Thèses sur le sujet « H3K4me1 »

La sindrome di Kabuki (KS) è una patologia caratterizzata da anomalie congenite multiple. Sintomi tipici sono anomalie scheletriche, disabilità cognitiva lieve-moderata e difetti nello sviluppo cranio-facciale. Recentemente, i geni KMT2D e KDM6A sono stati identificati come geni causativi della malattia nel 60-80% dei casi. Questi geni codificano enzimi che modificano gli istoni e sono parte del complesso multiproteico COMPASS-like MLL4. Questo complesso è respondabile del rimodellamento della cromatina delle regioni enhancers. Uno dei maggiori ostacoli per lo studio dei meccanismi molecolari della patologia è la mancanza di modelli sperimentali, sia in vitro che in vivo. Per poter comprendere gli effetti causati dalle mutazioni sul gene KMT2D, proponiamo lo sviluppo di un modello cellulare della patologia attraverso l’utilizzo di techniche di genome editing. Abbiamo deciso di concentrarci sul gene KMT2D, responsabile della monometilazione di H3K4, perchè è stato trovato più frequentemente mutato nei pazienti Kabuki. Come modello cellulare, abbiamo utilizzato le cellule staminali mesenchimali (MSC) perchè sono i precursori di osteoblasti ed condrociti, cellule da cui derivano due dei maggiori tessuti alterati nella patologia (ossa e cartilagini). Attraverso la tecnologia CRISPR/Cas abbiamo introdotto mutazioni sul gene KMT2D. Queste mutazioni portano alla produzione di una forma tronca della proteina che manca del dominio metiltransferasico. Le cellule mutate mostrano livelli ridotti di H3K4me1, ma non di H3K4me3, confermando il ruolo di mono-metilasi di KMT2D. Analizzando il fenotipo delle cellule mesenchimali nello stato indifferenziato, abbiamo osservato solo piccole differenze presenti nelle cellule mutate rispetto alle cellule non mutate. In particolare sono presenti alterazioni nella morfologia. Infatti, le cellule mutate sono più piccole e hanno un citoscheletro di actina meno strutturato. Parallelamente, abbiamo osservato che le cellule mutate non sono in grado di completare il differenziamento condrocitario, sia in termini di morfologia ma anche in termini di produzione della matrice extracellulare. Considerando anche che le cellule staminali mesenchimali mutate hanno un’alterata espressione dei fattori di trascrizione responsabili per il differenziamento condrocitario, noi ipotiziamo che uno sbilanciamento dell’attività di KMT2D causi un’alterazione strutturale e trascrizionale nelle cellule staminali mesenchimali che, a loro volta, non sono poi in grado di differenziare completamente in condrociti. La difficoltà delle cellule mutate nel completare il differenziamento condrocitario è anche confermato dall’analisi del ciclo cellulare. Infatti, le cellule mutate non sono in grado di uscire dalla fase S del ciclo cellulare, che è un passaggio importante durante il differenziamento condrocitario. Il coinvolgimento di KMT2D nel differenziamento condrocitario e nella patologia è inoltre confermato in vivo poichè la down regolazione, mediata da morfolino, di KMT2D causa alterazioni nello sviluppo craniofacciale nel modello animale di medaka. In conclusione, abbiamo sviluppato un modello in vitro della sindrome di Kabuki il quale mostra alterazioni nelle cellule staminali mesenchimali che a loro volta non sono in grado di differenziare in condrociti, i cui tessuti che ne derivano sono alterati nella patologia. Il modello animale, inoltre, conferma questo risultato mostrandosi utile per confermare e rinforzare successivi studi. Questo modello non solo può essere utile per lo studio della patologia, ma potrebbe anche essere utilizzato per lo studio di approcci terapeutici.
Kabuki Syndrome (KS) is a rare multiple malformation disease characterized by intellectual disability, short stature and peculiar facial gestalt. Recently, mutations of KMT2D and KDM6A genes have been identified as causative genes in 60 to 80% of KS cases. These two genes encode for histone modifying enzymes that are specific subunit of the COMPASS-like MLL4 complex, which has been described to possess a gene-specific function by modulating the chromatin state of enhancers. The lack of any in vitro or animal disease model for KS represents a major obstacle to understand the mechanisms by which KMT2D and KDM6A gene alterations causes the disorder. We propose the development an in vitro disease model of KS through CRISPR/Cas9 system. In particular, we focused our attention ok KMT2D, a mono methyltransferase of H3K4, because it was found mutated in the majority of Kabuki patients. We used mesenchymal stem cells (MSCs) as cellular model since they are able to differentiate into osteocyte and chondrocyte, whose derived tissues are affected in Kabuki patients. In these cells, we introduced frame shift mutations that lead to the formation of a truncated form of KMT2D protein which lose the catalytic domain. Mutated MSCs show a reduction in the H3K4me1 level, but not in H3K4me3, confirming the role of KMT2D as mono methyltransferase. Analyzing the phenotype of undifferentiated MSCs, very slight differences are present between WT and mutated cells. Mutated cells appear smaller and with a less structured actin cytoskeleton. Also, KMT2D mutations impair iMSCs differentiation through chondrocyte lineages. Indeed these cells fail in chondrocyte differentiation, in terms of morphology and in terms of synthesis of extracellular matrix. Considering also that mutated iMSCs show an altered expression of chondrogenic specific transcription factors, we hypothesize that KMT2D impairment cause an alteration in undifferentiated stem cells structure and transcriptional program that, in turn, alters the differentiation process. The altered differentiation process is also confirmed by the cell cycle analysis that reveals how mutated cells are not able to exit from cell cycle, an important step during chondrogenesis. The involvement of KMT2D in chondrocyte lineage, and also in the pathology, was also confirmed in vivo because morpholino mediated down-regulation of KMT2D results in aberration of craniofacial development of medaka animal model. In conclusion, we developed a tool that will allow us to study at the molecular level the effects of KMT2D frame-shift mutations both in the undifferentiated state of MSCs but also during the differentiation process. Moreover, our results could be reinforced and confirmed in the medaka animal model. These models could be therefore a good candidate for the study of disease pathogenesis but also for drug screening approaches.

Des études récentes ont mis en évidence qu’au moins 70% du génome humain est transcrit et produit une myriade d’ARN non codants. Au début de ma thèse j’ai utilisé des données de RNA-Seq sens-spécifique pour identifier les transcrits divergents dans les tissus primaires de souris. J’ai utilisé aussi des données ChIP-Seq afin d’analyser leurs caractéristiques épigénétiques. Nous avons trouvé que la transcription divergente est associée de manière significative à des gènes liés à la régulation de la transcription et le développement.Dans un deuxième temps, je me suis intéressé à l'identification et la caractérisation des lncRNA chez l'homme. J’ai appliqué des approches statistiques pour quantifier leur expression et identifier ceux qui sont (dé)régulés dans un contexte normal ou leucémique Dans un troisième temps. Au cours de ma thèse, je me suis attaché à étudier le mécanisme moléculaire épigénomique ainsi qu'à développer un pipeline bioinformatique permettant d'identifier les gènes (codant ou non codant) associés à des profils H3K4me2/3 étendus. Ainsi, j’ai mis en évidences que ces profils étendus étaient directement dépendants d'un processus transcriptionelle impliquant des nouveaux mécanismes de régulation. Cette étude a donné aussi lieu à une publication dont je suis cosignataire en premier auteur. (Zacarias, Belhocine et al. Journal of Immunology 2015). Cette nouvelle approche devrait s'avérer très utile dans d'autres modèles développementaux et/ou pathologiques et peuvent être utilisé comme outil de prioritisation des candidats les plus relevant dans des approches plus globale
Recent studies indicate that at least 70% of the human genome is transcribed into a myriad of both coding and non-coding RNAs. at the beginning of my thesis I used RNA-Seq data to identify divergent transcripts in mouse primary tissues. I also used the ChIP-Seq data to analyze their epigenetic characteristics. The results demonstrated that divergent transcription was significantly associated with genes related to transcription regulation and development. In a second phase, I was interested in the LncRNAs identification and characterization during the development of human T lymphocytes and in T acute lymphoblastic leukemia (T-ALL). I applied statistical approaches to quantify their expression and identify those that are regulated in a normal or leukemic contextSubsequently, I determined the most appropriate approach to prioritize the functional role of LncRNAs. Indeed, I focused on studying the molecular epigenomic mechanism marking and developed a bioinformatics pipeline to identify genes (coding or non-coding) associated with the extended profiles of H3K4me2/3. Evidence generated through the pipeline demonstrated that these extended profiles were directly dependent on specific transcriptional process involving new regulatory mechanisms.In conclusion, this body of work has resulted in a more comprehensive approach to determining the true functional role of LncRNAs in both normal biological context and in human disease

During multicellular development, cells must make fate decisions that reproducibly generate the correct cell type proportions. It is remarkable that in certain developmental scenarios, seemingly identical cells in a homogenous environment can achieve this. It is thought that this is possible because cell populations exhibit reproducible cell-cell variation in gene expression. How these differences are generated has been intensely studied over the past decade, with transcriptional bursting emerging as an important factor for driving variability between cells. Furthermore, it is thought that chromatin structure around gene promoters is a key regulator of transcriptional bursting. However, key questions remain. What factors regulate chromatin structure at the molecular level? Is the activity of chromatin regulators governed by random processes or entrained by one of many hidden factors such as cell cycle positioning, cell volume, metabolism? Are the proportions of cells exhibiting different bursting patterns regulated to ensure normal cell fate choice and proportioning? To address these questions, we have investigated whether different regulators of chromatin structure affect the pre-stalk/pre-spore fate decision in the social amoebae D. discoideum. We have identified that set1, a methyl-transferase responsible for generating methylation on histone 3 at position lysine 4 (H3K4me), plays a key role in controlling the balance of cell types in multicellular development as in its absence cells become autonomously primed towards a pre-stalk fate. Single cell RNA-sequencing has revealed that genes normally regulated by this modification represent a specific class of hyper-variable genes. We find that this variability is generated by specific set1 dependent repression at these loci, as upon deletion of this enzyme we see an active recruitment of more cells to an expressing state. Our data suggest that set1 activity itself is controlled by the external source of the cell cycle. This cell cycle dependent regulation robustly ensures the correct proportions of cells within the population contain levels of set1 activity that prime 25% of cells towards the pre-stalk lineage and the other 75% to the pre-spore fate. As such we believe our study reveals a novel mechanism linking specific regulation of transcriptional bursting through the activity of set1 to cell fate propensity.

Neural stem cells (NSCs) participate in a delicate balance between maintaining cellular identity through self-renewal and differentiating into myriad neural cell types. Understanding exactly how epigenetic mechanisms regulate this balance and the subsequent differentiation process in adult mammalian brain is an ongoing effort. We conducted a genome wide association study to elucidate the roles of genes in neural progenitors regulated by chromatin modifications. Neural progenitors of baboon SVZ were examined using ChIP-Seq (chromatin immuneprecipitation followed by deep sequencing) to determine genome wide gene targets of three histone modifications: H3K4me3, H3K9me3 and H3K27me3. Our data suggest these chromatin marks are associated with genes responsible for cellular organization and morphology, proliferation and survival, neuron development. Taken together these processes suggest histone modifications, predominantly H3K27me3, are responsible for maintenance of NSC identity. Our findings also highlight the importance of using in vivo models to study the SVZ neurogenic niche and compel examination of the H3K27me3 catalyzing enzyme EZH2. In the future, the role of EZH2 will be determined by EZH2 conditional knockout and overexpression models, using stereotaxic injections of novel Cre protein and lentiviral delivery of EZH2, respectively.

Leaf senescence, which is the final process of leaf development, involves a complex regulation of thousands of genes to recover and recycle valuable nutrients and mobilize them to growing part of the plant for high yield of fruits and grains. A greater understanding of the complex senescence gene regulation could be helpful for higher crop yield. This study is focused on three genes (ATX1, ATX3 and ATX4) that code for H3K4me3 methyl transferases to investigate their effect on flowering transition time, and their importance during senescence by assaying total chlorophyll and protein levels, and quantifying the mRNA expression of senescence marker gene WRKY75. An additive early flowering phenotype was observed for double mutants. However, no senescence alteration was found for double mutants. An increased level of total chlorophyll was shown by single mutant atx4. Significant differences for total protein were observed in leaf 6 vs. leaf 7 for double mutants atx1atx3 and atx1atx4, suggesting a faster protein degradation rate or smaller variability (reduced confidence interval) in leaf 7 data. Due to the gene redundancy of the ten-member ATX family, knocking out two genes may not adequately affect the function of H3K4 methyltransferase activity. Therefore, phenotypic analyses of triple and quadruple mutants of senescence-expressed H3K4me3 methyltransferase coding genes may show stronger senescence phenotypes. Of importance, these data show that significantly early flowering does not dictate early leaf senescence.

Increasing evidences demonstrate that adapting to different environmental conditions is mediated by epigenetic changes, which can participate in cellular processes. In particular, the adaptation to the different caloric intakes is of great relevance as it is crucial for the organism’s fitness. Moreover, the phenotypic remodeling induced by different diets determine the susceptibility to life-threatening diseases. For example, refined sugar, fat and meat enriched diet, typical of Western countries, is thought to be responsible for about 30-35% of cancer cases, in addition to increased incidence of type 2 diabetes and cardiovascular diseases. On the other hand, caloric restriction has been shown to be the most powerful way to prolong lifespan and reduce cancer incidence in different experimental models. Based on the hypothesis that epigenetic changes represents the mechanistic link between diet and disease risk, the aim of this work is to investigate chromatin modifications induced by different diets in murine models to identify specific epigenetic profiles associated with fat enriched diets and caloric restriction. For this purpose, 8 weeks old C57Bl/6 female mice were divided in three groups and fed for 10 months with 3 different diets: Standard laboratory mouse Diet, Calorie Restriction without malnutrition, High Fat Diet. Then, livers were extracted and investigated by chromatin immunoprecipitation (anti-H3K4me3, anti-H3K27me3) and transcriptomic approach for gene expression analysis. Despite the presence of moderate technical and biological variability, data analysis demonstrated that specific epigenetic profiles were associated to different diets. In particular, the distribution and frequency of H3K4me3 enabled the clustering of samples by diet-group. Moreover, functional annotation of genes showing an increased signal of H3K4me3 for HF or CR respect to SD on their promoter regions, resulted in significantly enriched “Type II diabetes mellitus”, for which obesity represents a critical risk factor, and “Circadian Rhythm” pathways, whose known to affect longevity. At mechanistic level, two DNA motifs related to the transcription and chromatin regulators ZSCAN4 and REST/NRSF were found enriched in correspondence of the regulative regions of the genes of the aforementioned pathways, suggesting these factors mediate the effects of diet on chromatin and gene expression.

Cells within multicellular organisms share the same genetic information, yet their shape and function can differ dramatically. This diversity of form and function is established by differential use of the genetic information. Early embryonic development describes the processes that lead to a fully differentiated embryo starting from a single fertilized cell - the zygote. Interestingly, in metazoan species this early development is governed by maternally provided factors (nutrients, RNA, protein), while the zygotic genome is transcriptionally inactive. Only at a specific developmental stage, the zygotic genome becomes transcriptionally active, and zygotic transcripts drive further embryonic development. This major change is called zygotic genome activation (ZGA). While major regulators of activation of early zygotic genes could be identified recently, the molecular mechanisms that contribute to robust global genome activation during embryonic development is not fully understood. In this study, I investigated whether the establishment of histone H3 lysine 4 trimethylation (H3K4me3) is involved in zebrafish zygotic transcription activation and early embryonic development. H3K4me3 is a chromatin modification that is implicated in transcription regulation. H3K4me3 has been shown to be enriched at Transcription Start Sites (TSS) of genes prior to their activation, and is postulated facilitate transcription activation of developmentally important genes. To interfere with H3K4me3 establishment, I generated histone methyltransferase mutants. I further inhibited H3K4me3 establishment by introduction of histones with lysine 4-to-methionine (K4-to-M) substitution, which act as dominant-negative inhibitors of H3K4me3 establishment. Upon H3K4me3 reduction, I studied the resulting effect on early transcription activation. I found that H3K4me3 is not involved in transcription activation during early zebrafish embryogenesis. Finally I analyzed possible cues in DNA sequence and chromatin environment that might favor early H3K4me3 establishment. These studies show that H3K4me3 is established during ZGA, yet it is not involved in transcription activation during early zebrafish development. Establishment of H3K4me3 might be a consequence of histone methyltransferase recruitment during a permissive chromatin state, and might be targeted to CpG-rich promoter elements that are enriched for the histone variant H2A.z
Jede Zelle eines multizellulären Organismus enthält dieselbe Erbinformation, und doch können Form und Funktion von Zellen untereinander sehr unterschiedlich sein. Diese Diversität wird durch unterschiedliches Auslesen - Transkribieren - der Erbinformation erreicht. Embryogenese beschreibt den Prozess, der aus einer einzelnen Zelle - der Zygote - einen multizellulären Embryo entstehen lässt. Interessanterweise laufen frühe Stadien der Embryogenese ohne Transkription der embryonalen Erbinformation ab, sondern werden durch maternal bereitgestellte Faktoren ermöglicht. Erst nach einer spezies-spezifischen Entwicklungsphase wird das Erbgut der Zygote aktiv transkribiert und ermöglicht die weitere Embryonalentwicklung. Obwohl bereits wichtige Regulatoren dieser globalen Genomaktivierung identifiziert werden konnten, sind viele molekulare Mechanismen, die zur Aktivierung des zygotischen Genoms beitragen, bisher unbekannt. In der hier vorliegenden Doktorarbeit habe ich die Rolle von Histon H3 Lysin 4 Trimethylierung (H3K4me3) während der frühen Embryogenese des Zebrafischs untersucht. H3K4me3 ist eine Chromatinmodifikation, die mit aktiver Transkription in Verbindung gebracht wird. H3K4me3 ist an Transkriptions-Start-Stellen von aktiv ausgelesenen Genen angereichert und es wird vermutet, dass diese Modifikation das Binden von Transkriptionsfaktoren und der Transkriptionsmaschinerie erleichtert. Während meiner Arbeit habe ich durch Mutation verschiedener Histon-Methyltransferasen beziehungsweise die Überexpression eines dominant-negativen Histonsubstrats versucht, die Etablierung von H3K4me3 in frühen Entwicklungsstadien des Zebrafischs zu verhindern. Anschliessend habe untersucht, welchen Effekt H3K4me3-Reduktion auf Tranksriptionsaktivität entsprechender Gene hat. Allerdings konnte ich keinen Zusammenhang zwischen H3K4me3-Reduktion und Transkriptionsaktivität beobachten. Um herauszufinden, weshalb H3K4me3 dennoch während früher Embryonalstadien etabliert wird, habe ich nachfolgend untersucht, ob möglicherweise bestimmte DNASequenzen oder Chromatin-Modifikationen zur Etablierung von H3K4me3 wahrend der Embryogenese des Zebrafischs beitragen. Aus der hier vorliegenden Arbeit lässt sich schlussfolgern, dass H3K4me3 in Tranksriptionsaktivierung während früher Embryonalstadien des Zebrafischs nicht involviert ist. Möglicherweise wird H3K4me3 in diesen Stadien in einer permissiven Chromatinumgebung etabliert, bevorzugt an Promotoren mit starker H2A.z-Anreicherung und CpG-reichen DNA-Elementen

Orientador: Luiz Fernando Rolim de Almeida
Resumo: Plantas que presenciam a seca podem armazenar informações sobre esta experiência, tal como uma memória ao estresse. As informações adquiridas com a memória do estresse podem ser utilizadas para aumentar a tolerância a futuros eventos de défice hídrico, porém, o crescimento das plantas pode ser limitado. O objetivo deste estudo foi investigar as respostas fisiológicas, bioquímicas e epigenéticas de Sorghum bicolor (L.) Moench durante e após o primeiro e segundo evento de défice hídrico, bem como as vantagens e desvantagens de eventos recorrentes de seca para a tolerância e crescimento. Neste estudo foram utilizados quatro tratamentos que foram: Controle, onde a irrigação foi mantida, Défice Hídrico na Fase Juvenil, onde as plantas foram submetidas a desidratação na fase juvenil seguido de reidratação, Défice Hídrico na Fase Adulta, onde a irrigação foi suspensa na fase adulta seguido de reidratação e Défice Hídrico Recorrente, onde a irrigação foi suspensa na fase juvenil e adulta seguido de reidratação. Foram avaliadas as trocas gasosas, fluorescência da clorofila a, conteúdo relativo de água na folha, densidade estomática, crescimento, enzimas atioxidativas, conteúdo de açucares totais e sacarose e ocorrência da H3K4me3 no gene Sb04g038610. Mesmo com o intervalo entre o primeiro e o segundo evento de seca, o que poderia resultar na remoção da memória do estresse formada no primeiro evento, as respostas fotossintéticas, antioxidativas, morfo-anatomicas e de estado hídrico dem... (Resumo completo, clicar acesso eletrônico abaixo)
Abstract: Plants that experience drought can store information about this experience, such as a memory of stress. Information acquired with stress memory can be used to increase tolerance to future water deficit events, however, plant growth may be limited. The objective of this study was to investigate the physiological, biochemical and epigenetic responses of Sorghum bicolor (L.) Moench during and after the first and second water deficit event, as well as the advantages and disadvantages of recurrent drought events for tolerance and growth. Four treatments were used: Control, where irrigation was maintained, Water Deficit in the Juvenile Phase, where the plants were submitted to dehydration in the juvenile phase followed by rehydration, Water Deficit in the Adult Phase, where irrigation was suspended in the adult phase followed by rehydration and Recurrent Water Deficit, where irrigation was suspended in the juvenile and adult phases followed by rehydration. Gas exchanges, chlorophyll a fluorescence, relative leaf water content, stomatal density, plant growth, atioxidative enzymes, total sugar and sucrose content and the occurrence of H3K4me3 in the Sb04g038610 gene were evaluated. Even with the interval between the first and second drought events, which could result in the removal of the stress memory, the photosynthetic, antioxidative, morpho-anatomical and water status responses demonstrated that S. bicolor plants showed increased tolerance to drought during recurrent water defici... (Complete abstract click electronic access below)
Doutor

Les gliomes sont les tumeurs primitives les plus fréquentes du cerveau et restent de mauvais pronostic en raison de l’inefficacité des traitements actuels. Des cellules souches cancéreuses ont été isolées à partir de gliomes de haut grade de l’adulte. Ces cellules souches de gliomes (GSC) peuvent fournir tous les sous-types cellulaires qui composent la tumeur. De nombreuses données indiquent que la résistance aux traitements est due en grande partie aux GSC. Cibler les GSC et leurs propriétés souches constitue donc un enjeu thérapeutique important. [...] Une solution pertinente de ciblage thérapeutique est de forcer les GSC à quitter leur état souche. Dans ce cadre, mes principaux travaux ont eu pour but de caractériser les changements épigénétiques des marques d’histones qui accompagnent la répression des propriétés des GSC par un groupe de micro-ARN, miR-302-367. [...] L’étude de cette plasticité par notre équipe a abouti à l’identification de miR-302-367. Son expression forcée, à l’aide de lentivirus, bloque de façon irréversible les propriétés souches et initiatrices de tumeur des GSC. L’effet suppresseur de tumeur exercé par miR offre la possibilité d’identifier les mécanismes qui régulent le maintien ou la perte des propriétés des GSC. A l’aide d’un modèle formé par une lignée de GSC et de sa contrepartie dépourvue des propriétés souches et tumorigènes GSC-miR-302-367, je me suis attachée à caractériser les méthylations de l’histone H3, qui font parties du code d’histone associé à une transcription génique respectivement active ou réprimée. Je me suis axée sur la triméthylation de la lysine 4 (H3K4me3) et de la lysine 27 (H3K27me3), respectivement permissive et répressive de la transcription. Une analyse par ChIP-seq (Immunoprécipitation de la chromatine-séquençage) des gènes associés à ces marques a été associée à la caractérisation des transcriptomes des cellules par exon-array. Nos résultats montrent que l’expression du groupe de miR-302-367 ne modifie pas de façon globale les taux des marques H3K4me3 et H3K27me3. Par contre, des changements dans des groupes de gènes circonscrits ont pu être identifiés. La corrélation positive observée entre les marques d’histones et les taux d’expression des gènes montre une conservation du code d’histone dans les cellules cancéreuses, au moins pour les marques étudiées. L’analyse des termes GO (Gene Ontology) indique que la perte des propriétés induites par miR-302-367 s’accompagne d’un engagement de GSC dans une voie de différenciation. Les gènes portant la marque répressive dans les GSC-miR-302-367 participent notamment à des catégories fonctionnelles associées à l’expression de propriétés souches et tumorigènes. L’analyse du groupe de gènes portant une marque permissive dans les GSC et répressive dans les GSC-miR-302-367, a révélé un réseau de facteurs de transcription susceptible de participer au contrôle des propriétés souches des GSC. La répression à l’aide de siRNA d’un des membres de ce réseau, le facteur de transcription ARNT2, nous a permis de révéler son rôle dans le maintien des capacités prolifératives des GSC issues de gliomes distincts et dans l’expression du facteur de transcription Nanog, connu pour son rôle central dans le contrôle des propriétés souches des GSC. Nos résultats montrent que l’analyse des changements de marques d’histone offre donc non seulement une vue d’ensemble des différents réseaux moléculaires associés au maintien ou au contraire à la répression des propriétés des GSC, mais permet d’identifier de nouveaux acteurs. L’effet stimulateur d’ARNT2 sur la croissance cellulaire et l’expression de Nanog, dans des GSC dérivées de gliomes différents aux altérations génomiques distinctes, indique que ce facteur de transcription tient une place centrale, insoupçonnée jusqu’à présent, dans la hiérarchie des gènes qui gouvernent les propriétés des GSC
Gliomas, the most frequent primary brain tumors, are resistant to current therapies and the survival rate of patients is very low. Within high-grade gliomas, a cell sub-population bearing stem-like properties has been isolated. These cells, called glioma stem cell (GSC), are capable of generating all glioma cellular sub-types. Recent data indicates that resistance of these aggressive tumors to therapies is mostly due to GSCs. Thus, targeting the GSCs and their stem-like properties is imperative in order to improve current therapies. [...] Another effective solution to treat GSCs is to force them to lose their stem-like properties. In this context, the aims of my major project were to characterize the epigenetic modifications of histone marks accompanying the loss of GSC stem-like properties under the influence of a cluster of micro-RNA, miR-302-367. GSCs are endowed with an exceptional plasticity, allowing them to gain or lose their stem-like state in response to modifications in their micro-environment. Our results identified the implication of miR-302-367 in the regulation of GSC plasticity. Its stable expression using lentivirus inhibits in an irreversible manner the stem-like and tumorigenic properties of GSC. The tumor-suppressor effect of this miR offers the possibility to decipher the mechanisms responsible for the maintenance or the loss of GSC stem-like properties. Using the model of GSC and their counterparts, GSC-miR-302-367, who lost their stem-like and tumorigenic properties, my aim was to identify the methylation status of histone H3 of the histone code which is known to be associated either to an active or to a repressive gene transcription. I focused on the trimethylation of lysine 4 (H3K4me3) and lysine 27 (H3K27me3), which are associated with an activation or repression of gene transcription, respectively. We performed a ChIP-seq (Chromatin-immunoprecipitation-sequencing) analysis of the respective associated genes followed by a transcriptomic (exon-array) analysis of both cell lines. Our results show that miR-302-367 expression does not alter in a global manner the expression levels of H3K4me3 and H3K27me3. On the contrary, we were able to detect modifications in a discrete group of genes. At least for the studied marks, the positive correlation between the identified histone marks and the gene expression levels indicates that the histone code is well preserved in cancer. GO (Gene Ontology) analysis indicates that miR-302-367-induced loss of stem-like properties is accompanied with activation of the differentiation process in GSC. Genes implicated in the regulation of stem-like and tumorigenic properties were found to bear the repressive histone mark in GSC-miR-302-367. From our analysis of the group of genes bearing the active histone mark in GSC and the repressive one in GSC-miR-302-367, emerged a network of transcription factors that could possibly participate in the regulation of GSC stem-like properties. Down-regulation using siRNA of a member of this network, namely ARNT2, highlighted its role in the maintenance of the proliferative dynamic, as well as the expression of the transcription factor Nanog (a major regulator of GSC stem-like properties), in GSC derived from distinct gliomas. Our histone mark modification analysis, not only elucidated the molecular pathways implicated in the maintenance or, on the contrary, in the loss of GSC stem-like properties, but also, highlighted the implication of new actors in these processes. The activator effect of ARNT2 on GSC proliferation, as well as on the expression of Nanog, observed in GSC bearing distinct genetic alterations and derived from different glioma, indicates that this transcription factor plays a major role, not documented thus far, in the regulation of GSC stem-like properties

Les facteurs environnementaux, tels que les pesticides, peuvent induire des changements phénotypiques dans une variété d'organisme incluant les mammifères. Nous avons étudié chez la souris les effets d'un pesticide largement utilisé, l'atrazine (ATZ), sur la méiose, une étape clé du processus de spermatogenèse. L'utilisation des méthodes de puces à ADN (Gene-Chip) et de séquençage de chromatine immunoprécipité (ChIP-seq) nous a permis de mettre en évidence l'effet de l'ATZ sur une variété de fonctions cellulaires, incluant l'activité GTPase, la fonction mitochondriale et le métabolisme des hormones stéroïdes. De plus, les souris traitées présentent un enrichissement des marques d'histone H3K4me3 au niveau des régions de forte recombinaison (sites de cassures double brin) de gènes très long et une réduction de ces mêmes marques au niveau des régions pseudo-autosomal du chromosome X. Nos données démontrent que l'exposition à l'ATZ interfère avec le déroulement normal de la méiose, ceci affectant la production des spermatozoïdes. Nous avons trouvé que les marques H3K4me3, chez la souris mâle, sont largement affectées par l'ATZ grâce à l'utilisation de technique de séquençage du génome entier. La reprogrammation embryonnaire nécessite l'action coordonnée d'un grand nombre de gène et de facteurs épigénétiques afin de permettre la transition de cellules somatique en cellules germinales. Les modifications épigénétiques imposées pendant la transition des cellules somatiques en cellules germinales et affectées par des expositions nocives, peuvent être héritées et transmises aux générations suivantes via les gamètes. Dans cette étude, nous avons examiné l'héritage des histones modifié aux générations suivantes. Nous avons exposés des femelles gestantes CD1 non consanguines à l'ATZ et les mâles issus de ces femelles ont été croisés pendant trois générations avec des femelles non traitées. Nous avons démontré ici que l'exposition à l'ATZ réduit le nombre de spermatozoïdes sans affecter la morphologie cellulaire ou la proportion des différents types cellulaires constituant l'épithélium séminifère chez les individus issus de la 3ème génération après traitement. Beaucoup de gènes associés avec la réparation de l'ADN, la reproduction et les fonctions mitochondriales sont dérégulés chez les mâles issus de la 3ème génération après traitement. De façon importante, l'exposition à l'ATZ change dramatiquement l'initiation de la transcription, l'épissage et la polyadénylation alternative des ARN. Nous avons aussi observé chez les mâles F3 issus de souris traitées à l'ATZ une altération de la localisation des marques H3K4me3 dans le promoteur de gène associé à la régulation de processus métaboliques cellulaires, à la régulation de la transcription et à la mitose. Les changements de localisation des marques H3K4me3 chez les mâles F3 issus de souris traitées à l'ATZ correspondent à des changements de la localisation de ces marques au niveau de gènes impliqués dans la différenciation des cellules de type souche de la génération F1.Nos données suggèrent que l'héritage transgénérationnel est permis grâce à de multiples voies et repose sur le statut épigénétique de gènes impliqués dans la différenciation des cellules de type souches tels que Pou5f1 et Sox2, l'action des facteurs de transcription et la rétention d'histones dans le sperme
Environmental factors such as pesticides can cause phenotypic changes in various organisms, including mammals. We studied the effects of the widely used herbicide atrazine (ATZ) on meiosis, a key step of gametogenesis, in male mice. We demonstrate that exposure to ATZ reduces testosterone levels and the number of spermatozoa in the epididymis and delays meiosis. Using Gene-Chip and ChIP-Seq analysis of H3K4me3 marks, we found that a broad range of cellular functions, including GTPase activity, mitochondrial function and steroid-hormone metabolism, are affected by ATZ. Furthermore, treated mice display enriched histone H3K4me3 marks in regions of strong recombination (double-strand break sites), within very large genes and reduced marks in the pseudoautosomal region of X chromosome. Our data demonstrate that atrazine exposure interferes with normal meiosis, which affects spermatozoa production.We found that the H3K4me3 marks in male mice are broadly affected by the widely used herbicide atrazine with genome wide ChIP-sequencing. Embryonic reprogramming requires the coordinated action of many genes and epigenetic factors to perform somatic to germline transition. The epigenetic modifications imposed during somatic to germline transition and affected by harmful exposure can be inherited and transferred to subsequent generations via the gametes. In this study, we examine the inheritance of altered histone modifications by subsequent generations. We exposed pregnant outbred CD1 female mice to the widely used herbicide atrazine (ATZ), and the male progeny were crossed for three generations with untreated females. We demonstrate here that exposure to ATZ reduces the number of spermatozoa without changing the cell morphology or types in testis tissue in the third generation after treatment. Many genes associated with DNA repair, reproduction and mitochondrial function became dysregulated in the third generation (F3) of males after treatment. Importantly, exposure to ATZ dramatically changes the transcription initiation, splicing and alternative polyadenylation of RNA. We also observed altered occupancy of H3K4me3 markers in the F3 generation of ATZ-derived males in gene promoters associated with the regulation of cellular metabolic processes, transcriptional regulation and mitosis. The changes in H3K4me3 occupancy in F3 ATZ-derived males correspond to changes in the H3K4me3 occupancy of stem cell differentiation genes in the F1 generation. Our data suggest that transgenerational inheritance is accomplished through multiple pathways and relies on the epigenetic state of stem cell differentiation genes such as Pou5f1 and Sox2, transcription factor action and sperm histone retention

Chromatin structure is regulated in part by the post-translational modification of histones. Histone methylation is highly conserved amongst eukaryotes, and is arguably one of the best characterized indicators of whether a gene is repressed or active. There are several unique states of histone methylation, each capable of specific downstream effects through the recruitment of highly specific methyl-histone binding domains and their associated chromatin-altering protein complexes. Histone H3 lysine 4 tri-methylation (H3K4me3) is a well known mark of actively transcribed genes, and co-localizes with histone H3 lysine 14 acetylation (H3K14ac), another mark of actively transcribed genes. The discovery that H3K4me3 is lost when H3K14 is substituted with another residue, led to the possibility of cross-talk between H3K4me3 and H3K14ac. The first part of this thesis demonstrates that H3K4me3 is indeed dependent on H3K14ac. Furthermore, we go on to show for the first time, that H3K14ac protects H3K4me3 from demethylation by the histone demethylase Jhd2. Though the mechanisms by which methyl-histone binding domains recognize methylated chromatin have been well studied, the specific physiological roles of the numerous methyl-histone binding domains have yet to be investigated. Isw1 is a highly conserved catalytic subunit of several ATP-dependent chromatin-modifying complexes. One of these complexes, Isw1b, has two putative methyl-histone binding domains, the PHD finger of Ioc2, and the PWWP domain of Ioc4. The second part of this thesis investigates the role that these domains play in the localization of the Isw1b complex to a specific region of the genome. Though we were unable to demonstrate a role for the PHD finger of Ioc2, we did demonstrate that the PWWP domain of Ioc4 is involved in chromatin localization. Additionally we found that Ioc2’s ability to bind chromatin is negatively affected by association with Ioc4 in the Isw1b complex, though the significance of this finding has yet to be determined.

In Drosophila oocytes, chromosomes undergo dynamic reorganisation during the prophase of the first meiotic division. This is essential to prepare chromatin for synapsis, recombination and consequent chromosome segregation. The progression of meiotic prophase I is well described, while the molecular mechanisms and regulation of these dramatic chromosomal reorganisations are not well understood. Histone modifying enzymes are major regulators of chromatin structure, however, our knowledge of their roles in meiotic prophase I is still limited. In this work, I investigated the role of the histone demethylase Kdm5/Lid, which removes one of the trimethyl groups at Lys4 of Histone 3 (H3K4me3). I showed that Kdm5/Lid is important for the assembly of the synaptonemal complex, pairing of homologous centromeres, and the karyosome formation. Additionally, Kdm5/Lid promotes crossing over and therefore ensures accurate chromosome segregation. Although loss of Kdm5/Lid dramatically increased the level of H3K4me3 in oocytes, catalytically inactive Kdm5/Lid rescued the above cytological defects. Thereby, I found that Kdm5/Lid regulates chromatin architecture in meiotic prophase I oocytes independently of its demethylase activity. To further identify the regulators of meiotic chromatin organisation during prophase I, I carried out a small-scale RNAi screen for karyosome defects. I found that depletion of ubiquitin ligase components, SkpA, Cul-3 and Ubc-6, disrupted the karyosome formation and the assembly of the synaptonemal complex. The success of the small-scale screen motivated me to initiate the genome-scale RNAi screen for karyosome defects. I found 40 new genes that, when depleted, strongly impaired karyosome morphology. Further studies are required to confirm and elucidate their role in chromatin organisation in oocytes. Overall, my findings have advanced our understanding of the regulation of chromatin reorganisation during oocyte development. Because of the conservation between Drosophila and human meiosis, this study provides novel insights into the regulation of meiotic progression in human oocytes.

Toute l'information génétique octroyant l'existence à une cellule est encodée par les milliards de paires de bases d'ADN retrouvées principalement à l'intérieur du noyau. Toutefois, pour des raisons d'espace et d'accessibilité, tout cet ADN est soumis à de nombreuses étapes de compaction en plus d'être fractionné dans le but de former ultimement les chromosomes. Malgré que l'extrême compaction de l'ADN permette la protection des acides nucléiques contre la dégradation, certaines structures demeurent vulnérables et nécessitent une protection toute particulière. C'est le cas de séquences se retrouvant à l'extrémité des chromosomes, les télomères. Les télomères sont constitués de répétitions en tandem d'ADN non codant associées avec de nombreuses protéines spécialisées permettant une protection efficace contre la perte d'informations génétiques dû à la dégradation enzymatique ou à l'érosion naturelle. Ces structures télomériques sont normalement maintenues par une machinerie spécialisée, la télomérase, qui composée d'une sous unité ARN et de partenaires protéiques permet l'ajout de séquences télomériques spécifiquement aux extrémités. Cette enzyme essentielle est régulée par de nombreuses protéines et parmi celles-ci, de nombreuses observations font état du rôle essentiel joué par deux protéines kinases, les protéines Tel1p et Mec1p. Ces kinases occupent une double fonction; elles sont importantes pour la régulation de la taille des télomères, mais sont également au coeur de la réponse cellulaire face aux dommages à l'ADN. Étant donné la nature des télomères, soit des extrémités d'ADN libres, ceux-ci sont identiques en de nombreux points aux cassures double brins d'ADN expliquant probablement la double implication de ces protéines. Durant mes études, je me suis particulièrement intéressé à la double fonction jouée par les kinases Tel1p et Mec1p au niveau des télomères et du processus de réponse aux dommages à l'ADN. Dans un premier temps, avec l'aide d'un collègue j'ai pu démontrer l'étendue des fonctions télomériques et de réponses aux dommages à l'ADN jouées par Tel1p via l'isolation et la caractérisation d'un allèle de séparation de fonctions. Dans un deuxième temps, en poursuivant mes analyses génétiques sur la kinase Tel1p, j'ai pu déterminer que cette protéine possédait des fonctions indépendantes à celles octroyées par son domaine kinase, observation allant à l'encontre de l'idée générale que Tel1p sans fonction kinase opérationnelle simulait un allèle nul. Dans la même ligne de pensée, mes études ont permis d'identifier un nouveau mécanisme de régulation de la télomérase essentiel joué par les kinases Mec1p et Tel1p. En parallèle, je me suis intéressé aux mécanismes cellulaires permettant une régulation de l'enroulement global de l'ADN permettant son accessibilité à toutes les machineries cellulaires de transcription, de réparation et de réplication de l'ADN. Mes travaux ont permis d'identifier qu'une modification des histones, protéines critiques dans le processus de compaction de l'ADN, était extrêmement importante dans le processus de réponse aux dommages à l'ADN. En effet la triméthylation de l'histone H3 sur sa lysine 4 permet à la cellule de pouvoir efficacement réparer les dommages via la réparation de bout non-homologue et permettait de stabiliser les fourches de réplications soumises à un stress cellulaire. Globalement mes résultats m'ont permis de mieux comprendre deux moyens distincts utilisés par les cellules pour maintenir l'intégrité de leur génome : les rôles joués par les kinases Tel1p et Mec1p aux télomères et dans la réparation des dommages à l'ADN, ainsi que l'implication de la modification d'histone H3K4me3 dans le processus de réponse aux dommages à l'ADN.

Eukaryotic organisms require the ability to respond to their environments. They do so by utilizing signal transduction pathways that allow for signals to effect final biological responses. Many times, these final responses require new gene expression events that have been stimulated or repressed within the nucleus. Thus, much of the understanding of signal transduction pathways converges on the understanding of how signaling affects gene expression alterations (Kumar et al., 2004). The regulation of gene expression involves the modification of chromatin between condensed (closed, silent) and expanded (open, active) states. Histone modifications, such as acetylation, can determine the open versus closed status of chromatin. The PHD (Plant HomeoDomain) finger is a structural domain primarily found in nuclear proteins across eukaryotes. This domain specifically recognizes the epigenetic marks H3K4me2 and H3K4me3, which are di- and tri-methylated lysine 4 residues of Histone H3 (Loewith et al., 2000; Kuzmichev et al., 2002; Vieyra et al. 2002; Shiseki et al., 2003; Pedeux et al., 2005, Doyon et al., 2006). It is estimated that there are ~150 proteins that contain the PHD finger in humans (Solimon and Riabowol, 2007). The PHD finger is conserved in yeast and plants, however an analysis of this domain has only been performed done in Arabidopsis thaliana (Lee et al., 2009). The work presented in this report aims to extend the analysis of this domain in plants by identifying the PHD fingers of the crop species Oryza sativa (rice). In addition, a phylogenetic analysis of all PHD fingers in Arabidopsis and rice was undertaken. From these analyses, it was determined that there are 78 PHD fingers in Arabidopsis and 70 in rice. In addition, these domains can be categorized into classes and groups by defining features within the conserved motif. In a separate study, I investigated the function of two of the PHD finger proteins from Arabidopsis, ING1 (INhibitor of Growth1) and ING2. In humans, these proteins can be found in complexes associated with both open and closed chromatin. They facilitate chromatin remodeling by recruiting histone acetyltransferases and histone deacetylases to chromatin (Doyon et al., 2006, Pena et al., 2006). In addition, these proteins recognize H3K4me2/3 marks and are believed to be â interpretersâ of the histone code (Pena et al., 2006, Shi et al., 2006). To understand the function of ING proteins in plants, I took a reverse genetics approach and characterized ing1 and ing2 mutants. My analysis revealed that these mutants are altered in time of flowering, as well as their response to nutrient and stress conditions. Lastly, I was able to show that ING2 protein interacts in vitro with SnRK1.1, a nutrient/stress sensor (Baena-Gonzalez et al., 2007). These results indicate a novel function for PHD proteins in plant growth, development and stress response.
Master of Science

Pour se défendre des effets délétères des éléments transposables, les pezizomycotina ont développé un système de défense génétique et épigénétique appelé « Repeat Induced Point Mutation » (RIP). Chez N. crassa, le RIP survient dans la cellule dicaryotique avant la caryogamie et conduit à la méthylation de novo des cytosines (5mC) inclues dans les séquences répétées de chacun des noyaux parentaux haploïdes. De plus, certaines de ces cytosines sont la cible d’un processus de mutation qui les transforme en thymines. Cette étape est suivie par la mise en place locale de l’hétérochromatine constitutive permettant une répression transcriptionnelle durable des séquences cibles du RIP au cours des divisions nucléaires. L’acteur majeur du RIP correspond à une cytosine méthyltransférase putative appelée RID (RIP Defective). Bien que son génome ne montre pas une quantité significative de 5mC, l’inactivation de PaRid chez Podospora anserina aboutit à un blocage du développement sexué survenant après la fécondation. Dans ce contexte, nous avons voulu déterminer si la fonction de PaRid dans le développement sexué consiste à éteindre l’expression de gènes cibles via l’installation de foyers d’hétérochromatine constitutive aux loci concernés. Pour ce faire, nous avons identifié les gènes PaKmt1 et PaHP1, codant respectivement l’histone méthyltransférase PaKmt1 (l’homologue de SU(VAR)39 qui catalyse la tri-méthylation du résidu H3K9 (H3K9me3) et PaHP1 (l’homologue de Heterochromatin Protein 1 qui se lie à H3K9me3). Les deux protéines interviennent dans une même voie de régulation qui aboutit à la mise en place de l’hétérochromatine constitutive. Par opposition, PaKmt6, homologue de l’histone méthyltransférase E(Z), correspond à la sous-unité catalytique du complexe PRC2 qui catalyse la marque H3K27me3 pour permettre l’établissement de l’hétérochromatine facultative. Nos résultats ont montré que l’absence de PaKmt1 et PaHP1 ne provoquent que des défauts mineurs. A l’inverse, l’inactivation du gène PaKmt6 conduit à un ensemble de défauts sévères : croissance végétative altérée, surproduction des gamètes mâles, malformations critiques des fructifications, production très réduite d’ascospores dont la germination est pour partie déficiente. Une étude d’épistasie a montré que les protéines PaRid et PaKmt6 interviennent chacune dans deux voies développementales distinctes. Par ailleurs, nous avons établi par immuno-précipitation de la chromatine les profils de distribution à l’échelle du génome entier des modifications H3K9me3, H3K27me3 et H3K4me3. Caractéristique rare, la marque H3K9me3 colocalise avec H3K27me3 sur des gènes transcriptionnellement réprimés et les séquences répétées ripées. Conformément à sa fonction canonique, H3K4me3 est présente en 5’ des gènes transcrits et est exclue des domaines H3K9me3 et H3K27me3. Comme attendue, PaKmt6 est essentielle à la mise en place de la marque H3K27me3, mais, de manière surprenante, elle serait aussi impliquée dans le dépôt et/ou le maintien d’une partie des marques H3K9me3, dévoilant ainsi une voie de méthylation non canonique de ces résidus
In pezizomycotina, transposable elements are targeted by a genome defense system named Repeat Induced Point Mutation (RIP). First described in Neurospora crassa, RIP occurs before karyogamy in each parental haploid nucleus of the dikaryotic cells and results, within the repeats, in de novo methylation of cytosine (5mC) and mutations, mainly C to T transitions. This initial step triggers local assembly of constitutive heterochromatin, which allows transcriptional gene silencing. RID (RIP Defective) is a putative cytosine methyltransferase essential for RIP. Despite the absence of 5mC in its genome, PaRid inactivation in Podospora anserina results in sexual reproduction arrest right after fertilization. In this context, we asked whether PaRid is required to silence expression of some of sexual development-specific genes by nucleation of constitutive heterochromatin. To this end, we identified PaKmt1 and PaHp1 genes encoding respectively the histone methyltransferase PaKmt1 (SU(VAR)39 homologue protein) and the heterochromatin protein 1 (PaHP1). To assemble constitutive heterochromatin, PaKmt1 catalyses tri-methylation of H3K9 (H3K9me3), latter on bound by PaHP1. By contrast, the E(Z) histone methyltransferase homologue PaKmt6, as part of the PRC2 complex, catalyses tri-methylation of H3K27 (H3K27me3) to form facultative heterochromatin. Our results showed that loss of either PaKmt1 or PaHP1 does not cause major defects. Conversely, PaKmt6 gene inactivation results in severe defects: altered mycelium and vegetative growth rate, overproduction of male gamete, development of crippled fructifications, reduced production ascospores, part of which does not germinate. Furthermore, epistatic study showed that PaRid and PaKmt6 likely act in two different developmental pathways, with respect to sexual reproduction. In addition, using chromatin immuno-precipitation we characterized H3K9me3, H3K27me3 and H3K4me3 genome-wide distribution patterns. We observed an uncommon overlapping distribution between H3K9me3 and H3K27me3 on transcriptionally repressed genes and RIP target repeats. As expected, H3K4me3 localizes in 5’ of the transcribed genes and is excluded from the H3K9me3 and H3K27me3 domains. As expected, PaKmt6 is essential for H3K27me3 modification, but surprisingly, could also be responsible for some of the H3K9me3 setting up or maintenance

Mammalian cell types and tissues have diverse functional roles within an organism but can be derived by the differentiation of the embryonic stem cells (ESCs). ESCs are pluripotent cells with self-renewal properties. During development subsets of genes in ESCs are activated or silenced for manifestation of the cell type specific function. Gene expression changes occur transiently in early developmental stages, through signals received and executed by a variety of transcription factors (TFs), regulatory elements (promoters, enhancers) and epigenetic modifications of chromatin. Post-translational modifications of the histone tails are regulated by chromatin modifiers and transform the chromatin architecture. Polycomb (PcG) and Trithorax (TrxG) group proteins are the most commonly studied histone modifiers. They were first discovered as repressors (H3K27me3) and activators (H3K4me3) respectively of Homeobox (Hox) genes in Drosophila and they are conserved in mammals. Bivalent chromatin is defined as the simultaneous presence of silencing (H3K27me3) and activating (H3K4me3) histone marks and was first discovered as a feature of many developmental gene promoters of ESCs. Bivalent promoters are thought to be in a ‘poised’ state for later activation or repression during differentiation due to the presence of the two counter-acting histone modifications and a pausing variant of RNA polymerase II (RNAPII) accompanied with intermediate-low levels of expression. By integrative analysis of publicly available ChIP sequencing (ChIP-seq) datasets in murine and human ESCs, we predicted 3,659 and 4,979 high–confidence (HC) bivalent promoters in mouse and human ESCs respectively. Using a peak-based method, we acquire a set of bivalent promoters with high enrichment for developmental regulators. Over 85% of Polycomb targets were bivalent and their expression was particularly sensitive to TF perturbation. Moreover, murine HC bivalent promoters were occupied by both Polycomb repressive component classes (PRC1 and PRC2) and grouped into four distinct clusters with different biological functions. HC bivalent and active promoters were CpG rich while H3K27me3-only promoters lacked CpG islands. Binding enrichment of distinct sets of regulators distinguished bivalent from active promoters and a ‘TCCCC’ sequence motif was specifically enriched in bivalent promoters. Using the recent technology of single cell RNA sequencing (scRNA-seq) we focused on gene expression heterogeneity and how it may affect the output of differentiation. We collected single cell gene expression profiles for 32 human and 39 murine ESCs and studied the correlation between diverse characteristics such as network connectivity and coefficient of variation (CV) across single cells. We further characterized properties unique to genes with high CV. Highly expressed genes tended to have a low CV and were enriched for cell cycle genes. In contrast, High CV genes were co-expressed with other High CV genes, were enriched for bivalent promoters and showed enrichment for response to DNA damage and DNA repair. Bivalent promoters in ESCs grouped in four distinct classes of variable biological functions according to Polycomb occupancy and three RNAPII variants. To study the dynamics of epigenetic and transcription control at promoters during development, we collected ChIPseq data for two chromatin modifications (H3K4me3 and H3K27me3) and RNAPII (8WG16 antibody) as well as expression data (RNA-seq) across 8 cell types (ESCs and seven committed cell types) in mouse. Hierarchical clustering of 22,179 unique gene promoters across cell types, showed that H3K4me3 peaks are in agreement with the expression data while H3K27me3 and RNAPII peaks were not highly consistent with the hierarchical tree of gene expression. Unsupervised clustering of ChIP-seq and RNA-seq profiles has resulted in 31 distinct profiles, which were subsequently narrowed down to nine major profile groups across cell types. TF enrichment at individual clusters using ChIP sequencing data did not fully agree with the classification of 8 major profile groups. Considering all the above results, three major epigenetic profiles (active, bivalent and latent) seem to be conserved across the species and cell types in our study. These states could recapitulate only a fraction of the transcriptional information - adding other chromatin marks could enrich it - since they are seemingly unaffected by their respective expression profiles. H3K27me3 only state has low CpG density and shows stronger signatures at differentiated cell types. Transcriptional control is tighter in active than bivalent promoters and the different occupancy levels of PcG subunits and RNAPII can be reflected at the expression variance of bivalent genes, where a fraction of them are involved in developmental functions while others are more tissue-specific. Last, there is a striking similarity in the pausing patterns of RNAPII in the progenitor cell types, which suggests that RNAPII pausing is correlated with the developmental potential of the cell type. Finally, this analysis will serve as a resource for future studies to further understand transcriptional regulation during development.

Orientador : Profª. Drª. Giseli Klassen
Coorientador : Profª. Edneia A. S. R. Cavalieri
Tese (doutorado) - Universidade Federal do Paraná, Setor de Ciências Biológicas, Programa de Pós-Graduação em Ciencias Biológicas (Microbiologia, Parasitologia e Patologia Básica). Defesa: Curitiba, 26/08/2016
Inclui referências : f. 71-86
Área de concentração: Patologia
Linha de pesquisa: Epigenética e câncer
Resumo: As metástases são a causa das mortes por câncer em 90% dos casos. No processo de metástases a destruição ou degradação da matriz extracelular é muito importante para o deslocamento das células tumorais malignas. Este processo é mediado por diversas enzimas, destacando-se a gelatinase B ou MMP-9. A epigenética estuda mecanismos de regulação da expressão gênica utilizando a metilação do DNA (em citosinas adjacentes a guaninas) e modificações pós-traducionais de histonas como principais mediadores. As ilhas de CpGs ao longo do promotor podem ser hipermetiladas e assim promover o silenciamento gênico e vice-versa. A partir deste conhecimento vem sendo utilizados diversos análogos de citosina a fim de inibir o processo de metilação de DNA. Nesse sentido esta em avançado estudo clínico o uso do 5-aza-2'- deoxicitosina (5-azadC) ou decitabine em alguns tipos de leucemias e doenças mielodisplásicas e provável inicio de utilização em tumores sólidos. Neste estudo o objetivo foi avaliar a expressão do gene MMP9 em linhagens de câncer de mama e com esses dados estudar o efeito da metilação do DNA e modificações de histonas no promotor e corpo do gene com e sem tratamento com decitabine. Para isso clonamos e sequenciamos uma região contendo CpGs da região promotora do gene MMP9 e também e ilhas de CpG no corpo do gene utilizando linhagens tumorais, PMC42, HeLa, MCF7 e MDA-MB-436. As linhagens MCF7 e MDA-MB-436 expressam baixos níveis de MMP9. Apos o tratamento destas 5- azadC foi observado aumento da expressão do gene e proteína MMP-9. O sequenciamento de CpGs na região promotora revelou que a metilação do DNA regula a expressão deste gene nas linhagens tumorais. Além disso a análise em amostras tumorais de pacientes que expressam MMP-9 também possuem estes CpGs desmetilados. A região intragênica contém 4 ilhas de CpG que foram clonadas em 2 fragmentos e denominadas CGI1 e CGI2. A CGI1 é altamente metiladas com ou sem tratamento com decitabine nas linhagens tumorais. Por outro lado a CGI2 apresentou alguns CpGs nas posições 12 a 30 que estavam metilados nas linhagens tumorais sem tratamento com decitabine, e que são desmetiladas após o tratamento. Novamente os resultados de contrapartida com amostras de tumores primários, estes mesmos CpGs encontraram-se desmetilados nos tumores mais agressivos e com presença de MMP-9 na imunohistoquímica. Afim de se avaliar o provável envolvimento de modificações de histonas foi realizada a imunoprecipitação de cromatina para as marcas de cromatina para abertura H3K4me3 e fechamento H3K27me3. Utilizando a linhagem MCF7 observou-se que após o tratamento com decitabine houve o enriquecimento da marca de abertura na região promotora onde se ligam os fatores de transcrição AP1 e NFkB. Além disso os CpGs 12-30 da CGI2 também apresentaram aumento da marca de abertura. Em conjunto esses resultados mostram um provável novo mecanismo de regulação da expressão gênica através de CpGs localizados em íntron no gene MMP9. Esses resultados são importantes no contexto do entendimento de mecanismos de expressão de MMP-9 em câncer de mama e também para o estudo de possível efeito de ativação de metástases com o uso do medicamento decitabine.
Abstract: Metastases are the cause of cancer deaths in 90% of cases. In the process of metastasis destruction or degradation of extracellular matrix it is important for the displacement of malignant tumor cells. This process is mediated by several enzymes, especially B-gelatinase or MMP-9. Epigenetic studies of regulatory mechanisms of gene expression using DNA methylation (adjacent cytosine to guanine) and post-translational modifications of histones as major mediators. The CpG islands along the promoter may be hypermethylated and thus promote gene silencing and vice versa. From this knowledge different cytosine analogues are used to inhibit the DNA methylation process. Accordingly this in advanced clinical study using 5-aza-2'-deoxicitosine (5-azadC) or decitabine in some types of leukemias and myelodysplastic diseases and probable beginning of use in solid tumors. In this study our goal was to evaluate the expression of MMP9 gene in breast cancer cell lines and study the effect of DNA methylation and histone modifications in the promoter gene and intragenic region with and without treatment with decitabine. To this we have cloned and sequenced a region containing CpGs of the MMP9 promoter region and CpG islands in the gene's body using tumor cell lines, PMC42, HeLa, MCF7 and MDA-MB-436. The lines MCF7 and MDA-MB-436 expressed low levels of MMP9. After treatment with 5-azadC was observed an increase in the gene expression and MMP-9 protein. The sequencing CpGs in the promoter region revealed that the DNA methylation regulates the expression of this gene in tumor cell lines. Further analysis of tumor samples from patients expressing MMP-9 also have these demethylated CpGs. The MMP9 intragenic region contains 4 CpG islands that were cloned in two fragments and called CGI1 and CGI2. The CGI1 was highly methylated with or without treatment with decitabine in tumor cell lines. On the other hand CGI2 have showed some CpGs in positions 12 to 30 that were methylated in tumor cell lines without treatment with decitabine, and are demethylated following treatment. Again counterpart results with primary tumor samples, the same CpG were demethylated in more aggressive tumors of MMP-9 positive in immunohistochemistry. In order to evaluate the probable involvement of histone modifications was performed chromatin immunoprecipitation to chromatin marks for H3K4me3 H3K27me3 opening and closing respectivelly. Using the MCF7 it was observed that after treatment with decitabine was enriching the opening tag in the promoter region which bind transcription factors NFkB and AP1. Additionally the CpG 12-30 of CGI2 also increased too. Together these results showed a possible new mechanism for regulation of gene expression through CpGs located in intron in MMP9 gene. These results are important in the context of understanding of MMP-9 expression mechanisms in breast cancer and also for the study of possible metastases activation with the use of decitabine drug.

Cells within multicellular organisms share the same genetic information, yet their shape and function can differ dramatically. This diversity of form and function is established by differential use of the genetic information. Early embryonic development describes the processes that lead to a fully differentiated embryo starting from a single fertilized cell - the zygote. Interestingly, in metazoan species this early development is governed by maternally provided factors (nutrients, RNA, protein), while the zygotic genome is transcriptionally inactive. Only at a specific developmental stage, the zygotic genome becomes transcriptionally active, and zygotic transcripts drive further embryonic development. This major change is called zygotic genome activation (ZGA). While major regulators of activation of early zygotic genes could be identified recently, the molecular mechanisms that contribute to robust global genome activation during embryonic development is not fully understood. In this study, I investigated whether the establishment of histone H3 lysine 4 trimethylation (H3K4me3) is involved in zebrafish zygotic transcription activation and early embryonic development. H3K4me3 is a chromatin modification that is implicated in transcription regulation. H3K4me3 has been shown to be enriched at Transcription Start Sites (TSS) of genes prior to their activation, and is postulated facilitate transcription activation of developmentally important genes. To interfere with H3K4me3 establishment, I generated histone methyltransferase mutants. I further inhibited H3K4me3 establishment by introduction of histones with lysine 4-to-methionine (K4-to-M) substitution, which act as dominant-negative inhibitors of H3K4me3 establishment. Upon H3K4me3 reduction, I studied the resulting effect on early transcription activation. I found that H3K4me3 is not involved in transcription activation during early zebrafish embryogenesis. Finally I analyzed possible cues in DNA sequence and chromatin environment that might favor early H3K4me3 establishment. These studies show that H3K4me3 is established during ZGA, yet it is not involved in transcription activation during early zebrafish development. Establishment of H3K4me3 might be a consequence of histone methyltransferase recruitment during a permissive chromatin state, and might be targeted to CpG-rich promoter elements that are enriched for the histone variant H2A.z.:Frontmatter II Acknowledgements VII Thesis Summary (English) IX Thesis Summary (German) X Table of Contents XIV List of Figures XVI List of Tables XVII List of Abbreviations XXIII 1 Introduction 1 1.1 Transcription regulation 2 1.1.1 Promoter elements - genetic information that guides transcription initiation 2 1.1.2 Enhancers - fine-tuning of transcription by distal DNA elements 3 1.1.3 CpG islands - DNA sequences that allow for epigenetic regulation 4 1.2 Chromatin 4 1.2.1 Histone variants 7 1.2.2 Posttranslational histone modifications 7 1.2.3 Histone Lysine methylation 8 1.2.4 H3K4me3 in embryonic development 10 1.3 Establishment and removal of H3K4me3 10 1.3.1 Set1 homologs - Set1a and Set1b 11 1.3.2 Trithorax homologs - Mll1 and Mll2 11 1.3.3 Homologs of Trithorax-related - Mll3 and Mll4 13 1.3.4 COMPASS complex proteins 13 1.3.5 H3K4me3 removal 14 1.4 Transcription activation in embryos 14 1.4.1 Zebrafish early embryonic development 15 1.4.2 H3K4me3 during early zebrafish development 17 1.5 Thesis aim 17 2 Materials and Methods 19 2.1 Materials 19 2.2 Methods 36 2.2.1 Zebrafish husbandry and care 36 2.2.2 Generation of zebrafish knock-out lines by TALEN mutagenesis 36 2.2.3 Generation of plasmids for mRNA production 38 2.2.4 Microinjection 39 2.2.5 Germline transplantation 39 2.2.6 Western Blot Assays 40 2.2.7 RNA extraction and quantification assays 41 2.2.8 Chromatin immunoprecipitation (ChIP) 43 2.3 Bioinformatics Analyses 46 2.3.1 Quality control, alignment and peak calling 46 2.3.2 Lambda normalization 46 2.3.3 Differential ChIP enrichment analysis 47 2.3.4 Data integration 47 2.3.5 Gene classification 48 3 Results I: H3K4me3 interference by Histone methyltransferase mutation 49 3.1 Generation and phenotypic description of histone methyl-transferase mutants 49 3.1.1 HMT TALEN mutagenesis workflow 49 3.1.2 Ash2l TALEN mutation does not result in a larval or adult phenotype 52 3.1.3 Mll2 mutation results in increased larval mortality, while adult fish are healthy and fertile 54 3.1.4 Mll1 mutation results in increased larval mortality and a severe adult phenotype 56 3.2 HMT mutations do not affect global H3K4me3 levels in early zebrafish embryos 60 3.3 Mll1 mutation results in local H3K4me3 reduction of a small subset of genes 62 3.4 Early embryonic transcription is not altered in mll1 maternal-zygotic mutants 67 3.5 Conclusion 70 4 Results II: H3K4me3 interference by introduction of HMT inhibitors 71 4.1 Establishing a Western Blot assay to monitor H3K4me3 reduction 71 4.2 Overexpression of H3K4-specific histone demethylases does not result in global H3K4me3 reduction 73 4.3 Global reduction of H3K4me3 could not be achieved by small-molecule inhibition of HMT activity 75 4.4 Overexpression of K4-specific methylation-defective H3 results in global H3K4me3 reduction 76 4.4.1 Overexpression of H3K4-to-E constructs does not affect global H3K4me3 establishment 76 4.4.2 H3K4-to-M constructs act as dominant-negative substrate for H3K4me3 establishment 77 4.5 H3K4me3 levels at gene promoters are reduced upon introduction of methylation-defective Histone H3 79 4.6 Early transcription activation is not altered upon K4M overexpression 88 4.7 Conclusion 92 5 Results III: Promoters rich in CpG and H2A.z gain H3K4me3 early 93 5.1 H3K4me3 levels increase over developmental time at all gene classes 93 5.2 H3K4me3 is gained at CpG-rich elements 98 5.3 H2A.z marks overlaps with H3K4me3 at promoters of non-transcribed genes 100 5.4 High CpG density and H2A.z enrichment are predictive for H3K4me3 establishment 101 5.5 Maternally provided genes are enriched for H2A.z and CpG content 103 5.6 Conclusion 104 6 Discussion 105 6.1 Neither Mll1 nor Mll2 are the main histone methyltransferase for H3K4me3 establishment in early zebrafish development 106 6.2 H3K4me3 reduction does not affect transcription initiation during genome activation 107 6.3 The timing of H3K4me3 establishment might be determined by a permissive chromatin state 109 6.4 H3K4me3 potentially gains importance during later developmental stages 111 6.5 CpG-content and H2A.z enrichment might be predictive for H3K4me3 establishment during genome activation 112 6.6 Conclusion 115 Appendix 117 Bibliography 139 Authorship Declaration 159
Jede Zelle eines multizellulären Organismus enthält dieselbe Erbinformation, und doch können Form und Funktion von Zellen untereinander sehr unterschiedlich sein. Diese Diversität wird durch unterschiedliches Auslesen - Transkribieren - der Erbinformation erreicht. Embryogenese beschreibt den Prozess, der aus einer einzelnen Zelle - der Zygote - einen multizellulären Embryo entstehen lässt. Interessanterweise laufen frühe Stadien der Embryogenese ohne Transkription der embryonalen Erbinformation ab, sondern werden durch maternal bereitgestellte Faktoren ermöglicht. Erst nach einer spezies-spezifischen Entwicklungsphase wird das Erbgut der Zygote aktiv transkribiert und ermöglicht die weitere Embryonalentwicklung. Obwohl bereits wichtige Regulatoren dieser globalen Genomaktivierung identifiziert werden konnten, sind viele molekulare Mechanismen, die zur Aktivierung des zygotischen Genoms beitragen, bisher unbekannt. In der hier vorliegenden Doktorarbeit habe ich die Rolle von Histon H3 Lysin 4 Trimethylierung (H3K4me3) während der frühen Embryogenese des Zebrafischs untersucht. H3K4me3 ist eine Chromatinmodifikation, die mit aktiver Transkription in Verbindung gebracht wird. H3K4me3 ist an Transkriptions-Start-Stellen von aktiv ausgelesenen Genen angereichert und es wird vermutet, dass diese Modifikation das Binden von Transkriptionsfaktoren und der Transkriptionsmaschinerie erleichtert. Während meiner Arbeit habe ich durch Mutation verschiedener Histon-Methyltransferasen beziehungsweise die Überexpression eines dominant-negativen Histonsubstrats versucht, die Etablierung von H3K4me3 in frühen Entwicklungsstadien des Zebrafischs zu verhindern. Anschliessend habe untersucht, welchen Effekt H3K4me3-Reduktion auf Tranksriptionsaktivität entsprechender Gene hat. Allerdings konnte ich keinen Zusammenhang zwischen H3K4me3-Reduktion und Transkriptionsaktivität beobachten. Um herauszufinden, weshalb H3K4me3 dennoch während früher Embryonalstadien etabliert wird, habe ich nachfolgend untersucht, ob möglicherweise bestimmte DNASequenzen oder Chromatin-Modifikationen zur Etablierung von H3K4me3 wahrend der Embryogenese des Zebrafischs beitragen. Aus der hier vorliegenden Arbeit lässt sich schlussfolgern, dass H3K4me3 in Tranksriptionsaktivierung während früher Embryonalstadien des Zebrafischs nicht involviert ist. Möglicherweise wird H3K4me3 in diesen Stadien in einer permissiven Chromatinumgebung etabliert, bevorzugt an Promotoren mit starker H2A.z-Anreicherung und CpG-reichen DNA-Elementen.:Frontmatter II Acknowledgements VII Thesis Summary (English) IX Thesis Summary (German) X Table of Contents XIV List of Figures XVI List of Tables XVII List of Abbreviations XXIII 1 Introduction 1 1.1 Transcription regulation 2 1.1.1 Promoter elements - genetic information that guides transcription initiation 2 1.1.2 Enhancers - fine-tuning of transcription by distal DNA elements 3 1.1.3 CpG islands - DNA sequences that allow for epigenetic regulation 4 1.2 Chromatin 4 1.2.1 Histone variants 7 1.2.2 Posttranslational histone modifications 7 1.2.3 Histone Lysine methylation 8 1.2.4 H3K4me3 in embryonic development 10 1.3 Establishment and removal of H3K4me3 10 1.3.1 Set1 homologs - Set1a and Set1b 11 1.3.2 Trithorax homologs - Mll1 and Mll2 11 1.3.3 Homologs of Trithorax-related - Mll3 and Mll4 13 1.3.4 COMPASS complex proteins 13 1.3.5 H3K4me3 removal 14 1.4 Transcription activation in embryos 14 1.4.1 Zebrafish early embryonic development 15 1.4.2 H3K4me3 during early zebrafish development 17 1.5 Thesis aim 17 2 Materials and Methods 19 2.1 Materials 19 2.2 Methods 36 2.2.1 Zebrafish husbandry and care 36 2.2.2 Generation of zebrafish knock-out lines by TALEN mutagenesis 36 2.2.3 Generation of plasmids for mRNA production 38 2.2.4 Microinjection 39 2.2.5 Germline transplantation 39 2.2.6 Western Blot Assays 40 2.2.7 RNA extraction and quantification assays 41 2.2.8 Chromatin immunoprecipitation (ChIP) 43 2.3 Bioinformatics Analyses 46 2.3.1 Quality control, alignment and peak calling 46 2.3.2 Lambda normalization 46 2.3.3 Differential ChIP enrichment analysis 47 2.3.4 Data integration 47 2.3.5 Gene classification 48 3 Results I: H3K4me3 interference by Histone methyltransferase mutation 49 3.1 Generation and phenotypic description of histone methyl-transferase mutants 49 3.1.1 HMT TALEN mutagenesis workflow 49 3.1.2 Ash2l TALEN mutation does not result in a larval or adult phenotype 52 3.1.3 Mll2 mutation results in increased larval mortality, while adult fish are healthy and fertile 54 3.1.4 Mll1 mutation results in increased larval mortality and a severe adult phenotype 56 3.2 HMT mutations do not affect global H3K4me3 levels in early zebrafish embryos 60 3.3 Mll1 mutation results in local H3K4me3 reduction of a small subset of genes 62 3.4 Early embryonic transcription is not altered in mll1 maternal-zygotic mutants 67 3.5 Conclusion 70 4 Results II: H3K4me3 interference by introduction of HMT inhibitors 71 4.1 Establishing a Western Blot assay to monitor H3K4me3 reduction 71 4.2 Overexpression of H3K4-specific histone demethylases does not result in global H3K4me3 reduction 73 4.3 Global reduction of H3K4me3 could not be achieved by small-molecule inhibition of HMT activity 75 4.4 Overexpression of K4-specific methylation-defective H3 results in global H3K4me3 reduction 76 4.4.1 Overexpression of H3K4-to-E constructs does not affect global H3K4me3 establishment 76 4.4.2 H3K4-to-M constructs act as dominant-negative substrate for H3K4me3 establishment 77 4.5 H3K4me3 levels at gene promoters are reduced upon introduction of methylation-defective Histone H3 79 4.6 Early transcription activation is not altered upon K4M overexpression 88 4.7 Conclusion 92 5 Results III: Promoters rich in CpG and H2A.z gain H3K4me3 early 93 5.1 H3K4me3 levels increase over developmental time at all gene classes 93 5.2 H3K4me3 is gained at CpG-rich elements 98 5.3 H2A.z marks overlaps with H3K4me3 at promoters of non-transcribed genes 100 5.4 High CpG density and H2A.z enrichment are predictive for H3K4me3 establishment 101 5.5 Maternally provided genes are enriched for H2A.z and CpG content 103 5.6 Conclusion 104 6 Discussion 105 6.1 Neither Mll1 nor Mll2 are the main histone methyltransferase for H3K4me3 establishment in early zebrafish development 106 6.2 H3K4me3 reduction does not affect transcription initiation during genome activation 107 6.3 The timing of H3K4me3 establishment might be determined by a permissive chromatin state 109 6.4 H3K4me3 potentially gains importance during later developmental stages 111 6.5 CpG-content and H2A.z enrichment might be predictive for H3K4me3 establishment during genome activation 112 6.6 Conclusion 115 Appendix 117 Bibliography 139 Authorship Declaration 159

By 2030, the global colorectal cancer burden is projected to approximately double. This highlights the immediate need to expand our understanding of the etiological origins of colorectal cancer, so that novel therapeutic strategies can be identified and validated. The putative tumor suppressor gene RNF20 encodes a histone H2B mono-ubiquitin ligase and has been found altered/mutated in colorectal and numerous other cancer types. Several studies suggest that RNF20, and by extension mono-ubiquitinated histone H2B (H2Bub1), play important roles in maintaining genome stability in human cells. Indeed, hypomorphic RNF20 expression and/or function have been shown to underlie several phenotypes consistent with genome instability, making aberrant RNF20 biology a potential driver in oncogenesis. Through an evolutionarily conserved trans-histone pathway, RNF20 and H2Bub1 have been shown to modulate downstream di-methylation events at lysines 4 (H3K4me2) and 79 (H3K79me2) of histone H3. Accordingly, understanding the biology associated with RNF20, H2Bub1, H3K4me2, and H3K79me2 is an essential preliminary step towards understanding the etiological origins of cancer-associated RNF20 alterations and identifying a novel therapeutic strategy to selectively kill RNF20-deficient cancers. In this thesis, I employ single-cell imaging, and multiple biochemical techniques to investigate the spatial and temporal patterning and characterize the biology of RNF20, H2Bub1, H3K4me2 and H3K79me2 throughout the cell cycle. In addition, I employ the CRISPR-Cas9 genome editing system to generate RNF20-deficient HCT116 cells. Finally, I employ synthetic lethal strategies to selectively kill RNF20-depleted cells. In conclusion, the research chapters contained within this thesis have characterized putative drivers in cancer (Chapter 3), generated a valuable research reagent for CRISPR-Cas9 ii genome editing experiments (Chapter 4), and identified a novel therapeutic strategy to selectively kill certain cancer cells (Chapter 5). This thesis has increased our understanding of the etiological origins of cancer and generated novel reagents and treatments strategies that after further validation and clinical studies, could be employed to reduce morbidity and mortality rates associated with cancer.
October 2016