Domesticated P elements in the Drosophila montium species subgroup have a new function related to a DNA binding property.

Molecular domestication of a transposable element is defined as its functional recruitment by the host genome. To date, two independent events of molecular domestication of the P transposable element have been described: in the Drosophila obscura species group and in the Drosophila montium species subgroup. These P neogenes consist to stationary, non repeated sequences, potentially encoding 66 kDa repressor-like proteins (RLs). Here we investigate the function of the montium P neogenes. We provide evidence for the presence of RLs proteins in two montium species (D. tsacasi and D. bocqueti) specifically expressed in adult and larval brain and gonads. We tested the hypothesis that the montium P neogenes function is related to the repression of the transposition of distant related mobile P elements which coexist in the genome. Our results strongly suggest that the montium P neogenes are not recruited to down regulate the P element transposition. Given that all the proteins encoded by mobile or stationary P homologous sequences show a strong conservation of the DNA Binding Domain, we tested the capacity of the RLs proteins to bind DNA in vivo. Immunstaining of polytene chromosomes in D. melanogaster transgenic lines strongly suggest that montium P neogenes encode proteins that bind DNA in vivo. RLs proteins show multiple binding to the chromosomes. We suggest that the property recruited in the case of the montium P neoproteins is their DNA binding property. The possible functions of these neogenes are discussed

Telomeric Trans-Silencing: An Epigenetic Repression Combining RNA Silencing and Heterochromatin Formation

The study of P-element repression in Drosophila melanogaster led to the discovery of the telomeric Trans-Silencing Effect (TSE), a repression mechanism by which a transposon or a transgene inserted in subtelomeric heterochromatin (Telomeric Associated Sequence or TAS) has the capacity to repress in trans in the female germline, a homologous transposon, or transgene located in euchromatin. TSE shows variegation among egg chambers in ovaries when silencing is incomplete. Here, we report that TSE displays an epigenetic transmission through meiosis, which involves an extrachromosomal maternally transmitted factor. We show that this silencing is highly sensitive to mutations affecting both heterochromatin formation (Su(var)205 encoding Heterochromatin Protein 1 and Su(var)3–7) and the repeat-associated small interfering RNA (or rasiRNA) silencing pathway (aubergine, homeless, armitage, and piwi). In contrast, TSE is not sensitive to mutations affecting r2d2, which is involved in the small interfering RNA (or siRNA) silencing pathway, nor is it sensitive to a mutation in loquacious, which is involved in the micro RNA (or miRNA) silencing pathway. These results, taken together with the recent discovery of TAS homologous small RNAs associated to PIWI proteins, support the proposition that TSE involves a repeat-associated small interfering RNA pathway linked to heterochromatin formation, which was co-opted by the P element to establish repression of its own transposition after its recent invasion of the D. melanogaster genome. Therefore, the study of TSE provides insight into the genetic properties of a germline-specific small RNA silencing pathway

Spatio-temporal requirements for transposable element piRNA-mediated silencing during Drosophila oogenesis

International audienceDuring Drosophila oogenesis, transposable element (TE) repression involves the Piwi-interacting RNA (piRNA) pathway which ensures genome integrity for the next generation. We developed a transgenic model to study repression of the Idefix retrotrans-poson in the germline. Using a candidate gene KD-approach, we identified differences in the spatio-temporal requirements of the piRNA pathway components for piRNA-mediated silencing. Some of them (Aub, Vasa, Spn-E) are necessary in very early stages of oogenesis within the germarium and appear to be less important for efficient TE silencing thereafter. Others (Piwi, Ago3, Mael) are required at all stages of oogenesis. Moreover, during early oogenesis, in the dividing cysts within the germarium, Idefix anti-sense transgenes escape host control, and this is associated with very low piwi expression. Silencing of P-element-based transgenes is also strongly weakened in these cysts. This region, termed the 'Piwiless pocket' or Pilp, may ensure that new TE insertions occur and are transmitted to the next generation, thereby contributing to genome dynamics. In contrast, piRNA-mediated silencing is strong in germline stem cells in which TE mobilization is tightly repressed ensuring the continued production of viable germline cysts

Telomeric Trans-Silencing in Drosophila melanogaster: Tissue Specificity, Development and Functional Interactions between Non-Homologous Telomeres

BACKGROUND: The study of P element repression in Drosophila melanogaster led to the discovery of the telomeric Trans-Silencing Effect (TSE), a homology-dependent repression mechanism by which a P-transgene inserted in subtelomeric heterochromatin (Telomeric Associated Sequences, "TAS") has the capacity to repress in trans, in the female germline, a homologous P-lacZ transgene located in euchromatin. TSE can show variegation in ovaries, displays a maternal effect as well as an epigenetic transmission through meiosis and involves heterochromatin and RNA silencing pathways. PRINCIPAL FINDINGS: Here, we analyze phenotypic and genetic properties of TSE. We report that TSE does not occur in the soma at the adult stage, but appears restricted to the female germline. It is detectable during development at the third instar larvae where it presents the same tissue specificity and maternal effect as in adults. Transgenes located in TAS at the telomeres of the main chromosomes can be silencers which in each case show the maternal effect. Silencers located at non-homologous telomeres functionally interact since they stimulate each other via the maternally-transmitted component. All germinally-expressed euchromatic transgenes tested, located on all major chromosomes, were found to be repressed by a telomeric silencer: thus we detected no TSE escaper. The presence of the euchromatic target transgene is not necessary to establish the maternal inheritance of TSE, responsible for its epigenetic behavior. A single telomeric silencer locus can simultaneously repress two P-lacZ targets located on different chromosomal arms. CONCLUSIONS AND SIGNIFICANCE: Therefore TSE appears to be a widespread phenomenon which can involve different telomeres and work across the genome. It can explain the P cytotype establishment by telomeric P elements in natural Drosophila populations

The Epigenetic Trans-Silencing Effect in Drosophila Involves Maternally-Transmitted Small RNAs Whose Production Depends on the piRNA Pathway and HP1

BACKGROUND: The study of P transposable element repression in Drosophila melanogaster led to the discovery of the Trans-Silencing Effect (TSE), a homology-dependent repression mechanism by which a P-transgene inserted in subtelomeric heterochromatin (Telomeric Associated Sequences, "TAS") has the capacity to repress in trans, in the female germline, a homologous P-lacZ transgene located in euchromatin. Phenotypic and genetic analysis have shown that TSE exhibits variegation in ovaries, displays a maternal effect as well as epigenetic transmission through meiosis and involves heterochromatin (including HP1) and RNA silencing. PRINCIPAL FINDINGS: Here, we show that mutations in squash and zucchini, which are involved in the piwi-interacting RNA (piRNA) silencing pathway, strongly affect TSE. In addition, we carried out a molecular analysis of TSE and show that silencing is correlated to the accumulation of lacZ small RNAs in ovaries. Finally, we show that the production of these small RNAs is sensitive to mutations affecting squash and zucchini, as well as to the dose of HP1. CONCLUSIONS AND SIGNIFICANCE: Thus, our results indicate that the TSE represents a bona fide piRNA-based repression. In addition, the sensitivity of TSE to HP1 dose suggests that in Drosophila, as previously shown in Schizosaccharomyces pombe, a RNA silencing pathway can depend on heterochromatin components

Etude du Trans-Silencing Effect, un mécanisme de répression en trans dépendant de l' homologie de séquence, et de son implication dans la régulation de l' élément P chez la drosophile

PARIS-BIUSJ-Thèses (751052125) / SudocPARIS-BIUSJ-Physique recherche (751052113) / SudocSudocFranceF

Régulation de l'élément P chez Drosophila melanogaster, Trans-Silencing Effect et Paramutation

L'étude de la répression de l'élément transposable P chez Drosophila melanogaster a été menée à l aide d'un modèle transgénique appelé Trans-Silencing Effect (TSE). Au cours, du TSE un transgène P-lacZ télomérique réprime l'expression germinale d'un transgène homologue inséré en trans dans le génome. Le TSE présente un effet maternel et une transmission épigénétique sur plusieurs générations. Le TSE apparaît faire intervenir une boucle fonctionnelle positive entre formation d'hétérochromatine et répression par les piRNAs. Mon travail de thèse s est articulé autour de trois axes. Le premier fut l affinement de l étude phénotypique et génétique du TSE. Des transgènes répresseurs ont été identifiés à tous les télomères, et qu ils peuvent interagir fonctionnellement entre eux pour établir le TSE. De plus, plusieurs transgènes cibles peuvent être réprimés simultanément par un seul transgène répresseur. Le second a été de tester si le TSE est un phénomène cellulaire général pouvant fonctionner via des séquences différentes de l élément P. Il y est montré qu un transgène P-lacZ peut réprimer un transgène piggyBac-lacZ, l unique homologie de séquence résidant dans les séquences lacZ. Le troisième axe a concerné l analyse des propriétés épigénétiques fines du TSE ; cela a permis de montrer l existence d un premier cas de paramutation stable dans le monde animal. La paramutation chez la Drosophile correspond à l émergence d un locus producteur de piRNAs et, fournit ainsi un modèle unique pour étudier cette étape inexplorée de la voie du piRNA.PARIS-BIUSJ-Biologie recherche (751052107) / SudocSudocFranceF

From Embryo to Adult: piRNA-Mediated Silencing throughout Germline Development in Drosophila

International audienceIn metazoan germ cells, transposable element activity is repressed by small noncoding PIWI-associated RNAs (piRNAs). Numerous studies in Drosophila have elucidated the mechanism of this repression in the adult germline. However, when and how transposable element repression is established during germline development has not been addressed. Here, we show that homology-dependent trans silencing is active in female primordial germ cells from late embryogenesis through pupal stages, and that genes related to the adult piRNA pathway are required for silencing during development. In larval gonads, we detect rhino-dependent piRNAs indicating de novo biogenesis of functional piRNAs during development. Those piRNAs exhibit the molecular signature of the " ping-pong " amplification step. Moreover, we show that Heterochromatin Protein 1a is required for the production of piRNAs coming from telomeric transpos-able elements. Furthermore, as in adult ovaries, incomplete, bimodal, and stochastic repression resembling variegation can occur at all developmental stages. Clonal analysis indicates that the repression status established in embryonic germ cells is maintained until the adult stage, suggesting the implication of a cellular memory mechanism. Taken together, data presented here show that piRNAs and their associated proteins are epigenetic components of a continuous repression system throughout germ cell development