De novo piRNA cluster formation in the Drosophila germ line triggered by transgenes containing a transcribed transposon fragment

PIWI-interacting RNAs (piRNAs) provide defence against transposable element (TE) expansion in the germ line of metazoans. piRNAs are processed from the transcripts encoded by specialized heterochromatic clusters enriched in damaged copies of transposons. How these regions are recognized as a source of piRNAs is still elusive. The aim of this study is to determine how transgenes that contain a fragment of the Long Interspersed Nuclear Elements (LINE)-like I transposon lead to an acquired TE resistance in Drosophila. We show that such transgenes, being inserted in unique euchromatic regions that normally do not produce small RNAs, become de novo bidirectional piRNA clusters that silence I-element activity in the germ line. Strikingly, small RNAs of both polarities are generated from the entire transgene and flanking genomic sequences—not only from the transposon fragment. Chromatin immunoprecipitation analysis shows that in ovaries, the trimethylated histone 3 lysine 9 (H3K9me3) mark associates with transgenes producing piRNAs. We show that transgene-derived hsp70 piRNAs stimulate in trans cleavage of cognate endogenous transcripts with subsequent processing of the non-homologous parts of these transcripts into piRNAs

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

Viral particles of the endogenous retrovirus ZAM from Drosophila melanogaster use a pre-existing endosome/exosome pathway for transfer to the oocyte

BACKGROUND: Retroviruses have evolved various mechanisms to optimize their transfer to new target cells via late endosomes. Here, we analyzed the transfer of ZAM, a retroelement from Drosophila melanogaster, from ovarian follicle cells to the oocyte at stage 9–10 of oogenesis, when an active yolk transfer is occurring between these two cell types. RESULTS: Combining genetic and microscopic approaches, we show that a functional secretory apparatus is required to tether ZAM to endosomal vesicles and to direct its transport to the apical side of follicle cells. There, ZAM egress requires an intact follicular epithelium communicating with the oocyte. When gap junctions are inhibited or yolk receptors mutated, ZAM particles fail to sort out the follicle cells. CONCLUSION: Overall, our results indicate that retrotransposons do not exclusively perform intracellular replication cycles but may usurp exosomal/endosomal traffic to be routed from one cell to another

In Drosophila melanogaster the COM Locus Directs the Somatic Silencing of Two Retrotransposons through both Piwi-Dependent and -Independent Pathways

BACKGROUND: In the Drosophila germ line, repeat-associated small interfering RNAs (rasiRNAs) ensure genomic stability by silencing endogenous transposable elements. This RNA silencing involves small RNAs of 26-30 nucleotides that are mainly produced from the antisense strand and function through the Piwi protein. Piwi belongs to the subclass of the Argonaute family of RNA interference effector proteins, which are expressed in the germline and in surrounding somatic tissues of the reproductive apparatus. In addition to this germ-line expression, Piwi has also been implicated in diverse functions in somatic cells. PRINCIPAL FINDINGS: Here, we show that two LTR retrotransposons from Drosophila melanogaster, ZAM and Idefix, are silenced by an RNA silencing pathway that has characteristics of the rasiRNA pathway and that specifically recognizes and destroys the sense-strand RNAs of the retrotransposons. This silencing depends on Piwi in the follicle cells surrounding the oocyte. Interestingly, this silencing is active in all the somatic tissues examined from embryos to adult flies. In these somatic cells, while the silencing still involves the strict recognition of sense-strand transcripts, it displays the marked difference of being independent of the Piwi protein. Finally, we present evidence that in all the tissues examined, the repression is controlled by the heterochromatic COM locus. CONCLUSION: Our data shed further light on the silencing mechanism that acts to target Drosophila LTR retrotransposons in somatic cells throughout fly development. They demonstrate that different RNA silencing pathways are involved in ovarian versus other somatic tissues, since Piwi is necessary for silencing in the former tissues but is dispensable in the latter. They further demonstrate that these pathways are controlled by the heterochromatic COM locus which ensures the overall protection of Drosophila against the detrimental effects of random retrotransposon mobilization

Functional Characteristics of a Highly Specific Integrase Encoded by an LTR-Retrotransposon

Background: The retroviral Integrase protein catalyzes the insertion of linear viral DNA into host cell DNA. Although different retroviruses have been shown to target distinctive chromosomal regions, few of them display a site-specific integration. ZAM, a retroelement from Drosophila melanogaster very similar in structure and replication cycle to mammalian retroviruses is highly site-specific. Indeed, ZAM copies target the genomic 59-CGCGCg-39 consensus-sequences. To enlighten the determinants of this high integration specificity, we investigated the functional properties of its integrase protein denoted ZAM-IN. Principal Findings: Here we show that ZAM-IN displays the property to nick DNA molecules in vitro. This endonuclease activity targets specific sequences that are present in a 388 bp fragment taken from the white locus and known to be a genomic ZAM integration site in vivo. Furthermore, ZAM-IN displays the unusual property to directly bind specific genomic DNA sequences. Two specific and independent sites are recognized within the 388 bp fragment of the white locus: the CGCGCg sequence and a closely apposed site different in sequence. Conclusion: This study strongly argues that the intrinsic properties of ZAM-IN, ie its binding properties and its endonuclease activity, play an important part in ZAM integration specificity. Its ability to select two binding sites and to nick the DNA molecule reminds the strategy used by some site-specific recombination enzymes and forms the basis for site-specifi

CONTROLE GENETIQUE PAR L'HOTE DE DEUX RETROVIRUS ENDOGENES, ZAM ET IDEFIX, CHEZ DROSOPHILA MELANOGASTER (DOCTORAT)

CLERMONT FD-BCIU-Santé (631132104) / SudocPARIS-BIUM (751062103) / SudocPARIS-BIUP (751062107) / SudocSudocFranceF

Etude du contrôle épigénétique de l'expression de trois rétrovirus endogènes ZAM, Idefix et Gypsy

Les éléments transposables sont des séquences d'ADN trouvées chez tous les organismes vivants, capables de se déplacer d'un site à un autre. Certains d'entre eux possèdent une structure génomique similaire à celle des rétrovirus, et sont appelés rétrovirus endogènes. Les éléments transposables sont une source de mutations et doivent donc être finement contrôlés par leurs hôtes. Afin de parer à leur mobilisation, les génomes ont mis en place des mécanismes de régulation impliquant de petits ARNs dont les siARNs sont la composante la plus connue. Récemment, il a été mis en évidence chez la drosophile une nouvelle classe de petits ARNs appelés piARNs qui contrôlent spécifiquement les éléments transposables dans les tissus reproducteurs. ZAM et Idefix sont 2 rétrotransposons à LTR chez la drosophile, isolés au sein du laboratoire. Habituellement quiescents et contrôlés par le génome de l'hôte, ils sont réactivés et mobilisés dans une lignée génétiquement instable où leur nombre de copies a été amplifié. L'étude des lignées de drosophile stables, réprimant l'expression de ZAM et Idefix, ainsi que de la lignée instable appelée Rev, a permis de localiser génétiquement le déterminant de la stabilité sur le chromosome X, dans la région 20A, un locus péricentromérique situé dans l'hétérochromatine. Cette région 20A est par ailleurs connue pour contrôler un autre rétrotransposon à LTR Gypsy et a donc été appelée "Centre Organisateur de la Mobilisation" : COM ou flam/COM. J'ai précisé les caractéristiques moléculaires du locus flam/COM dans différentes lignées contrôlant ou non l'expression de ZAM, Idefix et Gypsy. Il apparaît que la structure génomique et la conformation de la chromatine sont différentes. Par ailleurs, des transcrits ont été détectés dans certaines régions du locus flam/COM. Ils correspondent à des transcrits complémentaires des messagers d'éléments transposables localisés dans l'euchromatine. Il a été montré au laboratoire qu'Idefix était régulé par un mécanisme post-transcriptionnel (PTGS) qui implique les piARNS dans les tissus reproducteurs de drosophiles. Je me suis intéressé à savoir si en plus de ce contrôle, il existait une régulation transcriptionnelle (TGS) entretenue sur Idefix. En étudiant le différentiel d'expression entre un transgène contenant Idefix et donc réprimé par un PTGS et le même transgène dont la répression par PTGS est levée, j'ai montré qu'une régulation transcriptionnelle était absente dans les cellules folliculaires de l'ovaire.Transposable elements are DNA sequences, that have been found in all studied organisms, able to move from a chromosomal site to another causing genomic alterations. Thus, elaborated genomic defenses have evolved to restrict their transposition. One of those involves mechanisms depending on small RNA (RNAi), whose siARNs are the most known constituent. Recently, a new class of small RNA has been identified in Drosophila and called piRNA. These piRNAs control specifically transposable elements in reproductive tissues. ZAM and Idefix are two LTR retrotransposon in Drosophila melanogaster isolated in our laboratory. They are usually quiescent but we isolated a drosophila line which lost this control, and where ZAM and Idefix express and transpose at high rate. The genetic locus responsible for this control is located on the X chromosome, at position 20A. This heterochromatic region is also known to regulate the expression of Gypsy an other LTR retrotransposon and thus, has been called "Centre Organisateur de la Mobilisation" : COM or flam/COM. Here I describe the moleclar determinants present in the flam/COM locus between different lines which control or not the expression of ZAM, Idefix and Gypsy. It has been shown that genomic and heterochromatic structures are different. Furthermore, transcripts from this locus have been detected in some regions. These transcripts are complementary to transposable element messengers which are located elsewhere in the drosophila genome. It has been shown in the laboratory that Idefix is controled by a post-transcriptional regulation (PTGS) involving piRNA in reproductive tissues of Drosophila. I was interested to known if, besides PTGS, another regulation which would implicate a transcriptional silencing (TGS) might also repress the expression of Idefix. By studying the differential of expression between a transgene repressed by PTGS, because it contains an Idefix fragment, and the same transgene from which the PTGS repression is released, I showed that a TGS doesn't occur in the somatic cells of the adult ovaries.CLERMONT FD-BCIU-Santé (631132104) / SudocSudocFranceF

During a short window of Drosophila oogenesis, piRNA biogenesis may be boosted and mobilization of transposable elements allowed.

International audience

Transposon Reactivation in the Germline May Be Useful for Both Transposons and Their Host Genomes

International audienceTransposable elements (TEs) are long-term residents of eukaryotic genomes that make up a large portion of these genomes. They can be considered as perfectly fine members of genomes replicating with resident genes and being transmitted vertically to the next generation. However, unlike regular genes, TEs have the ability to send new copies to new sites. As such, they have been considered as parasitic members ensuring their own replication. In another view, TEs may also be considered as symbiotic sequences providing shared benefits after mutualistic interactions with their host genome. In this review, we recall the relationship between TEs and their host genome and discuss why transient relaxation of TE silencing within specific developmental windows may be useful for both