RESUMEN
BACKGROUND: A fraction of all genomes is composed of transposable elements (TEs) whose mobility needs to be carefully controlled. In gonads, TE activity is repressed by PIWI-interacting RNAs (piRNAs), a class of small RNAs synthesized by heterochromatic loci enriched in TE fragments, called piRNA clusters. Maintenance of active piRNA clusters across generations is secured by maternal piRNA inheritance providing the memory for TE repression. On rare occasions, genomes encounter horizontal transfer (HT) of new TEs with no piRNA targeting them, threatening the host genome integrity. Naïve genomes can eventually start to produce new piRNAs against these genomic invaders, but the timing of their emergence remains elusive. RESULTS: Using a set of TE-derived transgenes inserted in different germline piRNA clusters and functional assays, we have modeled a TE HT in Drosophila melanogaster. We have found that the complete co-option of these transgenes by a germline piRNA cluster can occur within four generations associated with the production of new piRNAs all along the transgenes and the germline silencing of piRNA sensors. Synthesis of new transgenic TE piRNAs is linked to piRNA cluster transcription dependent on Moonshiner and heterochromatin mark deposition that propagates more efficiently on short sequences. Moreover, we found that sequences located within piRNA clusters can have different piRNA profiles and can influence transcript accumulation of nearby sequences. CONCLUSIONS: Our study reveals that genetic and epigenetic properties, such as transcription, piRNA profiles, heterochromatin, and conversion efficiency along piRNA clusters, could be heterogeneous depending on the sequences that compose them. These findings suggest that the capacity of transcriptional signal erasure induced by the chromatin complex specific of the piRNA cluster can be incomplete through the piRNA cluster loci. Finally, these results have revealed an unexpected level of complexity that highlights a new magnitude of piRNA cluster plasticity fundamental for the maintenance of genome integrity.
Asunto(s)
Elementos Transponibles de ADN , Drosophila melanogaster , Animales , Elementos Transponibles de ADN/genética , Drosophila melanogaster/genética , Heterocromatina , Inmunización , Células Germinativas , ARN de Interacción con PiwiRESUMEN
Genome integrity of the animal germline is protected from transposable element activity by PIWI-interacting RNAs (piRNAs). While piRNA biogenesis is intensively explored, little is known about the genetical determination of piRNA clusters, the genomic sources of piRNAs. Using a bimodal epigenetic state piRNA cluster (BX2), we identified the histone demethylase Kdm3 as being able to prevent a cryptic piRNA production. In the absence of Kdm3, dozens of coding gene-containing regions become genuine germline dual-strand piRNA clusters. Eggs laid by Kdm3 mutant females show developmental defects phenocopying loss of function of genes embedded into the additional piRNA clusters, suggesting an inheritance of functional ovarian "auto-immune" piRNAs. Antagonizing piRNA cluster determination through chromatin modifications appears crucial to prevent auto-immune genic piRNAs production.
Asunto(s)
Proteínas de Drosophila , Drosophila , Animales , Femenino , Drosophila/genética , Drosophila/metabolismo , ARN de Interacción con Piwi , ARN Interferente Pequeño/genética , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Elementos Transponibles de ADN/genéticaRESUMEN
Transposable element (TE) activity is repressed in animal gonads by PIWI-interacting RNAs (piRNAs) produced by piRNA clusters. Current models in flies propose that germinal piRNA clusters are functionally defined by the maternal inheritance of piRNAs produced during the previous generation. Taking advantage of an inactive, but ready to go, cluster of P-element derived transgene insertions in Drosophila melanogaster, we show here that raising flies at high temperature (29°C) instead of 25°C triggers the stable conversion of this locus from inactive into actively producing functional piRNAs. The increase of antisense transcripts from the cluster at 29°C combined with the requirement of transcription of euchromatic homologous sequences, suggests a role of double stranded RNA in the production of de novo piRNAs. This report describes the first case of the establishment of an active piRNA cluster by environmental changes in the absence of maternal inheritance of homologous piRNAs. Editorial note: This article has been through an editorial process in which the authors decide how to respond to the issues raised during peer review. The Reviewing Editor's assessment is that all the issues have been addressed (see decision letter).
Asunto(s)
Drosophila melanogaster/genética , Drosophila melanogaster/efectos de la radiación , Exposición a Riesgos Ambientales , Epigénesis Genética , ARN Interferente Pequeño/metabolismo , Temperatura , Animales , Perfilación de la Expresión GénicaRESUMEN
Two Telomeric Associated Sequences, TAS-R and TAS-L, form the principal subtelomeric repeat families identified in Drosophila melanogaster. They are PIWI-interacting RNA (piRNA) clusters involved in repression of Transposable Elements. In this study, we revisited TAS structural and functional dynamics in D. melanogaster and in related species. In silico analysis revealed that TAS-R family members are composed of previously uncharacterized domains. This analysis also showed that TAS-L repeats are composed of arrays of a region we have named "TAS-L like" (TLL) identified specifically in one TAS-R family member, X-TAS. TLL were also present in other species of the melanogaster subgroup. Therefore, it is possible that TLL represents an ancestral subtelomeric piRNA core-cluster. Furthermore, all D. melanogaster genomes tested possessed at least one TAS-R locus, whereas TAS-L can be absent. A screen of 110 D. melanogaster lines showed that X-TAS is always present in flies living in the wild, but often absent in long-term laboratory stocks and that natural populations frequently lost their X-TAS within 2 years upon lab conditioning. Therefore, the unexpected structural and temporal dynamics of subtelomeric piRNA clusters demonstrated here suggests that genome organization is subjected to distinct selective pressures in the wild and upon domestication in the laboratory.
Asunto(s)
Simulación por Computador , Drosophila melanogaster/metabolismo , Evolución Molecular , Genes de Insecto , ARN Interferente Pequeño/genética , Animales , Elementos Transponibles de ADN , Drosophila melanogaster/genética , Femenino , Masculino , TelómeroRESUMEN
In 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 transposable 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.
Asunto(s)
Proteínas Cromosómicas no Histona/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Células Germinativas/crecimiento & desarrollo , Larva/genética , ARN Interferente Pequeño/genética , Animales , Elementos Transponibles de ADN/genética , Drosophila melanogaster/crecimiento & desarrollo , Desarrollo Embrionario , Epigénesis Genética/genética , Regulación del Desarrollo de la Expresión Génica , Silenciador del Gen , Células Germinativas/metabolismo , Gónadas/crecimiento & desarrollo , Gónadas/metabolismo , Larva/crecimiento & desarrolloRESUMEN
Transposable element activity is repressed in the germline in animals by PIWI-interacting RNAs (piRNAs), a class of small RNAs produced by genomic loci mostly composed of TE sequences. The mechanism of induction of piRNA production by these loci is still enigmatic. We have shown that, in Drosophila melanogaster, a cluster of tandemly repeated P-lacZ-white transgenes can be activated for piRNA production by maternal inheritance of a cytoplasm containing homologous piRNAs. This activated state is stably transmitted over generations and allows trans-silencing of a homologous transgenic target in the female germline. Such an epigenetic conversion displays the functional characteristics of a paramutation, i.e., a heritable epigenetic modification of one allele by the other. We report here that piRNA production and trans-silencing capacities of the paramutated cluster depend on the function of the rhino, cutoff, and zucchini genes involved in primary piRNA biogenesis in the germline, as well as on that of the aubergine gene implicated in the ping-pong piRNA amplification step. The 21-nt RNAs, which are produced by the paramutated cluster, in addition to 23- to 28-nt piRNAs are not necessary for paramutation to occur. Production of these 21-nt RNAs requires Dicer-2 but also all the piRNA genes tested. Moreover, cytoplasmic transmission of piRNAs homologous to only a subregion of the transgenic locus can generate a strong paramutated locus that produces piRNAs along the whole length of the transgenes. Finally, we observed that maternally inherited transgenic small RNAs can also impact transgene expression in the soma. In conclusion, paramutation involves both nuclear (Rhino, Cutoff) and cytoplasmic (Aubergine, Zucchini) actors of the piRNA pathway. In addition, since it is observed between nonfully homologous loci located on different chromosomes, paramutation may play a crucial role in epigenome shaping in Drosophila natural populations.
Asunto(s)
Núcleo Celular/genética , Citoplasma/genética , Genes de Insecto , ARN Interferente Pequeño/biosíntesis , Animales , Proteínas Cromosómicas no Histona/genética , Cruzamientos Genéticos , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Endorribonucleasas/genética , Epigénesis Genética , Femenino , Silenciador del Gen , Masculino , Mutación , Factores de Iniciación de Péptidos/genética , Proteínas de Unión al ARN/genética , TransgenesRESUMEN
Paramutation was initially described in maize and was defined as an epigenetic interaction between two alleles of a locus, through which one allele induces a heritable modification of the other allele without modifying the DNA sequence [1,2]. Thus it implies that the paramutated allele conserves its new properties on the long term over generations even in the absence of the paramutagenic allele and that it turns paramutagenic itself, without undergoing any changes in the DNA sequence. Some epigenetic interactions have been described in two non-vertebrate animal models, which appear to exhibit similar properties. Both systems are linked to trans-generational transmission of non-coding small RNAs. In Drosophila melanogaster, paramutation is correlated with transmission of PIWI-Interacting RNAs (piRNAs), a class of small non-coding RNAs that repress mobile DNA in the germline. A tandem repeated transgenic locus producing abundant ovarian piRNAs can activate piRNA production and associated homology-dependent silencing at a locus that was previously stably devoid of such capacities. The newly converted locus is then perfectly stable in absence of the inducer locus (>100 generations) and becomes fully paramutagenic. In Caenorhabditis elegans, paramutation is correlated with transmission of siRNAs, which are produced by transgenes targeted by piRNAs in the germline. Indeed, a transgenic locus, targeted by the piRNA machinery, produces siRNAs that can induce silencing of homologous transgenes, which can be further transmitted in a repressed state over generations despite the absence of the inducer transgenic locus. As in fly, the paramutated locus can become fully paramutagenic, and paramutation can be mediated by cytoplasmic inheritance without transmission of the paramutagenic locus itself. Nevertheless, in contrast to flies where the induction is only maternally inherited, both parents can transmit it in worms. In addition, a reciprocal phenomenon - (from off toward on) - appears to be also possible in worms as some activated transgenes can reactivate silent transgenes in the germline, and this modification can also be transmitted to next generations, even so it appears to be only partially stable. Thus, in a given system, opposite paramutation-like phenomena could exist, mediated by antagonist active pathways. As in plants, paramutation in flies and worms correlates with chromatin structure modification of the paramutated locus. In flies, inheritance of small RNAs from one generation to the next transmits a memory mainly targeting loci for repression whereas in worms, small RNAs can target loci either for repression or expression. Nevertheless, in the two species, paramutation can play an important role in the epigenome establishment.
Asunto(s)
Drosophila melanogaster/genética , Epigénesis Genética , Mutación , ARN Pequeño no Traducido/genética , AnimalesRESUMEN
Heterochromatin is made of repetitive sequences, mainly transposable elements (TEs), the regulation of which is critical for genome stability. We have analyzed the role of the heterochromatin-associated Su(var)3-7 protein in Drosophila ovaries. We present evidences that Su(var)3-7 is required for correct oogenesis and female fertility. It accumulates in heterochromatic domains of ovarian germline and somatic cells nuclei, where it co-localizes with HP1. Homozygous mutant females display ovaries with frequent degenerating egg-chambers. Absence of Su(var)3-7 in embryos leads to defects in meiosis and first mitotic divisions due to chromatin fragmentation or chromosome loss, showing that Su(var)3-7 is required for genome integrity. Females homozygous for Su(var)3-7 mutations strongly impair repression of P-transposable element induced gonadal dysgenesis but have minor effects on other TEs. Su(var)3-7 mutations reduce piRNA cluster transcription and slightly impact ovarian piRNA production. However, this modest piRNA reduction does not correlate with transposon de-silencing, suggesting that the moderate effect of Su(var)3-7 on some TE repression is not linked to piRNA production. Strikingly, Su(var)3-7 genetically interacts with the piwi and aubergine genes, key components of the piRNA pathway, by strongly impacting female fertility without impairing transposon silencing. These results lead us to propose that the interaction between Su(var)3-7 and piwi or aubergine controls important developmental processes independently of transposon silencing.
Asunto(s)
Proteínas Argonautas/metabolismo , Elementos Transponibles de ADN/genética , Proteínas de Drosophila/metabolismo , Fertilidad/fisiología , Oogénesis/fisiología , Solanum melongena/metabolismo , Animales , Proteínas Argonautas/genética , Drosophila , Proteínas de Drosophila/genética , Femenino , Fertilidad/genética , Silenciador del Gen , Oogénesis/genética , Solanum melongena/genéticaRESUMEN
During 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 retrotransposon 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.
Asunto(s)
Drosophila/genética , Silenciador del Gen , Oogénesis/genética , ARN Interferente Pequeño/metabolismo , Retroelementos , Animales , Proteínas Argonautas/metabolismo , Drosophila/crecimiento & desarrollo , Drosophila/metabolismo , Proteínas de Drosophila/metabolismo , Femenino , Mutación , TransgenesRESUMEN
Transposable element (TE) activity is repressed in the Drosophila germline by Piwi-Interacting RNAs (piRNAs), a class of small non-coding RNAs. These piRNAs are produced by discrete genomic loci containing TE fragments. In a recent publication, we tested for the existence of a strict epigenetic induction of piRNA production capacity by a locus in the D. melanogaster genome. We used 2 lines carrying a transgenic 7-copy tandem cluster (P-lacZ-white) at the same genomic site. This cluster generates in both lines a local heterochromatic sector. One line (T-1) produces high levels of ovarian piRNAs homologous to the P-lacZ-white transgenes and shows a strong capacity to repress homologous sequences in trans, whereas the other line (BX2) is devoid of both of these capacities. The properties of these 2 lines are perfectly stable over generations. We have shown that the maternal transmission of a cytoplasm carrying piRNAs from the first line can confer to the inert transgenic locus of the second, a totally de novo capacity to produce high levels of piRNAs as well as the ability to induce homology-dependent silencing in trans. These new properties are stably inherited over generations (n>50). Furthermore, the converted locus has itself become able to convert an inert transgenic locus via cytoplasmic maternal inheritance. This results in a stable epigenetic conversion process, which can be performed recurrently--a phenomenon termed paramutation and discovered in Maize 60 y ago. Paramutation in Drosophila corresponds to the first stable paramutation in animals and provides a model system to investigate the epigenetically induced emergence of a piRNA-producing locus, a crucial step in epigenome shaping. In this Extra View, we discuss some additional functional aspects and the possible molecular mechanism of this piRNA-linked paramutation.
Asunto(s)
Drosophila melanogaster/genética , Epigénesis Genética , ARN Interferente Pequeño/fisiología , Animales , Citoplasma/metabolismo , Femenino , Regulación de la Expresión Génica , Genoma de los Insectos , Masculino , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismoRESUMEN
Piwi-interacting RNAs (piRNAs) ensure transposable element silencing in Drosophila, thereby preserving genome integrity across generations. Primary piRNAs arise from the processing of long RNA transcripts produced in the germ line by a limited number of telomeric and pericentromeric loci. Primary piRNAs bound to the Argonaute protein Aubergine then drive the production of secondary piRNAs through the "ping-pong" amplification mechanism that involves an interplay with piRNAs bound to the Argonaute protein Argonaute-3. We recently discovered that clusters of P-element-derived transgenes produce piRNAs and mediate silencing of homologous target transgenes in the female germ line. We also demonstrated that some clusters are able to convert other homologous inactive transgene clusters into piRNA-producing loci, which then transmit their acquired silencing capacity over generations. This paramutation phenomenon is mediated by maternal inheritance of piRNAs homologous to the transgenes. Here we further mined our piRNA sequencing data sets generated from various strains carrying transgenes with partial sequence homology at distinct genomic sites. This analysis revealed that same sequences in different genomic contexts generate highly similar profiles of piRNA abundances. The strong tendency of piRNAs for bearing a U at their 5' end has long been recognized. Our observations support the notion that, in addition, the relative frequencies of Drosophila piRNAs are locally determined by the DNA sequence of piRNA loci.
Asunto(s)
Drosophila melanogaster/genética , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Animales , Proteínas Argonautas , Secuencia de Bases , Drosophila melanogaster/metabolismo , Femenino , Silenciador del Gen , Sitios Genéticos , Células Germinativas , ARN Interferente Pequeño/química , Análisis de Secuencia de ADN , Análisis de Secuencia de ARN , Homología de Secuencia de Ácido Nucleico , Transgenes , Uridina/metabolismoAsunto(s)
Drosophila/genética , Epigénesis Genética/fisiología , Patrón de Herencia/fisiología , Mutación/fisiología , Plantas/genética , Animales , Epigénesis Genética/genética , Sitios Genéticos , Patrón de Herencia/genética , Modelos Genéticos , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/fisiologíaRESUMEN
A paramutation is an epigenetic interaction between two alleles of a locus, through which one allele induces a heritable modification in the other allele without modifying the DNA sequence. The paramutated allele itself becomes paramutagenic, that is, capable of epigenetically converting a new paramutable allele. Here we describe a case of paramutation in animals showing long-term transmission over generations. We previously characterized a homology-dependent silencing mechanism referred to as the trans-silencing effect (TSE), involved in P-transposable-element repression in the germ line. We now show that clusters of P-element-derived transgenes that induce strong TSE can convert other homologous transgene clusters incapable of TSE into strong silencers, which transmit the acquired silencing capacity through 50 generations. The paramutation occurs without any need for chromosome pairing between the paramutagenic and the paramutated loci, and is mediated by maternal inheritance of cytoplasm carrying Piwi-interacting RNAs (piRNAs) homologous to the transgenes. The repression capacity of the paramutated locus is abolished by a loss-of-function mutation of the aubergine gene involved in piRNA biogenesis, but not by a loss-of-function mutation of the Dicer-2 gene involved in siRNA production. The paramutated cluster, previously producing barely detectable levels of piRNAs, is converted into a stable, strong piRNA-producing locus by the paramutation and becomes fully paramutagenic itself. Our work provides a genetic model for the emergence of piRNA loci, as well as for RNA-mediated trans-generational repression of transposable elements.
Asunto(s)
Drosophila melanogaster/genética , Silenciador del Gen , Sitios Genéticos/genética , ARN Interferente Pequeño/biosíntesis , ARN Interferente Pequeño/genética , Alelos , Animales , Citoplasma/genética , Elementos Transponibles de ADN/genética , Proteínas de Drosophila/deficiencia , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Herencia Extracromosómica/genética , Femenino , Células Germinativas/metabolismo , Masculino , Modelos Genéticos , Familia de Multigenes/genética , Mutación , Ovario/metabolismo , Factores de Iniciación de Péptidos/deficiencia , Factores de Iniciación de Péptidos/genética , Factores de Iniciación de Péptidos/metabolismo , ARN Helicasas/deficiencia , ARN Helicasas/genética , Ribonucleasa III/deficiencia , Ribonucleasa III/genética , Transgenes/genéticaRESUMEN
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) represses in trans, in the female germline, a homologous P-lacZ transgene inserted in euchromatin. TSE shows variegation in ovaries and displays a maternal effect as well as epigenetic transmission through meiosis. In addition, TSE is highly sensitive to mutations affecting heterochromatin components (including HP1) and the Piwi-interacting RNA silencing pathway (piRNA), a homology-dependent silencing mechanism that functions in the germline. TSE appears thus to involve the piRNA-based silencing proposed to play a major role in P repression. Under this hypothesis, TSE may also be established when homology between the telomeric and target loci involves sequences other than P elements, including sequences exogenous to the D. melanogaster genome. We have tested whether TSE can be induced via lacZ sequence homology. We generated a piggyBac-otu-lacZ transgene in which lacZ is under the control of the germline ovarian tumor promoter, resulting in strong expression in nurse cells and the oocyte. We show that all piggyBac-otu-lacZ transgene insertions are strongly repressed by maternally inherited telomeric P-lacZ transgenes. This repression shows variegation between egg chambers when it is incomplete and presents a maternal effect, two of the signatures of TSE. Finally, this repression is sensitive to mutations affecting aubergine, a key player of the piRNA pathway. These data show that TSE can occur when silencer and target loci share solely a sequence exogenous to the D. melanogaster genome. This functionally supports the hypothesis that TSE represents a general repression mechanism which can be co-opted by new transposable elements to regulate their activity after a transfer to the D. melanogaster genome.
RESUMEN
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.
Asunto(s)
Drosophila/genética , Epigénesis Genética , Silenciador del Gen , Impresión Genómica , Heterocromatina/genética , ARN/genética , Animales , Femenino , Ovario/metabolismo , Fenotipo , TransgenesRESUMEN
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.
Asunto(s)
Drosophila melanogaster/genética , Silenciador del Gen , Telómero/ultraestructura , Animales , Mapeo Cromosómico , Cruzamientos Genéticos , Elementos Transponibles de ADN , Epigénesis Genética , Regulación de la Expresión Génica , Modelos Biológicos , Modelos Genéticos , Fenotipo , Interferencia de ARN , Temperatura , TransgenesRESUMEN
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.
Asunto(s)
Epigénesis Genética , Silenciador del Gen , Heterocromatina/genética , ARN/genética , Telómero , Animales , Homólogo de la Proteína Chromobox 5 , Elementos Transponibles de ADN , Proteínas de Drosophila , Drosophila melanogaster , Femenino , Mutación , ARN Interferente Pequeño , TransgenesRESUMEN
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 of stationary, nonrepeated sequences, potentially encoding 66-kDa repressor-like (RL) proteins. Here we investigate the function of the montium P neogenes. We provide evidence for the presence of RL 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 distantly related mobile P elements which coexist in the genome. Our results strongly suggest that the montium P neogenes are not recruited to downregulate 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 RL proteins to bind DNA in vivo. Immunostaining of polytene chromosomes in D. melanogaster transgenic lines strongly suggests that montium P neogenes encode proteins that bind DNA in vivo. RL 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.
Asunto(s)
Elementos Transponibles de ADN/genética , ADN/metabolismo , Drosophila/clasificación , Drosophila/genética , Animales , Encéfalo/metabolismo , Cromatina/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Evolución Molecular , Exones/genética , Femenino , Gónadas/metabolismo , Proteínas de Insectos/análisis , Proteínas de Insectos/genética , Proteínas de Insectos/metabolismo , Larva/metabolismo , Masculino , Mutagénesis Insercional , Ovario/metabolismo , Regiones Promotoras Genéticas/genética , Testículo/citología , Transcripción GenéticaRESUMEN
In Drosophila, relocation of a euchromatic gene near centromeric or telomeric heterochromatin often leads to its mosaic silencing. Nevertheless, modifiers of centromeric silencing do not affect telomeric silencing, suggesting that each location requires specific factors. Previous studies suggest that a subset of Polycomb-group (PcG) proteins could be responsible for telomeric silencing. Here, we present the effect on telomeric silencing of 50 mutant alleles of the PcG genes and of their counteracting trithorax-group genes. Several combinations of two mutated PcG genes impair telomeric silencing synergistically, revealing that some of these genes are required for telomeric silencing. In situ hybridization and immunostaining experiments on polytene chromosomes revealed a strict correlation between the presence of PcG proteins and that of heterochromatic telomeric associated sequences (TASs), suggesting that TASs and PcG complexes could be associated at telomeres. Furthermore, lines harboring a transgene containing an X-linked TAS subunit and the mini-white reporter gene can exhibit pairing-sensitive repression of the white gene in an orientation-dependent manner. Finally, an additional binding site for PcG proteins was detected at the insertion site of this type of transgene. Taken together, these results demonstrate that PcG proteins bind TASs in vivo and may be major players in Drosophila telomeric position effect (TPE).
Asunto(s)
Proteínas de Drosophila/metabolismo , Drosophila/genética , Regulación de la Expresión Génica , Telómero , Factores de Transcripción , Animales , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Proteínas de Drosophila/genética , Genes Reporteros , Complejo Represivo Polycomb 1RESUMEN
Autonomous P elements, inserted in heterochromatic telomeric associated sequences (TAS) at the X chromosome telomere (site 1A) have strong P element regulatory properties that include repression of P-induced hybrid-dysgenesis and of P-lacZ expression in the germline. P-lacZ insertions or defective P elements at 1A in TAS can also repress in trans a euchromatic P-lacZ in the germline. This property has been called a trans-silencing effect (TSE). It requires some sequence-homology between the telomeric insertion and the euchromatic transgene. When repression is partial, variegating lacZ expression is observed, suggesting a chromatin-based component. TSE is observed only when the silencer transgenes are maternally inherited and occurs only in the female germline. We have evidence that this silencing also works in the presence of homologous non-P element sequences suggesting that homology-dependent silencing could be a general phenomenon in the female germline; such a system might have been subsequently adopted by the P element family, allowing its own repression.