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.

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.

No description available.

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.

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.

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.

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 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.

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

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.