La rétine est constituée de plusieurs types de neurones incluant les cellules amacrines, ganglionnaires, bipolaires et les photorécepteurs. Les photorécepteurs, qui englobent les cônes et les bâtonnets, sont des neurones sensoriels hautement spécialisés qui permettent la conversion de la lumière en signaux électriques par le mécanisme de phototransduction. Les mécanismes moléculaires par lesquels les progéniteurs rétiniens (RPCs) se différencient en différents neurones spécialisés comme les photorécepteurs sont encore peu connus. Le gène Polycomb Bmi1 appartient à la famille des gènes Polycomb qui forment des complexes multimériques impliqués dans la répression de l’expression génique via le remodelage de la chromatine. Au niveau biologique, le gène Bmi1 régule, entre autre, le contrôle de la prolifération cellulaire, le métabolisme des radicaux libres, et la réparation de l’ADN. Récemment, il a été démontré que Bmi1 joue un rôle critique dans la prolifération et l’auto-renouvellement d’un groupe de RPCs immatures. De plus, Bmi1 est essentiel au développement post-natal de la rétine. L'objectif de cette étude est d'analyser le rôle de Bmi1 dans le développement et la survie des photorécepteurs chez la souris. Nos résultats révèlent un phénotype de dégénérescence des photorécepteurs de types cônes chez notre modèle de souris déficiente pour Bmi1. Les bâtonnets sont insensibles à la mutation. De plus, Bmi1 est exprimé de façon prédominante dans les cônes. Nos expériences de culture de cellules rétiniennes suggèrent que le phénotype est cellule-autonome. Par ailleurs, la co-délétion du gène Chk2, membre de la réponse aux dommages à l'ADN, permet de ralentir la progression du phénotype. Les rétines Bmi1-/- et Bmi1-/-Chk2-/- présentent une augmentation importante des dommages oxydatifs à l'ADN. Ces résultats suggèrent que le stress oxydatif pourrait jouer un rôle important dans la survie des cônes. L'étude du rôle du gène Polycomb Bmi1 dans les photorécepteurs est importante pour une meilleure compréhension des mécanismes contribuant à la survie des cônes et pourrait mener à la découverte de nouveaux traitements des maladies dégénératives des cônes. / The retina is composed of several types of neurons such as amacrin, ganglion, bipolar and photoreceptor cells. Photoreceptors, which include cones and rods, are highly specialized neurons that convert light into electrical signals by phototransduction. The molecular mechanisms involved in differentiation of retinal progenitors (RPCs) into specialized neurons such as photoreceptors are poorly understood. The polycomb gene Bmi1 is a member of the Polycomb gene family that forms multimeric complexes involved in chromatin remodeling leading to gene repression. Biological functions of Bmi1 include regulation of cell proliferation, free radical metabolism, and DNA repair. Recently, it was shown that Bmi1 plays a critical role in the proliferation and self-renewal of a specific immature RPC group. Moreover Bmi1 is essential for post-natal retinal development. The objective of the current study is to analyze Bmi1 function in photoreceptor development and survival. Our results show that Bmi1 deficiency in mice causes degeneration of cone photoreceptors, but not of rods. Furthermore, Bmi1 is predominantly expressed in cones. Experiments using primary retinal cell cultures suggest a cell-autonomous phenotype. In addition, codeletion of Bmi1 and the critical DNA damage response protein Chk2 resulted in partial rescue and slow-down of cone degeneration. Bmi1-/- and Bmi1-/-Chk2-/- retinas also exhibit an important increase in oxidative DNA damage, suggesting that cellular redox state could play an important role in cone survival. Our studies on the role of Bmi1 in photoreceptors elucidate the mechanisms contributing to cone survival, and could lead to the development of new treatments for cone degenerative diseases.

No description available.

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. / In multicellular organism, the identity of cell is determined by several factors playing on genesexpression. Once established, the gene expression pattern is transmitted to daughter cells through aprocess involving epigenetic mechanisms that locally reshape the structure of chromatin. In Drosophilamelanogaster, the Polycomb (PcG) and trithorax (trxG) group genes are involved in the maintenanceof gene expression profile during development. Inside multimeric complexes, PcG proteins maintaingenes in repressed state whereas TrxG maintain genes active. A third class of proteins, calledEnhancers of Trithorax and Polycomb, regulate PcG and TrxG activities. Dorsal Switch Protein 1(DSP1) is a High Mobility Group B protein acting as an ETP. But DSP1 has not yet been identified inPcG or TrxG complexes. On the basis of gel filtration analysis of protein complexes in embryo nuclearextracts, it appears that the majority of DSP1 is present in complex(es) from 100 kDa to 1MDa. Aimsof present work are the identification of DSP1 protein partners in drosophila embryo and thecharacterization of biochemical properties of DSP1. Firstly, I used immunopurification from drosophilaembryonic nuclear extracts. The proteins purified with DSP1 were characterized through sequencingof peptides from individual protein bands by mass spectrometry. Among identified proteins, wefocused on the DEAD Box RNA helicase, Rm62. The role of interaction between DSP1 and Rm62 hasbeen characterized. Secondly, I have identified a new physicochemical aspect of DSP1 protein.

Analyse fonctionnelle de la protéine Enhancer of Zeste, SlEZ2, chez la tomate, Solanum lycopersicumLes protéines Polycomb, initialement découvertes chez la drosophile, ont récemment caractérisées chez les plantes où elles remplissent des fonctions essentielles au cours du développement de la plante. Chez la drosophile, les protéines polycomb (PcG) agissent sous forme de trois complexes multi-protéiques : PRC1, PRC2 et PhoRC. Seulement, deux de ces complexes ont été identifiés chez les plantes : un orthologue fonctionnel du complexe PRC1 (PRC1-like) et PRC2. Le complexe PRC2 maintien la chromatine dans un état condensé et intervient dans le contrôle du développement des fleurs, des graines, des fruits et des feuilles. Chez la tomate Solanum lycopersicum, le complexe PRC2 est composé de trois protéines polycomb : SlEMF2 (EMbryotic Flower), SlFIE (Fertilization Independent Endosperm) and SlE(Z) (Enhancer of Zeste). Les protéines SlE(Z) portent l’activité histone méthyl transférase qui permet la mise en place de la marque répressive H3K27me3. Chez la plante modèle, Arabidopsis thaliana, cette marque joue un rôle essentiel au cours du développement de la plante Afin d’étudier le rôle du complexe PRC2 dans le développement du fruit et de la plante de tomate, et plus particulièrement de la protéine SlE(Z), nous avons identifié trois gènes codant les protéines SlE(Z) : SlEZ1, SlEZ2 et SlEZ3. Au laboratoire, il a récemment été montré que la protéine SlEZ1 intervient au cours du développement floral (How Kit et al., 2010). L’objectif de ce travail est de déterminer la fonction de la protéine SlEZ2 au cours du développement du fruit et de la plante de tomate. Pour cela, nous avons analysé des plantes transgéniques sous exprimant le gène SlEZ2, orthologue au gène CURLY LEAF d’A. thaliana, par stratégie RNAi. Ce travail indique que la protéine SlEZ2 est impliquée dans la croissance de la plante de tomate, ainsi que dans le développement des feuilles, des fleurs et des fruits. Les plantes transgéniques présentent des phénotypes pléiotropes tels que des fleurs et des feuilles modifiées, un fort taux d’avortement des fruits, des fruits de texture et de couleur altérées ainsi qu’une réduction de la taille des plantes. De plus, nous avons identifiés quatre gènes ciblés par la protéine SlEZ2 dont l’expression est dérégulée dans les feuilles. Il s’agit de deux gènes à MADS box, TAG1 et TAGL1, ainsi que de deux gènes KNOX, LeT6 et TKN4. / Functional analysis SlEZ2, a tomato Enhancer of zeste proteinPolycomb proteins, first discovered in Drosophila, have been identified in plants and play essential functions in plant development. In Drosophila, polycomb proteins (PcG) acts as a complex and three have been identified: PRC1, PRC2 and PhoRC. However, only two polycomb complexes have been identified in plants: like-PCR1 and PRC2. The PCR2 complex maintain chromatin in a closed state and control flower, seed, fruit and leaf development.In tomato Solanum lycopersicum, PRC2 is composed by three polycomb proteins SlEMF2 (EMbryotic Flower), SlFIE (Fertilization Independent Endosperm) and SlE(Z) (Enhancer of Zeste)(Enhancer of Zeste). SlE(Z) proteins have a methyltransferase activity that puts in place an repressive epigenetic mark a trimethylation of lysine 27 histone 3. In plant model, Arabidopsis thaliana, this mark plays an essential role in plant development but little is known about PRC2 role in plant and fruit development of tomato. In order to unravel the function of the E(z) protein in the control of tomato fruit and plant development, we have characterized three E(z) encoding genes, namely SlEz1, SlEz2 and SlEZ3. In a recent work, we reported that SlEZ1 protein plays a role in flower development (How Kit at al., 2010). The aim of this present study was to determine the function of the SlEZ2 protein in plant and fruit development. We present our results focusing on RNAi transgenic plants which underexpressed SlEZ2 gene, homologue of Curly Leaf Arabidopsis gene. This analysis indicates that SlEZ2 protein is implicated in tomato plant growth and affects also leaf, flower and fruit development. Phenotypes include abnormal flowers and leafs, fruit development abortion, altered fruit colour and texture and plant of reduced size. Moreover, we characterize four target genes of SlEZ2 genes in leaves which present a deregulated expression : TAG1, TAGL1, LeT6 and TKN4.

L’établissement et le maintien du patron d’expression génique sont d’une importance critique pour l’identité cellulaire. Les protéines du groupe Polycomb (PcG) agissent sur la chromatine afin de maintenir la répression génique de ses gènes cibles à travers les cycles cellulaires de façon épigénétique. Toutefois, durant la mitose, la structure de la chromatine est grandement altérée par la répression de la transcription, la condensation de la chromatine et le relâchement de nombreux facteurs de transcription. Une question se pose alors : comment les protéines PcG peuvent-elles maintenir leur fonction à travers la mitose ? En interphase, les protéines PcG sont liées à leurs cibles sur la chromatine. Durant la mitose, la majorité des protéines PcG se libèrent de la chromatine mais une petite fraction persiste. Selon l’hypothèse du mitotic bookmarking, cette fraction agirait comme un ensemble de marqueurs guidant le recrutement des protéines PcG en fin de mitose pour maintenir le profil d’expression génique de la cellule. Cependant, nous ne savons pas comment ce recrutement à lieu, ni comment une fraction de protéines PcG est retenue à la chromatine. Afin de répondre à ces questions, un crible à ARN interférent a été établi pour identifier des facteurs contrôlant la liaison des protéines PcG à la chromatine à travers le cycle cellulaire. Quoiqu’une confirmation soit nécessaire, les facteurs spécifiques à l’interphase sont enrichis en protéines co-purifiant avec la protéine PcG testée et en hélicases alors que ceux spécifiques à la mitose sont enrichis en candidats liés aux protéines du groupe Trithorax (TrxG). / A critical part of cell identity is the establishment and maintenance of gene expression patterns. Polycomb group proteins (PcG) act on chromatin to maintain gene repression through cell cycles (epigenetically). However, during mitosis, chromatin structure is greatly altered by transcription repression, chromatin condensation, and the release of many transcription factors. A question then arises: how can PcG proteins maintain their function through mitosis? During interphase, PcG proteins are bound to their chromatin targets. During mitosis, most PcG proteins are released from chromatin, but a small fraction remains bound to chromatin. According to the mitotic bookmarking hypothesis, this fraction acts as a set of markers to guide the recruitment of PcG proteins at the end of mitosis to maintain the gene expression profile. However, we do not know how this recruitment takes place, nor do we know how a fraction of PcG proteins is retained on chromatin. To address these questions, an RNAi screen was established to identify factors that control the binding of PcG proteins to chromatin across the cell cycle. Although a confirmation is necessary, factors identified from interphase cells were enriched in proteins co-purifying with the tested PcG protein and in helicases while mitosis specific factors were enriched in Trithorax group (TrxG) protein related candidates.

The neocortex is considered the seat of higher cognitive function in humans. It develops from a sheet of neural progenitor cells, most of which eventually give rise to neurons. This process of cell fate determination is controlled by precise temporal and spatial gene expression patterns that in turn are affected by epigenetic mechanisms including Polycomb group (PcG) regulation. PcG proteins assemble in multiprotein complexes and catalyze repressive posttranslational histone modifications. Their association with neurodevelopmental disease and various types of cancer of the central nervous system, as well as observations in mouse models, has implicated these epigenetic modifiers in controlling various stages of cortex development. The precise mechanisms conveying PcG-associated transcriptional repression remain incompletely understood and are an active field of research. PcG activity appears to be highly context-specific, raising the question of species-specific differences in the regulation of neural stem and progenitor regulation. In this review, we will discuss our growing understanding of how PcG regulation affects human cortex development, based on studies in murine model systems, but focusing mostly on findings obtained from examining impaired PcG activity in the context of human neurodevelopmental disorders and cancer. Furthermore, we will highlight relevant experimental approaches for functional investigations of PcG regulation in human cortex development.

Polycomb-group (Pc-G) proteins regulate homeotic gene silencing associated with the repressive covalent histone modification, trimethylation of histone H3 lysine 27 (H3K27me3). Pc-G mediated silencing is believed to remodel chromatin, rendering target genes inaccessible to transcription factors. Pc-G mediated silencing might result in irreversible changes in chromatin structure, however, there has been little analysis addressing whether Pc-G mediated silencing is reversible. In this work we focused on CURLY LEAF (CLF), the first Pc-G homologue discovered in Arabidopsis. CLF mediated repression of the floral homeotic gene AGAMOUS (AG) was challenged during early and late leaf development. AG was activated by the late leaf promoter, revealing that Pc-G mediated silencing can be overcome in old leaves in the presence of CLF. AG was also activated in young leaf primordia, yet did not persist in older leaves, revealing that transient activation of a Pc-G target is not epigenetically stable. To address the mechanism of Pc-G action within an endogenous environment, the histone dynamics at the APETALA1 (AP1) locus were characterized by Chromatin Immunoprecipitation. Unexpectedly, we found that the activation of AP1 in leaves did not require the removal of H3K27me3, questioning whether H3K27me3 is sufficient to silence. The roles of CLF in leaf and flower development are masked due to partial redundancy with SWINGER (SWN). clf- swn- mutants form a callus-like mass on sterile-tissue culture with no distinguishable plant organs. The role of CLF in regulating flowering time in natural populations of A. thaliana was investigated by complementing clf- mutants with CLF alleles from two accessions. We found that natural variation in CLF did not affect flowering time. To dissect the roles of CLF and SWN in late leaf and flower development, two approaches were developed for targeted expression. Firstly, CLF was introduced into the LhG4/ pOp transactivation system to provide CLF during early plant development. For mosaic analysis, CLF was introduced into the CRE lox recombination system in order to create clf- sectors surrounded by CLF+ SWN+ and CLF+ swn- cells.

During tissues development multipotent progenitor cells establish tissue-specific gene expression programmes, leading to differentiation into specialized cell types. It has been previously shown that the transcription factor p63, a master regulator of skin development, controls the expression of adhesion molecules and essential cytoskeleton components. It has also been shown that p63 plays an important role in establishing distinct three-dimensional conformations in the Epidermal Differentiation Complex (EDC) locus (Fessing et al., 2011). Here we show that in p63-null mice about 32% of keratinocytes showed altered nuclear morphology. Alterations in the nuclear shape were accompanied by decreased expression of nuclear lamins (Lamin A/C and Lamin B1), proteins of the LINC complex (Sun-1, nesprin-2/3) and Plectin. Plectin links components of the nuclear envelope (nesprin-3) with cytoskeleton and ChIP-qPCR assay with adult epidermal keratinocytes showed p63 binding to the consensus binding sequences on Plectin 1c, Sun-1 and Nesprin-3 promoters. As a possible consequence of the altered expression of nuclear lamins and nuclear envelope-associated proteins, changes in heterochromatin distribution as well as decrease of the expression of several polycomb proteins (Ezh2, Ring1B, Cbx4) has been observed in p63-null keratinocytes. Moreover, recent data in our lab have showed that p63 directly regulates Cbx4, a component of the polycomb PRC1 complex. Here we show that mice lacking Cbx4 displayed a skin phenotype, which partially resembles the one observed in p63-null mice with reduced epidermal thickness and keratinocyte proliferation. All together these data demonstrate that p63-regulated gene expression program in epidermal keratinocytes includes not only genes encoding adhesion molecules, cytoskeleton proteins (cytokeratins) and chromatin remodelling factors (Satb1, Brg1), but also polycomb proteins and components of the nuclear envelope, suggesting the existence of a functional link between cytoskeleton, nuclear architecture and three dimensional nuclear organization. Other proteins important for proper epidermal development and stratification, are cytokeratins. Here, we show that keratin genes play an essential role in spatial organization of other lineage-specific genes in keratinocytes during epidermal development. In fact, ablation of keratin type II locus from chromosome 15 in epidermal keratinocytes led to changes in the genomic organization with increased distance between the Loricrin gene located on chromosome 3 as well as between Satb1 gene located on chromosome 17 and keratin type II locus, resulting in a more peripheral localization of these genes in the nucleus. As a possible consequence of their peripheral localization, reduced expression of Loricrin and Satb1 has also been observed in keratins type II-deficient mice. These findings together with recent circularized chromosome conformation capture (4C) data, strongly suggest that keratin 5, Loricrin and Satb1 are part of the same interactome, which is required for the proper expression of these genes and proper epidermal development and epidermal barrier formation. Taken together these data suggest that higher order chromatin remodelling and spatial organization of genes in the nucleus are important for the establishment of lineage-specific differentiation programs in epidermal progenitor cells. These data provide an important background for further analyses of nuclear architecture in the alterations of epidermal differentiation, seen in pathological conditions, such as psoriasis and epithelial skin cancers.

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.

Li, Chi Han Samson. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 158-175). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.