Les piwi-interacting RNAs (piRNA) interagissent avec les protéines qui font partie de la branche PIWI de la famille des Argonautes. Ils participent à la répression des transposons dans la ligne germinale. Chez la souris, les protéines PIWI sont indispensables pour la fertilité des mâles et sont responsables de la méthylation de l'ADN au niveau des promoteurs des transposons. Les piRNA sont produis à partir de deux mécanismes: la biogenèse primaire et le cycle d'amplification ping-pong. Nous avons fait des études fonctionnelles sur deux protéines qui participent à la voie des piRNA: l'hélicase d'ARN MOV10L1 et l'immunophiline FKBP6. Nous avons décrit le rôle de MOV10L1 en tant que facteur de biogenèse primaire. La disruption génétique du domaine hélicase de MOV10L mène à une activation des transposons et à une perte de la méthylation de l'ADN. Ainsi, les piRNA ne sont pas produits chez le mutant, ce qui suggère que MOV10L1 est essentielle pour la biogenèse des piRNA. Tdrd1 interagit avec les protéines PIWI et joue un rôle dans la voie des piRNA en recrutant quelques facteurs à partir de son domaine MYND N-terminal. Nous avons montré que le domaine MYND de Tdrd1 interagit avec FKBP6, une protéine qui appartient à une famille d'isomérases de prolines qui sont présentes dans des complexes de chaperonnes. Les études biochimiques réalisées indiquent que FKBP6 est inactive et qu'elle utilise son domaine isomérase comme un module structural qui interagit avec les protéines PIWI, alors qu'elle interagit avec Hsp90 avec son domaine TPR. L'analyse d'une souris mutante pour fkbp6 a révélé une dérépression des transposons et une perte de la méthylation de l'ADN, mais peu d'effets sur la biogenèse primaire. Ces résultats sont en accord avec un rôle de FKBP6 en aval de Tdrd1, et nous pouvons penser que ce rôle pourrait être le recrutement de Hsp90 pour participer au cycle d'amplification ping-pong. / Piwi-interacting RNAs (piRNAs) associate to members of the PIWI clade of Argonaute proteins and are responsible for silencing of transposable elements in animal germ lines. In mouse, PIWI proteins mediate DNA methylation at the level of transposon promoters and are required for male fertility. piRNAs are produced through two biogenesis pathways, known as primary processing and ping-pong amplification cycle. This study focuses on the functions of two PIWI-associated proteins: the putative RNA helicase MOV10L1 and the immunophilin FKBP6. Here, the role of MOV10L1 as a primary piRNA processing factor is described. Genetic disruption of MOV10L1's RNA helicase domain results in male-specific sterility and derepression of retrotransposons due to reduction of DNA methylation. A complete loss of piRNAs is observed in the mutant, pointing to a pivotal role of MOV10L1 in piRNA biogenesis. Tdrd1 associates with PIWI proteins and has been suggested to play a role in the piRNA pathway by recruiting some factor through its N-terminal MYND domain. We show that Tdrd1's MYND domain interacts with FKBP6, which belongs to a family of prolyl isomerases present in chaperone complexes. Biochemical studies suggest FKBP6 is inactive and uses its prolyl isomerase domain as a structural module for binding PIWI proteins, while binding Hsp90 through its TPR domain. Analysis of an fkbp6 mutant mouse reveals transposon derepression and loss of DNA methylation, but little defects in primary piRNA biogenesis. These results are consistent with a role of FKBP6 downstream of Tdrd1, and we speculate that it might recruit Hsp90 to PIWI complexes to mediate ping-pong amplification cycle.

Texte remanié de: Diss.--Technische Universität, Dresden, 1971. / Bibliogr. p. 253-274.

Unter diesem Motto startete die Stadtbibliothek Pirna in das neue Jahr. Gleich um acht Stunden wöchentlich wurde die Öffnungszeit erweitert.

Les éléments transposables (ETs) sont des séquences d'ADN mobiles retrouvées dans les génomes de toutes les espèces où ils ont été recherchés. Moteurs de l'évolution, ces éléments mobiles, présents en de nombreuses copies dans les génomes, ont joué un rôle majeur dans la dynamique des génomes en engendrant des mutations et des réarrangements chromosomiques.Néanmoins, étant des constituants majeurs des génomes, ils doivent être finement régulés dans le but de préserver l'intégrité génomique, et ainsi de conserver l'équilibre entre variabilité et stabilité des génomes. Afin de protéger l'information génétique de l'hôte transmise à la descendance, la régulation des ETs au niveau des gonades est effectuée par la voie des piRNAs, voie d'ARN interférent conservée chez les animaux. Bien qu'elle soit relativement bien décrite chez la drosophile et la souris, certaines étapes de cette voie restent encore incomprises. Durant ma thèse, j’ai exploré différents aspects de la biologie des clusters de piRNAs, en prenant comme modèle d’étude le locus flamenco. Le cluster de piRNAs flamenco est le producteur majeur de piRNAs dans les cellules folliculaires des ovaires de Drosophila melanogaster. Tout d'abord, j'ai analysé les fenêtres spatio-temporelles de l’expression du cluster de piRNAs flamenco tout au long du développement de la drosophile,de l'embryon à l'âge adulte. Ensuite, j'ai recherché, in vivo, la séquence des transcrits de flamenco qui serait suffisante pour induire l'adressage d'un transcrit chimérique à la voie de maturation des piRNAs. J'ai également exploré l'impact de certains facteurs sur la prise en charge de transcrits artificiels par la voie des piRNAs. Enfin, je me suis intéressée à la régulation génique que pourraient effectuer les piRNAs provenant de flamenco dans les ovaires de drosophile en recherchant, par des approches bioinformatiques et de biologie moléculaire, les gènes potentiellement reconnus, et par conséquent, régulés par les piRNAs de flamenco. L'ensemble de ces axes de recherche in vivo permettront d'avancer dans la compréhension de la biologie des clusters de piRNAs ainsi que sur les mécanismes moléculaires mis en jeu lors de la biogenèse des piRNAs chez la drosophile. / Transposable elements (TEs) are defined such as mobile DNA sequences found in genomes ofall species where they were searched. As evolutionary drivers, these mobile elements, presentin many copies in genomes, have played a major role in the genome dynamics by generatingmutations and chromosomal rearrangements. Nevertheless, being major genome constituents,they must be finely regulated in order to preserve the genomic integrity, and thus, to maintainthe balance between variability and stability of genomes. In order to protect the geneticinformation of the host transmitted to the offspring, the gonadal TE regulation is carried outby the piRNAs pathway, an interfering RNA pathway conserved in animals. Although this isrelatively well described in Drosophila and in mouse, some steps of piRNA pathway are stillmisunderstood. During my thesis, I explored various aspects of piRNA cluster biology, usingthe flamenco locus as a model. This piRNA cluster is the main piRNA producer in thefollicular cells of Drosophila melanogaster ovaries. First, I analyzed the spatio-temporalwindows of flamenco piRNA cluster expression throughout the Drosophila development,from embryo to adulthood. Then, I searched, in vivo, the flamenco transcript sequence thatwould be sufficient to induce the addressing of a chimeric transcript to the piRNA processingpathway. I also explored the impact of some factors on the management of artificialtranscripts by piRNAs. Finally, I was interested in the gene regulation that flamenco-derivedpiRNAs could make in Drosophila ovaries by searching, through bioinformatics andmolecular biology approaches, the potentially recognized genes, and therefore, regulated byflamenco piRNAs. All of these in vivo research axes will advance in the understanding of thebiology of piRNA clusters as well as the molecular mechanisms involved in the piRNAbiogenesis in Drosophila.

Unter diesem Motto startete die Stadtbibliothek Pirna in das neue Jahr. Gleich um acht Stunden wöchentlich wurde die Öffnungszeit erweitert.

The advancement of Nucleic acids (DNA and RNA) sequencing technology has enabled many projects targeted towards the identification of genome structure and transcriptome complexity of organisms. The first conclusions of the human and mouse projects have underscored two important, yet unexpected, findings. First, while almost the entire genome is transcribed, only 5% of it encodes for proteins. Thereby, most transcripts are noncoding RNA. This includes both short RNA (<200 nucleotides (nt)) comprising piRNAs; microRNAs (miRNAs); endogenous Short Interfering RNAs (siRNAs) among others, and includes lncRNA (>200nt). Second, a significant portion of the mammalian genome (45%) is composed of Repeat Elements (REs). RE are mostly relics of ancestral viruses that during evolution have invaded the host genome by producing thousands of copies. Their roles within their host genomes have yet to be fully explored considering that they sometimes produce lncRNA, and have been shown to influence expression at the transcriptional and post-transcriptional levels. Moreover, because some REs can still mobilize within host genomes, host genomes have evolved mechanisms, mainly epigenetic, to maintain REs under tight control. Recent reports indicate that REs activity is regulated in somatic cells, particularily in the brain, suggesting a physiological role of RE mobilization during normal development. In this thesis, I focus on the analysis of ncRNAs, specifically REs; piRNAs; lncRNAs in human and mouse post-mitotic somatic cells. The main aspects of this analysis are: Using sRNA-Seq, I show that piRNAs, a class of ncRNAs responsible for the silencing of Transposable elements (TEs) in testes, are present also in adult mouse brain. Furthermore, their regulation shows only a subset of testes piRNAs are expressed in the brain and may be controlled by known neurogenesis factors. To investigate the dynamics of the transcriptome during cellular differentiation, I examined deep RNA-Seq and Cap Analysis of Gene Expression (CAGE) data from time-course progression program of primary human skeletal muscle cell differentiation. I contrasted this program with Duchenne Muscular Dystrophy (DMD) donors. I identified novel candidates, protein-coding genes and lncRNAs, that may be involved in myogenesis and reaffirmed known myogenic players. Using RNA-Seq data, I designed a novel pipeline to identify possible de novo insertion sites during muscular differentiation, which I have also tested on embryonic mouse cerebral cortex.

Im Jahr 2010 begann die Stadtbibliothek Pirna aktiv mit der Suche nach ehrenamtlichen Mitarbeitern und Mitarbeiterinnen. Hintergrund war die zu diesem Zeitpunkt erstmals erstellte Bibliothekskonzeption für die Jahre 2010 bis 2015. Aus der Umfeldanalyse ergaben sich eine Vielzahl von Problemlagen und Maßnahmen. Den wichtigsten wollten sich die Bibliotheksmitarbeiterinnen stellen. Doch nur aus eigener Kraft schien das nicht lösbar. Der dringendste Handlungsbedarf zeigte sich in der Förderung der Sprach- und Lesekompetenz von Vor- und Grundschulkindern. Die Bibliothek startete einen Aufruf und suchte nach Vorlesepaten für diese Zielgruppe. Dem öffentlichen Vorlesen stellten sich acht Kandidaten und Kandidatinnen, zwei von ihnen sind bis heute dabei. Die zuhörenden Kinder, Eltern und die Leiterin der Kinderbibliothek wählten drei Personen aus, die in den folgenden Monaten die ehrenamtlichen Projekte aufbauten. In der Zwischenzeit kamen zwei weitere Frauen auf die Bibliothek zu, um sich ehrenamtlich einzubringen. Daraus erwuchsen die Projekte der „Vorlesestunden für Krabbel- und Vorschulkinder“ und der monatliche „Tagesmuttitreff “. 2011 erhielt die Stadtbibliothek Pirna den Sächsischen Bibliothekspreis für ihre „gelungene Integration des Ehrenamtes in die Bibliotheksarbeit“, wie es im Ausschreibungstext hieß. Inzwischen arbeitet die Pirnaer Stadtbibliothek mit zehn ehrenamtlich tätigen Frauen zusammen. Eine von ihnen ist Petra Hagestedt, 65 Jahre, Rentnerin aus Pirna.

The evolution of genes and genomes has attracted great interest. The research presented here is an examination of genomes at three distinct levels, protein evolution, gene family evolution, and TE content regulation. First at a genetic level, I conducted an analysis of the salivary androgen-binding proteins (ABPs). I focused on comparing patterns of molecular evolution between the Abpa gene expressed in the submaxillary glands of species of New World and Old World muroids and found that in both sets of rodents, the Abpa gene expressed in the submaxillary glands appear to be evolving under sexual selection, suggesting ABP might play a similar biological role in both systems. Thus, ABP could be involved with mate recognition and species isolation in New World as well as Old World muroids. Second I examined the largest gene family in vertebrate olfactory receptors (ORs) among birds and reptiles. I found that the number of intact OR genes in sauropsid genomes analyzed ranged over an order of magnitude, from 108 in the lizard to over 1000 in turtles. My results suggest that different sauropsid lineages have highly divergent OR repertoire compositions. These differences suggest that varying rates of gene birth and death, together with selection related to diverse natural histories, have shaped the unique OR repertoires observed across sauropsid lineages. Lastly, I studied the interactions between transposable elements (TEs) and PIWI-interacting RNAs (piRNAs) among laurasiatherian mammals. piRNAs are predominantly expressed in germlines and reduce TE expression and risks associated with their mobilization. I found that within TE types, families that are the most highly transcribed appear to elicit the strongest ping-pong response. This was most evident among LINEs, but the relationships between expression and PPE was more complex among SINEs. I also found that the abundance of insertions within piRNAs clusters strongly correlated with genome insertions and there was little evidence to suggest that piRNA clusters regulated TE silencing. In summary, the piRNA response is efficient at protecting the genome against TE mobility, particularly LINEs, and can have an evolutionary impact on the TE composition of a genome.

The Anopheles gambiae complex is comprised of the most important vectors of malaria in Sub-Saharan Africa. Most current control methods involve the use of chemicals that help to limit potential contact with these mosquitoes. However, these control methods still have risks that include insect resistance, environmental toxicity, human health, as well as animal health. In order to develop new strategies that either produce novel targeted insecticides or transgenic mosquitoes that can replace current mosquito populations, it is important to acquire as much biological information about the vector as possible. The reduction in cost and speed of high-throughput sequencing has brought forth many new sequenced genomes that can provide a wealth of information about individual populations as well as their respective evolutionary histories. However, in order to fully understand a genome, these sequences must be assembled properly. One of the largest challenges toward fully assembling a genome is the abundance of repetitive sequences. These sequences, typically part of gene poor regions known as heterochromatin, are generally left as unassembled scaffolds that are neglected in many genomic studies. Heterochromatin is a biologically important chromatin state that has roles in gene regulation and genome stability. Exclusion of these chromatin domains from experimental assays can provide an incomplete picture in regards to organismal biology. A lack of information regarding heterochromatin, even in An. gambiae, necessitates further understanding and characterization of this chromatin type that can provide valuable information about the mosquito's biology. Heterochromatin is organized differently amongst different species. Some species with compact genomes, like Drosophila melanogaster, exhibit rigid organization of heterochromatin, with repetitive elements being confined to peri-centromeric and sub-telomeric regions of the chromosome. Larger genomes such as Aedes aegypti, have a much less structured heterochromatin pattern, with repetitive elements being dispersed across the genome. However, An. gambiae's genome is more intermediate in size as well as transposable element content. These factors may have an impact in controlling how heterochromatin is organized within the An. gambiae genome. Does An. gambiae compensate for the increased genome size by expanding past the peri-centromeric heterochromatin into new intercalary compartments? In An. gambiae, heterochromatin had yet to be identified separately from euchromatin. Morphologically, some regions of An. gambiae chromosomes exhibited characteristics similar to transcriptionally active puffs or peri-centromeric heterochromatin. We characterize these regions, as well as the rest of the genomic landscape, by using morphological and genetic features to identify various chromatin types. Peri-centromeric heterochromatin and new regions of intercalary heterochromatin were identified. Genomic coordinates representing the transition from euchromatin to heterochromatin were also identified. By finding these heterochromatin-euchromatin boundaries, various genetic features could be assigned to either heterochromatin or euchromatin. Critical genes associated with heterochromatin formation and basic genomic functions were identified. These data help to better understand features that are associated with the different environments created by chromatin compaction. This study also looks at the Piwi-interacting RNA (piRNA) pathway and its role in An. gambiae. The piRNA pathway is associated with transposable element (TE) suppression in many species, where clusters of vestigial TEs provide some of the RNA necessary for the pathway to function. These clusters are primarily associated with heterochromatin in Drosophila melanogaster. We identify piRNA clusters in An. gambiae and see a similar shift from primarily peri-centromeric compartmentalization toward the presence of intercalary regions located within the euchromatin. Transposable elements are maintained in secondary heterochromatin regions that exhibit similar morphology and features to peri-centromeric heterochromatin. The piRNA pathway also has implications in gene regulation, germline development, and anti-viral immunity. Three candidate genes associated with spermatogenesis and embryogenesis have been identified. These genes showed piRNA enrichment, and upon further analysis show up-regulation after a blood meal is taken. These genes could potentially prove useful in vector control as targets of transgenic experiments. Heterochromatin is an important, yet neglected aspect of the genome. These studies attempt to provide data to stimulate the study of heterochromatin through characterization of heterochromatin-related genomic features. / Ph. D.

Les cellules, et tout particulièrement les cellules germinales, maintiennent l'intégrité de leur génome en prévenant d'éventuelles mutations comme celles dues à la mobilité des éléments transposables (ET). Dans la lignée germinale des animaux, une classe particulière de petits ARN régulateurs, les PIWI-interacting RNA (piRNA), sont les acteurs majeurs du contrôle des ET. Chez Drosophila melanogaster, il existe des souches dites réactives, dépourvues de copies actives de l'élément I, ET exprimé dans la lignée germinale femelle. Les femelles de ces souches voient leur capacité à réprimer l'invasion de leur génome par l'élément I augmenter avec l'âge. Des données antérieures ont montré qu'une fois acquise, la capacité à réprimer l'élément I est transmise maternellement au travers des générations. Mes travaux de thèse ont permis de montrer que la transmission de la capacité à réprimer l'élément I n'est pas corrélée à des modifications de l'activité transcriptionnelle des loci producteurs de piRNA, mais semble uniquement véhiculée par les piRNA. En effet, les piRNA de l'élément I déposés dans l'embryon vont amorcer la production de piRNA complémentaires dans les ovaires de la descendance, ce qui induit une forte accumulation de piRNA antisens à l'élément I. Ainsi, les piRNA maternellement déposés assurent la transmission de la capacité à réprimer l'élément I, acquise suite au vieillissement des ascendants maternels. Mes résultats mettent en évidence le rôle des piRNA comme support moléculaire d'une composante non génétique de l'information héritable, indépendante de la chromatine et déterminante pour le maintien de l'intégrité du génome. / Cells, especially germinal stem cells, maintain genomic integrity by averting the propagation of mutations, generated as a consequence of DNA damage. In particular, they must avoid the deleterious activity of transposable elements (TEs). In animal germlines, one of the key players of the TE repression involves a specific class of small regulatory RNAs, the PIWI-interacting RNAs (piRNAs). In Drosophila melanogaster, there are reactive strains that are devoid of functional copies of the I element, a TE specifically expressed in the female germ line. When they get older, females of these strains can acquire a strong capacity to repress the I element invasion. Anterior works have shown that once acquired, this capacity to repress the I element is maternally transmitted over generations. The results obtained during my thesis revealed that the transmission of the capacity to repress the I element is not correlated with increased transcriptional activity of piRNA producer loci but seems only mediated by the piRNAs. Indeed, I element piRNAs deposition in the embryo after aging treatment correlates with the production of complementary piRNAs in the ovaries of the progeny. This results in a strong accumulation of antisense I element piRNAs. The maternally deposited piRNAs ensure the transmission of the capacity to repress the I element acquired after ancestor aging. My results highlight the molecular support of a DNA- and chromatin-independant component of heritable information essential for the maintenance of genome integrity.