Repair of P element ends following hybrid element excision leads to recombination in Drosophila melanogaster

P elements are thought to replicate themselves starting with the association of the left and right ends, followed by a cut-copy-paste process. An abnormal form of this process has been shown to occur when the associated left and right ends come from sister elements rather than from the same element, leading to formation of a 'hybrid element.' These ends can insert nearby in the genome to produce recombination, with associated structural changes. We have previously increased the frequency of such 'hybrid element insertion' by combining end-deleted elements in trans in a genotype with a left-end on one chromosome and a right-end on the homologous chromosome. Although many recombinants produced by this genotype have structural changes expected with insertion, nearly 50% of the predicted insertional recombinants contain no structural change. We present evidence using RFLP markers closely linked to the end-deleted elements that in these cases the P element ends dissociate before insertion, and are subsequently ligated together following a process analogous to synthesis-dependent strand annealing. The results suggest that broken ends containing P elements are resolved by the same repair process as ends not containing P elements, and that such repair from hybrid element events may occur in the majority of cases.     

Cross-Species RNAi Rescue Platform in Drosophila melanogaster

RNAi-mediated gene knockdown in Drosophila melanogaster is a powerful method to analyze loss-of-function phenotypes both in cell culture and in vivo. However, it has also become clear that false positives caused by off-target effects are prevalent, requiring careful validation of RNAi-induced phenotypes. The most rigorous proof that an RNAi-induced phenotype is due to loss of its intended target is to rescue the phenotype by a transgene impervious to RNAi. For large-scale validations in the mouse and Caenorhabditis elegans, this has been accomplished by using bacterial artificial chromosomes (BACs) of related species. However, in Drosophila, this approach is not feasible because transformation of large BACs is inefficient. We have therefore developed a general RNAi rescue approach for Drosophila that employs Cre/loxP-mediated recombination to rapidly retrofit existing fosmid clones into rescue constructs. Retrofitted fosmid clones carry a selection marker and a phiC31 attB site, which facilitates the production of transgenic animals. Here, we describe our approach and demonstrate proof-of-principle experiments showing that D. pseudoobscura fosmids can successfully rescue RNAi-induced phenotypes in D. melanogaster, both in cell culture and in vivo. Altogether, the tools and method that we have developed provide a gold standard for validation of Drosophila RNAi experiments.RNAi-mediated gene knockdown, whereby an exogenous double stranded RNA (dsRNA) is used to trigger homology-dependent suppression of the target gene, is an effective loss-of-function method to interrogate gene function. The RNAi technology in Drosophila melanogaster is widely used for genomewide RNAi screens in cell culture (see review by Perrimon and Mathey-Prevot 2007a), and more recently has been extended to large scale in vivo studies (Dietzl et al. 2007; Ni et al. 2009; Mummery-Widmer et al. 2009). Gene knockdown by RNAi is achieved by the introduction of dsRNAs into cultured cells or by inducible overexpression of “hairpin” dsRNAs in transgenic flies. In the context of in vivo RNAi screening, the combination of a tissue-specific GAL4 driver with a GAL4-responsive hairpin dsRNA transgene allows knockdown of the target gene only in the desired cells, thus providing a powerful way of probing biological processes that have been so far difficult to investigate.Analysis of the specificity of long dsRNAs in Drosophila cells has revealed that these reagents, depending on their sequences and levels of expression, can knock down genes others than the intended target (Kulkarni et al. 2006; Ma et al. 2006). This phenomenon is not specific to long dsRNAs and has also been commonly observed with 21-nt long siRNAs and shRNAs used in mammalian RNAi screens. In fact the rate of false positives associated with off-target effects observed in mammalian screens is usually higher than those observed with long dsRNAs (Echeverri and Perrimon 2006). Unwanted false positives created by off-target effects are a major problem in RNAi screens and require lengthy secondary validation tests (Echeverri and Perrimon 2006; Perrimon and Mathey-Prevot 2007b; Ramadan et al. 2007). Further, false positives associated with RNAi reagents are not limited to tissue culture experiments, as they have also been reported in the context of transgenic RNAi. For example, ∼25% of the hairpins targeting nonessential genes cause lethality when driven by the constitutively expressed Act5C-GAL4 driver (Dietzl et al. 2007; Ni et al. 2009).A number of approaches can be used to validate the specificity of RNAi-induced phenotypes (Echeverri and Perrimon 2006). These include validation by multiple dsRNAs that target the same gene but that do not overlap in sequence, comparison of knockdown efficiencies of multiple dsRNAs and the phenotypic strengths, and rescue of the phenotype by either cDNAs or genomic DNAs. Rescue of RNAi phenotypes constitutes the gold standard in the field as it provides unambiguous proof that the targeted gene is indeed responsible for the phenotype observed. In Drosophila cell culture experiments, cDNAs that lack the original 3′-untranslated region (UTR) have been used to rescue phenotypes induced by dsRNAs targeting the 3′-UTR (Yokokura et al. 2004; Stielow et al. 2008). In mammalian cell culture experiments, cDNAs that have a silent point mutation in the region targeted by an siRNA are commonly used (Lassus et al. 2002). The intrinsic problem of these approaches, however, is that overexpression of cDNAs alone can evoke abnormal cellular responses on their own, complicating interpretation of the results. A cleaner method is based on cross-species rescue that uses genomic DNA from a different species whose sequence is divergent enough from the host species to make it refractory to the RNAi reagent directed against the host gene. This approach effectively addresses the issue of overexpression artifact, as the rescue transgene is expressed from its endogenous promoter, ensuring proper levels and precise spatiotemporal regulation of gene expression. Cross-species rescue methods that use bacterial artificial chromosome (BACs) retrofitted with an appropriate selection marker have been described for mammals and C. elegans (Kittler et al. 2005; Sarov et al. 2006). However, the BAC strategies are not realistic for large-scale studies, because transformation of BACs, which are typically larger than 100 kb, is inefficient, albeit not impossible, in Drosophila (Venken et al. 2006).To provide a feasible way to validate large-scale RNAi screening results, we decided to develop a universal method for cross-species RNAi rescue in Drosophila. We chose to use fosmids, which are single-copy bacterial vectors with a cloning capacity of ∼40 kb, rather than BACs because (1) transformation of plasmids around this size is relatively efficient (Venken et al. 2006) and (2) end-sequenced fosmid clones for 11 different Drosophila species generated by the Drosophila species genome project are now publicly available (Richards et al. 2005; Drosophila 12 Genomes Consortium 2007).     

Adaptation to P element transposon invasion in Drosophila melanogaster

Transposons evolve rapidly and can mobilize and trigger genetic instability. Piwi-interacting RNAs (piRNAs) silence these genome pathogens, but it is unclear how the piRNA pathway adapts to invasion of new transposons. In Drosophila, piRNAs are encoded by heterochromatic clusters and maternally deposited in the embryo. Paternally inherited P element transposons thus escape silencing and trigger a hybrid sterility syndrome termed P-M hybrid dysgenesis. We show that P-M hybrid dysgenesis activates both P elements and resident transposons and disrupts the piRNA biogenesis machinery. As dysgenic hybrids age, however, fertility is restored, P elements are silenced, and P element piRNAs are produced de novo. In addition, the piRNA biogenesis machinery assembles, and resident elements are silenced. Significantly, resident transposons insert into piRNA clusters, and these new insertions are transmitted to progeny, produce novel piRNAs, and are associated with reduced transposition. P element invasion thus triggers heritable changes in genome structure that appear to enhance transposon silencing.     

Differential gene silencing by trans-heterochromatin in Drosophila melanogaster.

The brown(Dominant) (bw(D)) allele contains a large insertion of heterochromatin leading to the trans-inactivation of the wild-type allele in bw(D)/bw(+) heterozygous flies. This silencing is correlated with the localization of bw(+) to a region of the interphase nucleus containing centric heterochromatin. We have used a series of transgene constructs inserted in the vicinity of the bw locus to demarcate both the extent of bw(D) influence along the chromosome and the relative sensitivities of various genes. Examples of regulatory regions that are highly sensitive, moderately sensitive, and insensitive were found. Additionally, by using the same transgene at increasing distances from the bw(D) insertion site in trans we were able to determine the range of influence of the heterochromatic neighborhood in terms of chromosomal distance. When the transgene was farther away from bw, there was, indeed, a tendency for it to be less trans-inactivated. However, insertion site also influenced silencing: a gene 86 kb away was trans-inactivated, while the same transgene 45 kb away was not. Thus location, distance, and gene-specific differences all influence susceptibility to trans-silencing near a heterochromatic neighborhood. These results have important implications for the ability of nuclear positioning to influence the expression of large blocks of a chromosome.     

RNAi living-cell microarrays for loss-of-function screens in Drosophila melanogaster cells

RNA interference (RNAi)-mediated loss-of-function screening in Drosophila melanogaster tissue culture cells is a powerful method for identifying the genes underlying cell biological functions and for annotating the fly genome. Here we describe the development of living-cell microarrays for screening large collections of RNAi-inducing double-stranded RNAs (dsRNAs) in Drosophila cells. The features of the microarrays consist of clusters of cells 200 mum in diameter, each with an RNAi-mediated depletion of a specific gene product. Because of the small size of the features, thousands of distinct dsRNAs can be screened on a single chip. The microarrays are suitable for quantitative and high-content cellular phenotyping and, in combination screens, for the identification of genetic suppressors, enhancers and synthetic lethal interactions. We used a prototype cell microarray with 384 different dsRNAs to identify previously unknown genes that affect cell proliferation and morphology, and, in a combination screen, that regulate dAkt/dPKB phosphorylation in the absence of dPTEN expression.     

Structural instability of 297 element in Drosophila melanogaster

297 element Southern pattern modifications previously detected in mutation accumulation lines of Drosophila melanogaster were further investigated by in situ hybridisation, Southern blotting with different combinations of genomic digest-probe, and PCR. Only one out of the nine pattern modifications studied could be interpreted as an excision and was detectable by in situ hybridisation to polytene chromosomes. Results were consistent with most pattern modifications being small rearrangements within the body of the element. In agreement with the existence of spontaneous rearrangements of this kind is the observation that many genomic copies of element 297 are defective and these are not limited to heterochromatin. These findings have important implications for the models of transposable element (TE) number regulation as well as for the study of genome evolution. This revised version was published online in July 2006 with corrections to the Cover Date.     

A simplified miRNA-based gene silencing method for Drosophila melanogaster

MicroRNA-based RNA interference is commonly used to produce loss-of-function phenotypes in mammalian systems, but is used only sparingly in invertebrates such as Caenorhabditis elegans and Drosophila melanogaster. Here, we evaluate this method in transgenic strains of D. melanogaster and cultured S2 cells. High throughput-ready expression vectors were developed that permit rapid cloning of synthetic hairpin RNAs. As proof of concept, this method was used for the efficient silencing of dpp gene activity in the adult wing, and the analysis of the general RNA Polymerase II (Pol II) elongation factor, Nelf-E.     

The interplay of transposon silencing genes in the Drosophila melanogaster germline

Complexes of Piwi proteins and Piwi-interacting RNAs (piRNAs) carry out the repression of transposable elements in animal gonads. The Piwi protein clade is represented in D. melanogaster by three members: Piwi, Aub and Ago3. Piwi protein functions in the nuclei of somatic and germinal ovarian cells, whereas Aub and Ago3 are cytoplasmic proteins of germinal cells. Aub and Ago3 interact with each other in the perinuclear nuage organelle to perform piRNA amplification via the ping-pong mechanism. Previously, derepression of several transposable elements as a result of mutations in the piRNA silencing system was shown. Here we quantify the increase in expression level of an enlarged number of retrotransposons due to the mutations in the piwi gene, nuage components coding aub, mael and spn-E genes and the RNA helicase armi gene mutation that impairs Piwi nuclear localization, but not the ping-pong cycle. We reveal that piwi, armi, aub, spn-E and mael genes participate together in the repression of several transposons (HMS-Beagle, Gate and HeT-A), whereas silencing of land G elements requires the same genes except piwi. We suggest that Armi has other functions besides the localizing of Piwi protein in the nuclei. Our data suggest also a role of cytoplasmic Aub, Spn-E and Mael nuage proteins in Piwi-mediated repression of Gate and HMS-Beagle transposons in the germline nuclei. As a whole, our results corroborate the idea that genome stabilization in the germline is realized by different silencing strategies specific for different transposable elements. At the same time, our data suggest the existence of yet unknown mechanisms of interplay between nuclear and cytoplasmic components of the piRNA machinery in the germline.     

Transvection and silencing of the Scr homeotic gene of Drosophila melanogaster

The Sex combs reduced (Scr) gene specifies the identities of the labial and first thoracic segments in Drosophila melanogaster. In imaginal cells, some Scr mutations allow cis-regulatory elements on one chromosome to stimulate expression of the promoter on the homolog, a phenomenon that was named transvection by Ed Lewis in 1954. Transvection at the Scr gene is blocked by rearrangements that disrupt pairing, but is zeste independent. Silencing of the Scr gene in the second and third thoracic segments, which requires the Polycomb group proteins, is disrupted by most chromosomal aberrations within the Scr gene. Some chromosomal aberrations completely derepress Scr even in the presence of normal levels of all Polycomb group proteins. On the basis of the pattern of chromosomal aberrations that disrupt Scr gene silencing, we propose a model in which two cis-regulatory elements interact to stabilize silencing of any promoter or cis-regulatory element physically between them. This model also explains the anomalous behavior of the Scx allele of the flanking homeotic gene, Antennapedia. This allele, which is associated with an insertion near the Antennapedia P1 promoter, inactivates the Antennapedia P1 and P2 promoters in cis and derepresses the Scr promoters both in cis and on the homologous chromosome.     

Different aubergine alleles confirm the specificity of different RNAi pathways in Drosophila melanogaster

The crystal-Stellate system is one of the best-known example of heterochromatin-euchromatin interaction. The components of this system are homologous repetitive sequences clustered in three regions: 12E1 and h27 on the X, h11 on the Y. The symptom of a disrupted crystal-Stellate interaction is the presence of crystals in spermatocytes of males lacking the crystal region. Stellate silencing is based on the RNAi process. Many modifiers of this system have been isolated and many of these are involved in RNAi. One of these modifiers is aubergine^sting; this is a "gain of function" allele in somatic tissues. Here we report the different behavior of two aubergine alleles with respect to the RNAi pathway: aub^sting and a "loss of function" heteroallelic combination aub^HN/aub^QC42. An increased amount of Aub interferes with the correct functioning of the somatic yellow hairpin RNAi, whereas the Aub reduction does not. We also demonstrate the different behavior of these alleles on the I trasposon silencing in ovaries. Intriguingly, both of these aubergine alleles do not silence the Stellate locus.

We can conclude that the crystal-Stellate system reveals different RNAi pathways even though much still remains to be done to completely explain the molecular bases of the crystal-Stellate interaction.