Les éléments transposables d’ADN sont presque ubiquitaires dans le monde vivant et leur mobilité peut être délétère pour le génome. Leur régulation dans les tissus germinaux animaux passe par la voie de silencing des piRNAs (PIWI-interacting RNAs). Les piRNAs sont produits à partir de loci contenant des fragments d’éléments transposables insérés en clusters. Nous étudions l’émergence de ces clusters de piRNAs chez la drosophile.Nous avons activé de novo un cluster de transgènes par héritage maternel de piRNAs homologues. Il s’agit d’un cas de paramutation, ou conversion épigénétique stable et récurrente.Nous avons montré que ce cluster paramuté produit de novo des piRNAs, et étonnement dessiRNAs.J’ai caractérisé de façon fonctionnelle et moléculaire ce phénomène de paramutation par l’utilisation de mutants. J’ai montré que les propriétés de silencing, ainsi que la production depiRNAs et de siRNAs, sont abolies en contexte mutant pour tous les gènes testés de la voie despiRNAs (voies primaire et secondaire). Parallèlement, j’ai étudié un cas de paramutation« partiellement homologue » dans laquelle le cluster reçoit des piRNAs homologues seulement àune partie de sa séquence. J’ai montré qu’il y a production de piRNAs par la totalité du cluster dès la 3e génération.J’ai montré, enfin, que des clusters activés de novo par la chaleur, présentent des propriétés fonctionnelles et moléculaires semblables aux clusters activés par les piRNAsmaternels.Ces travaux apportent des éléments clés pour la compréhension de la mise en place de l’épigénome, tant d’un point de vue mécanistique qu’évolutif. / DNA transposable elements are almost ubiquitous in the living world and their mobility can be deleterious for the genome. Their regulation in germaria is mediated by the piRNAsilencing pathway (PIWI-interacting RNAs). piRNAs are produced by loci formed by clusters of fragments of transposable elements. We are studying the emergence of these piRNA-producing clusters in Drosophila.We have de novo activated a cluster of transgenes via maternal inheritance of homologous piRNAs. This is a case of paramutation i.e. a stable and recurrent epigenetic conversion process.We have shown that this paramutated cluster produces de novo piRNAs and, surprisingly, also siRNAs. I have characterized this paramutation functionally and molecularly, by a mutant approach. I have shown that its silencing properties, as well as piRNA and siRNA production are abolished in mutant contexts for all the genes from the primary and secondary piRNA pathways I have tested. At the same time, I have studied the case of a partially homologous paramutation, in which piRNAs maternally inherited by the cluster are homologous to only a part of its sequence. I have shown that piRNA are produced all along the cluster as early as the 3rdgeneration.Finally, I have shown that a cluster activated de novo by an environmental stress shows the same functional and molecular properties as a cluster paramutated via maternal piRNA inheritance.These studies provide key elements for understanding the emergence of the epigenome from a mechanistic and an evolutionary perspective.

Transposons constitute much of the animal genome. While many transposons are ancient and inactivated, numerous others are intact and must be actively repressed. Uncontrolled transposons can cause genomic instability through DNA damage or mutations and must be carefully silenced in the germline or risk sterility or mutations that are passed on to offspring. In Drosophila melanogaster, 23–30 nt long piRNAs direct transposon silencing by serving as guides for Aubergine, Argonaute3, and Piwi, the three fly PIWI proteins. piRNAs derive from piRNA clusters—large heterochromatic DNA loci comprising transposons and transposon fragments. piRNAs are loaded into PIWI proteins via the ping-pong cycle which serves to amplify guide piRNAs. Loaded Piwi then enters the nucleus to transcriptionally repress transposons by establishing heterochromatin. Therefore, to silence transposons, transposon sequences must also be expressed. To bypass this paradox, the HP1 homolog Rhino (Rhi) allows non-canonical, promoter-independent, transcription of transposons embedded in heterochromatin. Transposon RNAs produced in this manner are “incoherent” and have little risk of being translated into transposon-encoded proteins required for transposition. This thesis focuses on understanding how piRNA clusters permit non-canonical transcription yet restrict canonical transcription. We found that although Rhi promotes non-canonical transcription in piRNA clusters, it also creates a transcriptionally permissive environment that is amenable to canonical transcription. In addition, we discovered that the conserved protein, Maelstrom, is required to repress promoter-driven transcription of individual, potentially active, transposons within piRNA clusters and allows Rhi to transcribe such transposon sequences into incoherent piRNA precursors.

Les piRNAs sont une population de petit ARNs très diverse, que l'on retrouve dqns la lignée germinales des animaux pour réprimer les éléments génétiques mobiles : agissant de pair avec les protéines Piwi, ils guident le clivage des transposons actif. Chez la Drosophile, 3 protéines Piwi sont présentes, dont deux d'entre elles, AUB et AGO3, sont cytoplasmique et la dernière, PIWI, est nucléaire cependant son mécanisme d'action reste inconnu. La source principale de piRNAs sont des régions du génome bien particulière, appelé cluster de piRNAs. Cependant, il n'est pas encore connu a ce jour qu'est ce qui différentie ces région du reste du génome. Durant mon doctorant mon travail s'est focalisé sur ces deux questions centrales :Quel est le rôle de PIWI dans le noyau? Nous avons montré que PIWI était responsable de répression transcriptionnelle des transposons par l'intermédiaire de la déposition de marques chromatiniennes répressive, H3K9me3, grâce à la spécificité des piRNAs.Comment sont définit les régions générant des piRNA et comment sont régules leur expression ?Nous avons trouvé que les piRNAs qui sont transmis par la mère aux progénitures sont responsables de l'identification des régions génomiques donnant naissances à de nouveau piRNAs, grâce à la déposition de H3K9me3 dans le noyau et par l'initiation du cycle ping-pong dans le cytoplasme.Nous avons aussi mis en évidence les régions promoteurs des clusters de piRNAs, et trouve qu'elles sont nécessaires pour la production de piRNAs. / PiRNAs are a diverse population of small RNA found in the animal germline to silence mobile genetic elements: loaded into Piwi proteins, they guide homology-dependent cleavage of active transposon mRNAs. In Drosophila, three Piwi proteins are expressed, from which two, AUB and AGO3, are known to destroy transposon transcripts in the cytoplasm. The third one, Piwi itself, is nuclear and the molecular mechanism of its function remains unknown. The main sources of piRNAs are discrete genomic loci called piRNA clusters, however it is not known what differentiate them from non-piRNA producing loci. During my PhD, I focused my work on two central questions:1) What is the role of Piwi in the nucleus? We showed that Piwi is responsible for transcriptional silencing by mediating installment of repressive marks, especially H3K9me3, over active transposons copies in a piRNA dependent manner.2) How are piRNA clusters defined, and what regulates their expression? Analyzing what features differentiate a piRNA producing loci from any non-producing loci in the genome, we were able to single out some specific characteristics: . We showed that maternally inherited piRNAs are responsible to define germline clusters at the next generation through two mechanisms: in the nucleus, by deposition of H3K9me3 onto complementary genomic sequence, and, in the cytoplasm, by initiating the ping-pong cycle using cluster transcripts as substrates, leading to their processing into mature piRNAs.. We found that cluster promoters are essential to mediate full cluster transcription, which is allowed thanks to a very specific chromatin signature necessary to ensure piRNA production.

This diploma thesis studies the evaluation of wine grape PIWI varieties for the production of red wines. The experiment took place in autumn 2016, specifically in September and October. Nine type of interspecific varieties, were studied and two control varieties were studied. All varieties come from the Mikulov wine-growing subregion from the Lednice village in Moravia. For all varieties, the basic analytical parameters of the qualitative characters were determined. Everything was statistically evaluated and compared. The literary part deals with problems of resistance, propagation, occurrence and description of the three most serious diseases that have just begun to breed these resistant varieties. For this research is a brief look and description of each variety separately, their origins, ampelographic features, suitable habitat and oenological properties.

This thesis focuses on the possibility of environmental protection of PIWI vine varieties against fungal diseases. The theoretical part of the thesis summarizes information about organic viticulture, vine resistance against fungal diseases, explains the importance of PIWI varieties and describes the most important fungal diseases of vine. In the experimental part, an experiment using ecological preparations against fungal diseases is described. The effectiveness of these formulations and the content of grapevine elements is evaluated here. Furthermore, qualitative parameters of grapes and aromatic substances in wine are analyzed and statistically processed.

PIWI-interacting RNAs (piRNAs) are small non-coding RNAs unique to animals that guard the germline genome integrity by regulating transposons, viruses, and genes. In mice, piRNAs are highly expressed in testis and guide one of the three PIWI proteins to regulate their targets. The purpose of the 3′ end 2′-O-methyl modification in piRNAs is unknown. It has been speculated that the modification increases stability and facilitates the function of piRNAs, but the direct evidence is lacking. My dissertation addresses two unanswered questions about mouse piRNAs: (1) how are piRNAs produced and how conserved is the piRNA pathway in all animals, and (2) why are mouse piRNAs 2′-O-methylated at their 3′ ends? How piRNAs are generated is still poorly characterized in several model organisms. Studies of these model organisms imply the mechanisms that produce piRNAs differ among animals, tissues and cell types. Here, we demonstrate that a single unified mechanism can explain piRNA production in most animals, from human to the non-bilateral animal hydra. Our analysis elucidated that, in male mouse and female fly germlines, PIWI proteins guided by the initiator piRNA slice long piRNA precursor transcripts, and this PIWI-guided slicing action starts the piRNA biogenesis. PIWI proteins also position the endonuclease to further fragment long piRNA precursor transcripts into a string of tail-to-head, phased trailing piRNAs in a stepwise manner. Our discovery shows the central role of PIWI proteins in the piRNA pathway: both initiating and sustaining the production of piRNAs. For the second question, we discovered that pre-piRNA trimming and piRNA 2′-O-methylation protect piRNAs from separate decay mechanisms. We showed that in the absence of 2′-O-methylation, mouse piRNAs with extensive complementarity to long RNAs are destabilized and destroyed by a mechanism similar to target-directed microRNA degradation (TDMD). On the other hand, untrimmed pre-piRNAs are destroyed by a different mechanism, independent of their extensive complementarity to long RNAs. In the absence of both 2′-O-methylation and trimming, the piRNA pathway collapses which supports the idea of piRNA trimming and methylation collaborating to stabilize piRNAs. Our work suggests that 2′-O-methylation and trimming are important for maintaining the steady-state abundance of piRNAs which is necessary for their function in either transposon silencing or gene regulation.

PIWI Proteine sind die zentralen Darsteller eines RNA-basierten Mechanismus, der die Mobilisierung transponierbarer Elemente im Genom unterdrückt, um genetische Stabilität zu gewährleisten. Demzufolge sind PIWI-Proteine für die langfristige Erhaltung verschiedener Stamzellpopulationen notwendig. Beispiele dafür sind verschiedene adulte somatische Stammzellen in Drosophila und die Stammzellen der Keimbahn aller bisher untersuchten Tierarten. Bei Säugetieren sind die beschriebenen Funktionen von PIWI Proteinen strikt auf die männliche Keimbahn beschränkt. Trotz Andeutungen auf eine Rolle von PIWI-Proteinen in somatischen Zellen von Säugetieren, wurde eine Funktion bisher nicht beschrieben. Ähnlich wie Stammzellen, können sich Makrophagen in verschiedenen Geweben selbst-erneuern, um ihre Populationen zu erhalten. Diese Selbsterneuerung beruht auf der geringen Expression der Transkriptionsfaktoren MafB und cMaf, was die Aktivierung eines stammzell-ähnliches Gen-Netzwerk, das die Proliferation vorantreibt. Makrophagen mit einer genetischen Deletion von MafB und cMaf (MafDKO-Makrophagen) oder Makrophagen mit natürlich niedriger Expression von MafB oder cMaf, wie z.B. alveoläre Makrophagen, weisen dementsprechend eine erweiterte Kapazität zur Selbsterneuerung auf. Wie haben festgestellt, dass eine kurze Isoform des Maus- Gens Piwil2, die wir ‚Piwito’ genannt haben, in MafDKO und alveolären Makrophagen exprimiert wird. Die Expression von Piwito ist für die normale Selbsterneuerung der untersuchten Makrophagen notwendig, wie die in vitro und in vivo Untersuchungen darlegen. Eine Abwesenheit von Piwito in alveolären Makrophagen führt zu einer Verkürzung derer Lebenspanne in Kultur. Außerdem beweisen wir, dass Piwito von MafB in nicht-proliferierenden Makrophagen gebunden und unterdrückt wird. Diese Studie ist somit der erste Bericht über eine somatische Funktion von PIWI-Proteinen in nicht transformierten Zellen von Säugetieren. / PIWI proteins are the main players of an RNA-based gene regulatory machinery that represses transposable elements in the genome to prevent their mobilization and ensure genetic stability. PIWI proteins have thus highly conserved stem-cell functions. They are indispensable for the long-term maintenance of the somatic stem cells that drive regeneration in invertebrates, of various adult somatic stem cells in Drosophila and, most prominently, of the germline of all species studied so far. In mammals, their described functions are strictly restricted to the male germline. Despite suggestive observations for a role of PIWI proteins in the mammalian soma, robust evidence remains absent. Similar to stem cells, tissue macrophages can locally self-renew to maintain their populations. Mechanistically, their self-renewal relies on low expression of the macrophage transcription factors MafB and cMaf, since it allows the induction of a stem cell-like network of genes that drives proliferation. Macrophages with a genetic deletion of MafB and cMaf (MafDKO macrophages) acquire therefore the capacity to self-renew, defined by an indefinite growth in culture that does not comprise their identity and does not involve cancerogenic transformation. Similarly, macrophages with naturally low levels of MafB or cMaf, such as alveolar macrophages, display an extended self-renewal capacity in vivo and in vitro. We have found that a short isoform of the murine Piwil2 gene, that we named ‘Piwito’, is expressed in MafDKO and alveolar macrophages. Piwito expression is necessary for the unaltered self-renewal of macrophages, as shown by in vitro and in vivo assays. To highlight is the fact that Piwito deficiency limits the extended lifespan of alveolar macrophages in culture. Additionally, we show that Piwito is bound and repressed by MafB in quiescent macrophages. This study thus represents the first report of a somatic function for mammalian PIWI proteins in non-transformed cells.

No description available.

No description available.

<p>PIWI proteins belong to an evolutionary conserved protein family as the sister sub-family of ARGONAUTE (AGO) proteins. While AGO proteins are functionally well-characterized and shown to mediate small-RNA guided gene regulation, the function of PIWI proteins remain elusive. Here we pursued functional characterization of PIWI proteins by studying MILI and MIWI, two PIWI proteins in the mouse.</p><p>We first show that both MIWI and MILI co-immunoprecipitate with a novel class of non-coding small RNAs from the post-natal mouse testis extract, which are named Piwi-interacting RNAs (piRNAs). Our cloning efforts identified thousands of different piRNA sequences, mostly derived from intergenic regions. Interestingly, both MILI and MIWI piRNAs correspond to the same regions on the genome and differ primarily in length. We propose piRNAs in the adult testis are produced by the processing of long, single stranded RNA precursors, based on the observation that piRNAs originate in clusters from a number of sites on the genome in a head-to-tail homology. In support, we bioinformatically predicted putative promoters, and yeast one hybrid analysis on two such regions found out that they interact with Krueppel C2H2 type zinc finger transcription factors. We did not observe the features of the "ping-pong" mechanism in their biogenesis: Both MILI and MIWI piRNAs are biased for 5` Uracil without an Adenine bias on the 10th nucleotide position, and do not significantly consist of sequences complementary to each other along their first 10nt. Moreover, MILI piRNAs are not down-regulated in Miwi-/- testis. These results indicate that the post-natal testicular piRNAs are produced independent of the ping-pong mechanism. </p><p>Although piRNAs are highly complex, PAGE and in situ analyses showed that piRNAs are germ cell-specific with predominant expression in spermatocytes and round spermatids, suggestive of a meiotic function. Correspondingly, we found that Miwi-/-; Mili-/- mice undergo only male infertility with terminal spermatogenic arrest during meiosis. piRNAs show a nucleo-cytoplasmic distribution, with enrichment in the chromatoid and dense bodies, two male germ cell-specific structures. The dense body has been implicated in synapsis and in the heterochromatinization of the sex chromosomes during male meiosis, a process known as meiotic sex chromosome inactivation (MSCI). Our histological analysis on Miwi-/-; Mili-/- testes showed that, while the overall synapsis is not affected, the sex chromosomes retain the euchromatin marker acetyl-H4K16 and lacks the heterochromatin marker H3K9-dimethyl. These observations indicate that murine PIWI proteins are necessary for MSCI. Moreover, we identified piRNA production from the X chromosome before MSCI, and propose PIWI proteins utilize piRNAs to target and silence unpaired chromosomal regions during meiosis.</p> / Dissertation