Tesi: "Complexe polycomb"

1

Campagne, Antoine. "Etude du complexe Polycomb PR-DUB : une approche mécanistique". Thesis, Paris 6, 2015. http://www.theses.fr/2015PA066624/document.

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Abstract (sommario):

BAP1 est un suppresseur de tumeurs dont le nombre de partenaires protéiques rend complexe l'appréhension de son rôle dans la cellule. Chez la Drosophile, BAP1 et ASX forment le complexe Polycomb PR-DUB, qui déubiquitine l'histone H2A sur la lysine 119 afin de maintenir une répression transcriptionnelle sur ses gènes cibles. Comprendre les mécanismes de régulation de BAP1 et définir son implication au sein de la machinerie Polycomb s'avèrent donc des enjeux cruciaux pour mieux appréhender son rôle au cours de la tumorigenèse. Par des approches biochimiques, nous avons montré l'existence de plusieurs complexes fonctionnellement distincts associés à BAP1. ASXL1 semble ainsi nécessaire à l'activité H2A deubiquitinase de BAP1, tandis qu'ASXL2 forme un complexe ternaire avec BAP1 et la déméthylase d'histones KDM1B. Par ailleurs, nous avons démontré le potentiel de répresseur transcriptionnel de BAP1, qui semble posséder différents domaines répresseurs. Afin d'étudier ces aspects à l'échelle du génome, des analyses du transcriptome et de différentes marques d'histone sont en cours, dans des cellules sauvages ou mutées pour différents membres de la famille Polycomb. Dans un deuxième temps, nous avons entrepris une recherche exhaustive des substrats de BAP1. Nos résultats préliminaires suggèrent que non seulement H2A mais également H2B sont des cibles de BAP1, de même qu'un complexe protéique responsable du contrôle de la prolifération cellulaire via la régulation post-transcriptionnelle de plusieurs cyclines. Ces observations ouvrent la voie à plusieurs projets qui pourraient contribuer à expliquer les conséquences des mutations de BAP1 dans le processus tumoral
BAP1 is as a tumor suppressor that associates to a variety of protein partners, thereby limiting the comprehension of its cellular functions. In Drosophila, BAP1 binds ASX to form the Polycomb PR-DUB complex, which deubiquitinates histone H2A on lysine 119 in order to maintain transcriptional repression on its target genes. Describing BAP1 mechanisms of action and defining how BAP1 cooperates with the Polycomb machinery are prerequisites to understand its role during tumorigenesis. Using a biochemical approach, we described the existence of several distinct subcomplexes associated with BAP1. Therefore, ASXL1 seems required for H2A deubiquitination, while ASXL2 forms a ternary complex of unknown function with BAP1 and the histone demethylase KDM1B. In addition, we demonstrated the transcriptional repressor function of BAP1, which possess several repressive domains. In addition, we are currently performing transcriptomic analysis combined with genome-wide mapping of different histone marks. These last analyses are performed in wild type cells or deficient in PR-DUB or other Polycomb components, which will help us to understand how BAP1 fits within the Polycomb machinery. In parallel, we engaged a comprehensive study aiming at the identification of new BAP1 substrates. Our preliminary results suggest that not only H2A but also H2B may be direct substrates of BAP1. In addition, we identified as a potential substrate the HNRNPM-IMP3 complex, which controls cell proliferation via post-transcriptional regulation of several cyclins. These observations pave the way for new projects that may contribute to explain the consequences of BAP1 mutations in cancer development

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2

Campagne, Antoine. "Etude du complexe Polycomb PR-DUB : une approche mécanistique". Electronic Thesis or Diss., Paris 6, 2015. https://accesdistant.sorbonne-universite.fr/login?url=https://theses-intra.sorbonne-universite.fr/2015PA066624.pdf.

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Abstract (sommario):

BAP1 est un suppresseur de tumeurs dont le nombre de partenaires protéiques rend complexe l'appréhension de son rôle dans la cellule. Chez la Drosophile, BAP1 et ASX forment le complexe Polycomb PR-DUB, qui déubiquitine l'histone H2A sur la lysine 119 afin de maintenir une répression transcriptionnelle sur ses gènes cibles. Comprendre les mécanismes de régulation de BAP1 et définir son implication au sein de la machinerie Polycomb s'avèrent donc des enjeux cruciaux pour mieux appréhender son rôle au cours de la tumorigenèse. Par des approches biochimiques, nous avons montré l'existence de plusieurs complexes fonctionnellement distincts associés à BAP1. ASXL1 semble ainsi nécessaire à l'activité H2A deubiquitinase de BAP1, tandis qu'ASXL2 forme un complexe ternaire avec BAP1 et la déméthylase d'histones KDM1B. Par ailleurs, nous avons démontré le potentiel de répresseur transcriptionnel de BAP1, qui semble posséder différents domaines répresseurs. Afin d'étudier ces aspects à l'échelle du génome, des analyses du transcriptome et de différentes marques d'histone sont en cours, dans des cellules sauvages ou mutées pour différents membres de la famille Polycomb. Dans un deuxième temps, nous avons entrepris une recherche exhaustive des substrats de BAP1. Nos résultats préliminaires suggèrent que non seulement H2A mais également H2B sont des cibles de BAP1, de même qu'un complexe protéique responsable du contrôle de la prolifération cellulaire via la régulation post-transcriptionnelle de plusieurs cyclines. Ces observations ouvrent la voie à plusieurs projets qui pourraient contribuer à expliquer les conséquences des mutations de BAP1 dans le processus tumoral
BAP1 is as a tumor suppressor that associates to a variety of protein partners, thereby limiting the comprehension of its cellular functions. In Drosophila, BAP1 binds ASX to form the Polycomb PR-DUB complex, which deubiquitinates histone H2A on lysine 119 in order to maintain transcriptional repression on its target genes. Describing BAP1 mechanisms of action and defining how BAP1 cooperates with the Polycomb machinery are prerequisites to understand its role during tumorigenesis. Using a biochemical approach, we described the existence of several distinct subcomplexes associated with BAP1. Therefore, ASXL1 seems required for H2A deubiquitination, while ASXL2 forms a ternary complex of unknown function with BAP1 and the histone demethylase KDM1B. In addition, we demonstrated the transcriptional repressor function of BAP1, which possess several repressive domains. In addition, we are currently performing transcriptomic analysis combined with genome-wide mapping of different histone marks. These last analyses are performed in wild type cells or deficient in PR-DUB or other Polycomb components, which will help us to understand how BAP1 fits within the Polycomb machinery. In parallel, we engaged a comprehensive study aiming at the identification of new BAP1 substrates. Our preliminary results suggest that not only H2A but also H2B may be direct substrates of BAP1. In addition, we identified as a potential substrate the HNRNPM-IMP3 complex, which controls cell proliferation via post-transcriptional regulation of several cyclins. These observations pave the way for new projects that may contribute to explain the consequences of BAP1 mutations in cancer development

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3

Molitor, Anne. "Caractérisation moléculaire et fonctionnelle du complexe PRC1 chez Arabidopsis thaliana". Thesis, Strasbourg, 2012. http://www.theses.fr/2012STRAJ053.

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Abstract (sommario):

Les protéines du groupe Polycomb sont des régulateurs épigénétiques impliqués dans divers processus développementaux et cellulaires. Le complexe Polycomb Répressif 1 (PRC1) est bien caractérisé chez les animaux, cependant sa composition et sa fonction restent énigmatiques dans les plantes. Sur base d'homologie de séquences trois homologues de la sous-unité de base BMI1 du complexe PRC1 animal ont été identifiés dans Arabidopsis: AtBMI1a, AtBMI1b et AtBMI1c. L'interaction de ces trois protéines avec les composantes PRC1 connues (i.e. AtRING1ab, et LHP1) a été démontrée. Des analyses génétiques et moléculaires ont permis d'attribuer aux protéines AtBMI1ab et AtRING1ab un rôle essentiel dans la répression des caractères embryonnaire lors de la croissance végétative. Un nouvel interactant d'AtRING1a, une protéine à domaine PHD de la famille AL (Alfine-Like) a été identifiée dans criblage d'une banque de ADNc. Par différentes techniques l'association entre les protéines de la famille AL et les membres de bases du complexe PRC1 (i.e. AtBMI1ab, AtRING1ab et LHP1) a été démontrée. Les protéines AL sont nucléaires et se lient in vitro à H3k4me3, une marque active de la chromatine. Des analyses génétiques ont révélé que les protéines AL et AtBMI1ab régulent la germination en réprimant l'expression de gènes impliqués dans le développement de la graine. Au niveau chromatinien, les protéines PRC1 interviennent dans la transition d'une chromatine active, marquée par du H3K4me3 vers une chromatine répressive enrichie en H3K27me3. Nous proposons que les protéines AL reconnaissent la marque active et recrutent la fonction répressive des protéines à domaine RING du complexe PRC1 afin d'induire la répression transcriptionelle
Polycomb group (PcG) proteins are critical epigenetic repressors implicated in various developmental and cellular processes. While the Polycomb Repressive Complex 2 (PRC2) is evolutionary conserved and its functions extensively studied in Arabidopsis, the PRC1 complex composition and function remain still enigmatic in plants. Our work focuses on several Arabidopsis RING-domain proteins to unravel PRC1-like functions in the regulation of various processes during plant development. Based on sequence similarity we identified three homologues of the animal PRC1 core subunit BMI1: AtBMI1a, AtBMI1b and AtBMI1c. These proteins were found to interact with other PRC1-like components, AtRING1a, AtRING1b and LHP1. Genetic and molecular analyses demonstrated that AtBMI1a/b and AtRING1a/b play crucial roles in stable repression of embryonic traits to allow proper somatic growth. Comparative transcriptome analyses were performed to uncover genetic networks underlying seedling growth and the flower development defects of several different PRC1-like and PRC2 Arabidopsis mutants. Our data revealed overlapping and non-overlapping gene categories of misregulated genes in Atring1a/b, Atbmi1a/b and lhp1 mutants. The Atring1a/b mutant showed particular disturbed expression of flower developmental genes. Accordingly, phenotypic and molecular analyses of the mutant flowers confirmed that AtRING1a/b play a critical role in cell fate determination and in different aspects of flower development. To better understand the broad function of AtRING1a/b, we performed yeast two-hybrid screen and identified PHD-domain proteins of the ALFIN-LIKE (AL) family as binding partners. In vitro AL proteins bind the active mark for gene transcription, H3K4me3. By various methods, both in vitro and in planta, we provided strong evidence for the physical interaction between AL and PRC1 RING-domain proteins. We uncovered that al6/7 similar to Atbmi1a/b mutants exhibit seed germination defects, which are associated with the derepression of several seed related genes. Consistently on the corresponding chromatin a delay of the remodeling from active H3K4me3 labeled to a repressive H3K27me3 marked chromatin could be detected. We propose that through binding to H3K4me3 AL6/7 function as scaffold proteins to target PRC1 RING-domain proteins to active chromatin in order to establish gene silencing. Taken together, the presented work contributes significantly to the knowledge of PRC1 complex(es) in Arabidopsis at both biological function and complex composition levels. It opens several exciting perspectives for future research in the field

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4

Netter, Sophie. "Identification d'un nouveau complexe de genes a homeoboite cible des proteines des groupes polycomb et trithorax : le complexe iroquois". Paris 6, 1998. http://www.theses.fr/1998PA066258.

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J'ai caracterise des insertions de genes rapporteurs w presentant un profil d'expression differentiel sur l'axe dorso-ventral (d/v) dans l'il de drosophile. Ce profil est stable et hereditaire. Cette etude m'a permis d'isoler des genes impliques dans des processus regulateurs du developpement. J'ai montre que tous les transgenes presentant un profil d/v etaient localises dans une region unique du genome s'etendant sur 140 kb au niveau du site 69d, decrit comme etant un site de fixation de plusieurs proteines du pc-g. Cette region contient au moins trois genes, araucan (ara), caupolican (caup) et mirror (mirr), qui codent des proteines a homeodomaine tres similaires et constituent le complexe iroquois (iro-c). Ces genes sont impliques dans des processus developpementaux communs, mais semblent egalement avoir des fonctions specifiques, notamment dans la mise en place de la polarite dorso-ventrale de l'embryon et le developpement de l'aile. J'ai montre que l'expression de ces trois genes dans l'il est controlee par les produits des genes des groupes polycomb (pc-g) et trithorax (trx-g). Le produit du gene polyhomeotic est requis pour maintenir la repression de l'expression de mirr dans la moitie ventrale du disque imaginal d'il au stade larvaire, mais pas dans les autres tissus larvaires, ni au cours de l'embryogenese. Enfin, j'ai etudie le role des genes du iro-c dans la formation des soies sensorielles du thorax et dans le developpement de l'aile. Ainsi, j'ai pu identifier un nouveau complexe de genes a homeoboite, cible des produits des genes du pc-g et du trx-g. L'ensemble des travaux presentes sur ce complexe indique que ces genes jouent un role central au cours du developpement embryonnaire et post-embryonnaire. Je propose que le maintien de la repression des genes du iro-c dans l'il serait mediee par un mecanisme de compaction locale de la structure de la chromatine impliquant les proteines du pc-g.

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5

Cedrone, L. "THE ROLE OF ENHANCED POLYCOMB REPRESSIVE COMPLEX 2 ACTIVITY IN TUMORIGENESIS". Doctoral thesis, Università degli Studi di Milano, 2017. http://hdl.handle.net/2434/468289.

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Polycomb Group of proteins are essential factors present in cells’ nuclei. These multiprotein complexes are key repressive chromatin factors that regulate cellular differentiation during development, contributing to the correct establishment of lineage-specific transcriptional programs. Moreover, they represent key factors of proliferation and deregulation of their levels and activity have been linked to the onset and development of several human cancers. Recently, gain of function heterozygous EZH2 mutations have been discovered in non-Hodgkin lymphomas and melanomas. These mutations cause an aminoacidic substitution within the EZH2 catalytic SET domain (Y641), resulting in increased H3K27me3 deposition. Very little is known about this mutated enzyme, therefore the aim of my thesis is trying to unravel the tumorigenic mechanisms underlying these mutations. To understand a general oncogenic role for this mutated enzyme, we used MEF as an alternative, simpler model system. We observed increased deposition of H3K27me3 without any relevant transcriptional alteration at steady state, confirming our results also in lymphoma cell lines. To investigate a cooperative transcriptional deregulation for mutant EZH2, we then subjected MEFs to three different stimuli (starvation, myc upregulation and reprogramming to pluripotency). Since we found this to be true only during cell-fate transition, we proposed a model in which the levels of the H3K27me3 are increasingly deposited where the mark is already present at steady state. This could be relevant in lymphomas, impeding centroblasts differentiation and resulting in tumorigenesis in the presence of concomitant oncogenic mutations. This observation could shed light on the molecular mechanisms underlying lymphomagenesis in patients.

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6

Jacobs, Chean Sern. "Role of PRC2-mediated chromatin regulation in fine tuning Arabidopsis root development". Thesis, Lyon, 2020. http://www.theses.fr/2020LYSEN085.

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La régulation de l’expression des gènes par la voie des mécanismes chromatiniennes sont critiques dans la modulation et la stabilisation de l’expression des programmes génétiques, essentielles pour l’organogenèse et le développement. Le complexe répresseur Polycomb 2 (PRC2) catalyse le triple méthylation de la lysine 27 de l’histone H3 auprès des gènes cibles. Ceci est un régulateur des programmes du développement, globalement conservé chez les eucaryotes multicellulaires. Afin de déterminer l’implication de PRC2 au cours des transitions de l’identité cellulaire, j’ai caractérisé le paysage chromatinien d’un type cellulaire unique de la niche des cellules souches racinaire. L’intégration quantitative des données épigénomique a révélé trois types chromatiniens qui corrèlent avec des niveaux d’activité transcriptionnelle, ainsi que des profils d’expression bien distincts, au cours de la différentiation cellulaire. Ces données suggèrent que la régulation par PRC2 est importante pour maintenir le contrôle temporel des gènes pendant l’avancé de la différentiation cellulaire. De plus, j’ai effectué des études fonctionnelles sur deux homologues de la sous unité catalytique de PRC2 qui indique que au moins deux complexes PRC2 de composition diffèrent peuvent coopérer afin de moduler finement la régulation des gènes clés du développement. En conclusion, le travail mené souligne l’importance de PRC2 dans le contrôle précis des profils d’expression des gènes, et aussi la capacité des données épigénétique d’un état précoce de différentiation de prédire l’activité transcriptionnelle dans les étapes plus tardives
Chromatin-based mechanisms are pivotal regulators of transcriptional patterns that are central to cell fate determination, organogenesis and development in multicellular organisms. The activity of Polycomb Repressive Complex 2 (PRC2) is involved in the maintenance of transcriptional gene repression by catalysing the trimethylation of histone H3 on lysine 27 at specific loci, and is a conserved modulator of developmental programs.To reveal the extent to which PRC2 shapes transcriptional decisions during cell fate specification, I have characterized the epigenome organization of a single cell type from the root stem cell niche (SCN). Quantitative integration of (epi)-genomic data revealed three main chromatin states that correlate with distinct gene expression levels as well as patterns along the differentiation gradient. These results indicate that PRC2 activity over specific genes within the SCN regulates their timing of expression in daughter cells, at successive differentiation stages.In addition, functional studies of PRC2 catalytic subunit homologues support the notion that distinct PRC2 complexes with different compositions cooperate to fine-tune the transcriptional regulation of key regulatory genes during root development. Taken together, this work highlights the importance of PRC2-regulated chromatin states in shaping expression patterns along a differentiation gradient. They also pinpoint the potential of such epigenetic studies in predicting, from an initial chromatin state, the timing of gene transcriptional activation in subsequent differentiation stages

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7

LA, MASTRA FEDERICA. "POLYCOMB GROUP PROTEINS RING1A/RING1B CONTROL PERIPHERAL B CELL HOMEOSTASIS AND TERMINAL DIFFERENTIATION". Doctoral thesis, Università degli Studi di Milano, 2019. http://hdl.handle.net/2434/609574.

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Polycomb Repressive Complex 1 and 2 (PRC1 and PRC2) modulate chromatin accessibility through covalent histone modifications. The ubiquitous expression of PRC1 catalytic subunits throughout B cell development hypothesized roles for PRC1 in B cell physiology. This study aimed to dissect the contribution of PRC1 to peripheral B cell maturation, homeostasis and terminal differentiation. We analyzed mutant mice allowing conditional Cre-dependent inactivation of PRC1 catalytic function starting from late transitional B cells. In response to induced PRC1 inactivation, peripheral B cells in secondary lymphoid organs were reduced in number and displayed alterations in the surface phenotype, which reflected a major disturbance of their transcriptional profile. Reduced fitness of PRC1 mutant resting B cells was associated to heightened sensitivity to pro-apoptotic signals, consequent of the higher levels in these cells of the BIM protein and of the sub-optimal activation of the AKT kinase in response to either BAFF-R or BCR engagement. PRC1 mutant mice displayed major defects in the marginal zone (MZ) B cell subset, both in number and localization. This phenotype correlated with reduced expression of the Sphingosine-1-phosphate receptor-1 (S1pr1), which is crucial for B cell migration to the MZ, and the increased expression of the Polycomb target microRNA mir-125b, which targets S1pr1 transcript. Moreover, PRC1 mutant B cells displayed premature de-repression of Prdm1 gene and facilitated plasma cell differentiation upon lipopolysaccharide stimulation. Our work provides evidence for a crucial role played by PRC1 in peripheral B cell subset differentiation, B cell homeostasis and timing of terminal B cell differentiation.

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8

Brule, Baptiste. "Caractérisation et modulation non pharmacologique des dérégulations épigénétiques associées à la maladie de Huntington : vers l’identification de nouvelles cibles thérapeutiques". Electronic Thesis or Diss., Strasbourg, 2024. http://www.theses.fr/2024STRAJ015.

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La maladie de Huntington (MH) est une maladie neurodégénérative génétique caractérisée par des symptômes moteurs, cognitifs et psychiatriques causés par une atteinte primaire du striatum. Le mécanisme pathogénique implique une dérégulation épigénétique et transcriptionnelle à l’origine d’une perte d’identité et de fonction des neurones. Cette thèse a consisté en la caractérisation épigénétique du striatum de modèles murins à une résolution type cellulaire-spécifique et à différent stades de la MH. Nous avons observé que les neurones striataux qui expriment le gène muté dans la MH présentent une érosion épigénétique traduisant un vieillissement accéléré qui implique une altération des complexes polycomb. Les régulations épigénétiques étant sensibles à l’environnement, nous avons caractérisé le phénotype comportemental et moléculaire de modèles murins de la MH hébergés en environnement enrichi (EE) afin de décrypter l’effet de l’EE sur les régulations épigénétiques et transcriptionnelles. Nos résultats permettent une meilleure compréhension des mécanismes pathogéniques de la MH, et offrent de nouvelles perspectives thérapeutiques
Huntington's disease (HD) is a neurodegenerative genetic disease characterized by motor, cognitive, and psychiatric disorders caused by primary damage to the striatum. The pathogenic mechanism is complex and involve epigenetic and transcriptional dysregulations leading to a loss of neuronal identity and cell function. This thesis aimed to characterize the striatal epigenetic signature in mouse models with a celltype-specific resolution at different stages of HD. We observed that striatal neurons expressing the HD mutation undergo epigenetic erosion, reflecting accelerated aging in HD, induced by alterations in polycomb complexes. As epigenetic regulations are sensitive to the environment, we characterized the behavioral phenotype and molecular alterations of HD mouse model after housing in an enriched environment (EE) to decipher the epigenetic and transcriptomic effects induced by EE. Our findings thus provide a better understanding of early pathogenic mechanisms in HD, opening new therapeutic perspectives

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9

Schivre, Geoffrey. "Transcriptome augmentation, Polycomb-mediated chromatin dynamics and their links to metabolism during Arabidopsis thaliana photomorphogenesis". Electronic Thesis or Diss., université Paris-Saclay, 2024. http://www.theses.fr/2024UPASB014.

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La lumière permet aux plantes de métaboliser le carbone atmosphérique grâce à la photosynthèse, constituant ainsi leur source d'énergie. Par ailleurs, les différentes propriétés de la lumière constituent une source d'informations essentielles sur leur environnement perçues par de multiples capteurs de lumière, les photorécepteurs, déclenchant des réponses adaptatives spécifiques. Parmi elles, l'une des adaptations développementales les plus spectaculaires des plantes, appelée photomorphogenèse, se produit lorsqu'une jeune plantule en cours de germination est exposée à la lumière pour la première fois. Les plantules germant sous terre, à l'abri de la lumière, suivent un développement étiolé ou skotomorphogénique, au cours duquel l'allongement rapide de l'hypocotyle facilite l'émergence de la plante à la surface, tandis que la maturation des cotylédons et la mise en place de la photosynthèse sont inhibés. La croissance skotomorphogénique repose donc entièrement sur les réserves métaboliques principalement stockées dans les cotylédons. Lorsque la surface du sol est atteinte, la détection de la lumière par les photorécepteurs déclenche l'expansion, sans division cellulaire, des cotylédons et la biogenèse des chloroplastes permettant l'acquisition de la photo-autotrophie. Au niveau moléculaire, le dé-étiolement des cotylédons est associé à une spécialisation du transcriptome et à une intensification de l'activité de l'ARN polymérase II (ARN Pol II). A l'échelle cytologique, de profonds réarrangements de la chromatine conduisent à l'élargissement du noyau et à la condensation des régions péricentromériques au sein de foyers hétérochromatiniens. Considérant qu'une grande partie de ces contrôles métaboliques, cellulaires, moléculaires et cytologiques sont réalisés de manière synchrone au cours de la transition, la photomorphogenèse d'A. thaliana constitue un modèle de choix pour caractériser les interactions entre la signalisation lumière, la régulation des gènes et les dynamiques chromatiniennes avec le statut énergétique de la plante. Au cours de ma thèse, j'ai développé une analyse de données de séquençage ARN normalisées grâce à une référence exogène pour revisiter les connaissances actuelles sur les changements transcriptomiques au vu de l'augmentation globale de l'activité de l'ARN Pol II. Ceci a permis d'identifier un doublement de l'abondance des transcrits pendant la photomorphogenèse des cotylédons qui résulte très probablement de l'augmentation de l'activité de l'ARN Pol II. J'ai ensuite exploré le rôle joué par le senseur métabolique Target Of Rapamycin (TOR) dans la régulation du régime transcriptionnel ainsi que dans la composition et l'organisation de la chromatine lors du dé-étiolement des cotylédons. Cette seconde partie de mon étude apporte un nouvel éclairage sur les liens fonctionnels entre la voie TOR et l'homéostasie d'une marque d'histone, la triméthylation de la lysine 27 de l'histone H3 (H3K27me3), catalysée par le complexe Polycomb Repressive Complexe 2 (PRC2). En particulier, elle révèle que H3K27me3 est moins abondant dans la chromatine des cotylédons étiolés que dans celle des cotylédons photomorphogéniques, un effet général qui s'avère sensible au sucre et à la signalisation TOR. Par conséquent, ces travaux mettent en évidence des rôles inattendus de la signalisation TOR et de la marque H3K27me3 dans la régulation du régime transcriptionnel général et ouvrent de nouvelles perspectives sur la régulation transcriptionnelle régie par TOR. De futures études visant à décrypter le rôle de l'homéostasie de H3K27me3, en particulier sur les gènes de réponse à la lumière, devraient permettre de mieux comprendre comment la signalisation métabolique interagit avec les dynamiques de l'épigénome et la répression transcriptionnelle régies par PRC2 avec des implications au-delà de la photomorphogenèse des plantes
Light fuels plant photosynthesis providing the energy source for growth. Light intensity and quality also convey essential information on the plant's immediate surroundings, which are integrated through multiple light sensors, the photoreceptors, enabling developmental and physiological adaptations. The photomorphogenic transition, or de-etiolation, occurs when a young germinating plantlet is exposed to light for the first time, and is one of the most spectacular plant developmental adaptations to light. Seedlings germinating underground, protected from light, undergo an etiolated development, or skotomorphogenesis, during which rapid hypocotyl elongation facilitates plant drilling through the soil, while cotyledon maturation is arrested and the plantlet remains non-photosynthetic. In the absence of photosynthesis, skotomorphogenic growth relies entirely on the plant metabolic reserves, predominantly stored in cotyledons. Upon reaching the soil surface, photoreceptor light sensing triggers the expansion and greening of cotyledons, independently from cell divisions. Inducing chloroplast biogenesis and the acquisition of photosynthesis, this developmental switch marks the transition toward photo-autotrophy. At the molecular scale, cotyledon de-etiolation associates with a specialization of the transcriptome and an intensification of RNA polymerase II (RNA Pol II) activity. At the cytological scale, chromatin rearrangements lead to nucleus enlargement and the condensation of pericentromeric regions in conspicuous heterochromatic foci. Considering that much of these metabolic, cellular, molecular and cytological controls are synchronously achieved during the transition, A. thaliana photomorphogenesis is a model of choice to characterize the interplays between light signaling, gene regulation and chromatin dynamics as well as their link to the plant energetic status. During my thesis, I first contributed to develop an RNA-seq normalization methodology to revisit transcriptome changes in light of the global increase in RNA Pol II activity. This identified a 2-fold increase in transcript abundance during cotyledon photomorphogenesis, which most likely results from the increase in RNA Pol II activity. I further explored the role played by the conserved metabolic sensor Target Of Rapamycin (TOR) in defining the transcriptional regime along with chromatin composition and organization during cotyledon photomorphogenesis. This notably shed a new light on the functional links between the TOR pathway and the homeostasis of a specific histone mark, trimethylation of histone H3 at lysine 27 (H3K27me3), mediated by Polycomb Repressive Complex 2 (PRC2). More precisely, this study revealed that H3K27me3 is less abundant at chromatin in etiolated cotyledons as compared to photomorphogenic ones, a global effect that was further shown here to be sensitive to sugar and TOR signaling. Hence, this work points towards unexpected roles of TOR signaling and the PRC2-regulated mark H3K27me3 in the global regulation of transcription and opens new perspectives on TOR-mediated gene regulation. Future studies aimed at deciphering the role of H3K27me3 homeostasis, especially at specific genes induced by light, should provide new insight on how metabolic signaling interplays with Polycomb-mediated chromatin dynamics and transcription with implications beyond plant photomorphogenesis

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10

Lamiable, Olivier. "Identification et caractérisation des partenaires protéiques de DSP1 chez Drosophila melanogaster". Phd thesis, Université d'Orléans, 2010. http://tel.archives-ouvertes.fr/tel-00558801.

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Chez les eucaryotes pluricellulaires, la différenciation des cellules repose en partie sur l'activation oula répression des gènes. Les profils d'expression génique mis en place vont perdurer d'une générationcellulaire à l'autre. Ce phénomène met en jeu des mécanismes épigénétiques qui remodèlentlocalement la structure de la chromatine. Chez Drosophila melanogaster, les protéines des groupesPolycomb (PcG) et Trithorax (TrxG) participent au maintien du profil d'expression des gènes au coursdu développement. Les protéines PcG maintiennent les gènes réprimés tandis que les protéines TrxGmaintiennent les gènes activés. Une troisième classe de protéines nommée Enhancers of Trithoraxand Polycomb (ETP) module l'activité des PcG et TrxG. Dorsal Switch Protein 1 (DSP1) est uneprotéine HMGB (High Mobility Group B) classée comme une ETP. Par tamisage moléculaire, nousavions montré que la protéine DSP1 était présente au sein de complexes de poids moléculaire de 100kDa à 1 MDa. Le travail de thèse présenté ici a pour but d'identifier les partenaires de la protéineDSP1 dans l'embryon et de mieux connaître les propriétés biochimiques de DSP1. Premièrement, j'aimis en place puis effectué l'immunopurification des complexes contenant DSP1 dans des extraitsprotéiques embryonnaires. Cette approche nous a permis d'identifier 23 partenaires putatifs de laprotéine DSP1. Parmi ces protéines, nous avons identifié la protéine Rm62 qui est une ARN hélicaseà boîte DEAD. Les relations biologiques entre DSP1 et Rm62 ont été précisées. Deuxièmement, j'aidéterminé, par une approche biochimique, de nouvelles caractéristiques physico-chimiques de laprotéine DSP1.

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11

Angulo, Parera Mireia. "Ash2 a Drosophila: anàlisi funcional i aproximació al complexe". Doctoral thesis, Universitat de Barcelona, 2006. http://hdl.handle.net/10803/1876.

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A l'inici del desenvolupament, les cèl·lules d'un organisme comencen a diferenciar-se en resposta a estímuls intrínsecs i del seu entorn cel·lular. Aquests estímuls provoquen l'expressió de factors reguladors de la transcripció que estableixen uns patrons d'expressió gènica específics per a cada tipus cel·lular. Malgrat l'expressió d'aquests factors és transitòria, els patrons d'expressió gènica establerts inicialment es mantenen al llarg del desenvolupament, transmetent-se de generació en generació a les cèl·lules filles. El fet que una cèl·lula expressi uns gens i no uns altres durant molts cicles de divisió cel·lular, li confereix una identitat pròpia i fa sobreentendre que hi ha d'haver un mecanisme, basat en senyals epigenètics, que reconegui i preservi el patró de gens que s'ha d'activar o silenciar.
Els gens del grup Polycomb (PcG) i trithorax (trxG) codifiquen per proteïnes reguladores de la cromatina implicades en el manteniment de les identitats cel·lulars. La seva funció com a reguladors dels gens homeòtics ha estat molt ben documentada però es coneix molt poc sobre els efectes que poden tenir en altres gens diana o processos del desenvolupament. En aquesta Tesi s'utilitza el patró de venes i intervenes de l'ala com a model per entendre la funció del gen trxG absent, small or homeotic discs 2 (ash2). Es mostra que ash2 es requereix per mantenir l'activació dels gens promotors d'intervena blistered i net, i per reprimir l'expressió de rhomboid, un component de la via de DER, necessària per la formació de venes. Els nostres resultats també mostren que ash2 actua com a repressor del gen knirps (kni), organitzador de la vena longitudinal L2. La inhibició que ash2 exerceix sobre kni és independent de spalt-major i spalt-related, els quals s'ha postulat que són els reguladors de kni. Tots aquests experiments indiquen que ash2 és essencial per dos processos durant el desenvolupament de l'ala: (1) mantenir el destí cel·lular d'intervena, ja sigui activant els gens promotors d'intervena o inhibint els de vena; i (2) mantenir kni en un estat reprimit en teixits fora de la vena L2.
A més, donat que els gens del grup PcG i trxG codifiquen per proteïnes que formen part de complexes multiproteics reguladors de la cromatina, en aquesta Tesi també s'ha intentat fer una aproximació al complexe d' ASH2. Mitjançant estudis de co-immunoprecipitació i marcatges en cromosomes politènics es va demostrar que les proteïnes ASH2 i Sin3A interaccionen amb la proteïna Host Cell Factor (HCF), suggerint que a Drosophila podria existir un complexe conservat, semblant al purificat en humans, amb capacitat histona metiltransferasa.
Differential gene expression results in cell diversity, although how different cell identities are established early in development and maintained throughout life is still poorly understood. Most of the transcription factors required for early developmental decisions are expressed transiently, but the gene expression patterns they trigger are maintained during cell division and inherited by daughter cells. Actively dividing cells must preserve individual genes in an on or off expression state after an initial commitment is made, especially given that some regulators disassemble from promoters during DNA replication or mitosis. Thus, developmental decisions may be maintained by the ability to deposit epigenetic marks involving chromatin modifying complexes to control the cellular memory of gene activity states. Genes of the Polycomb (PcG) and trithorax group (trxG) encode proteins that are engaged in the regulation of cellular memory.
absent, small or homeotic discs 2 (ash2) is a trxG protein that belongs to a 0.5 MDa complex thought to be involved in chromatin remodelling. In this thesis, to gain more insight into the function of ash2, we have used the Drosophila wing as a model system to examine whether vein- and intervein-specific genes and vein positioning genes act as putative targets of ash2 function. We found that ash2 is involved in activating intervein-promoting genes and downregulating the Egfr pathway. Moreover, ash2 also acts as a kni repressor independently of sal-C. These results strongly support a role for ash2 in maintaining vein/intervein developmental decisions and vein patterning in the developing wing.
We have also tried to do an approximation to the Drosophila ASH2 complex. We have demonstrated that ASH2 and Sin3A were found to colocalize on polytene chromosomes and coimmunoprecipitate with the Host Cell Factor (HCF). Together with the localization of ASH2 at sites of H3K4 trimethylation we propose that Drosophila might contain an ASH2 complex with histone methyltransferase activity similar to the one found in humans.

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12

Herranz, Martín Nicolás. "New insights in the epigenetic control of EMT". Doctoral thesis, Universitat Pompeu Fabra, 2011. http://hdl.handle.net/10803/80660.

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The epithelial to mesenchymal transition (EMT) is a highly conserved cellular program that allows well-‐differentiated epithelial cells to convert to motile mesenchymal cells. EMT is critical for appropriate embryogenesis and plays a crucial role in tumorigenesis and cancer progression. At this regard, it has become increasingly evident that, in addition to genetic alterations, tumour development involves the alteration of gene expression patterns owing to epigenetic changes. Taking this into account, this thesis mainly addresses the description of new molecular epigenetic mechanisms underlying one of the hallmark processes governing EMT, the Snail1-‐mediated E-‐cadherin repression. Indeed, our results demonstrate that both Polycomb group (PcG) proteins and the LOXL2 protein are involved in this process. Apart from providing novel insights into the significance of these proteins in tumor progression, our work uncovers the characterization of a new epigenetic modification carried out by LOXL2; H3K4 deamination.
La transició epiteli-‐mesènquima (EMT) és un programa cel·lular molt conservat que permet a les cèl·lules epitelials convertir-‐se en cèl·lules mesenquimals indiferenciades. La EMT és un procés crucial pel desenvolupament embrionari i per la progressió tumoral. A aquest respecte, ha esdevingut cada cop més evident que el desenvolupament tumoral no només està associat a alteracions genètiques, sinó també a l'alteració de l’expressió gènica causada per canvis epigenètics. Tenint això en compte, aquesta tesi es centra en la descripció de nous mecanismes moleculars en l’àmbit de l’epigenètica associats a un dels processos clau en la EMT, la repressió de la E-‐ cadherina mitjançada pel factor de transcripció Snail1. De fet, els nostres resultats demostren que tant les proteïnes del grup Polycomb (PcG) com la proteïna LOXL2 estan implicades en aquest procés. A part de proporcionar nova informació respecte la importància d'aquestes proteïnes en la progressió tumoral, la nostra feina ha permès la caracterització d'una nova modificació epigenètica duta a terme per la proteïna LOXL2; la deaminació de H3K4.

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13

Violot, Sébastien. "Etudes fonctionnelles et structurales de la protéine EED, partenaire cellulaire du virus VIH-1 et de la cellulase « froide » Cel5G de Pseudoalteromonas haloplanktis". Phd thesis, Université Claude Bernard - Lyon I, 2005. http://tel.archives-ouvertes.fr/tel-00091916.

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La protéine humaine EED appartient à la famille des «Polycomb». Elle intervient dans la régulation génique. Elle jouerait aussi un rôle dans le cycle du virus de l'immunodéficience humaine (VIH-1) en participant au transport du complexe de préintégration et en facilitant la réaction d'intégration. EED a été surproduite afin d'être cristallisée seule et en complexe avec les protéines virales IN, MA et Nef. Un modèle de EED a été construit par homologie structurale. D'après nos expériences de « phage display », les régions susceptibles d'interagir avec les partenaires viraux se situent sur des boucles de ce modèle.
La bactérie psychrophile P. haloplanktis produit la cellulase Cel5G. Les structures de cette cellulase seule et en complexe avec le cellobiose, ont été déterminées à haute résolution par remplacement moléculaire. La cellulase Cel5A de la bactérie mésophile E. chrysanthemi a été utilisée comme modèle. Nos résultats permettent de mieux comprendre les déterminants structuraux responsables de l'adaptation des enzymes aux basses températures.

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14

Preissner, Tanja Stephanie. "The Polycomb-repressive complex 2 in X-inactivation". Thesis, Imperial College London, 2007. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.445872.

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15

Courel, María F. (María Federica). "The function of E2F6 in the Polycomb complex". Thesis, Massachusetts Institute of Technology, 2005. http://hdl.handle.net/1721.1/86281.

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Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biology, 2005.
Cataloged from PDF version of thesis.
Includes bibliographical references.
The E2F family of transcription factors are known cell cycle regulators that function at the G1/S transition. Unlike other E2Fs, E2F6 does not activate transcription and is not regulated by pocket protein binding. Instead, this protein appears to repress transcription through the recruitment of the Polycomb Group (PcG) complex. This complex is responsible for the maintenance of Hox gene expression patterns during development and thus ensures the correct anterior-posterior segmentation of the embryo. Genetic ablation of PcG proteins leads to posterior transformations of the axial skeleton as well as other developmental abnormalities such as hematopoietic, cerebellar and smooth muscle defects. The PcG complex has been implicated in cell cycle control since several of its members, including the oncoprotein Bmi 1, appear to repress the transcription of p1 6INK4A and pI 9 ARF. In order to determine the biological function of E2F6, we have generated and characterized E2f6'- mice and mouse embryonic fibroblasts (MEFs). The mutant mice are viable and survive into adulthood with similar lifespan as their littermate controls. Furthermore, the E2f6 null MEFs are indistinguishable from wild-type MEFs in asynchronous proliferation, cell cycle re-entry from quiescence, senescence and E2F target genes expression levels. These findings suggest that E2F6 does not play a major role in cell cycle control or that its function can be compensated by the action of other factors. In fact, preliminary results from combined loss of E2f6 and Bmil suggest that E2F6 may take part in the Bmi 1-mediated control of the cell cycle. Furthermore, we found that the loss of E2F6 results in posterior axial skeleton transformations that are reminiscent of the Bmil-deficient mice defects. The study of the E2f6;Bmil compound mutant mice revealed a dosage-dependent synergism between E2F6 and Bmi 1. These results indicate that E2F6 participates in segmentation during murine development. As a whole, our work has provided proof that E2F6 is a bonafide Polycomb Group protein and, at the same time, has opened the field to a number of interesting questions.
by María F. Courel.
Ph. D.

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16

Grijzenhout, Anne Elizabeth. "Characterisation of AEBP2 : a polycomb repressive complex 2 component". Thesis, University of Oxford, 2013. http://ora.ox.ac.uk/objects/uuid:159716a1-a03c-44f3-9fd1-0e88328caef6.

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17

Chandler, H. "Association of CK2 with Polycomb complexes and its functional implications". Thesis, University College London (University of London), 2013. http://discovery.ucl.ac.uk/1383781/.

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Polycomb group (PcG) proteins are important for establishing the patterns of gene expression in different cell types and are critical for the maintenance of pluripotency. They participate in multi-component complexes, such as Polycomb repressive complex 1 (PRC1), which modify, and bind to, histone tails. A number of auxiliary proteins consistently associate with PRC1, including the three subunits of protein kinase CK2 (CK2). The work described in this thesis investigates the interaction of CK2 with PRC1 components and the implications for PRC1 function. The data suggest that CK2 can directly bind to members of the CBX family, the mammalian orthologues of Polycomb in Drosophila. In the case of CBX7, residues within the conserved Pc box, near the C-terminus, were critical for this interaction. Interestingly, these residues were also required for the interaction between CBX7 and RING2, another core component of mammalian PRC1. Whether CBX7 is phosphorylated by CK2 remains equivocal and likewise, it has been difficult to demonstrate a role for CK2 in the ability of CBX7 to function as a transcriptional repressor. In addition to their role as regulators of gene expression, PcG proteins have been recently implicated in the DNA damage response (DDR). Moreover, several proteins involved in the DDR are known to be CK2 substrates. To explore the link between CK2 and PRC1 in the context of DNA damage, a cell system was established in which multiple sequence-specific double-strand breaks (DSBs) could be induced in human diploid fibroblasts. Interestingly, detection of PRC1 proteins by both immunofluorescence and genome-wide ChIP-seq suggests that they are not recruited to DSBs in this system. Furthermore, the data indicate that extensive DNA damage does not mobilise PRC1 complexes from known binding sites.

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18

Weaver, Tyler M. "Regulation of the polycomb repressive complexes by histone reader domains". Diss., University of Iowa, 2019. https://ir.uiowa.edu/etd/6877.

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Histone post-translational modifications (PTMs) are key determinants of the local chromatin landscape and critical for regulation of eukaryotic gene expression. These histone marks are deposited by a vast number of chromatin modifying enzymes and preferentially recognized by specific associated histone reader domains. Recognition of histone PTMs by histone reader domains is important for either targeting these complexes to chromatin or regulating their enzymatic activity once there. The Polycomb repressive complex 1 and 2 (PRC1 and PRC2) are two such chromatin modifying complexes that are critical for developmental gene repression. The enzymatic activity of PRC2 is tightly regulated by many histone reader domains whereas the PRC1 complex is targeted to chromatin through these domains. In this thesis, I explore how PRC1 and PRC2 functions are regulated by histone reader domains. I identify a previously unrecognized histone reader domain within the PRC2 complex, the EZH2 SANT1 domain, which has important implications for regulating PRC2 enzymatic activity. In addition, I explore the mechanism through which the CBX8 chromodomain targets the PRC1 complex to chromatin. Together, these studies provide significant insight into the regulation of chromatin modifying complexes by histone reader domains and how this occurs via multiple mechanisms.

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19

Palau, de Miguel Anna. "Polycomb Repressive Complex 1 functions in differentiation and myelodysplastic syndromes". Doctoral thesis, Universitat de Barcelona, 2016. http://hdl.handle.net/10803/400293.

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Polycomb proteins are important epigenetic regulators involved in the maintenance of stemness and differentiation. In this thesis, I focused on the role of some Polycomb Repressive Complex 1 (PRC1) components. On the one hand, I studied the role of Cbx8 PRC1 component protein in the differentiation of mouse embryonic stem cells (mESCs). On the other hand, I analyzed the role of PRC1 components in a hematological disease-related context which implies a defect in differentiation, the myelodysplastic syndrome (MDS). Specifically, I focused on RING1A PRC1-component function in this disease. Our previous data showed that upon addition of retinoic acid (RA) during 3 days to E14 mouse ESC cell line, by which cells were prompted into the neuronal lineage, Cbx8 was upregulated both at mRNA and protein level. We performed a genome-wide chromatin immunoprecipitation of endogenous Cbx8 coupled to direct massive parallel sequencing (ChIP-seq) to assess the binding sites of Cbx8 genome- wide using IgG and Cbx8 ChIP in untreated mESC as negative controls. Our analysis identified 171 high confidence peaks. To our surprise, by crossing our data with previously published microarray analysis, we showed that several differentiation genes transiently recruit Cbx8 during their early activation. Depletion of Cbx8 by 2 different shRNA partially impaired the transcriptional activation of these genes as well as diminish Cbx8 recruitment to its target genes. Both interaction analysis, as well as chromatin immunoprecipitation experiments supported the idea that activating Cbx8 acts in the context of an intact PRC1 complex. Prolonged gene activation resulted in eviction of PRC1 despite persisting H3K27me3 and H2A ubiquitination. The composition of PRC1 is highly modular and changes when embryonic stem cells commit to differentiation. We further demonstrated that the exchange of Cbx7 for Cbx8 is required for the effective activation of differentiation genes. Taken together, our results establish a function for a Cbx8-containing complex in facilitating the transition from a Polycomb- repressed chromatin state to an active state. In order to characterize the function of PRC1 in the pathogenesis of MDS we used publicly available expression datasets of PRC1 components from MDS patients and during normal myeloid differentiation to identify and quantify the level of relevant PRC1 complexes. From this data mining we selected four PRC1 components (CBX6, BMI1, RING1A and CBX7) and two PRC2 components (EZH2 and ASXL1) for further analysis. To study these PRC components we wished to use cell lines that are MDS-related. For this reason, we extensively characterized 5 MDS/AML derived cell lines by conventional cytogenetics, single nucleotide polymorphism arrays, mutational panel of 83 MDS/AML relevant genes and immunoprofile. After this study, we selected SKK-1 cell line as the most suitable model to study the function of the selected PRC1 components. Based on the finding that RING1A is highly expressed in hematopoietic stem cells and further overexpressed in patients with high risk MDS, we have analyzed the role of RING1A in cells. We found that RING1A inhibits differentiation in MDS-derived AML cells and in primary human hematopoietic stem cells (HSCs). We further provide first evidence that pharmacological inhibition of RING1A could be therapeutic strategy by showing that the treatment of HSCs favors differentiation.
Les proteïnes Polycomb són importants reguladors epigenètics implicats en el manteniment de la pluripotència i la diferenciació. En aquesta tesi, m'he centrat en el paper d'alguns components del Polycomb Repressive Complex 1 (PRC1). D'una banda, he estudiat el paper de la proteïna Cbx8, component del PRC1, en la diferenciació de les cèl·lules mare embrionàries de ratolí (mESCs). D'altra banda, he analitzat el paper dels components del PRC1 en un una malaltia hematològica que implica un defecte en la diferenciació, la síndrome mielodisplàstica (SMD). En concret, m'he centrat en la funció de RING1A, component del PRC1, en aquesta malaltia. Les nostres dades anteriors van mostrar que després de l'addició d'àcid retinoic (RA) durant 3 dies a la línia cel·lular de mESCs Cbx8 es sobreexpressava, tant a nivell d'ARNm com de proteïnes. Vam realitzar una immunoprecipitació de cromatina del Cbx8 endogen a nivell de tot el genoma seguit de seqüenciació massiva (ChIP-seq) per avaluar els punts d'unió de Cbx8 en tot el genoma utilitzant els ChIPs IgG i Cbx8 de mESC sense tractar com a controls negatius. La nostra anàlisi va identificar 171 pics d'alta confiança. Sorprenentment, en creuar les nostres dades amb l'anàlisi de microarrays publicat prèviament, es va demostrar que diversos gens de diferenciació transitòriament recluten Cbx8 durant la seva activació primerenca. El knockdown de Cbx8 per 2 shRNA diferents va afectar parcialment l'activació transcripcional d'aquests gens, així com va disminuir el reclutament de Cbx8 als seus gens diana. Tant l’anàlisi d'interacció per espectrometria de masses com els experiments de immunoprecipitació de la cromatina van donar suport a la idea que l'activació de Cbx8 actua en el context d'un complex PRC1 intacte. L’activació gènica prolongada va resultar en l’expulsió de PRC1 amb un H3K27me3 i H2AK119ub persistents. La composició del PRC1 és altament modular i canvia quan les cèl·lules mare embrionàries es diferencien. A més, vam demostrar que es requereix l'intercanvi de Cbx7 per Cbx8 per a l'activació efectiva dels gens de diferenciació. En conjunt, els nostres resultats estableixen una funció per a un complex que conté Cbx8 a l'hora de facilitar la transició d'un estat de cromatina reprimida per Polycomb a un estat actiu. Per tal de caracteritzar la funció de PRC1 en la patogènesi de SMD vam utilitzar dades d’expressió públicament disponibles de pacients amb SMD i durant la diferenciació mieloide normal per tal d’identificar i quantificar el nivell dels components de PRC1. A partir d'aquesta anàlisi es van seleccionar quatre components del PRC1 ( CBX6, BMI1, RING1A i CBX7) i dos components del PRC2 (EZH2 i ASXL1) per al seu posterior estudi. Vam decidir treballar amb línies cel·lulars relacionades amb MDS per tal d’estudiar aquests components PRC. Per aquesta raó, hem caracteritzat àmpliament 5 línies cel·lulars de leucèmia mieloide aguda (LMA) derivades de síndromes mielodisplàstiques (SMD) per citogenètica convencional, single nucleotide polymorphism arrays, un panell mutacional de 83 gens relacionats amb SMD /LMA i immunofenotip. Després d'aquest estudi, vam seleccionar la línia cel·lular SKK-1 com el model més adequat per estudiar la funció dels components PRC1 seleccionats. Basant- nos en la troballa que RING1A està altament expressat en cèl·lules mare hematopoètiques i a més es sobreexpressa en pacients de SMD amb alt risc, hem analitzat la funció de RING1A. Vam trobar que RING1A inhibeix la diferenciació de la línia cel·lular de SMD/LMA i en cèl·lules mare hematopoètiques primàries. Proporcionem a més la primera evidència que la inhibició farmacològica de RING1A podria ser una estratègia terapèutica ja que el tractament en cèl·lules mare hematopoètiques afavoreix la diferenciació.

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20

Semprich, Claudia. "Regulation of polycomb repressive complexes at the neural differentiation gene Pax6". Thesis, University of Dundee, 2016. https://discovery.dundee.ac.uk/en/studentTheses/51529b2a-9763-424b-acea-808700b1d1a9.

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During body axis elongation spinal cord neural tissue in the posterior of the embryo is progressively generated from a pool of bipotent progenitors of the stem zone/caudal lateral epiblast, termed neuromesodermal progenitors. Neural differentiation gene transcription begins in a sharply defined position due to loss of fibroblast growth factor (FGF) signalling. This highly coordinated onset of gene expression implies the involvement of a higher order mechanism to co-ordinately regulate hundreds of genes at the same time. One possible mechanism underlying this phenomenon is the regulation of chromatin structure around neural differentiation genes. The analysis of chromatin structure by fluorescence in situ hybridisation uncovered that chromatin compaction at the neural differentiation gene Pax6 is maintained by FGF signalling in the stem zone/caudal lateral epiblast of the mouse embryo (Patel et al., 2013). Here I show that this process is mediated by ERK signalling, with just one hour of ERK inhibition leading to a decompaction of the Pax6 locus in the mouse embryo. One way in which local chromatin compaction is regulated is by polycomb repressive complexes. They build a machinery that deposit the histone modifications H3K27me3 and H2AK119Ub at target genes and were shown to impose chromatin compaction. To test the possibility of FGF/ERK regulated polycomb mediated chromatin compaction at neural differentiation gene loci in the stem zone/ caudal lateral epiblast a chromatin immunoprecipitation (ChIP) experiment was performed for the histone modification H3K27me3. Preliminary data showed that the Pax6 transcription start site in the stem zone caudal lateral epiblast is marked by H3K27me3, comparable to the known unexpressed polycomb target HoxD11. This indicated the potential for polycomb mediated chromatin compaction at the Pax6 locus. A human embryonic stem (ES) cell based in vitro model for the generation of spinal cord neural progenitors used to analyse further whether polycomb mediated chromatin compaction determines neural differentiation gene expression and to evaluate its regulation by FGF/ERK signalling. To that end human ES cells were exposed to FGF and Wnt signals for 3 days to generate neuromesodermal progenitors (hNMPs) (Gouti et al., 2014), these were then differentiated into neural progenitors (hNPs) of posterior spinal cord fate. ChIP assays showed that during the differentiation of hNMPs to hNPs the polycomb components Jarid2 and Ring1B dissociate from the Pax6 locus (transcription start site and gene body) and the H3K27me3 mark is removed in order to facilitate Pax6 transcription from a now decompacted locus. When ERK signalling was blocked in the hNMPs for 12 hours the polycomb components Jarid2 and Ring1B dissociated from the locus whereas the H3K27me3 remained correlating with chromatin decompaction; however, no Pax6 gene transcription was observed. These observations suggest that ERK promotes polycomb mediated chromatin compaction. Interestingly, upon only 3 hours of ERK inhibition only Jarid2 was found to have dissociated from the Pax6 locus whereas Ring1B occupancy and H3K27me3 levels remained, thereby opening the possibility for a step wise dissociation/unloading of the polycomb machinery upon FGF/ERK signalling loss. Taken together the data in the hES in vitro system implies that during neural differentiation the loss of FGF/ERK signalling leads to the dissociation of polycomb neural differentiation gene loci and the removal of the H3K27me3 mark in order to facilitate transcription from these loci. In pluripotent mouse ES cells it was recently revealed that the recruitment of polycomb complexes to differentiation genes relies on activated ERK (Tee et al., 2014). Furthermore, a few polycomb components were shown to be potential phosphorylation targets of FGF/ERK downstream pathways. The data in this thesis suggest a FGF/ERK mediated polycomb regulation that could potentially involve either or both of these mechanisms to regulate gene expression in the stem zone\caudal lateral epiblast of the mouse embryo. During body axis elongation FGF signalling is attenuated by retinoic acid signalling in the posterior of the mouse embryo (Diez del Corral et al., 2003). The work of this thesis suggests that this retinoic acid mediated attenuation of FGF signalling leads to the dissociation of the polycomb machinery at neural differentiation genes and thereby the de-repression of target gene transcription.

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21

Delest, Anna. "Ciblage dynamique et différentiel des complexes Polycomb au cours du développement de Drosophila melanogaster". Thesis, Montpellier 1, 2012. http://www.theses.fr/2012MON13520.

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Les protéines du groupe Polycomb (PcG) sont évolutivement conservées et sont des régulateurs chromatiniens responsables du maintien de la répression transcriptionnelle des gènes homéotiques (HOX) au cours du développement. Elles assurent ainsi une mémoire cellulaire. Cependant, ces protéines peuvent aussi cibler des gènes contrôlant le cycle cellulaire et la détermination du destin cellulaire. Au laboratoire, il a été montré que dans le disque imaginal d'œil de drosophile, plusieurs gènes de la voie de signalisation Notch sont réprimés par les protéines du PcG. La perte de fonction de ces dernières résulte en l'activation ectopique de Notch et en la formation de tumeurs néoplasiques. De manière intéressante, Notch n'est pas une cible des protéines du PcG dans les embryons. Ceci suggère qu'au cours du développement, les protéines du PcG pourraient être impliquées dans un contrôle dynamique de l'expression génique.L'objectif de ma thèse a été d'étudier le ciblage dynamique des protéines du PcG au cours du développement et de la différentiation tissulaire. Pour cela, j'ai effectué des expériences de ChIP dirigées contre des protéines du complexe PRC1 et pour la marque répressive H3K27me3 (typique du complexe PRC2) à partir de tissus larvaires : les disques imaginaux d'œil et d'aile. En comparant ces données aux données embryonnaires, nous avons découvert un néo-recrutement du système Polycomb spécifique du stade larvaire. Etonnamment, il existe plusieurs catégories de gènes cibles qui se distinguent sur la base de leurs profils de ChIP, ce qui suggère de nouveaux mécanismes de régulation par les protéines du PcG. En effet, certains gènes sont fixés uniquement par les protéines du PRC1 en l'absence de la marque H3K27me3 et inversement. Les 2 complexes PRCs pourraient donc agir indépendamment dans la régulation de l'expression génique
Polycomb group (PcG) proteins are an evolutionarily conserved set of chromatin regulators implicated in stable long-term homeotic gene silencing. PcG proteins additionally bind and regulate genes implicated in cell cycle control or cellular fate determination, suggesting that PcG proteins can be involved in more dynamic regulation of target genes. Recent studies in Drosophila eye imaginal discs showed that PcG proteins can control cellular proliferation by repression of signalling genes, and that abrogation of this process promotes tumours. Interestingly, one of the regulated genes was not found to be a PcG target in embryonic tissues, suggesting that PcG-mediated gene regulation is dynamic throughout development. To gain a comprehensive view of the targeting of PcG proteins throughout development and to understand its role during tissue differentiation, we performed ChIP experiments in eye and wing imaginal discs for components of the PcG complex, PRC1, and the repressive histone mark H3K27me3 (deposited by the PcG complex, PRC2). Compared to embryo datasets, we find many novel PcG target genes, several with tissue-specific recruitment in eye or wing discs. Furthermore, we report new classes of PcG target genes based on their ChIP profiles, which may have implications for their modes of regulation. For example, some genes are bound only by PRC1 components (Pc, Ph), without the presence of H3K27me3, or vice versa, indicating that these complexes may play more independent roles in gene regulation than previously appreciated

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22

Ragazzini, Roberta. "Identification of a tissue-specific cofactor of polycomb repressive complex 2". Thesis, Paris 6, 2017. http://www.theses.fr/2017PA066196/document.

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Abstract (sommario):

Répression des genes par le dépôt de la marque H3K27me3. Divers cofacteurs contrôlent sa fonction dans des cellules de différentes origines, comme les gametes. Au cours de ma thèse, j'ai utilisé des modèles murins ou un tag a été introduit dans les gènes Ezh2 et Ezh1, j'ai isolé des extraits nucléaires de testicules adultes entiers et identifié un nouveau polypeptide interagissant avec PRC2. Ce dernier est spécifiquement exprimé dans les gonades et sa fonction est inconnue. J'ai confirmé son interaction avec PRC2 et montré qu'il pourrait recruter PRC2 à la chromatine. Grâce à un modèle de souris knock-out, j'ai démontré que la protéine est nécessaire pour la fertilité féminine, alors que son ablation apporte une augmentation globale de la marque associée à PRC2, dans les cellules germinales masculines avec peu de conséquences sur la fertilité. J'ai également contribué à la caractérisation de l'interaction entre le long ARN non-codant HOTAIR et PRC2. Nombreux ARNnc ont été proposés pour moduler l'action des complexes modifiant la chromatine. Avec l'aide d'un nouveau système de recrutement artificiel d'ARN, l'expression induite par HOTAIR provoque une répression transgénique indépendamment de PRC2. La surexpression forcée de HOTAIR a également peu d'impact sur le transcriptome dans des cellules cancéreuses. En conclusion, la liaison PRC2 à l'ARN n'est pas requise pour le ciblage de la chromatine
The Polycomb Repressive Complex 2 (PRC2) plays an essential role in development by maintaining gene repression through the deposition of H3K27me3. A variety of cofactors have been shown to control its function in cells of various origins however little is known about PRC2 regulation during gametogenesis. During my PhD, I took advantage of murine models where Ezh2 and Ezh1 were knocked-in, I isolated nuclear extracts from whole adult testis and, identified a new polypeptide interacting with PRC2. This protein is specifically expressed in gonads, is of unknown function and does not contain any conserved domain. I have confirmed its interaction with PRC2, identified the domain of interaction with PRC2 and shown that it could tether PRC2 to chromatin. Thanks to a knockout mouse model, I demonstrated that the protein is required for female fertility, whereas its ablation brings to a global increase of H3K27me3 PRC2-associated mark in male germ cells with little consequences on male fertility. I also contributed to the characterization of the interplay between the long non-coding RNA (lncRNA) HOTAIR and PRC2 complex. Many lncRNAs have been proposed to modulate chromatin-modifying complexes action on chromatin. With the help of novel RNA-tethering system, HOTAIR inducible expression causes transgene repression independently from PRC2. Forced overexpression of HOTAIR also has little impact on transcriptome in breast cancer cells. Generally, PRC2 binding to RNA is not required for chromatin targeting. Taken together these results shed light to the mechanism of a new-identified cofactor regulating PRC2 in the gonads and contribute to dissect PRC2-RNA relationship at molecular level

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23

Ragazzini, Roberta. "Identification of a tissue-specific cofactor of polycomb repressive complex 2". Electronic Thesis or Diss., Paris 6, 2017. https://accesdistant.sorbonne-universite.fr/login?url=https://theses-intra.sorbonne-universite.fr/2017PA066196.pdf.

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Abstract (sommario):

Répression des genes par le dépôt de la marque H3K27me3. Divers cofacteurs contrôlent sa fonction dans des cellules de différentes origines, comme les gametes. Au cours de ma thèse, j'ai utilisé des modèles murins ou un tag a été introduit dans les gènes Ezh2 et Ezh1, j'ai isolé des extraits nucléaires de testicules adultes entiers et identifié un nouveau polypeptide interagissant avec PRC2. Ce dernier est spécifiquement exprimé dans les gonades et sa fonction est inconnue. J'ai confirmé son interaction avec PRC2 et montré qu'il pourrait recruter PRC2 à la chromatine. Grâce à un modèle de souris knock-out, j'ai démontré que la protéine est nécessaire pour la fertilité féminine, alors que son ablation apporte une augmentation globale de la marque associée à PRC2, dans les cellules germinales masculines avec peu de conséquences sur la fertilité. J'ai également contribué à la caractérisation de l'interaction entre le long ARN non-codant HOTAIR et PRC2. Nombreux ARNnc ont été proposés pour moduler l'action des complexes modifiant la chromatine. Avec l'aide d'un nouveau système de recrutement artificiel d'ARN, l'expression induite par HOTAIR provoque une répression transgénique indépendamment de PRC2. La surexpression forcée de HOTAIR a également peu d'impact sur le transcriptome dans des cellules cancéreuses. En conclusion, la liaison PRC2 à l'ARN n'est pas requise pour le ciblage de la chromatine
The Polycomb Repressive Complex 2 (PRC2) plays an essential role in development by maintaining gene repression through the deposition of H3K27me3. A variety of cofactors have been shown to control its function in cells of various origins however little is known about PRC2 regulation during gametogenesis. During my PhD, I took advantage of murine models where Ezh2 and Ezh1 were knocked-in, I isolated nuclear extracts from whole adult testis and, identified a new polypeptide interacting with PRC2. This protein is specifically expressed in gonads, is of unknown function and does not contain any conserved domain. I have confirmed its interaction with PRC2, identified the domain of interaction with PRC2 and shown that it could tether PRC2 to chromatin. Thanks to a knockout mouse model, I demonstrated that the protein is required for female fertility, whereas its ablation brings to a global increase of H3K27me3 PRC2-associated mark in male germ cells with little consequences on male fertility. I also contributed to the characterization of the interplay between the long non-coding RNA (lncRNA) HOTAIR and PRC2 complex. Many lncRNAs have been proposed to modulate chromatin-modifying complexes action on chromatin. With the help of novel RNA-tethering system, HOTAIR inducible expression causes transgene repression independently from PRC2. Forced overexpression of HOTAIR also has little impact on transcriptome in breast cancer cells. Generally, PRC2 binding to RNA is not required for chromatin targeting. Taken together these results shed light to the mechanism of a new-identified cofactor regulating PRC2 in the gonads and contribute to dissect PRC2-RNA relationship at molecular level

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24

González, Morao Ana Karina. "Rôle des complexes PRC2 dans la régulation de la différenciation cellulaire chez Arabidopsis thaliana". Thesis, Université Paris-Saclay (ComUE), 2017. http://www.theses.fr/2017SACLS153.

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Abstract (sommario):

Les protéines du groupe Polycomb (PcG) ont initialement été identifiées chez la Drosophile, en tant que facteurs nécessaires au maintien de l’expression spatio-temporelle de gènes homéotiques le long de l’axe antéro-postérieur. Depuis, leur rôle en tant que régulateurs du développement a été mis en évidence chez la plupart des métazoaires ainsi que chez les plantes, chez lesquelles elles orchestrent les transitions développementales, l’organogenèse et la différenciation cellulaire. Les protéines PcG sont nécessaires au maintien de la répression transcriptionnelle de gènes cibles, par la régulation de leur structure chromatinienne via des modifications post-traductionnelles des histones. Elles forment des complexes multiprotéiques, notamment les Complexes Répressifs Polycomb PRC1 et PRC2. PRC2 est responsable de la tri-méthylation de la lysine 27 de l’histone H3 (H3K27me3) et est constitué de 4 sous-unités principales qui, pour la plupart, sont présentes sous forme de familles multigéniques dans le génome d’Arabidopsis thaliana. Ainsi, il existe plusieurs complexes PRC2 constitués de combinaisons alternatives de ces sous-unités, qui sont potentiellement présents au sein d’une même cellule et dont les rôles sont considérés comme partiellement redondants. En utilisant des approches moléculaires, génétiques et génomiques, nous avons analysé le rôle des sous-unités PRC2 exprimées dans la pointe racinaire d’Arabidopsis. Nous avons montré que l’interaction entre différents PRC2 est nécessaire pour réguler l’activité du méristème, le déroulement temporel de la différenciation cellulaire, ainsi que pour le maintien de l’identité des cellules matures. De plus, notre travail montre que les complexes PRC2 contenant l’une ou l’autre des deux méthyltransférases, CLF et SWN, régulent des groupes de gènes communs ainsi que distincts, à travers des mécanismes différents incluant une fonction non-canonique. Par ailleurs, nos résultats indiquent que les différences fonctionnelles entre CLF-PRC2 et SWN-PRC2 reposent, au moins en partie, sur les sous-unités non-catalytiques avec lesquelles la méthyltransférase interagit. Pour identifier les gènes régulés dynamiquement par PRC2 durant la différenciation cellulaire, nous avons développé des approches permettant d’accéder à la résolution des types cellulaires afin d’analyser les états chromatiniens à l’intérieur de la niche de cellules souches et de la zone de maturation de la racine. Nos données suggèrent que PRC2 participe au maintien de l’identité du Centre Quiescent (QC) en réprimant des voies de signalisations spécifiques. De plus, la différenciation cellulaire en direction de la zone de maturation est accompagnée par un accroissement du répertoire des cibles PRC2, incluant des régulateurs méristématiques ainsi que des gènes spécifiquement exprimés dans différents types cellulaires. Enfin, nos résultats suggèrent qu’une proportion significative des cibles PRC2 sont présentes sous la forme de domaines bivalents H3K27me3-H3K4me3 dans les cellules souches végétales, cette proportion étant moins importante que celle décrite chez les cellules souches embryonnaires de mammifères. Dans l’ensemble, ce travail apporte une vue intégrée de la fonction, la dynamique et la multiplicité de l’activité PRC2 au cours du processus de différenciation cellulaire, dans le contexte d’un organe en développement. Nos résultats mettent en évidence le rôle de PRC2 en tant que régulateur majeur de la différenciation cellulaire, qui apporte à la fois robustesse et plasticité aux programmes transcriptionnels qui sous-tendent l’acquisition spatio-temporelle et le maintien de l’identité cellulaire
The Polycomb group (PcG) proteins were originally identified in Drosophila as factors required for maintaining the spatio-temporal expression of homeotic genes along the head-to-tail axis. Since then, their role as developmental regulators has been highlighted in most metazoans as well as plants, in which they orchestrate developmental transitions, organogenesis and cell differentiation. PcG proteins are required to maintain the transcriptional repression of target genes by regulating their chromatin structure via post-translational histone modifications. They are found in multiprotein complexes, including Polycomb Repressive Complexes PRC1 and PRC2. PRC2 is responsible for the trimethylation of histone H3 at lysine 27 (H3K27me3) and consists of four core subunits, most of which are represented by multigene families in Arabidopsis thaliana. Thus, distinct PRC2 complexes formed by alternative subunit combinations exist, possibly in the same cell, and are thought to play partly overlapping roles. By combining molecular, genetic and genomic approaches, we have analyzed the role of the PRC2 subunits expressed in the Arabidopsis root tip used as a model. We show that the interplay between distinct PRC2s is necessary to regulate the activity of the meristem and the timing of cell differentiation, as well as the maintenance of cell identity. In addition, our work reveals that PRC2 complexes containing either of the two related methyltransferases CLF or SWN regulate common as well as specific sets of genes through distinct mechanisms, including a non-canonical function. Furthermore, our results indicate that the functional differences between CLF-PRC2 and SWN-PRC2 rely, at least in part, on the non-catalytic subunit they are interacting with. To identify the genes dynamically regulated by PRC2 during cell differentiation, we have developed cell type-specific approaches to analyze chromatin marks in cell populations within the stem cell niche and the maturation zone of the root. Our data suggest that PRC2 participates in the maintenance of the quiescent center (QC) identity by repressing specific signaling pathways. In addition, cell differentiation towards the maturation zone is accompanied by an increase of the repertoire of PRC2 targets including stem cell and meristem regulators, as well as cell type-specific genes. Finally, our findings suggest that bivalent H3K27me3-H3K4me3 domains in the QC represent a significant, though smaller proportion of PRC2 targets in plant stem cells compared to what has been described in mammalian embryonic stem cells. Overall, this work provides an integrated view of the function, dynamics and multiplicity of PRC2 activity during the cell differentiation process, in the context of a developing organ. Our results highlight the role of PRC2s as major regulators of cell differentiation that provide both robustness and plasticity to the transcriptional programs underlying cell fate acquisition and identity maintenance

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25

Moffat, Michael. "Aberrant DNA modification profiles in embryonic stem cells lacking polycomb repressive complexes". Thesis, University of Edinburgh, 2016. http://hdl.handle.net/1842/25711.

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Abstract (sommario):

Transcriptional repression is maintained by many molecular processes, including DNA methylation and polycomb repression. These two systems are both associated with chromatin modification at the promoters of silent genes, and are both essential for mammalian development. Previous work has shown that DNMT proteins are required for correct targeting of polycomb repressive complexes (PRCs). In this thesis, I investigate whether targeting of DNA modification has a reciprocal dependence on the polycomb machinery by mapping DNA modification in wild-type and PRC-mutant ES cells (Ring1B null, EED null, and Ring1B/EED duble null). I find that the loss of PRCs results in increased DNA modification at sites normally targeted by de novo DNA methyltransferase which lose H3K4 methylation upon PRC removal. This increased DNA modificaiton is associated with increased gene expression when found at CpG island shores of genes marked by the PRC-mediated histone modifications H3K27me3 and H2AK119ub, but not genes lacking these marks. Gene misregulation may be further linked to DNA modification changes by increased DNA modification at enhancers. While loss of either Ring1B or EED led primarily to increases in DNA modification at regions dependant on DNMT3A/DNMT3B, the combined loss of Ring1B and EED results in widespread loss of DNA modification at sites more dependent on DNMT1 activity. This thesis suggests an interplay between PRCs and DNA modification placement which is relevant to the cntrol of gene expression.

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26

Sanulli, Serena. "Polycomb repressive complex 2 and jarid2 in the establishment of repressive chromatin state". Paris 6, 2013. http://www.theses.fr/2013PA066429.

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RC2 contribue au maintien de la répression transcriptionnelle au cours du développement par la di- et tri- méthylation de H3K27. PRC2 est nécessaire à de nombreux processus biologiques tels que le renouvellement et différenciation des cellules souches et le maintien de l’identité cellulaire. Malgré de nombreuses recherches, les mécanismes qui régulent la dynamique du recrutement de PRC2 à la chromatine sont peu compris. Des études récentes ont montré que Jarid2 est un cofacteur de PRC2 qui régulerait son ciblage à la chromatine. Pendant ma thèse, je me suis concentrée sur l’étude des mécanismes moléculaires de régulation du recrutement de PRC2 dépendents de Jarid2. J’ai montré que Jarid2 est méthylé par PRC2 et que cette méthylation stimule l’activité enzymatique de PRC2. Des expériences in vitro et in vivo ont permis de montrer que la méthylation de Jarid2 est impliquée dans la relocalisation de PRC2 à de nouvelles régions du génome, initialisant l’activité enzymatique de PRC2. J’ai également contribué à la caractérisation d’une nouvelle protéine contenant un domaine SET codée par la bactérie L. Pneumophila. Cette protéine, qui est secrétée par la bactérie lors de l’infection, modifie la chromatine des cellules hôtes en méthylant H3K14, une modification normalement absente de la chromatine des cellules hôtes. En empêchant l’acétylation de H3K14, cette modification induirait une répression transcriptionnelle globale afin de limiter les défenses de la cellule hôte. Ces découvertes ouvrent de nouvelles perspectives sur les mécanismes de régulation et la fonction des enzymes méthyltransférases lors du développement et aussi en réponse aux infections cellulaires
Polycomb Repressive complex 2 (PRC2) contributes to the maintenance of epigenetic silencing established during development through the di- and trimethylation of H3K27. PRC2 complex is crucial for several biological processes, including stem cell self-renewal and differentiation, and maintenance of cell identity. Despite intensive research, the mechanisms that dynamically regulate PRC2 recruitment to the chromatin are still poorly understood. Recent studies identified Jarid2 as a cofactor of PRC2 and proposed this protein as a regulator of PRC2 targeting. During my PhD, I focused on the molecular mechanisms responsible for PRC2 chromatin targeting mediated by Jarid2 cofactor. I demonstrated that Jarid2 is methylated by PRC2 and that its methylation stimulates PRC2 enzymatic activity. Biochemical and in vivo approaches revealed that Jarid2 methylation acts during the de novo targeting of PRC2 complex to prime PRC2 activity and ensure the establishment of H3K27me3 at new genomic sites. I also contributed to the characterization of a novel SET-domain containing protein encoded by the bacteria L. Pneumophila. This protein, secreted by the bacteria after cellular infection, is targeted to the host chromatin to induce a unique modification, H3K14me. This mark, normally not present in mammalian host cells, prevents H3K14 acetylation and causes global transcriptional repression to circumvent cellular defense. These findings provided new perspectives about the regulation and function of histone-methyltransferase proteins during development and cell fate decision, as well as during cellular infections

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Asamaowei, Inemo E. "The Role of Polycomb Repressive Complex 2 in Epidermal Homeostasis and Hair Growth". Thesis, University of Bradford, 2017. http://hdl.handle.net/10454/16844.

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Polycomb repressive complex 2 (PRC2) catalyses the methylation of ‘Lys-27’ of histone H3, leading to transcriptional repression of target genes through its catalytic subunit Enhancer of zeste homolog 1/2 (EZH1/2). PRC2 functions as a critical regulator of stem cells in mouse embryonic and adult tissues. However, the role of PRC2 in human skin remains largely unknown. This study investigated the role of PRC2 in human epidermal homeostasis and hair growth. The expression of EZH2 was elevated in differentiating suprabasal layers of the human epidermis. Consistently, EZH1/2 expression and enzymatic activity was upregulated in differentiating primary human keratinocytes (NHEKs) in vitro. Inhibition of EZH2 and Embryonic ectoderm development (EED) in NHEKs stimulated the expression of differentiation-associated genes, therefore leading to their premature differentiation; while inhibition of EZH1/2 reduced cell proliferation and promoted apoptosis. Silencing of EZH2 in NHEKs induced complex changes in gene expression programmes, including the upregulation of terminal differentiation genes, such as Filaggrin. EZH2 expression was downregulated in aged keratinocytes accompanied with upregulation of senescence-associated genes, p16INK4A and p19INK4D, suggesting EZH2 involvement in epidermal aging. In human anagen hair follicle (HF), EZH2 was detected in stem and progenitor cells; and hair matrix keratinocytes. Silencing EZH2 in HFs accelerated anagen-catagen transition and retarded hair growth accompanied by decreased proliferation and increased apoptosis. Silencing EZH2 in outer root sheath keratinocytes resulted in upregulation of p14ARF and K15, suggesting EZH2 involvement in regulating proliferation and stem cell activity. Thus, this study demonstrates that PRC2-mediated repression is crucial for epidermal homeostasis and hair growth. Modulating the activities of PRC2 in skin might offer a new therapeutic approach for disorders of epidermal differentiation and hair growth.

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Alzrigat, Mohammad. "Targeted Inhibition of Polycomb Repressive Complexes in Multiple Myeloma : Implications for Biology and Therapy". Doctoral thesis, Uppsala universitet, Experimentell och klinisk onkologi, 2017. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-312250.

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Multiple myeloma (MM) is a hematological malignancy of antibody producing plasmablasts/plasma cells. MM is characterized by extensive genetic and clonal heterogeneity, which have hampered the attempts to identify a common underlying mechanism for disease establishment and development of appropriate treatment regimes. This thesis is focused on understanding the role of epigenetic regulation of gene expression mediated by the polycomb repressive complexes 1 and 2 (PRC1 and 2) in MM and their impact on disease biology and therapy. In paper I the genome-wide distribution of two histone methylation marks; H3K27me3 and H3K4me3 were studied in plasma cells isolated from newly diagnosed MM patients or age-matched normal donors. We were able to define targets of H3K27me3, H3K4me3 and bivalent (carry both marks) which are, when compared to normal individuals, unique to MM patients. The presence of H3K27me3 correlated with silencing of MM unique H3K27me3 targets in MM patients at advanced stages of the disease. Notably, the expression pattern of H3K27me3-marked genes correlated with poor patient survival. We also showed that inhibition of the PRC2 enzymatic subunit EZH2 using highly selective inhibitors (GSK343 and UNC1999) demonstrated anti-myeloma activity using relevant in vitro models of MM. These data suggest an important role for gene repression mediated by PRC2 in MM, and highlights the PRC2 component EZH2 as a potential therapeutic target in MM. In paper II we further explored the therapeutic potential of UNC1999, a highly selective inhibitor of EZH2 in MM. We showed that EZH2 inhibition by UNC1999 downregulated important MM oncogenes; IRF-4, XBP-1, BLIMP-1and c-MYC. These oncogenes have been previously shown to be crucial for disease establishment, growth and progression. We found that EZH2 inhibition reactivated the expression of microRNAs genes previously found to be underexpressed in MM and which possess potential tumor suppressor functions. Among the reactivated microRNAs we identified miR-125a-3p and miR-320c as predicted negative regulators of the MM-associated oncogenes. Notably, we defined miR-125a-3p and miR-320c as targets of EZH2 and H3K27me3 in MM cell lines and patients samples. These findings described for the first time PRC2/EZH2/H3K27me3 as regulators of microRNA with tumor suppressor functions in MM. This further strengthens the oncogenic features of EZH2 and its potential as a therapeutic target in MM. In paper III we evaluated the therapeutic potential of targeting PRC1 in MM using the recently developed chemical PTC-209; an inhibitor targeting the BMI-1 subunit of PRC1. Using MM cell lines and primary cells isolated from newly diagnosed or relapsed MM patients, we found that PTC-209 has a potent anti-MM activity. We showed, for the first time in MM, that PTC-209 anti-MM effects were mediated by on-target effects i.e. downregulation of BMI-1 protein and the associated repressive histone mark H2AK119ub, but that other subunits of the PRC1 complex were not affected. We showed that PTC-209 reduced MM cell viability via significant induction of apoptosis. More importantly, we demonstrated that PTC-209 shows synergistic anti-MM activity with other epigenetic inhibitors targeting EZH2 (UNC1999) and BET-bromodomains (JQ1). This work highlights the potential use of BMI-1 and PRC1 as potential therapeutic targets in MM alone or in combination with other anti-MM agents including epigenetic inhibitors.

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29

Liabeuf-Le, Goff Emilie. "Implications des complexes Polycomb et Trithorax au cours du développement précoce chez Ciona intestinalis". Thesis, Montpellier 2, 2012. http://www.theses.fr/2012MON20193.

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Abstract (sommario):

Implications des complexes Polycomb et Trithorax au cours du développement précoce chez Ciona intestinalisLes protéines des groupes Polycomb (PcG) et Trithorax (TrxG) ont été initialement découvertes chez Drosophila melanogaster. Ces deux groupes sont classiquement connus pour leurs rôles respectifs de répresseurs et d'activateurs épigénétiques qui contrôlent et maintiennent les états chromatiniens au cours du temps. Ces facteurs régulent de nombreux gènes cibles dont les gènes homéotiques. Au cours de ma thèse, j'ai étudié trois composants de ces deux groupes : Enhancer of zeste (E(z)), appartenant au complexe PRC2 du PcG et responsable du dépôt de la marque de répression génique H3K27me3, Polyhomeotic (Ph), appartenant au complexe PRC1 du PcG et dont le rôle exact reste à déterminer, et Trithorax (Trx), appartenant au complexe TAC1 du TrxG et responsable du dépôt de la marque d'activation génique H3K4me3. Jusqu'à présent, aucune étude n'a abordé la régulation épigénétique via les PcG et TrxG chez l'ascidie solitaire Ciona intestinalis. Cette espèce présente un cluster des gènes Hox désorganisé et ne possède pas la protéine Polycomb (Pc) du PRC1, responsable de la reconnaissance de la marque de répression H3K27me3 déposée par la protéine E(z).Nos travaux montrent que la protéine E(z) est fonctionnelle et conserve son activité méthyltransférase sur le résidu H3K27 chez Ciona intestinalis. Nous avons ensuite observé, par des expériences de knockdown par micro-injection de morpholinos, que les inhibitions protéiques d'E(z), Ph et Trx ont des conséquences dramatiques sur la différenciation et la mise en place des différents tissus au cours du développement larvaire, notamment sur la mise en place de la notochorde puisque celle-ci est totalement absente chez les morphants E(z) et Ph. Les défauts de phénotype du morphant E(z) sont corrélés à la perte du dépôt d'H3K27me3 et nous avons mis en évidence, lors de l'inhibition d'E(z), une dérépression des gènes tissu-spécifiques impliqués dans le développement embryonnaire précoce alors que les gènes tardivement exprimés sont réprimés. De plus, l'expression des gènes Hox n'est pas significativement modifiée au cours du développement embryonnaire lorsque la protéine E(z) est inhibée, à l'exception du gène Hox12 qui est déréprimé, comme attendu.L'ensemble de ces résultats permet d'émettre l'idée innovante selon laquelle les protéines des PcG et TrxG jouent un rôle déterminant dans la régulation de l'expression génique lors de l'embryogénèse de Ciona intestinalis tout en ayant une implication mineure dans la régulation de l'expression des gènes Hox à ce stade du développement
Implications of Polycomb and Trithorax complexes in the early development of Ciona intestinalisPolycomb and Trithorax group (PcG and TrxG) proteins were discovered originally in Drosophila melanogaster. Both groups are classically known for their roles in the maintenance of silenced and active chromatin states over time, respectively. These factors regulate many target genes including the homeotic genes. During my PhD, I studied three components of these two groups: Enhancer of zest (E(z)), belonging to the PRC2 complex of PcG and responsible for H3K27me3 mark deposit for gene repression, Polyhomeotic (Ph), belonging to the PRC1 complex of PcG whose role remains to be determined, and Trithorax (Trx), belonging to the TAC1 complex of TrxG and responsible for H3K4me3 mark deposit for gene activation. Until now, no study addresses the epigenetic regulation mediated by PcG and TrxG in the solitary ascidian Ciona intestinalis. This specie has a disorganized Hox cluster and in which the Polycomb (Pc) protein of PRC1, responsible for the recognition of the repressive H3K27me3 mark, is absent.Our work shows that the E(z) protein is functional and retains its methyltransferase activity on H3K27 residue in Ciona intestinalis. Then, we demonstrated, by knockdown experiments with morpholino microinjection, that the inhibition of E(z), Ph and Trx has dramatic consequences on differentiation and on the establishment of different tissues during larval development, particularly on the notochord establishment since it is totally absent in E(z) and Ph morphants. E(z) morphant phenotypic defects are correlated with lack of H3K27me3 mark deposit and we highlighted that, during the E(z) inhibition, tissue-specific genes implied in early development are de-repressed while late-expressed genes are down-regulated. In addition among Hox genes, only Hox12 expression is significantly modified and found to be de-repressed in E(z) morphant context, as expected.Altogether, our results present the innovative idea that the PcG and TrxG proteins play a major role in the gene expression regulation during embryogenesis of Ciona intestinalis while having a minor involvement in the regulation of Hox genes expression at this stage of development

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Sharif, Azar. "Structural characterization of the polycomb repressor complex 1 binding partner ubiquitin specific protease 11". Thesis, Imperial College London, 2013. http://hdl.handle.net/10044/1/39355.

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Abstract (sommario):

Ubiquitin Specific Protease 11 (USP11), USP4 and USP15 are highly conserved and are characterised by an N-terminal 'domain present in ubiquitin specific proteases' (DUSP) and 'ubiquitin-like' (UBL) domains. This DUSP-UBL (DU) domain is thought to be involved in substrate recognition. It was shown that USP11 co-purifies with human Polycomb Repressive Complex type 1 (PRC1) and regulates the stability of the E3 ligase component of PRC1 (Maertens et al, 2010). PRC1 repress transcription from the INK4a tumour suppressor locus. Hence knockdown of USP11 in primary human fibroblasts causes de-repression of INK4a, followed by a senescence-like proliferative arrest. In this project we aimed to map the interaction between USP11 and PRC1 components (BMI1, RING2, MEL18 and CBX8). We used two methods to investigate their interactions; yeast two-hybrid and in vitro pull down. Unexpectedly, we could not confirm a direct interaction between USP11 and any PRC1 component. We hypothesize that the lack of post-translation modifications, the presence of fusion tags and/or the need of a multi-subunit PRC1 complex might be needed to observe a high affinity interaction. We also aimed to map the interaction between three PRC1 components; RING2, BMI1 and RYBP, with the ultimate aim of solving the X-ray structure of the complex. The main obstacle in this project was to express, extract and purify these proteins at high levels in bacterial culture. Preliminary data suggests that RYBP and BMI1 do not interact directly. Here we report the 3.6 Å resolution X-ray structure of the human USP11 DU. The sequence linking the DUSP and UBL domains, the DU finger, could not be assigned in the electron density map due to low resolution. Comparison with the related USP4 DU crystal structure reveals that the structures are mostly conserved.

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Pereira, João Duarte Tavares da Silva. "The role of the polycomb repressive complex 2 in the regulation of neocortical neurogenesis". Thesis, University of Cambridge, 2012. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.610489.

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32

Vandamme, Julien. "Analyse protéomique des complexes associés aux membres de la famille CBX (HP1 et Polycomb) dans des cellules humaines". Thesis, Lille 1, 2009. http://www.theses.fr/2009LIL10160/document.

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Abstract (sommario):

Dans le noyau des cellules eucaryotes, l’ADN est associé aux histones pour former la chromatine. Les extrémités terminales des histones peuvent subir un grand nombre de modifications posttraductionnelles réversibles (méthylation, acétylation, phosphorylation, ubiquitinylation…). Certaines de ces marques épigénétiques définissent des domaines chromatiniens particuliers du noyau. Les triméthylations des lysines 9 et 27 de l’histone H3 (H3K9me3 et H3K27me3) correspondent respectivement à des domaines d’hétérochromatine constitutive et d’hétérochromatine facultative. Les proteines qui possèdent un chromo-domaine sont capables de se lier à des lysines méthylées, et permettent de recruter des complexes qui ont une activité enzymatique ou mécanique sur la chromatine. Les protéines de la famille CBX (ChromoBoX) possèdent toutes un chromodomaine, elles sont subdivisées en 2 groupes: les protéines du groupe HP1 (CBX1, CBX3 et CBX5) et celles du groupe Polycomb (CBX2, CBX4, CBX6, CBX7 et CBX8) qui se fixent respectivement sur H3K9me3 et H3K27me3, respectivement. Afin de mieux comprendre comment s’organise la chromatine au niveau des régions hétérochromatiques, nous avons purifié, en condition native, les complexes associés à ces 8 protéines dans des cellules humaines en culture. Pour ce faire, nous avons opté pour la purification d’affinité en tandem (TAP). Les protéines co-éluées ont été identifiées par spectrométrie de masse. Nos résultats confirment que, d’une part les protéines du groupe Polycomb sont associées au complexe PRC1 (Polycomb Repressive Complex 1). Toutefois, ces protéines sont mutuellement exclusives au sein du complexe PRC1, indiquant qu’il existe plusieurs complexes PRC1 de composition distincte dans la cellule. D’autre part, de nouveaux partenaires associés aux protéines du groupe HP1 ont été identifiés. La mise au point et le développement de la technologie TAP sur des cellules humaines en culture nous ont permis de purifier des complexes associés à la chromatine. Cette technique demeure un outil biochimique efficace pour la purification de complexes protéiques
In the nucleus of eukaryotic cells, DNA is wrapped around histones to form chromatin. The terminal ends of histones may undergo many reversible post-translational modifications (methylation, acetylation, phosphorylation, ubiquitinylation...). Some of these epigenetic marks define specific chromatic regions in the nucleus. The tri-methylation of lysines 9 and 27 of histone H3 (H3K9me3 and H3K27me3) correspond respectively to constitutive and facultative heterochromatin. Chromo-domain containing proteins can bind to methylated lysines, and can recruit complexes having enzymatic or mechanical activity on chromatin. All the members of the CBX (ChromoBoX) family contain a chromodomain, they are divided into 2 groups: the HP1 group (CBX1, CBX3 and CBX5) and the Polycomb group (CBX2, CBX4, CBX6, CBX7 and CBX8) able to bind to H3K9me3 and H3K27me3, respectively. To better understand how the chromatin is organized in heterochromatic regions we purified, in native condition, the complexes associated with these 8 proteins from human cells in culture. To this end, we opted for the tandem affinity purification (TAP). The co-eluted proteins were identified by mass spectrometry. Our results confirm that firstly, Polycomb group proteins are involved in PRC1 complex (Polycomb Repressive Complex 1). However, these proteins are mutually exclusive in the PRC1 complex, indicating that several PRC1 of distinct compositions co-exist in the cell. On the other hand, new partners associated with HP1 group were identified. The development of the TAP technology to cultured human cells allowed us to purify complexes associated with chromatin. This technique remains an effective tool for the biochemical purification of protein complexes

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33

Choi, Jeong-Yoon [Verfasser], e Elena [Akademischer Betreuer] Conti. "Structural and functional analysis of polycomblike-polycomb repressive complex-2 / Jeongyoon Choi ; Betreuer: Elena Conti". München : Universitätsbibliothek der Ludwig-Maximilians-Universität, 2016. http://d-nb.info/1141053810/34.

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34

Choi, Jeongyoon Verfasser], e Elena [Akademischer Betreuer] [Conti. "Structural and functional analysis of polycomblike-polycomb repressive complex-2 / Jeongyoon Choi ; Betreuer: Elena Conti". München : Universitätsbibliothek der Ludwig-Maximilians-Universität, 2016. http://d-nb.info/1141053810/34.

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35

Jiang, Ying. "Fasting alters histone methylation in paraventricular nucleus of chick through regulating of polycomb repressive complex 2". Diss., Virginia Tech, 2013. http://hdl.handle.net/10919/51751.

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Abstract (sommario):

The developing brain is highly sensitive to environmental influences. Unfavorable nutrition is one kind of stress that can cause acute metabolic disorders during the neonatal period [1,2,3] and severe diseases in later life [4,5]. These early life experiences occurring during heightened periods of brain plasticity help determine the lifelong structural and functional aspects of brain and behavior. In humans, for example, weight gain during the first week of life increased the propensity for developing obesity several decades later [5]. This susceptibility is, if not all, related to the dynamic reversible epigenetic imprints left on the histones [6,7,8], especially during the prenatal and postpartum period [9]. Histones are highly dynamic and responsive towards environmental stress [10,11]. Through covalent modification of the histone tail, histones are able to direct DNA scaffolding and regulate gene expression [10,12]. Thus far, various types of post translational modifications have been identified on various histones tails [12]. Among them, the methylation and acetylation on lysine residue (K) 27 on histone 3 (H3) has been tightly linked to gene repression [13,14] and activation [15], respectively. EZh2 (enhancer of zeste 2) in the polycomb repressive complex 2 (PRC2) is the only methyltransferase that has been linked to catalyze this methylation reaction. In addition, SUZ (suppressor of zeste) and EED (embryonic ectoderm development) are two other key proteins in PRC2 function core that help EZH2. As previous reported, increased H3K27 methylation was monitored after fasting stress during neonatal period in chicks' paraventricular nucleus (PVN). In this study, we investigated the detailed mechanism behind changes in H3K27 methylation following fasting stress. After 24 hours fasting on 3 days-of-age (D3), chicks exhibited elevated mRNA levels of PRC2 key components, including EZH2, SUZ and EED, in the PVN on D4. Western blots confirmed this finding by showing increased global methylation status at the H3K27 site in the PVN on D4. In addition, until 38 days post fasting, SUZ and EZH2 remained inhibited. A newly identified anorexigenic factor, Brain-derived neurotrophic factor (BDNF), was used as an example of multiple hormones expressed in PVN to verify this finding. Both BDNF protein and mRNA exhibited compatible changes to global changes of tri- (me3) and di-methylated (me2) H327. Furthermore, by using chromatin immunoprecipitation assays (ChIP), we were able to monitor the changes of H3K27me2/me3 deposition along the Bdnf gene. Fasting significantly increased H3K27me2/me3 as well as EZH2 at the Bdnf's promoter, transcription start site and 3'-untranslated region. These data show that fasting stress during the early life period could leave epigenetic imprinting in PVN for a long time. Next, we tried to understand the function of this epigenetic imprinting in the chicks' PVN. Thus, we compared naive chicks (never fasted) to chicks that received either a single 24 hour fast on D3 or two 24 hour fast on both D3 and 10 days-of-age (D10). We found that the D3 fasted group significantly increased the level of PRC2 key components and its product H3K27me2/me3 compared to the naive group. However, D3 fasting and D10 fasting together decreased the surges of H3K27me2/me3, SUZ and EED (not EZH2) compared to the naive group. We called this phenomenon "epigenetic memory". The Western blot, qPCR and CHIP assay results from BDNF all confirmed the existence of "epigenetic memory" for PRC2. These data suggested that fasting stress during the early period of brain development could leave long term epigenetic modifications in neurons. These changes could be beneficial to the body, which keeps homeostasis of inner environment and prevent massive response to future same stress. The EZH2 protein was knocked down and the H3K27 methylation status changes were monitored after applying the same treatment. We first confirmed that EZH2 antisense oligonucleotides (5.5 ug), but not EZH2 siRNA and artificial cerebrospinal fluid (ACSF), inhibit EZH2 protein by 86 % in the PVN. Then, on D3, chicks were subjected to a 24 hour fasting stress (D3-fasting) post either EZH2 antisense or ACSF injection. The EZH2 antisense blocked the surge of both EZH2 mRNA and H3K27 methylation after D3-fasting. At the same time, BDNF exhibited elevated expression levels and less methylated H3K27 deposition along the Bdnf gene. In addition, we were also interested in the changes of "epigenetic memory" post EZH2 antisense injection. We found that after EZH2 antisense injection, chicks' PVN no longer exhibited any "epigenetic memory" to repetitive fasting stress. While EZH2 mRNA was constantly inhibited, SUZ, EED and H3K27me2/3 levels were unpredictable. These findings suggested that neurons in the PVN utilized PRC2 as a major H3K27 methylation tool. Knockdown of EZH2 in the PRC2 impaired the proper response in PVN to fasting stress and PVN's ability to acclimate to repetitive fasting stresses. Thus, EZH2 is an important H3K27 methyltransferase inside chicken hypothalamus to maintain homeostasis. In conclusion, fasting stress during the early life period could leave epigenetic markers on chromosomes of neurons in the feeding regulation center. These epigenetic markers will be left on chromosomes for a long period of time and have a beneficial role in keeping homeostasis when individuals face future fasting stress again. H3K27 methylation is one of these epigenetic markers and inhibits expression of various genes inside neurons. EZH2 is so far the only detected methyltransferases for H3K27 that form the PRC2. Thus EZH2 plays a key function in the body's response to fasting.
Ph. D.

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36

Abdelfettah, Souhila. "Conséquences fonctionnelles de la surexpression de l’isoforme courte de la protéine Polycomb-like hPCL3, hPCL3S dans les tumeurs prostatiques". Thesis, Lille 1, 2019. http://www.theses.fr/2019LIL1S112.

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Abstract (sommario):

Le complexe Polycomb PRC2 permet le dépôt de la marque épigénétique répressive H3K27me3 par sa sous unité catalytique, EZH2. Selon les types de cancers, EZH2 est soit surexprimée (prostate) ou fait l’objet de mutations perte ou gain de fonction, ce qui aboutit à des niveaux aberrants d’H3K27me3. In vitro, un tetramère constitué du « coeur » de PRC2, EZH2, SUZ12, EED et RBBP4 est suffisant pour catalyser la triméthylation d’H3K27. Par contre, in vivo, plusieurs facteurs modulant l’activité enzymatique du complexe PRC2 ou participant à son recrutement et/ou à sa stabilisation au niveau des régions génomiques adéquates ont été identifiés. Parmi ceux-ci, se trouve la protéine PCL3/PHF19, l’une des trois orthologues humains avec PHF1 et PCL2 de l’unique protéine Polycomb-like (PCL) de Drosophile. Ces protéines partagent un domaine N-terminal structuré consistant en l’enchaînement d’un domaine TUDOR, de deux domaines PHD (Plant Homeo Domain) suivi d’un domaine de type «Winged-helix » impliqué dans la fixation à l’ADN. En outre, hPCL3 fixe la marque activatrice H3K36me3 via son domaine TUDOR grâce à une «cage aromatique» constituée par les Acides Aminés W50, Y56, F74 et Y80 et permet ainsi l’intrusion de PRC2 dans l’euchromatine, l’activation d’EZH2 et le dépôt d’H3K27me3. En raison des différents sites de polyadénylation et d’événements d’épissage alternatifs, le locus humain hPCL3 / PHF19 code pour deux isoformes : une protéine de longueur totale hPCL3L/ PHF19L (580 AA) et une courte isoforme, hPCL3S/PHF19S (207 AA) contenant uniquement le domaine TUDOR, le premier des deux domaines PHD, le PHD1 et une région C-terminale spécifique à hPCL3S. Le domaine PHD1 est faiblement conservé parmi les 3 orthologues humains et pourrait être associé à des fonctions spécifiques de chaque orthologue, comme la stabilisation de P53 dans le cas de PHF1.Des expériences de RT-qPCR sur une cohorte de 25 tumeurs prostatiques ont révélé que hPCL3S est surexprimée dans 75% des cas. De plus, hPCL3S est surexprimée dans les lignées hormono-insensibles DU145 et PC3 mais pas dans la lignée hormono-sensible LNCaP. Dans des tests de blessure sur tapis confluents de cellules «Wound Healing assays », nous avons montré que l’inactivation spécifique par siARN de hPCL3S ralentit la prolifération et la migration des cellules DU145 qui le surexpriment. Inversement, la transfection stable de hPCL3S dans des LNCaP accroit ces propriétés. Ces effets reposent en partie sur la surexpression de gènes connus pour être importants pour la prolifération et/ou la migration de cellules cancéreuses de la prostate telles que S100A16, PlexinA2 et Spondin1. La transfection stable d’un mutant ponctuel de hPCL3S, W50A, incapable de fixer H3K36me3 se traduit par une prolifération accrue des LNCaP comme dans le cas de hPCL3S-WT. Ceci suggère que cet effet ne dépend pas de la fixation à la marque épigénétique H3K36me3 du domaine TUDOR. Par contre, une mutation dans le domaine PHD1 abolit l’effet sur la croissance. Ce domaine PHD1 est un domaine d’interaction protéine-protéine qui est très divergent entre les 3 orthologues Polycomb-like, et pourrait donc avoir des fonctions différentes. Ces résultats nous ont permis de mettre en évidence un rôle de hPCL3S dans la progression tumorale prostatique et suggèrent que hPCL3S soit une nouvelle cible thérapeutique potentielle dans les cancers de la prostate résistant à la castration
The Polycomb PRC2 complex allows the deposition of the repressive epigenetic mark H3K27me3 by its catalytic subunit, EZH2. According to the type of cancers, EZH2 either is overexpressed (prostate) or is subject to loss or gain of function mutations, which lead to aberrant levels of H3K27me3. In vitro, a tetramer consisting of the "core" PRC2 subunits, EZH2, SUZ12, EED and RBBP4 is sufficient to catalyze the trimethylation of H3K27. In vivo, several factors modulating the enzymatic activity of the PRC2 complex or participating in its recruitment and/or its stabilization at the promoters of target genes have been identified. Among them, the three human orthologs of the unique Polycomb-like protein (PCL) of Drosophila, PHF1, PCL2 and PCL3/PHF19 have recently gained much attention. These proteins share a structured N-terminal domain consisting of a TUDOR domain and two PHD domains (Plant Homeo Domain) followed by a "Winged-helix" domain involved in DNA binding. In addition, PHF1 and PHF19 bind H3K36me3 via their TUDOR domain through an "aromatic cage" and thus allow the intrusion of PRC2 into euchromatin, the activation of EZH2 and H3K27me3 deposition. Owing to different polyadenylation sites and alternative splicing events, the human hPCL3/PHF19 locus encodes two isoforms: a full-length protein, hPCL3L/PHF19L and a shorter isoform, hPCL3S/PHF19S, which contains only the domain TUDOR, PHD1-the first of two PHD- domains and a small specific C-terminal region. The PHD1 domain, which is very divergent between the three orthologues, could be associated with specific functions of each orthologue. For example, PHF1 is the only one capable of inducing cell quiescence by interacting with and stabilizing P53 through its PHD1 domain and independently of its TUDOR domain. Our RT-qPCR experiments on a cohort of 25 prostate tumors revealed that hPCL3S is overexpressed in 75% of the cases. In addition, hPCL3S is overexpressed in the DU145 and PC3 hormone-insensitive cell lines, but not in the hormone-sensitive LNCaP cell line. In Wound-healing and proliferation assays, we have shown that the specific siRNA inactivation of hPCL3S decreases the proliferation and migration of DU145 cells that over-express it. Conversely, the stable transfection of hPCL3S into LNCaP increases these properties. These effects partially relied on the up-regulation of genes known to be important for the proliferation and/or migration of prostate cancer cells such as S100A16, PlexinA2 and Spondin1. Stable transfection of a punctual mutant of hPCL3S, W50A, is unable to bind H3K36me3 and results in increased proliferation of LNCaP as in the case of hPCL3S-WT. suggesting that this effect is not dependent on the reading H3K36me3 by the TUDOR domain.By contrast, a mutation in the PHD1 domain abolishes the effect on growth. This PHD1 domain is a protein-protein interaction domain that is very divergent between the 3 Polycomb-like orthologs, and could therefore have different functions. These results allow us to highlight the role of hPCL3S in prostate tumor progression and suggest that hPCL3S is a potential new therapeutic target in castration-resistant prostate cancer

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Rossi, A. "ROLE OF THE POLYCOMB GROUP PROTEINS IN THE ADULT INTESTINAL STEM CELLS HOMEOSTASIS". Doctoral thesis, Università degli Studi di Milano, 2015. http://hdl.handle.net/2434/260390.

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Abstract (sommario):

Polycomb group proteins (PcG) are among the most important gatekeepers that ensure the correct establishment and maintenance of cellular identity in metazoans. This occurs by modifying chromatin through the activity of two Polycomb Repressive Complexes (PRC1 and PRC2) that deposit H2A ubiquitylation and H3K27 methylation respectively, in order to guarantee repression of their target genes. Although the development of PRC2 inhibitory compounds is becoming a very promising strategy for specific cancer treatment, the controversial role of PcG proteins, acting as oncogenes or tumor suppressors in a tissue/cancer specific manner, prompt us to further investigate the role PcG proteins in regulating adult tissue homeostasis. Using different genetic models, we have found that PRC1 activity is required for the integrity of the mouse intestinal epithelia. More in detail, PRC1 activity is required for the self-renewal of the intestinal stem cells (ISCs) via a cell-autonomous mechanism that is independent of Ink4a-Arf expression. Using high-throughput transcription and location analysis, we have dissected the direct transcriptional pathways regulated by PRC1 in ISC showing that PRC1 inactivation induces a loss of ISC identity as a result of a massive up-regulation of non-lineage specific transcription factors that can directly inhibit the transcriptional activity of the ß-Catenin/Tcf4 complex. Overall, we propose that PRC1 control the self-renewal of ISC by positively sustaining Wnt transcriptional activity also in the presence of oncogenic mutations that constitutively activate the Wnt pathway in intestinal tumors.

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38

PIVETTI, SILVIA. "POLYCOMB REPRESSIVE COMPLEX 1 IS REQUIRED TO MAINTAIN STEM CELL IDENTITY AND TO PRESERVE ADULT TISSUE HOMEOSTASIS". Doctoral thesis, Università degli Studi di Milano, 2020. http://hdl.handle.net/2434/688743.

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Abstract (sommario):

Polycomb Repressive Complex 1 (PRC1) is an evolutionary conserved transcriptional repressive complex, fundamental for lineage fate decisions and maintenance during development. PRC1 acts by depositing a moiety of Ubiquitin on lysine 119 on histone H2A, promoting nucleosome compaction and transcriptional repression. Cell identity is a fundamental feature, not only during development, but it has to be maintained throughout the entire individual lifespan. Importantly, loss of cell identity is associated with pathologies, primarily with cancer. We recently described that, in homeostatic condition, PRC1 loss of function in LGR5 expressing intestinal stem cells, led to severe defects in tissue maintenance due to loss of stem cell identity and stem cell self-renewal. This places PRC1 as a fundamental complex in stem cell preservation. In this context, PRC1 is necessary to repress non-lineage specific transcription factors, whose upregulation not only perturbs the transcriptome of stem cells, thus leading to identity loss, but also impairs the transcriptional control of the WNT pathway that is essential for stem cell self-renewal. The landscape is even more complicated by the evidence that tissue context plays a critical role in protein function. In pathological conditions, primarily in cancer, several reports have described opposite roles for epigenetic players, including PRC1 subunits, depending on the cancer type. This highlights the importance of context dependency for the correct choice of the therapeutic approach. This opens up the possibility that PRC1 function, or PRC1 activity loss, could be different among different tissue types. To address this point we evaluated PRC1 activity and function in a different LGR5 expressing stem cell populations that derives from a different embryonic layer, the hair follicle, analyzing the phenotype of loss of PRC1 activity in this context and comparing the transcriptional outcomes in the different stem cell populations to describe the influence of context on PRC1-loss. Our data support a general role of PRC1 in stem cell identity maintenance, that is accomplished through the regulation of the same targets. However, we show that, differently from intestinal stem cells, PRC1 activity loss in the hair follicle leads to the activation of a specific epidermal program, showing that the pool of transcription factors present in different stem cell population alters the transcriptional outcome of PRC1 loss. To add a layer of complexity to the Polycomb field and to its role in identity maintenance, PRC1 is composed by several subunits that define at least 5 different biochemical sub-complexes. These complexes are specified by 6 different mutually exclusive PCGF proteins (PCGF1-6) that determine the ancillary subunits composing the complexes. Their role in embryonic development is matter of several studies, however their involvement in adult tissue maintenance is still obscure. Exploiting different PCGFs conditional knock out mouse models we aim to address the specific role of different sub complexes in the maintenance of tissue homeostasis, in order to define the contribution of these Polycomb complexes in the phenotypic outcome observed in PRC1 loss of function intestinal and hair follicle LGR5 stem cells.

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39

Gleason, Emily Jean. "Conserved Genetic Modules Controlling Lateral Organ Development: Polycomb Repressive Complex 2 and ASYMMETRIC LEAVES1 Homologs in the Lower Eudicot Aquilegia (Columbine)". Thesis, Harvard University, 2013. http://dissertations.umi.com/gsas.harvard:10983.

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Development in multicellular organisms relies on establishing and maintaining gene expression profiles that give cells identity. Transcription factors establish gene expression profiles by integrating positional, temporal, and environmental cues to regulate genes essential for a cell's identity. These signals are often short lived while the differentiated state may persist for a long time. Epigenetic factors maintain these gene expression profiles by making heritable chemical alterations to target gene chromatin to stabilize transcriptional patterns. Here we explore the evolution and function of an epigenetic regulator, the Polycomb Repressive Complex 2 (PRC2), and a transcription factor, ASYMMETRIC LEAVES 1 (AS1) , in the lower eudicot Aquilegia. PRC2 is an important and deeply conserved epigenetic regulator, which is critical to many plant developmental processes, including the regulation of major developmental transitions and lateral organ development. We find that Aquilegia has a relatively simple complement of PRC2 genes that are expressed throughout development. Contrary to findings in other plant species, two members of the Aquilegia PRC2, AqSWN and AqCLF, are not imprinted in Aquilegia endosperm. Using virusinduced gene silencing (VIGS), we determined that Aquilegia PRC2 regulates aspects of lateral organ development, including branching within the leaf and lamina expansion, along with caroteinoid production in floral organs. PRC2 targeting of several floral MADS box genes may be conserved in Aquilegia, but other known targets such as the class I KNOX gene are not. AS1 is a transcription factor that plays a conserved role in controlling differentiation and polarity of lateral organs. In species with simple leaves, AS1 promotes cell determination by suppressing the expression of the class I KNOX genes in leaf primordia and regulates abaxial-adaxial polarity in the developing leaf. However, in species with compound leaves, KNOX genes and AS1 often work together to control leaflet initiation and arrangement. In Aquilegia, AqAS1 appears to primarily contribute to proper regulation of class I KNOX genes with a more minor role in leaflet polarity and positioning. Most interestingly, these combined datasets suggest that contrary to the widely held model, class I KNOX genes are neither necessary nor sufficient for leaf complexity in Aquilegia.

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Dimitrova, Emilia Atanasova. "Recruitment and interplay between distinct polycomb repressive complexes 1 in embryonic stem cells and during neural lineage commitment". Thesis, University of Cambridge, 2013. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.648170.

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41

Adhikari, Abhinav. "THE ROLE OF POLYCOMB REPRESSIVE COMPLEX-2 (PRC2) MEDIATED REGULATION OF SKELETAL MUSCLE PROLIFERATION AND DIFFERENTIATION BY JARID2". OpenSIUC, 2019. https://opensiuc.lib.siu.edu/dissertations/1739.

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Eukaryotic DNA is packaged into highly ordered structures knows as chromatin that further packs the DNA into higher-order structures, limiting the accessibility of the underlying genetic information for the processes like transcription, replication, and repair. However, eukaryotic cells have evolved proteins called chromatin regulators that regulate the accessibility of the genetic information when needed. This dissertation aims to characterize the role of two such proteins, JARID2 and the polycomb repressive complex-2 (PRC2), during skeletal muscle proliferation and differentiation.JARID2 is an inactive yet evolutionarily conserved histone demethylase that is shown to be a sub-stoichiometric component of the PRC2 complex. The PRC2 complex represses gene expression through the trimethylation of lysine 27 of histone 3 (H3K27me) tails. H3K27 methylation leads to chromatin compaction. JARID2 helps in targeting of the PRC2 complex to its target loci. JARID2 is shown to be required for the normal development of mice, as loss of Jarid2 leads to lethality in utero. We, for the first time, show that JARID2 is required for the normal skeletal muscle differentiation. We show that the JARID2 regulates the expression of myogenic regulatory factor, Myod1, both through direct repression and activation through the modulation of canonical Wnt signaling pathway. JARID2, in association with the PRC2 complex, represses Wnt antagonist Sfrp1 to modulate the activity of the canonical Wnt signaling pathway. The translocation of Wnt effector protein, b-catenin, from the cytoplasm to the nucleus modulates the activity of the canonical Wnt signaling pathway during activation. We also show that b-catenin directly regulates the expression of Myod1 gene through its direct binding in the distal regulatory region.We further extend the role of JARID2 during skeletal muscle proliferation. We show that JARID2 also plays an essential role in restraining the skeletal muscle proliferation through its direct repression of positive cell cycle regulators cyclin D1 (Ccnd1) and cyclin E1 (Ccne1). Furthermore, we show that retinoblastoma protein 1 (Rb1), a negative regulator of cell proliferation that promotes cell cycle exit and differentiation, is also directly regulated by JARID2 in PRC2 dependent manner. Together, we show that JARID2 precisely controls cell proliferation and differentiation during skeletal muscle differentiation.Further, we show that the regulation of cell proliferation by JARID2 is PRC2 complex dependent. When the PRC2 complex was depleted or inhibited to a modest level, the cells have an increased cell proliferation ability compared to severe loss or inhibition of EZH2, the catalytic subunit of the PRC2 complex, that leads to the apoptosis of the cells. It is also marked by increased expression of known PRC2 targets genes. We show that the increased proliferation upon modest inhibition or depletion of EZH2 is through direct de-repression of positive cell cycle genes, Ccnd1, and Ccne1. It is the first work that shows a context-dependent role of the PRC2 complex during skeletal muscle proliferation and differentiation.My dissertation also makes an extraordinary discovery as to why myogenin is required for the proper function of MyoD during skeletal muscle differentiation, even though both proteins share a large set of overlapping target genes. We show that myogenin is required for the nucleosome disassembly and reassembly at the target genes through recruitment of the FACT complex, a histone chaperone. We also show that myogenin is required for the assembly of the basic transcription machinery and RNA polymerase II to the target muscle genes during differentiation. Surprisingly, we also show that myogenin reinforces its own expression through the activation of Myod1 expression during skeletal muscle differentiation. Myogenin is a known target of MyoD.Taken together, this dissertation provides a molecular mechanism for the crosstalk between a signaling pathway with chromatin regulatory proteins, JARID2, and the PRC2 complex in regulating skeletal muscle differentiation. It also extends the role of JARID2 and the PRC2 complex - known oncogenes, in precise, context-dependent control of cell proliferation and differentiation in skeletal muscle.

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Deng, Hou Liang. "Characterizing a novel component of polycomb repressive complex 1 (PRC1) and the functions of CBX6 in breast cancer". Thesis, University of Macau, 2018. http://umaclib3.umac.mo/record=b3953848.

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43

Nowak, Agnieszka. "Étude structurale de la protéine Nurf55 : une chaperonne d'histone et une sous-unité de Polycomb Repressive Complex 2". Grenoble 1, 2009. http://www.theses.fr/2009GRE10194.

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Les gènes du groupe Polycomb (PcG) ont été identifiés chez D. Melanogaster. Leur rôle est de maintenir les états réprimés des gènes homéotiques. Les produits des gènes PcG agissent en complexes multi-protéiques, les Polycomb Repressive Complexes, qui interagissent au niveau de la chromatine affectant sa structure. L'objectif du projet a été l'étude structurale du Polycomb Repressive Complex 2 (PRC 2), qui possède une activité histone methyltransferase, spécifique pour la lysine 27 du histone H3. Ce manuscrit décrit les études structurales et fonctionnelles d'une sous-unité de PRC2, une chaperon d'histone Nurf55. Par cristallographie aux rayons X nous avons obtenu la structure atomique de Nurf55 en complexe avec un fragment hélical de l'histone H4 à une résolution de 1. 7Â. Nous avons identifié les acides aminés nécessaires à l'interaction de ces deux protéines puis validé les observations par calorimétrie isotherme à titration (ITC). Nous avons aussi observé que cette même région de Nurf55 interagit avec la partie N-terminale de l'histone H3 et que cette interaction implique deux sites. On a proposé un modèle d'interaction entre Nurf55 et un dimère composé des histones H3 et H4. Finalement, l'interaction de Nurf55 avec la partie N-terminale d'une autre protéine composante de PRC2, Su(z)12, a été charterisée. Cette interaction implique les mêmes résidus de Nurf55 responsables de l'interaction avec les histones H3 et H4. On propose l'hypothèse que Nurf55 interagirait avec une hélice a de la partie N-terminale de Su(z)12 d'une manière similaire qu'avec l'hélice a de l'histone H4 observée dans notre structure atomique
Polycomb genes (PcG) were first discovered by genetic screens in D. Melanogaster, where they act as repressors of key developmental genes, known as homeotic genes. The protein products of PcG genes form large multi protein complexes, called Polycomb Repressive Complexes, which regulate the expression of target genes via modification of the local chromatin structure and probably via direct interaction with the transcription machinery. The objective of the project described here was structural characterization of the Polycomb Repressive Complex 2 (PRC2), which has a histone methyltransferase activity specific for lysine 27 of histone H3. Ln this manuscript 1 present a high resolution X¬ray structure (1. 7 Â) of one of the PRC2 subunits, the histone-chaperone Nurf55, in complex with a fragment of histone H4. The importance of individual interactions observed in the crystal structure was confirmed by ITC measurements with mutant Nurf55 proteins and mutant H4 peptides. Additionally, a novel interaction between Nurf55 and the N¬terminal tail of histone H3 has been characterized. This interaction is mediated by two binding sites on the surface of Nurf55, one of which overlaps with the H4 binding site. Based on these observations we propose a model of how Nurf55 may bind the dimer of histones H31H4 and perform its role of a histone chaperone. We also found out that in the context of the PRC2 Nurf55 interacts with the N-terminal part ofanother subunit of the complex, protein Su(z)12 and that this interaction is mediated by the same binding pocket of Nurf55"which also mediates the binding of the H4 and H3 peptides

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Chang, Eun Hyuk. "The role of polycomb repressive complex 2 in postnatal subventricular zone neural stem/progenitor cell self-renewal and multipotency". Thesis, University of Oxford, 2012. http://ora.ox.ac.uk/objects/uuid:1ddbd108-0256-4a4a-b40a-35818197ca39.

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The murine subventricular zone (SVZ) in a brain contains a population of stem cells and daily produces tens of thousands of neurons throughout lifetime. However, the mechanisms of SVZ neural stem/progenitor cell (NSPC) maintenance, differentiation and cell-fate specification are still not clear. To understand these parameters via histone methylations with bivalent mechanism, the SVZ NSPCs were first isolated by using a culture technique called neurosphere assay (NSA). It has been a challenge to culture pure cell populations of SVZ subtypes, so the NSA was initially validated. The H3K27me3 mark, which has a dominant role in the bivalent mechanism, has not been studied in postnatal and adult SVZ in vivo, yet their role has been implicated to control the shift of embryonic cortical neurogenesis to gliogenesis. Therefore, we have first investigated whether H3K27me3 marks are present in the postnatal and adult SVZ NSPC population and whether their marks have been changed after stroke or demyelination in central nervous system (CNS) by immunohistrochemistry. With the confirmation of H3K27me3 mark present in SVZ NSPCs, the presence of H3K27me3 catalyzer, called polycomb repressive complex 2 (PRC2) core components (Eed, Ezh2, Suz12) including Jarid2, was investigated and confirmed in postnatal SVZ in vitro by qRT-PCR and Western blot. To understand the role of PRC2 enzymatic activity in postnatal SVZ neurosphere self-renewal and multipotency, Eed was down-regulated by using lentiviral mediated delivery of shRNA. Also, PRC2 dependent or independent function of Jarid2 was examined via knockdown approach. The lack of Eed in the neurospheres resulted the attenuation of self-renewal and oligodendrogenesis, whereas the Jarid2 knockdown neurospheres showed the decreased proliferation with no SVZ NSPC differentiation. Based on these knockdown studies, it suggests Eed and Jarid2 might not share their function in the postnatal SVZ NSPCs to govern postnatal SVZ NSPC self-renewal and multipotency.

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Poisot, Emilie. "Recrutement des complexes des groupes Polycomb et trithorax sur la chromatine : Dissection fonctionnelle des complexes liant les séquences d(GA)n des éléments de mémoire épigénétique". Versailles-St Quentin en Yvelines, 2010. http://www.theses.fr/2010VERS0022.

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Au moins trois mécanismes sont connus pour influer sur le maintien des profils d’expression des gènes: l’état chromatinien, les modifications épigénétiques liées aux protéines du groupe Polycomb (PcG) et Trithorax (trxG), et les ARN non codants (ARNnc). Les gènes du PcG et trxG maintiennent l’identité au cours du temps par des processus épigénétiques figeant un état transcriptionnel déterminé qui constitue la « mémoire cellulaire ». Les trois protéines Batman (BAN), Trithorax-like (GAF) et Pipsqueak (PSQ) font partie de complexes de mémoire cellulaire qui lient l’ADN. Le premier objectif de ma thèse était d’affiner la description des complexes contenant BAN, GAF et PSQ fixés sur leurs cibles. J’ai montré l’existence de plusieurs complexes, indiquant qu’il existe une combinatoire dans ces complexes en fonction de la cible. En réalisant une extinction ciblée de l’une ou l’autre de ces protéines, j’ai étudié leur hiérarchie de recrutement. L’élargissement de cette étude à d’autres protéines PcG et trxG a permis ensuite de modéliser le recrutement des différents complexes de mémoire. J’ai parallèlement étudié le lien existant entre la formation de l’hétérochromatine et les ARN non codants (ARNnc). A l’aide de mutants affectant des voies de production d’ARNnc, j’ai montré qu’ils participent à la localisation d’HP1 sur chromosomes polytènes et donc à la formation de hétérochromatine. L’ensemble de ces études a permis de caractériser la complexité des relations entre BAN, GAF et PSQ et de définir leur importance respective dans le recrutement différentiel de complexes PcG ou trxG, mais aussi de contribuer à établir le lien entre les ARNnc et la formation de l’hétérochromatine
At least three important mechanisms are known to maintain gene expression patterns: the chromatine state, the epigenetic modifications due to the Polycomb ( PcG) and Trithorax ( TrxG ) groups proteins, and non coding RNAs (ncRNAs). The PcG and trxG genes maintain cell identity through epigenetic modifications of chromatin, thereby maintaining a predefined transcriptional state that establishes "cell memory". Three proteins, i. E. Batman (BAN), Trithorax-like (GAF) and Pipsqueak (PSQ) are components of a DNA-binding cell memory complex. The first goal of my thesis was to refine the description of complexes containing BAN, GAF and PSQ bound to their targets. I showed the existence of several complexes, suggesting a combinatory composition of these complexes depending on the target. Using dsRNA-driven extinction of each of the three proteins, I determined the hierarchy of their recruitment. The extension of this approach to other PcG and trxG proteins lead us to propose a new model for the recruitment of several of the memory complexes. I also studied the link between heterochromatin formation and ncRNAs. By using mutants affecting pathways of ncRNAs production, I showed that they participate in the localization of the heterochromatin protein HP1 on polytene chromosomes and thus in the formation of heterochromatin. All these studies allowed us to characterize the complexity of relations between BAN, GAF and PSQ and to define their respective contribution in the differential recruitment of PcG or TrxG complexes, but also to contribute to establishing the link between the ncRNAs and heterochromatin

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Londhe, Priya V. "Unraveling the molecular mechanisms of the class II transactivator, CIITA in skeletal muscle". OpenSIUC, 2013. https://opensiuc.lib.siu.edu/dissertations/773.

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AN ABSTRACT OF THE DISSERTATION OF Priya Londhe, for the Doctor of Philosophy degree in Biochemistry and Molecular Biology, presented on 30th July, 2013 at Southern Illinois University Carbondale. TITLE: UNRAVELING THE MOLECULAR MECHANISMS OF THE CLASS II TRANSACTIVATOR IN SKELETAL MUSCLE MAJOR PROFESSOR: Dr. Judy Davie The inflammatory cytokine, interferon gamma, IFN-gamma orchestrates a diverse array of fundamental physiological processes and exhibits complex effects on myogenesis. IFN-gamma also induces the class II transactivator, CIITA, which is a critical mediator of IFN-gamma mediated repression and activation. The aims in my dissertation are directed towards understanding the role of IFN-gamma and CIITA in muscle. Stimulation by IFN-gamma in skeletal muscle cells induces CIITA expression as well as MHC class II gene expression. We show that the IFN-gamma induced inhibition of myogenesis is mediated by CIITA, which specifically interacts with myogenin. CIITA acts by both, repressing the expression and inhibiting the activity of myogenin at different stages of myogenesis. The IFN-gamma mediated repression is reversible, with myogenesis proceeding normally upon removal of IFN-gamma. We also show that CIITA is indispensible for the inhibition of myogenesis. To gain a mechanistic insight into the IFN-gamma induced repression of myogenesis, we have discovered that IFN-gamma and CIITA inhibit myogenesis by modifying gene regulation in a muscle cell subject to inflammation. We show that CIITA first interacts with JARID2, a non catalytic subunit of PRC2 complex, which induces a paused RNAPII, phosphorylated at serine 5 and then interacts with the catalytic subunit EZH2, in a JARID2 dependent manner. Our data show that both CIITA and IFN-gamma block myogenesis by the induction and recruitment of the PRC2 complex, which is normally silenced in a differentiating muscle cell. One of my dissertation aims sheds light on the silencing of CIITA in Rhabdomyosarcoma. Silencing of CIITA prevents the expression of MHC Class I and II genes. We have found that IFN-gamma signaling is intact in these cells, but pSTAT1 and IRF1 do not bind to the CIITA PIV promoter. The CIITA promoter is not hypermethylated in RD (ERMS) cells, but shows a modestly enhanced methylation status in SJRH30 (ARMS) cells. We have also observed that histone acetylation, which normally increases on the CIITA PIV promoter following IFN-gamma treatment, is blocked in both types of RMS cells. Further, our studies also impart a novel role for IFN-gamma and CIITA in inhibiting the IGF induced activation of muscle specific genes. Our data show that IFN-gamma does not block the signaling cascade of IGF. However, blocking exogenous IFN-gamma restores IGF activation of muscle specific genes. My dissertation also reveals an important role for the FACT complex in the early steps of gene activation through its histone chaperone activities that serve to open chromatin structure and facilitate transcription promoting muscle differentiation. We show that myogenin interacts with the FACT complex and the recruitment of FACT complex to muscle specific genes is dependent on myogenin. The final aim in my dissertation highlights the distinct binding profiles of the MRFs and E proteins during proliferation and differentiation. Our sequential ChIP assays show that MYOD, MYOG, and MYF5 co-occupy promoters. Taken together, my dissertation provides a comprehensive understanding of the molecular mechanisms during myogenesis and reveals the deleterious effects of chronic inflammation in skeletal muscle.

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Peruzzi, Pierpaolo. "MiR-128 controls the activity of Polycomb Repressor Complexes 1 and 2 in Neural Stem Cells: Implications of its loss in gliomagenesis". The Ohio State University, 2013. http://rave.ohiolink.edu/etdc/view?acc_num=osu1366055621.

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Siebold, Alexander Paul King. "Investigation into the role of Polycomb Repressive Complex 2 in the modulation of life span and stress resistance in Drosophila melanogaster". Case Western Reserve University School of Graduate Studies / OhioLINK, 2010. http://rave.ohiolink.edu/etdc/view?acc_num=case1274371273.

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Beckedorff, Felipe César Ferrarezi. "Recrutamento do complexo repressivo polycomb 2 pelo RNA não codificador longo antissenso ANRASSF1 modula a expressão do gene RASSF1A e a proliferação celular". Universidade de São Paulo, 2012. http://www.teses.usp.br/teses/disponiveis/46/46131/tde-23042013-083641/.

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O gene supressor tumoral RASSF1A tem sido associado com redução da proliferação celular em diversos tumores. Sua expressão é regulada por eventos epigenéticos que envolvem o complexo repressivo polycomb (PRC2), no entanto os mecanismos moleculares da modulação do recrutamento deste modificador epigenético para este locus ainda são desconhecidos. Neste trabalho identificamos e caracterizamos ANRASSF1, um RNA não codificador longo (lncRNA) intrônico unspliced, que é transcrito na fita oposta do gene RASSF1A, em várias linhagem celulares e tecidos, e se liga a PRC2. ANRASSF1 é transcrito pela RNAPII, possui cap-5´ e cauda poli-A, além de localizar-se no núcleo e possuir uma meia-vida em média quatro vezes menor comparada com outros lncRNAs ligados à PRC2. A super-expressão ectópica de ANRASSF1 reduziu os níveis de RASSF1A e aumentou a taxa de proliferação em células HeLa, enquanto seu silenciamento provocou efeito oposto. Essas mudanças nos níveis de ANRASSF1 não afetaram a abundância da isoforma RASSF1C em nenhuma das condições. A super-expressão de ANRASSF1 provocou um grande aumento tanto da ocupação de PRC2 como da marca de histona repressiva H3K27me3 especificamente na região promotora RASSF1A. Nenhum efeito da super-expressão de ANRASSF1 foi detectado na ocupação de PRC2 e na histona H3K27me3 nas regiões promotoras de RASSF1C e de outros quatro genes vizinhos, incluindo dois genes supressores tumorais bem caracterizados. Além disso, foi demonstrado que ANRASSF1 forma um híbrido de RNA/DNA e recruta SUZ12, um componente do PRC2, para o promotor de RASSF1A. Notavelmente, foi detectado pelo ensaio de RNase-ChIP que a degradação de ANRASSF1 diminui a ocupação de PRC2 neste promotor. Esses resultados demonstram um novo mecanismo de repressão epigenética do supressor tumoral RASSF1A, envolvendo um lncRNA unspliced antissenso, onde ANRASSF1 reprime seletivamente a expressão da isoforma de RASSF1 que sobrepõe o transcrito antissenso de modo local e específico. Considerando uma perspectiva mais ampla, nossos resultados sugerem que outros lncRNAs intrônicos unspliced não caracterizados no genoma humano podem contribuir para uma modulação epigenética local e específica de cada região em que os lncRNAs são transcritos.
Tumor-suppressor RASSF1A gene down-regulation has been implicated in increasing cell proliferation in several tumors. Its expression is regulated by epigenetic events involving polycomb repressive complex 2 (PRC2), however the molecular mechanisms modulating recruitment of this epigenetic modifier to the locus remain largely unknown. Here, we identify and characterize ANRASSF1, an endogenous unspliced long noncoding RNA (lncRNA) that is transcribed from the opposite strand of RASSF1 gene in several cell lines and tissues, and binds to PRC2. ANRASSF1 is transcribed by RNA Polymerase II, 5\'-capped, polyadenylated, displays nuclear localization, and has on average a four-fold shorter half-life compared to other lncRNAs that bind PRC2. ANRASSF1 ectopic overexpression decreases RASSF1A abundance and increases the proliferation rate of HeLa cells, whereas its silencing causes opposite effects. These changes in NRASSF1 levels do not affect RASSF1C isoform abundance. ANRASSF1 overexpression causes a marked increase both in PRC2 occupancy and in histone H3K27me3 repressive mark specifically at the RASSF1A promoter region. No effect of ANRASSF1 overexpression is detected on PRC2 occupancy and on histone H3K27me3 at the promoter regions of RASSF1C and of four other neighbor genes, including two well-characterized tumor suppressor genes. Additionally, we demonstrate that ANRASSF1 forms an RNA/DNA hybrid, and recruits SUZ12, a PRC2 component, to the RASSF1A promoter. Notably, depletion of ANRASSF1 disrupts SUZ12 occupancy on RASSF1A promoter as measured by RNAse-ChIP assay. Together, these results show a new mechanism of epigenetic repression of RASSF1A tumor suppressor gene involving an antisense unspliced lncRNA, in which ANRASSF1 selectively represses expression of the RASSF1 isoform overlapping the antisense transcript in a location-specific manner. In a broader perspective, our findings suggest that other non-characterized unspliced intronic lncRNAs transcribed in the human genome may contribute to a location-specific epigenetic modulation of genes.

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Schäfer, Vivien [Verfasser], Thomas [Gutachter] Bach, Frank-Dietmar Gutachter] Böhmer e Alice [Gutachter] [Fabarius. "Mutationen und Promotormethylierung des Polycomb Repressive Complex 2 bei akuter lymphoblastischer Leukämie im Kindesalte / Vivien Schäfer ; Gutachter: Thomas Ernst, Frank-Dietmar Böhmer, Alice Fabarius". Jena : Friedrich-Schiller-Universität Jena, 2017. http://d-nb.info/1177833867/34.

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