Les protéines du groupe Polybomb (PcG) sont conservées de la drosophile jusqu’à l’homme et assurent la « mémoire cellulaire » d’un état transcriptionnel réprimé au cours du développement. Un modèle a été proposé pour expliquer leur fonctionnement, qui propose que les deux principaux complexes, PRC1 et PRC2 (Polycomb Repressive Complexes 1 & 2), sont recrutés ensemble au niveau de séquences spécifiques appelés PREs (Polycomb Responsive Elements) où ils collaborent pour maintenir la chromatine dans un été réprimé.Au cours de ma thèse, j’ai voulu tester ce modèle en utilisant les disques imaginaux d’œil-antenne de drosophile, qui sont des structures larvaires préfigurant l’œil adulte. Étonnamment, alors que les mutants PRC1 et PRC2 présentent des phénotypes similaires dans l’embryon, seuls les clones mutants PRC1 présentent une transformation néoplasique et une surcroissance dans l’œil. Pour comprendre les mécanismes moléculaires qui sous-tendent ce découplage fonctionnel, nous avons réalisé des ChIP-Seq contre plusieurs marques d’histones actives et répressives, ainsi que contre des protéines du PcG. La comparaison de ces ChIP-Seq avec les profils embryonnaires a d’abord révélé un redéploiement majeur du PRC1 au stade larvaire, sur environ 1000 promoteurs actifs. Cette nouvelle classe de cibles, que nous avons appelée « Neo-PRC1 », se trouve au niveau de gènes actifs où la marque H3K27me3 normalement déposée par le PRC2 est remplacée par la marque active H3K27Ac. Ces gènes sont impliqués dans la régulation de la polarité, la prolifération ou encore la signalisation cellulaires, et un nombre substantiel d’entre eux est surexprimé dans les mutants PRC1, mais pas PRC2. Ces résultats suggèrent que l’activité suppresseur de tumeurs du PRC1 au stade larvaire découle de la régulation précise de gènes classiquement dérégulés dans les cancers, et ce en l’absence du PRC2En plus des sites situés sur des promoteurs actifs, nous avons détecté des sites PRC1 sans PRC2 au niveau de régions enrichies pour des marques de séquences amplificatrices (« Enhancers » en anglais) actives. Ces sites correspondent à des séquences amplificatrices spécifiquement actives au stade larvaire, et sont localisés à proximité de gènes codant pour des facteurs de transcription cruciaux pour le développement de l’œil, tels que les gènes du réseau de détermination de la rétine (RDGN). Pour mieux comprendre l’action du PRC1 sur ces cibles, j’ai réalisé des expériences de Hi-C (High-throughput Chromosome Conformation Capture) dans l’œil et l’embryon, révélant ainsi que ces séquences amplificatrices contactent les promoteurs proches spécifiquement au stade larvaire. De plus, la fréquence des contacts est positivement corrélée au niveau de PRC1 fixé. Étonnamment, ces gènes cibles sont sous-exprimés dans les mutants PRC1 mais pas dans les mutants PRC2, ce qui suggère que les contacts PRC1-dépendants entre ces séquences amplificatrices et leurs promoteurs cibles promeuvent la transcription. Pour vérifier cette hypothèse, j’ai étudié l’impact de la délétion via CRISPR de deux sites PRC1 impliqués dans une boucle régulatrice ; l’un situé au promoteur d’un gène du RDGN appelé dac et l’autre sur une séquence amplificatrice putative située en aval du gène. Des expériences de 3D-FISH révèlent que leur délétion entraîne la diminution des contacts entre la séquence amplificatrice et le promoteur, avec pour effet la sous-expression de dac. Ces résultats suggèrent que le PRC1 est impliqué dans la formation de boucles entre les séquences amplificatrices et leurs promoteurs cibles, et que cette topologie est nécessaire pour l’activation de ces gènes au cours du développement.Ma thèse a donc contribué à la découverte de nouvelles fonctions pour le PRC1, qui acquiert de nouvelles cibles au cours du développement et régule la transcription de gènes impliqués dans le cancer ou le développement indépendamment du PRC2, via des mécanismes dédiés. / Polycomb Group (PcG) are a set of highly conserved proteins implicated in cellular memory of transcriptional gene silencing throughout development. A classical model of PcG mode of action proposes that the two main Polycomb Repressive Complexes (PRC), PRC1 and PRC2, are co-recruited at specific DNA sequences called PREs (Polycomb Responsive Elements) where they collaborate to stably maintain a repressed chromatin state.My PhD work has challenged this collaborative model, by using as an experimental system the Drosophila larval Eye-Antennal imaginal Disc (EAD) that prefigures the adult eye. Surprisingly, while PRC1 and PRC2 mutants exhibit similar phenotypes in embryos, only PRC1 mutant clones show neoplastic transformation and massive overgrowth in EAD, while PRC2 mutant clones do not. To understand the molecular basis of this functional uncoupling, we generated ChIP-Seq directed against a large set of repressive and active Histone Marks (HTMs) as well as against core PcG proteins in EAD. A comparative analysis with Chip-Seq embryonic profiles firstly identified a massive de novo redeployment of PRC1 proteins at mostly 1000 active promoters that occurs only at larval stage. This new class of transcriptionally active PcG target genes, that we named “Neo-PRC1”, is devoid of the H3K27me3 epigenetic mark normally deposited by PRC2 and carry instead the active H3K27Ac mark. Moreover, this Neo-PRC1 category of PcG targets is enriched in ontologies linked to cell polarity, proliferation or signalling. A substantial subset of neo-PRC1 targets is up-regulated in PRC1 but not in PRC2 mutants, suggesting that the tumour-suppressor activity of PRC1 during Drosophila development might be exerted by fine-tuning the expression of cancer-related genes independently of PRC2.In addition to neo-PRC1 sites located at promoters, we next detected an enrichment of PRC1, but not PRC2, at regions enriched for active enhancer marks. These neo-sites which correspond to larval stage-specific enhancers are found in the vicinity of genes encoding for transcription factors playing a key role in EAD development, like genes implicated in the Retinal Determination Gene Network (RDGN). To understand the function of PRC1 at these enhancers, we performed comparative Hi-C (High-throughput Chromosome Conformation Capture) experiments between embryos and EADs, and discovered differential chromatin contacts occurring between the stage-specific neo-PRC1 enhancers and their closest promoters. The intensity of these 3D contacts is positively correlated with the PRC1-binding levels. Unexpectedly, in PRC1, but not in PRC2 mutants, these genes are down-regulated, suggesting that PRC1-dependent enhancer-promoter loops promote transcription. To study if larval 3D chromatin loops are PcG-dependent and functionally relevant, we analyzed the topological and transcriptional impact of two CRISPR-generated deletions affecting two PRC1 binding sites known to form a regulatory loop. These two PREs are respectively located close to the promoter and a putative 3’ enhancer of the dac locus encoding for a crucial member of the RDGN. 3D FISH experiments demonstrate that the removal of the dac endogenous PRC1 binding sites is sufficient to significantly decrease dac enhancer-promoter contacts as well as to trigger down-regulation of dac expression. Altogether, these results suggest that PRC1 might contribute to enhancer-promoter contacts at crucial developmental genes in EAD and that these PRC1-dependent long-range interactions could be necessary to allow a proper transcriptional induction during development.To summarize, my PhD project contributed in opening a new perspective, namely that in addition to conveying cellular memory, a main function of PcG correlates with a second wave of PRC1 recruitment during larval stage to subtly regulate and coordinate the expression of cancer-related and developmental genes through non-canonical molecular mechanisms.

At the beginning of mitosis, chromosomes are condensed and segregated to facilitate correct alignment later in cytokinesis. Condensin is the pentameric enzyme responsible for this DNA compaction and is composed of two structural maintenance of chromosomes (SMC) subunits and three non-SMC subunits. Condensin mutations generate chromosomal abnormalities due to improper segregation, leading to genome instability and eventual malignant transformation of the cell. Cdc2 phosphorylation of the non-SMC subunits, CAP-G, CAP-D2, and CAP-H, has been demonstrated to be important for condensin supercoiling activity and function. While these subunits are thought to be phosphorylated by Cdc2, the exact sites have not yet been identified and characterized. The basis of this research was to determine the Cdc2 phosphorylation sites in the CAP-G subunit of the condensin enzyme and to characterize the functional significance of the sites in the regulation of condensin activity using site-directed mutagenesis and immunofluoresence microscopy. While DNA condensation represents a critical step early in mitosis, formation of the mitotic spindle represents a vital event leading to the division of a cell into two daughter cells in a process known as cytokinesis. Protein regulating cytokinesis 1 (PRC1) is a mitotic protein essential for cytokinesis that participates in formation of the mitotic spindle in a phosphorylation dependent manner. PRC1 possesses microtubule bundling properties. Loss of PRC1 leads to mis-segregation of chromosomes and abnormal cytokinesis. HSF2 is a transcription factor known to be important in development and differentiation. Previous research has determined that HSF2 plays a significant mechanistic role in the process of hsp70i gene bookmarking during mitosis. Bookmarking is an epigenetic phenomenon whereby certain gene promoters remain uncompacted, in contrast to the majoritiy of genomic DNA during mitosis. This lack of compaction allows quick reassembly to a transcriptionally competent in G1 of the cell cycle and ensures the ability of the cell to induce expression of the cytoprotective hsp70i protein. HSF2 and PRC1 were found to interact in a yeast-two hybrid screen. Given the importance of both of these proteins during mitosis, this study seeks to characterize the HSF2/PRC1 interaction and determine the potential role for PRC1 in hsp70i gene bookmarking.

L’acquisition et le maintien de l’identité d’un neurone sont assurés par des facteurs de transcription terminaux exprimés durant toute la vie du neurone. Cependant le processus d’expression génique peut être très bruité. L’objectif de mon projet de thèse est de déterminer comment un neurone peut acquérir et maintenir son identité de manière fiable malgré ce bruit intrinsèque, en utilisant le modèle C. elegans. En combinant des techniques récentes d’ingénierie du génome par CRISPR et des méthodes d’imagerie quantitative in vivo, j’ai observé que l’expression endogène des facteurs de transcription terminaux est fortement bruitée. J’ai également établi que des mutations dans le complexe chromatinien PRC1 induisent une perte stochastique de l’identité de certains neurones au cours du temps. Le complexe PRC1 agit directement au sein des neurones. Il affecte le niveau d’initiation de l’expression des facteurs de transcription terminaux durant l’embryogenèse ainsi que la fiabilité de la maintenance de leur expression aux stades larvaires et adultes. En conclusion, mon travail suggère que le complexe PRC1 joue un rôle important dans la protection des neurones contre le bruit génique, les aidant ainsi à acquérir et maintenir de manière fiable leur identité. / The acquisition and maintenance of neuronal identity is driven by terminal transcription factors expressed throughout the life of the neuron. However, the gene expression process can be noisy. The aim of my PhD work is to determine how a neuron can acquire and maintain its identity in a reliable manner despite this intrinsic noise, using C. elegans as a model system. Combining recent techniques of genome engineering by CRISPR with in vivo quantitative imaging, I observed that the endogenous expression of terminal transcription factors is highly noisy. I also established that mutations in the chromatin complex PRC1 induce a stochastic loss of the identity of some neurons over time. The PRC1 complex directly acts in the neurons. It affects the levels of initiation of the terminal transcription factors during embryogenesis as well as the reliability of their maintenance at larval and adult stages. To conclude, my work suggests that the PRC1 complex plays an important role to protect neurons against gene expression noise, helping them to acquire and maintain their identity in a reliable manner.

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.

Cardiomyocyte perinatal development is characterized by the transition from a hyperplastic to a hypertrophic growth. We hypothesize that genetic factors and processes in the cardiac perinatal transitional program can be identified by a systematic analysis of different stages in heart development. Microarray expression patterning of mRNAs and microRNAs uncovered a perinatal cardiogenomic switch between 5 and 7 days post-birth. Gene ontology analysis revealed cellular and metabolic processes as highly representative Biological Processes. Moreover, approximately 40% of known mice transcription factors are significantly (p<0.05) fluctuating between embryonic day 19 and 10 days post-birth. As the heart matures, cardiomyocytes progressively exit cell cycle with day 5 as a pivotal point. Hypertrophy entails cardiomyocyte binucleation which may be promoted by Protein Regulator of Cytokinesis (Prc1) and its interactors. Temporal cardiac transcription expression analysis provides insight into underlining effectors within the cardiac perinatal transitional program as well as cardiac pathology.

Cardiomyocyte perinatal development is characterized by the transition from a hyperplastic to a hypertrophic growth. We hypothesize that genetic factors and processes in the cardiac perinatal transitional program can be identified by a systematic analysis of different stages in heart development. Microarray expression patterning of mRNAs and microRNAs uncovered a perinatal cardiogenomic switch between 5 and 7 days post-birth. Gene ontology analysis revealed cellular and metabolic processes as highly representative Biological Processes. Moreover, approximately 40% of known mice transcription factors are significantly (p<0.05) fluctuating between embryonic day 19 and 10 days post-birth. As the heart matures, cardiomyocytes progressively exit cell cycle with day 5 as a pivotal point. Hypertrophy entails cardiomyocyte binucleation which may be promoted by Protein Regulator of Cytokinesis (Prc1) and its interactors. Temporal cardiac transcription expression analysis provides insight into underlining effectors within the cardiac perinatal transitional program as well as cardiac pathology.

Cardiomyocyte perinatal development is characterized by the transition from a hyperplastic to a hypertrophic growth. We hypothesize that genetic factors and processes in the cardiac perinatal transitional program can be identified by a systematic analysis of different stages in heart development. Microarray expression patterning of mRNAs and microRNAs uncovered a perinatal cardiogenomic switch between 5 and 7 days post-birth. Gene ontology analysis revealed cellular and metabolic processes as highly representative Biological Processes. Moreover, approximately 40% of known mice transcription factors are significantly (p<0.05) fluctuating between embryonic day 19 and 10 days post-birth. As the heart matures, cardiomyocytes progressively exit cell cycle with day 5 as a pivotal point. Hypertrophy entails cardiomyocyte binucleation which may be promoted by Protein Regulator of Cytokinesis (Prc1) and its interactors. Temporal cardiac transcription expression analysis provides insight into underlining effectors within the cardiac perinatal transitional program as well as cardiac pathology.

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.

Cardiomyocyte perinatal development is characterized by the transition from a hyperplastic to a hypertrophic growth. We hypothesize that genetic factors and processes in the cardiac perinatal transitional program can be identified by a systematic analysis of different stages in heart development. Microarray expression patterning of mRNAs and microRNAs uncovered a perinatal cardiogenomic switch between 5 and 7 days post-birth. Gene ontology analysis revealed cellular and metabolic processes as highly representative Biological Processes. Moreover, approximately 40% of known mice transcription factors are significantly (p<0.05) fluctuating between embryonic day 19 and 10 days post-birth. As the heart matures, cardiomyocytes progressively exit cell cycle with day 5 as a pivotal point. Hypertrophy entails cardiomyocyte binucleation which may be promoted by Protein Regulator of Cytokinesis (Prc1) and its interactors. Temporal cardiac transcription expression analysis provides insight into underlining effectors within the cardiac perinatal transitional program as well as cardiac pathology.

京都大学 / 0048 / 新制・課程博士 / 博士(生命科学) / 甲第22864号 / 生博第447号 / 新制||生||59(附属図書館) / 京都大学大学院生命科学研究科高次生命科学専攻 / (主査)教授 北島 智也, 教授 上村 匡, 教授 豊島 文子 / 学位規則第4条第1項該当 / Doctor of Philosophy in Life Sciences / Kyoto University / DGAM