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.

No description available.

Le raccourcissement progressif des télomères, en grande partie dû à la réplication incomplète des télomères, est l’une des caractéristiques principales du vieillissement. De façon encore plus frappante, une attrition trop marquée ou rapide des télomères est l’une des causes majeures d’un vieillissement prématuré. Les patients diagnostiqués avec un tel syndrome présentent généralement des mutations délétères dans un gène de maintien de l’homéostasie des télomères. Parmi les symptômes physiologiques, on remarque chez ces patients, des dégénérescences dans les tissus hautement prolifératifs, dus à l’exhaustion des cellules souches. Les cellules souches adultes, spécialisées et présentes dans nos tissus, permettent normalement la régénération des organes grâce à leur potentiel prolifératif et de différenciation. Afin de maintenir leur intégrité génomique, les cellules souches expriment une enzyme, la télomérase, capable de rallonger les extrémités terminales des chromosomes, et ainsi de maintenir intègre les télomères de ces cellules subissant des divisions cellulaire et subsistant dans l’organisme durant la vie de l’individu. En revanche, l’expression de la télomérase s’amenuise au cours du temps, les télomères se raccourcissent et les fonctions des cellules souches sont altérées. Nous étudions ce phénomène en utilisant comme modèle cellulaire, les cellules souches embryonnaires de souris, dont le gène de la télomérase réverse transcriptase a été anéanti génétiquement (mESC Tert -/-). Notamment, ces cellules, qui possèdent des télomères extrêmement courts n’arrivent pas à se différencier correctement. D’une étude précédente, mon laboratoire avait montré que ces cellules ne réussissaient pas à réprimer l’expression des gènes de pluripotence, comme Pou5F1/Oct4 et Nanog, et donc montraient des difficultés à sortir de l’état indifférencié. Au cours de mes recherches, nous avons montré que ce défaut était en fait dû à une altération en profondeur de l’épigénétique de ces cellules. Après avoir observé une déméthylation globale de l’ADN, nous avons constaté une augmentation de la marque d’histone H3K27me3 à travers le génome. De plus, moduler la présence de H3K27me3 via des petits inhibiteurs du complexe PRC2, ou par une approche génétique, modifie également le potentiel de différenciation des cellules souches. À la suite de cette étude, nous avons voulu en savoir davantage sur le signal liant attrition des télomères et défaut de différenciation. Lorsque les télomères sont très courts, la voie de réparation de l’ADN les reconnait comme une cassure double-brin. Parmi les acteurs de la réparation de l’ADN, la protéine p53 joue un rôle central puisqu’elle influence le destin cellulaire. Candidat idéal, nous avons cherché à savoir si p53 influençait la différenciation des mESC Tert -/- en utilisant une approche génétique. Nous avons été surpris de constater que l’absence de p53 restaurait le potentiel de différenciation des mESC Tert -/-. Ainsi et pour la première fois, nous avons montré que le raccourcissement des télomères pouvait avoir un effet très global sur les cellules. Nos recherches permettront de mieux appréhender certaines problématiques, notamment en matière de vieillissement. / The progressive decline of telomere length, mainly due to incomplete DNA replication at telomeres, is one of the main features of aging. Even more strikingly, excessive or rapid telomere attrition is one of the major causes of premature aging. Patients diagnosed with such syndromes have deleterious mutations in genes that maintain telomere homeostasis. For these patients, physiological manifestations include degeneration in highly proliferative tissues due to stem cell exhaustion. Adult stem cells, present in our tissues, typically allow the regeneration of organs due to their proliferative and differentiation potential. To maintain their replicative potential, stem cells express an enzyme, called telomerase, that is capable of lengthening the terminal ends of chromosomes. The maintenance of telomere length and, consequently favoring genomic stability, is crucial for stem cells that undergo cell division and remain in the organism throughout the individual's life. However, in stem cells, telomerase expression decreases over time, leading to telomere attrition and defects in stem cell functions. We studied this phenomenon using mouse embryonic stem cells disrupted for telomerase reverse transcriptase (mESC Tert -/-) as a model. In particular, these cells, which have extremely short telomeres, are unable to differentiate appropriately. Previously, my colleagues had shown that these cells failed to repress the expression of pluripotency genes, such as Pou5f1/Oct4 and Nanog, and therefore were refractory to differentiation. Here, we uncovered that profound epigenetic alterations influenced mESC Tert-/- cell fate. In addition to global DNA demethylation, we found an increase in H3K27me3 throughout the genome. Furthermore, modulating the levels of H3K27me3 via small inhibitors of the PRC2 complex or by a genetic approach also altered the differentiation potential of stem cells. Following this study, we wanted to learn more about the signals linking telomere attrition and differentiation failure. When telomeres are very short, the DNA repair pathway recognizes them as a double-strand break. Among the actors in DNA repair, the p53 protein plays a central role in influencing cell fate. As an ideal candidate, we investigated whether p53 influenced the differentiation of Tert-/- mESCs using a genetic approach. We were surprised to find that the absence of p53 restored the differentiation potential of Tert-/- mESCs. Thus, for the first time, we have shown that telomere shortening can have a very global effect on stem cells. Our research will allow us to better understand specific problems, particularly in the field of aging.

No description available.

Organoids retain the morphological and molecular patterns of their tissue of origin, are self-organizing, relatively simple to handle and accessible to genetic engineering. Thus, they represent an optimal tool for studying the mechanisms of tissue maintenance and aging. Long-term expansion under standard growth conditions, however, is accompanied by changes in the growth pattern and kinetics. As a potential explanation of these alterations, epigenetic drifts in organoid culture have been suggested. Here, we studied histone tri-methylation at lysine 4 (H3K4me3) and 27 (H3K27me3) and transcriptome profiles of intestinal organoids derived from mismatch repair (MMR)-deficient and control mice and cultured for 3 and 20 weeks and compared them with data on their tissue of origin. We found that, besides the expected changes in short-term culture, the organoids showed profound changes in their epigenomes also during the long-term culture. The most prominent were epigenetic gene activation by H3K4me3 recruitment to previously unmodified genes and by H3K27me3 loss from originally bivalent genes. We showed that a long-term culture is linked to broad transcriptional changes that indicate an ongoing maturation and metabolic adaptation process. This process was disturbed in MMR-deficient mice, resulting in endoplasmic reticulum (ER) stress and Wnt activation. Our results can be explained in terms of a mathematical model assuming that epigenetic changes during a long-term culture involve DNA demethylation that ceases if the metabolic adaptation is disturbed.

<p dir="ltr">H3K27me3 is a transcriptional repressive epigenetic mark that plays vital roles in many biological processes in <i>Arabidopsis thaliana</i>. A number of biochemical and functional characterizations of PKL, an ATP-dependent CHD chromatin remodeler, suggest that PKL contributes to maintain the homeostasis of H3K27me3. To identify other factors that act with PKL together to contribute to the homeostasis of H3K27me3, we undertook an EMS-mutagenesis screen for <i>pkl</i>-associated phenotypes. This genetic screen suggests that PKL may contribute to maintaining the homeostasis of H3K27me3 in an H2A.Z associated or a Mediator associated pathway.</p><p dir="ltr">Here, we took advantage of a combined genetic and bioinformatic method to characterize the contribution of PKL in these two pathways as described above. Our analysis revealed a robust genetic interaction between <i>HTA9</i>, <i>HTA11</i>, and <i>PKL</i> in maintaining proper H2A.Z distribution and enrichment of H3K27me3. In addition, the characterization also sheds light on unexpected roles of PKL in promoting the homeostasis of H3K4me3 and acting with histone demethylases to promote removal of H3K27me3 in an H2A.Z dependent manner. Furthermore, our result also raised the possibility that the tail module of the Mediator complex also plays a critical role in the homeostasis of H3K27me3. While we were examining <i>PKL</i>-dependent chromatin features, we largely optimized the protocol for preparation ChIP-seq samples and libraries and implemented a gene-centric ChIP-seq bioinformatics pipeline for providing robust analysis.</p><p dir="ltr">Ultimately, the work presented in this thesis highlights several divergent pathways that PKL contributes to maintain chromatin homeostasis. By and large, the combined observation from this thesis advances our knowledge of how PKL interacts with other chromatin-associated machineries together to maintain proper epigenetic states and promote other more emergent DNA-templated processes, including replication and transcription.</p>

Nitrogen (N), an essential plant macronutrient, is among the most limiting factors of crop yield. To sustain modern agriculture, N is often amended in soil in the form of chemical N fertilizer, a major anthropogenic contributor to nutrient pollution that affects climate, biodiversity and human health. To achieve agricultural sustainability, a comprehensive understanding of the regulation of N response in plants is required, in order to engineer crops with higher N use efficiency. Recently, epigenetic mechanisms, such as histone modifications, have gained increasing importance as a new layer of regulation of biological processes. However, our understanding of how epigenetic processes regulate N uptake and assimilation is still in its infancy. To fill this knowledge gap, we first performed a meta-analysis that combined functional genomics and network inference approaches to identify a set of N-responsive epigenetic regulators and predict their effects in regulating epigenome and transcriptome during plant N response. Our analysis suggested that histone modifications could serve as a regulatory mechanism underlying the global transcriptomic reprogramming during plant N response. To test this hypothesis, I applied chromatin immunoprecipitation-sequencing (ChIP-Seq) to monitor the genome-wide changes of four histone marks (H3K27ac, H3K4me3, H3K36me3 and H3K27me3) in response to N supply in tomato plants, followed by RNA-Seq to profile the transcriptomic changes. To investigate the organ specificity of histone modifications, I assayed shoots and roots separately. My results suggest that up to two-thirds of differentially expressed genes (DEGs) are modified in at least one of the four histone marks, supporting an integral role of histone modification in regulating N response. I observed a synergistic modification of active histone marks (H3K27ac, H3K4me3 and H3K36me3) at gene loci functionally relevant to N uptake and assimilation. Surprisingly, I uncovered a non-canonical role of H3K27me3, which is conventionally associated with repressed genes, in modulating active gene expression. Interestingly, such regulatory role of H3K27me3 is specifically associated with highly expressed genes or low expressed genes, depending on the organ context. Overall, I revealed the multi-faceted role of histone marks in mediating the plant N response, which will guide breeding and engineering of better crops with higher N use efficiency