition, the decrease of 18:3 containing PC is no longer observed. Taken together these results suggest, that ALA10 contributes in chloroplast-distal ER interacting domains, to reduce the 18:3 desaturation of PC and that PUB11 is involved in reconditioning of ALA10 from chloroplast-proximal to chloroplast-distal ER interacting domains.

Published
2020
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3. Stepwise Biogenesis of Subpopulations of Lipid Droplets in Nitrogen Starved Phaeodactylum tricornutum Cells

Author

Antoine Jaussaud, Josselin Lupette, Juliette Salvaing, Juliette Jouhet, Olivier Bastien, Marina Gromova, and Eric Maréchal

Subjects
diatoms, Phaeodactylum, Triacylglycerol, lipid droplet, lipids, Plant culture, SB1-1110
Abstract

Diatoms are unicellular heterokonts, living in oceans and freshwaters, exposed to frequent environmental variations. They have a sophisticated membrane compartmentalization and are bounded by a siliceous cell-wall. Formation of lipid droplets (LDs), filled with triacylglycerol (TAG), is a common response to stress. The proteome of mature-LDs from Phaeodactylum tricornutum highlighted the lack of proteins involved in early-LD formation, TAG biosynthesis or LD-to-LD connections. These features suggest that cytosolic LDs might reach a size limit. We analyzed the dynamics of LD formation in P. tricornutum (Pt1 8.6; CCAP 1055/1) during 7 days of nitrogen starvation, by monitoring TAG by mass spectrometry-based lipidomics, and LD radius using epifluorescence microscopy and pulse field gradient nuclear magnetic resonance. We confirmed that mature LDs reach a maximal size. Based on pulse field gradient nuclear magnetic resonance, we did not detect any LD-LD fusion. Three LD subpopulations were produced, each with a different maximal size, larger-sized LDs (radius 0.675 ± 0.125 µm) being generated first. Mathematical modeling showed how smaller LDs are produced once larger LDs have reached their maximum radius. In a mutant line having larger cells, the maximal size of the first LD subpopulation was higher (0.941 ± 0.169 µm), while the principle of stepwise formation of distinct LD populations was maintained. Results suggest that LD size is determined by available cytosolic space and sensing of an optimal size reached in the previous LD subpopulation. Future perspectives include the unraveling of LD-size control mechanisms upon nitrogen shortage. This study also provides novel prospects for the optimization of oleaginous microalgae for biotechnological applications.

Published
2020
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4. PRC1 Prevents Replication Stress during Chondrogenic Transit Amplification

Author

Frank Spaapen, Lars M. T. Eijssen, Michiel E. Adriaens, Tim J. Welting, Peggy Prickaerts, Juliette Salvaing, Vivian E. H. Dahlmans, Donald A. M. Surtel, Frans Kruitz, Roel Kuijer, Yoshihiro Takihara, Hendrik Marks, Hendrik G. Stunnenberg, Bradly G. Wouters, Miguel Vidal, and Jan Willem Voncken

Subjects
polycomb, topoisomerase, transit amplification, chromatin, DNA replication, transcription, chondrogenesis, differentiation, DNA damage, hypertrophy, senescence, Genetics, QH426-470, Biotechnology, TP248.13-248.65
Abstract

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.

Published
2017
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5. Lipid droplets in plants: More than a simple fat storage

Author

Damien Le Moigne, Nolwenn Guéguen, Juliette Salvaing, LIPID, Physiologie cellulaire et végétale (LPCV), Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Université Grenoble Alpes (UGA)-Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Université Grenoble Alpes (UGA), Fabrice Rebeille, Eric Marechal, and ANR-17-EURE-0003,CBH-EUR-GS,CBH-EUR-GS(2017)

Subjects
[SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, ComputingMilieux_MISCELLANEOUS
Abstract

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.

Published
2022
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6. Quantitative proteomic analyses reveal the impact of nitrogen starvation on the proteome of the model diatom Phaeodactylum tricornutum

Author

Josselin Lupette, Marianne Tardif, Sabine Brugière, Yohann Couté, Juliette Salvaing, Eric Maréchal, LIPID, Physiologie cellulaire et végétale (LPCV), Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Université Grenoble Alpes (UGA)-Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Université Grenoble Alpes (UGA), Laboratoire de biogenèse membranaire (LBM), Université de Bordeaux (UB)-Centre National de la Recherche Scientifique (CNRS), Etude de la dynamique des protéomes (EDyP), BioSanté (UMR BioSanté), Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes (UGA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes (UGA), ANR-10-LABX-0049,GRAL,Grenoble Alliance for Integrated Structural Cell Biology(2010), ANR-17-EURE-0003,CBH-EUR-GS,CBH-EUR-GS(2017), ANR-10-INBS-0008,ProFI,Infrastructure Française de Protéomique(2010), ANR-15-IDEX-0002,UGA,IDEX UGA(2015), Salvaing, Juliette, Grenoble Alliance for Integrated Structural Cell Biology - - GRAL2010 - ANR-10-LABX-0049 - LABX - VALID, CBH-EUR-GS - - CBH-EUR-GS2017 - ANR-17-EURE-0003 - EURE - VALID, Infrastructure Française de Protéomique - - ProFI2010 - ANR-10-INBS-0008 - INBS - VALID, and IDEX UGA - - UGA2015 - ANR-15-IDEX-0002 - IDEX - VALID

Subjects
Diatoms, Proteomics, Proteome, Nitrogen, Nitrogen starvation, Phaeodactylum, Fatty Acids, Diatom, Biochemistry, Carbon, Proteome remodelling, [SDV.MP]Life Sciences [q-bio]/Microbiology and Parasitology, [SDV.BBM.GTP]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Genomics [q-bio.GN], Quantitative proteomics, [SDV.BBM.GTP] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Genomics [q-bio.GN], Molecular Biology, [SDV.MP] Life Sciences [q-bio]/Microbiology and Parasitology, Triglycerides
Abstract

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.

Published
2022
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7. Lipid Droplets in Unicellular Photosynthetic Stramenopiles

Author

Juliette Salvaing, Alberto Amato, Damien Le Moigne, Eric Maréchal, Nolwenn Guéguen, LIPID, Physiologie cellulaire et végétale (LPCV), Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes (UGA)-Centre National de la Recherche Scientifique (CNRS)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes (UGA)-Centre National de la Recherche Scientifique (CNRS), ANR-11-BTBR-0008,OCEANOMICS,Biotechnologies et bioressources pour la valorisation des écosystèmes marins planctoniques(2011), ANR-10-LABX-0049,GRAL,Grenoble Alliance for Integrated Structural Cell Biology(2010), ANR-17-EURE-0003,CBH-EUR-GS,CBH-EUR-GS(2017), Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Université Grenoble Alpes (UGA)-Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), and Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Université Grenoble Alpes (UGA)

Subjects
0106 biological sciences, Membrane lipids, Phaeodactylum, Review, Plant Science, 01 natural sciences, SB1-1110, 03 medical and health sciences, Lipid droplet, Endomembrane system, Phaeodactylum tricornutum, Nannochloropsis, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], Plastid, heterokont, triacylglycerol (TAG), 030304 developmental biology, 0303 health sciences, biology, Chemistry, Heterokont, food and beverages, Plant culture, [SDV.BV.BOT]Life Sciences [q-bio]/Vegetal Biology/Botanics, biology.organism_classification, seipin, Biochemistry, stramenopile, Microchloropsis, Biogenesis, 010606 plant biology & botany, lipid droplet (LD)
Abstract

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.

Published
2021
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8. The architecture of lipid droplets in the diatom Phaeodactylum tricornutum

Author

Khawla Seddiki, Juliette Salvaing, Fabrice Rébeillé, Juliette Jouhet, Denis Falconet, Sabine Brugière, Yohann Couté, Eric Maréchal, Marcel Kuntz, Pierre-Henri Jouneau, Hubert Schaller, Antoine Jaussaud, Josselin Lupette, Marianne Tardif, Christian Morabito, Jean-Luc Putaux, LIPID, Physiologie cellulaire et végétale (LPCV), Institut National de la Recherche Agronomique (INRA)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Recherche Agronomique (INRA)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Etude de la dynamique des protéomes (EDyP ), Laboratoire de Biologie à Grande Échelle (BGE - UMR S1038), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Institut de biologie moléculaire des plantes (IBMP), Centre National de la Recherche Scientifique (CNRS)-Université de Strasbourg (UNISTRA), Dynamique du protéome et biogenèse du chloroplaste (ChloroGenesis), Centre de Recherches sur les Macromolécules Végétales (CERMAV ), Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019]), Laboratoire d'Etude des Matériaux par Microscopie Avancée (LEMMA ), Modélisation et Exploration des Matériaux (MEM), Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019]), Flagship program from the CEA High Commissioner, French National Research Agency (ANR) ANR-11-BTBR-0008 ANR-15-IDEX-02 ANR-10-INBS-08, ANR-11-BTBR-0008,OCEANOMICS,Biotechnologies et bioressources pour la valorisation des écosystèmes marins planctoniques(2011), ANR-15-IDEX-0002,UGA,IDEX UGA(2015), ANR-10-INBS-0008,ProFI,Infrastructure Française de Protéomique(2010), Institut National de la Recherche Agronomique (INRA)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes (UGA)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Recherche Agronomique (INRA)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes (UGA)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Etude de la dynamique des protéomes (EDyP), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes (UGA)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes (UGA), Université de Strasbourg (UNISTRA)-Centre National de la Recherche Scientifique (CNRS), Centre de Recherches sur les Macromolécules Végétales (CERMAV), Université Joseph Fourier - Grenoble 1 (UJF)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes (UGA), Laboratoire d'Etude des Matériaux par Microscopie Avancée (LEMMA), Université Grenoble Alpes (UGA)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes (UGA)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), ANR-11-BTBR-0008/11-BTBR-0008,OCEANOMICS,Biotechnologies et bioressources pour la valorisation des écosystèmes marins planctoniques(2011), ANR-15-IDEX-02,DATA@UGA,Grenoble Alpes Data Institute(2016), ANR-10-INBS-08-01/10-INBS-0008,ProFI,Infrastructure Française de Protéomique(2010), Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Jouneau, Pierre-Henri, Biotech - Bioressources - Biotechnologies et bioressources pour la valorisation des écosystèmes marins planctoniques - - OCEANOMICS2011 - ANR-11-BTBR-0008 - BTBR - VALID, IDEX UGA - - UGA2015 - ANR-15-IDEX-0002 - IDEX - VALID, and Infrastructure Française de Protéomique - - ProFI2010 - ANR-10-INBS-0008 - INBS - VALID

Subjects
0106 biological sciences, 0301 basic medicine, Betaine lipid, Phaeodactylum, [SDV.BC.BC]Life Sciences [q-bio]/Cellular Biology/Subcellular Processes [q-bio.SC], 01 natural sciences, Ribosome, Triacylglycerol, [SDV.BV.BOT] Life Sciences [q-bio]/Vegetal Biology/Botanics, Histones, 03 medical and health sciences, Lipid droplet, Protein biosynthesis, ERAD pathway, Phaeodactylum tricornutum, Plastid, Secondary plastid, ComputingMilieux_MISCELLANEOUS, Diatoms, biology, Chemistry, Endoplasmic reticulum, fungi, [SDV.BV.BOT]Life Sciences [q-bio]/Vegetal Biology/Botanics, biology.organism_classification, Lipid droplets, Carotenoids, 030104 developmental biology, Biochemistry, Proteome, Agronomy and Crop Science, 010606 plant biology & botany
Abstract

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.

Published
2019
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9. Antibody-Based Detection of Global Nuclear DNA Methylation in Cells, Tissue Sections, and Mammalian Embryos

Author

Sari Pennings, Juliette Salvaing, Nur Annies Abd Hadi, Nathalie Beaujean, Biologie du Développement et Reproduction (BDR), École nationale vétérinaire d'Alfort (ENVA)-Institut National de la Recherche Agronomique (INRA), Physiologie cellulaire et végétale (LPCV), Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Recherche Agronomique (INRA)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), BHF / University Centre for Cardiovascular Science, University of Edinburgh, REVIVE Labex (Investissement d'Avenir, ANR-10-LABX-73), Tost J., Jörg Tost, Institut National de la Recherche Agronomique (INRA), Institut National de la Recherche Agronomique (INRA)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes (UGA)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), École nationale vétérinaire - Alfort (ENVA)-Institut National de la Recherche Agronomique (INRA), and Institut National de la Recherche Agronomique (INRA)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG)

Subjects
0301 basic medicine, MESH: Cell Nucleus, Fluorescence immunocytochemistry, Immunofluorescence, 03 medical and health sciences, MESH: DNA Methylation, medicine, MESH: Animals, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Epigenetics, MESH: Epigenesis, Genetic, Microscopy, DNA methylation, MESH: Humans, biology, medicine.diagnostic_test, Chemistry, MESH: Antibodies, MESH: Embryo, Mammalian, Methylation, Primary and secondary antibodies, Immunohistochemistry, Nuclear DNA, Cell biology, 030104 developmental biology, biology.protein, MESH: 5-Methylcytosine, Antibody, Immunostaining, MESH: Cells, Cultured
Abstract

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.

Published
2018
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10. Antibody-Based Detection of Global Nuclear DNA Methylation in Cells, Tissue Sections, and Mammalian Embryos

Author

Nathalie, Beaujean, Juliette, Salvaing, Nur Annies Abd, Hadi, and Sari, Pennings

Subjects
Cell Nucleus, 5-Methylcytosine, Animals, Humans, DNA Methylation, Embryo, Mammalian, Antibodies, Cells, Cultured, Epigenesis, Genetic
Abstract

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.

Published
2017

11. PRC1 Prevents Replication Stress during Chondrogenic Transit Amplification

Author

Donald A. M. Surtel, Frans Kruitz, Juliette Salvaing, Peggy Prickaerts, Jan Willem Voncken, Lars M. T. Eijssen, Vivian E. H. Dahlmans, Tim J. M. Welting, Michiel E. Adriaens, Miguel Vidal, Yoshihiro Takihara, Bradly G. Wouters, Frank Spaapen, Hendrik G. Stunnenberg, Roel Kuijer, Hendrik Marks, Consejo Superior de Investigaciones Científicas (España), European Molecular Biology Laboratory, Ministerio de Economía y Competitividad (España), Comunidad de Madrid, Netherlands Organization for Scientific Research, Reumafonds, Transnational University of Limburg, Department of Molecular Genetics, Maastricht University Medical Center, Department of Bioinformatics, Maastricht University Medical Center (BiGCaT), Maastricht Centre for Systems Biology, Maastricht University Medical Center (MaCSBio), Department of Orthopedic Research, Maastricht University Medical Center, Physiologie cellulaire et végétale (LPCV), Institut National de la Recherche Agronomique (INRA)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes (UGA)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), University Medical Center Groningen [Groningen] (UMCG), Stem Cell Biology, Research Institute for Radiation Biology and Medicine, Centre for Molecular Life Sciences (NCMLS), Department of Molecular Biology, Radboud university [Nijmegen], Departments of Laboratory Medicine, Pathobiology & Radiation Oncology, Maastricht Radiation Oncology Clinic (MAASTRO), Maastricht University [Maastricht], Centro de Investigaciones Biologicas, Consejo Superior de Investigaciones Científicas [Spain] (CSIC), Institut National de la Recherche Agronomique (INRA)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Radboud University [Nijmegen], Consejo Superior de Investigaciones Científicas [Madrid] (CSIC), Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Recherche Agronomique (INRA)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Radiotherapie, Psychiatrie & Neuropsychologie, RS: MHeNs - R3 - Neuroscience, Bioinformatica, Maastricht Centre for Systems Biology, RS: FHML MaCSBio, RS: FSE MaCSBio, Orthopedie, MUMC+: MA Orthopedie (9), RS: CAPHRI - R3 - Functioning, Participating and Rehabilitation, Promovendi ODB, Molecular Genetics, and RS: GROW - R2 - Basic and Translational Cancer Biology

Subjects
senescence, DNA damage, Health, Toxicology and Mutagenesis, [SDV]Life Sciences [q-bio], lcsh:Medicine, polycomb, topoisomerase, transit amplification, chromatin, DNA replication, transcription, chondrogenesis, differentiation, hypertrophy, macromolecular substances, Biochemistry, Genetics and Molecular Biology (miscellaneous), Biochemistry, 03 medical and health sciences, 0302 clinical medicine, Transcription (biology), Genetics, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, lcsh:QH301-705.5, 030304 developmental biology, 0303 health sciences, biology, Topoisomerase, lcsh:R, Geminin, Chondrogenesis, 3. Good health, Cell biology, Chromatin, lcsh:Biology (General), BMI1, 030220 oncology & carcinogenesis, biology.protein
Abstract

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.).

Published
2017
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12. Impact of assisted reproductive technologies on early embryo epigenome: in vitro culture affects embryonic genome demethylation and hydroxymethylation

Author

Juliette Salvaing, Peynot, Nathalie N., Maulny, Linda L., Sophie Veniel, Mohamed Negash Bedhane, Ruffini, Sylvie S., Nathalie Beaujean, Daniel, Nathalie N., Véronique Duranthon, Biologie du Développement et Reproduction (BDR), École nationale vétérinaire d'Alfort (ENVA)-Institut National de la Recherche Agronomique (INRA), Agence de la Biomédecine, and European Cooperation in Science and Technology (COST). BEL.

Subjects
[SDV.BDLR]Life Sciences [q-bio]/Reproductive Biology, [SDV.BDD]Life Sciences [q-bio]/Development Biology
Abstract

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.

Published
2015

13. MK3 Modulation Affects BMI1-Dependent and Independent Cell Cycle Check-Points

Author

Peggy Prickaerts, Bradly G. Wouters, Claudia Geijselaers, Ernst-Jan M. Speel, Jolien Vanhove, Hanneke E.C. Niessen, Juliette Salvaing, Iris Partouns, Yoshihiro Takihara, Frank Spaapen, Jan Willem Voncken, Stefanie J. J. Bartels, Dietbert Neumann, Vivian E. H. Dahlmans, Department of Molecular Genetics, Maastricht University Medical Center, Laboratoire de physiologie cellulaire végétale (LPCV), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Recherche Agronomique (INRA)-Université Joseph Fourier - Grenoble 1 (UJF)-Centre National de la Recherche Scientifique (CNRS), Interdepartmental Stem Cell Institute Leuven, Nijmegen Centre for Molecular and Life Sciences, Radboud University Nijmegen Medical Centre, Maastricht University Medical Centre/Caphri, Stem Cell Biology, Research Institute for Radiation Biology and Medicine, Princess Margaret Cancer Centre [Toronto, Canada], Maastro Lab, Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Recherche Agronomique (INRA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Moleculaire Genetica, MUMC+: MA Alg Interne Geneeskunde (9), Radiotherapie, Ondersteunend personeel NTM, Pathologie, RS: CARIM - R3 - Vascular biology, RS: GROW - Developmental Biology, Genetica & Celbiologie, RS: GROW - R3 - Innovative Cancer Diagnostics & Therapy, RS: GROW - R2 - Basic and Translational Cancer Biology, and RS: GROW - R4 - Reproductive and Perinatal Medicine

Subjects
MAPK/ERK pathway, Senescence, Cell cycle checkpoint, MAP Kinase Signaling System, Cell, lcsh:Medicine, Polycomb-Group Proteins, macromolecular substances, [SDV.BC.BC]Life Sciences [q-bio]/Cellular Biology/Subcellular Processes [q-bio.SC], Biology, Protein Serine-Threonine Kinases, medicine.disease_cause, 03 medical and health sciences, 0302 clinical medicine, Cell Line, Tumor, medicine, Humans, lcsh:Science, Cellular Senescence, Mitogen-Activated Protein Kinase 7, 030304 developmental biology, Cell Proliferation, 0303 health sciences, Multidisciplinary, Cell growth, lcsh:R, Intracellular Signaling Peptides and Proteins, Cell Cycle Checkpoints, Fibroblasts, Cell biology, medicine.anatomical_structure, BMI1, 030220 oncology & carcinogenesis, Mutation, lcsh:Q, Carcinogenesis, Cell aging, Research Article
Abstract

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.

Published
2015
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