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BACKGROUND Genetic manipulation of sex determination pathways in insects provides the basis for a broad range of strategies to benefit agricultural security and human health. The P-element somatic inhibitor (PSI) protein, an exon splicing silencer that promotes male-specific splicing of dsx, plays a critical role in male sexual differentiation and development. The functions of PSI have been characterized in the lepidopteran model species Bombyx mori. However, the molecular mechanism and functions of PSI in Plutella xylostella, a worldwide agricultural pest and taxonomically basal species, are still unknown. RESULTS Here we identified PxPSI transcripts and analyzed their spatiotemporal expression pattern in P. xylostella. Multiple sequence alignment revealed that PxPSI contains four KH domains and is highly conserved in lepidopterans. We used the CRISPR-Cas9 system to generate mutations of the PxPSI genomic locus. Disruptions of PxPSI caused male-specific defects in internal and external genitals. In addition, we detected female-specific Pxdsx transcripts in PxPSI male mutants. Mutations also caused changes in expression of several sex-biased genes and induced male sterility. CONCLUSION Our study demonstrates that PxPSI plays a key role in male sex determination in P. xylostella and suggests a potential molecular target for genetic-based pest management in lepidopteran pests. © 2021 Society of Chemical Industry.

The use of transposons to create mutations has been the cornerstone of Drosophila genetics in the past few decades. Second-site mutations caused by transpositions are often devoid of transposons and thereby affect subsequent analyses. In a P-element mutagenesis screen, a second site mutation was identified on chromosome 3, wherein the homozygous mutants exhibit classic hallmarks of tumor suppressor mutants, including brain tumor and lethality; hence the mutant line was initially named as lethal (3) tumorous brain [l(3)tb]. Classical genetic approaches relying on meiotic recombination and subsequent complementation with chromosomal deletions and gene mutations mapped the mutation to CG6169, the mRNA decapping protein 2 (DCP2), on the left arm of the third chromosome (3L). Thus the mutation was renamed as DCP2l(3)tb. Fine mapping of the mutation further identified the presence of a Gypsy-LTR like sequence in the 5′UTR coding region of DCP2, along with the expansion of the adjacent upstream intergenic AT-rich sequence. The mutant phenotypes are rescued by the introduction of a functional copy of DCP2 in the mutant background, thereby establishing the causal role of the mutation and providing a genetic validation of the allelism. With the increasing repertoire of genes being associated with tumor biology, this is the first instance of mRNA decapping protein being implicated in Drosophila tumorigenesis. Our findings, therefore, imply a plausible role for the mRNA degradation pathway in tumorigenesis and identify DCP2 as a potential candidate for future explorations of cell cycle regulatory mechanisms.

Transposable elements (TEs) are repetitive regions of DNA that are able to self-replicate and reinsert themselves throughout host genomes. Since the discovery of TEs, a prevalent question has been whether increasing TE copy number has an effect on the fitness of their hosts. P-elements (PEs) in Drosophila are a well-studied TE that has strong phenotypic effects. When a female without PEs (M) is crossed to a male with them (P), the resulting females are often sterile, a phenomenon called hybrid dysgenesis (HD). Here, we used short- and long-read sequencing to infer the number of PEs in the genomes of dozens of isofemale lines from two Drosophila species and measured whether the magnitude of HD was correlated with the number of PEs in the paternal genome. Consistent with previous reports, we find evidence for a positive correlation between the paternal PE copy number and the magnitude of HD in progeny from ♀M × ♂ P crosses for both species. Other crosses are not affected by the number of PE copies. We also find that the correlation between the strength of HD and PE copy number differs between species, which suggests that there are genetic differences that might make some genomes more resilient to the potentially deleterious effects of TEs. Our results suggest that PE copy number interacts with other factors in the genome and the environment to cause HD and that the importance of these interactions is species specific.

Peer reviewed: True, P elements were first discovered in the fruit fly Drosophila melanogaster as the causative agents of a syndrome of aberrant genetic traits called hybrid dysgenesis. This occurs when P element-carrying -males mate with females that lack P elements and results in progeny displaying sterility, mutations and chromosomal rearrangements. Since then numerous genetic, developmental, biochemical and structural studies have culminated in a deep understanding of P element transposition: from the cellular regulation and repression of transposition to the mechanistic details of the transposase nucleoprotein complex. Recent studies have revealed how piwi-interacting small RNA pathways can act to control splicing of the P element pre-mRNA to modulate transposase production in the germline. A recent cryo-electron microscopy structure of the P element transpososome reveals an unusual DNA architecture at the transposon termini and shows that the bound GTP cofactor functions to position the transposon ends within the transposase active site. Genome sequencing efforts have shown that there are P element transposase-homologous genes (called THAP9) in other animal genomes, including humans). This review highlights recent and previous studies, which together have led to new insights, and surveys our current understanding of the biology, biochemistry, mechanism and regulation of P element transposition.

The use of transposons to create mutants has been the cornerstone ofDrosophilagenetics in the past few decades. Transpositions often create second-site mutations, devoid of transposon insertion and thereby affect subsequent phenotype analyses. In aP-element mutagenesis screen, a second site mutant was discovered on chromosome 3 wherein the homozygous mutant individuals show the classic hallmarks of mutations in tumor suppressor genes including brain tumour and lethality, hence the mutant line was initially named aslethal (3) tumorous brain[l(3)tb]. Classical genetic approaches relying on meiotic recombination and subsequent complementation with chromosomal deletions and gene mutations mapped the mutation to CG6169, the mRNA decapping protein 2 (DCP2), on the left arm of the third chromosome (3L), and thus the mutation was renamed asDCP2l(3)tb. Fine mapping of the mutation further identified the presence of aGypsy-LTR like sequence in the 5’UTR coding region ofDCP2, alongwith expansion of the adjacent upstream intergenic AT-rich sequence. The mutant phenotypes are rescued by Introduction of a functional copy ofDCP2in the mutant background, thereby establishing the causal role of the mutation and providing a genetic validation of the allelism. With the increasing repertoire of genes being associated with tumor biology this is the first instance that the mRNA decapping protein is being implicated inDrosophilatumourigenesis. Our findings therefore imply a plausible role for mRNA degradation pathway in tumorigenesis and identifyDCP2as a potential candidate for future explorations of cell cycle regulatory mechanisms.

Efficient P element transposition is dependent on the expression of a transposon-encoded transposase and the recruitment of endogenous Drosophila DNA repair proteins to sites of transposase induced DNA breaks. Previous biochemical analysis of proteins bound to the 31 bp inverted repeats identified an 18 kDa basic leucine zipper protein (bZIP) (CG6272) termed Inverted Repeat Binding Protein 18 (IRBP18). IRBP18 forms a functional heterodimer with another bZIP protein Xrp1/CG17836, which is sufficient to bind the 31bp terminal inverted repeats in vitro. Together these proteins, in cell culture models, repress transcription from the P element promoter, as well as facilitate efficient DNA repair after transposase-mediated cleavage. Additionally flies homozygous for null mutations of IRBP18 show increased killing after somatic mobilization of P elements: consistent with an inability to repair post cleavage DNA breaks. In the absence of P elements, IRBP18-null flies are male sterile and display increased sensitivity to DNA damaging agents. IRBP18/Xrp1 heterodimer forms the site-specific binding core of a larger IRBP complex; of which several of the co-purifying proteins function in RNA interference (RNAi) pathways. Recently RNAi pathways have been shown to be integral components of general genome maintenance due to regulation of heterochromatin formation of repeated DNA elements, such as ribosomal DNA genes. In addition in this thesis Rm62, the novel role of the DEAD/H-box RNA helicase, in DNA repair is examined. Flies homozygous for a hypomorphic allele of Rm62 display similar sensitivity to DNA damaging agents as IRBP18 mutants. Interestingly, the majority of the DNA repair phenotype of Rm62 is caused by a nucleolar localized isoform of the protein. Furthermore RNAi-knockdown of the Rm62 interaction partner, Argonaute 2, also resulted in decrease in DNA repair efficiency. Taken together, these data implicate the IRBP complex as a critical component in maintaining genomic integrity and thereby connecting a new site-specific DNA binding protein complex to genome stability and DNA repair.

Using the FISH method and PCR analysis, the presence of canonical P element was studied in the genomes of a number of laboratory lines isolated from nature in different years from the Zaprionus genus (Z. indianus) and Drosophila genus, Sophophora subgenus (D. ananassae, D. eugracilis, D. simulans, D. immigrans), Drosophila subgenus (D. virilis, D. mercatorum, D. hydei, D. funebris, D. pseudoobscura), Lordiphosa subgenus (L. magnipectinata), and Dorsilopha subgenus (D. busckii) in a search for new cases of horizontal transfer. According to our data, the L. magnipectinata genome contains sequences homologous to terminal regions of canonical P element, as well as the sequence with a weak homology from the central part of canonical P element. The P-element hybridization sites adjacent to the chromocenter were found in the D. pseudoobscura genome; this can indicate an ancient origin of the sequences homologous to the P element. The P element is absent in old D. simulans lines, except for the line isolated from nature in 2014 (in which the P element was found); this confirms data of other researchers about recent cases of horizontal P-element transfer in this species. No new cases of horizontal transfer were detected in the analyzed lines.

Transposable elements (TEs) are virtually ubiquitous components of genomes, yet they often impose significant fitness consequences on their hosts. In addition to producing specific deleterious mutations by insertional inactivation, TEs also impose general fitness costs by inducing DNA damage and participating in ectopic recombination. These latter fitness costs are often assumed to be dosage-dependent, with stronger effects occurring in the presence of higher TE copy numbers. We test this assumption in Drosophila melanogaster by considering the relationship between the copy number of two active DNA transposons, P-element and hobo element, and the incidence of hybrid dysgenesis, a sterility syndrome associated with transposon activity in the germline. By harnessing a subset of the Drosophila Genetic Reference Panel (DGRP), a group of fully-sequenced D. melanogaster strains, we describe quantitative and structural variation in P-elements and hobo elements among wild-derived genomes and associate these factors with hybrid dysgenesis. We find that the incidence of hybrid dysgenesis is associated with both P-element and hobo element copy number in a dosage-dependent manner. However, the relationship is weak for both TEs, suggesting that dosage alone explains only a small part of TE-associated fitness costs.

The moso bamboo genome contains the two structurally intact and thus potentially functional mariner-like elements Ppmar1 and Ppmar2. Both elements contain perfect terminal inverted repeats (TIRs) and a full-length intact transposase gene. Here we investigated whether Ppmar1 is functional in yeast (Saccharomyces cerevisiae). We have designed a two-component system consisting of a transposase expression cassette and a non-autonomous transposon on two separate plasmids. We demonstrate that the Ppmar1 transposase Pptpase1 catalyses excision of the non-autonomous Ppmar1NA element from the plasmid and reintegration at TA dinucleotide sequences in the yeast chromosomes. In addition, we generated 14 hyperactive Ppmar1 transposase variants by systematic single amino acid substitutions. The most active transposase variant, S171A, induces 10-fold more frequent Ppmar1NA excisions in yeast than the wild type transposase. The Ppmar1 transposon is a promising tool for insertion mutagenesis in moso bamboo and may be used in other plants as an alternative to the established transposon tagging systems.

Without transposon-silencing Piwi-interacting RNAs (piRNAs), transposition causes an ovarian atrophy syndrome in Drosophila called gonadal dysgenesis (GD). Harwich (Har) strains with P-elements cause severe GD in F1 daughters when Har fathers mate with mothers lacking P-element-piRNAs (i.e. ISO1 strain). To address the mystery of why Har induces severe GD, we bred hybrid Drosophila with Har genomic fragments into the ISO1 background to create HISR-D or HISR-N lines that still cause Dysgenesis or are Non-dysgenic, respectively. In these lines, we discovered a highly truncated P-element variant we named ‘Har-P’ as the most frequent de novo insertion. Although HISR-D lines still contain full-length P-elements, HISR-N lines lost functional P-transposase but retained Har-P’s that when crossed back to P-transposase restores GD induction. Finally, we uncovered P-element-piRNA-directed repression on Har-P’s transmitted paternally to suppress somatic transposition. The Drosophila short Har-P’s and full-length P-elements relationship parallels the MITEs/DNA-transposase in plants and SINEs/LINEs in mammals., eLife digest DNA provides the instructions needed for life, a role that relies on it being a very stable and organized molecule. However, some sections of DNA are able to move from one place in the genome to another. When these “mobile genetic elements” move they may disrupt other genes and cause disease. For example, a mobile section of DNA known as the P-element causes a condition called gonadal dysgenesis in female fruit flies, leading to infertility. Only certain strains of fruit flies carry P-elements and the severity of gonadal dysgenesis in their daughters varies. For example, when male fruit flies of a strain known as Harwich (or Har for short) is crossed with female fruit flies that do not contain P-elements, all of their daughters develop severe gonadal dysgenesis and are infertile. However, if the cross is done the other way around, and female Har flies mate with males that do not contain P-elements, the daughters are fertile because the Har mothers provide their daughters with protective molecules that silence the P-elements. But it was a mystery as to why the P-elements from the Har fathers always caused such severe gonadal dysgenesis in all the daughters. Here, Srivastav et al. bred fruit flies to create offspring that had different pieces of Har DNA in a genetic background that was normally free from P-elements; they then analyzed the ‘hybrid’ offspring to identify which pieces of the Har genome caused gonadal dysgenesis in the daughter flies. These experiments showed that Har flies possess a very short variant of the P-element (named “Har-P”) that is more mobile than other variants. However, the Har-P variants still depended on an enzyme known as P-transposase encoded by the full-length P-elements to move around the genome. Further experiments showed that other strains of fruit flies that cause severe gonadal dysgenesis also had very short P-element variants that were almost identical to Har-P. These findings may explain why Har and some other strains of fruit flies drive severe gonadal dysgenesis. In the future, it may be possible to transfer P-transposase and Har-P into mosquitoes, ticks and other biting insects to make them infertile and help reduce the spread of certain diseases in humans.

Without transposon-silencing Piwi-interacting RNAs (piRNAs), transposition causes an ovarian atrophy syndrome inDrosophilacalled gonadal dysgenesis (GD).Harwich(Har) strains withP-elements cause severe GD in F1 daughters whenHarfathers mate with mothers lackingP-element-piRNAs (i.e.ISO1strain). To address the mystery of whyHarinduces severe GD, we bred hybridDrosophilawithHargenomic fragments into theISO1background to createHISR-D or HISR-Nlines that still causeDysgenesis or areNon-dysgenic, respectively. In these lines, we discovered a highly truncatedP-element variant we named “Har-P” as the most frequentde novoinsertion. AlthoughHISR-Dlines still contain full-lengthP-elements,HISR-Nlines lost functionalP-transposase but retainedHar-P’s that when crossed back toP-transposase restores GD induction. Finally, we uncoveredP-element-piRNA-directed repression onHar-P’stransmitted paternally to suppress somatic transposition. TheDrosophilashortHar-P’sand full-lengthP-elements relationship parallels the MITEs/DNA-transposase in plants and SINEs/LINEs in mammals.

Transposons are highly abundant in eukaryotic genomes, but their mobilization must be finely tuned to maintain host organism fitness and allow for transposon propagation. Forty percent of the human genome is comprised of transposable element sequences, and the most abundant cut-and-paste transposons are from the hAT superfamily. We found that the hAT transposase TcBuster from Tribolium castaneum formed filamentous structures, or rodlets, in human tissue culture cells, after gene transfer to adult mice, and ex vivo in cell-free conditions, indicating that host co-factors or cellular structures were not required for rodlet formation. Time-lapsed imaging of GFP-laced rodlets in human cells revealed that they formed quickly in a dynamic process involving fusion and fission. We delayed the availability of the transposon DNA and found that transposition declined after transposase concentrations became high enough for visible transposase rodlets to appear. In combination with earlier findings for maize Ac elements, these results give insight into transposase overproduction inhibition by demonstrating that the appearance of transposase protein structures and the end of active transposition are simultaneous, an effect with implications for genetic engineering and horizontal gene transfer.

In Drosophila , P-element transposition causes mutagenesis and genome instability during hybrid dysgenesis. The P-element 31-bp terminal inverted repeats (TIRs) contain sequences essential for transposase cleavage and have been implicated in DNA repair via protein–DNA interactions with cellular proteins. The identity and function of these cellular proteins were unknown. Biochemical characterization of proteins that bind the TIRs identified a heterodimeric basic leucine zipper (bZIP) complex between an uncharacterized protein that we termed “Inverted Repeat Binding Protein (IRBP) 18” and its partner Xrp1. The reconstituted IRBP18/Xrp1 heterodimer binds sequence-specifically to its dsDNA-binding site within the P-element TIRs. Genetic analyses implicate both proteins as critical for repair of DNA breaks following transposase cleavage in vivo. These results identify a cellular protein complex that binds an active mobile element and plays a more general role in maintaining genome stability.

In Drosophila melanogaster, P element transposition has been a productive means of insertional mutagenesis. Thousands of genes have been tagged with natural and engineered P element constructs. Nevertheless, chromosomes carrying P element insertions tend to have high levels of background mutations from P elements inserting and excising during transposition. Consequently, the phenotypes seen when P element-bearing chromosomes are homozygous are often not attributable to the P insertions themselves. In this study, 178 strains in the Bloomington Drosophila Stock Center collection with P insertions on the second chromosome were complementation tested against molecularly defined chromosomal deletions and previously characterized single-gene mutations to determine if recessive lethality or sterility is associated with the P insertions rather than background mutations. This information should prove valuable to geneticists using these strains for experimental studies of gene function.

In Drosophila melanogaster, the mini-w+transgene in Pci is normally expressed throughout the adult eye; however, when other P or KP elements are present, a variegated-eye phenotype results, indicating random w+silencing during development called P-element-dependent silencing (PDS). Mutant Su(var)205 and Su(var)3-7 alleles act as haplo-suppressors/triplo-enhancers of this variegated phenotype, indicating that these heterochromatic modifiers act dose dependently in PDS. Previously, we recovered a spontaneous mutation of P{lacW}ciDplaccalled P{lacW}ciDplacE1(E1) that variegated in the absence of P elements, presumably due to the insertion of an adjacent gypsy element. From a screen for genetic modifiers of E1 variegation, we describe here the isolation of five mutations in ash1 and three in trx that enhance the E1 variegated phenotype in a dose-dependent and cumulative manner. These mutant alleles enhance PDS at E1, and in E1/P{lacW}ciDplac, but suppress position effect variegation (PEV) at In(1)wm4. This opposite action is consistent with a model where ASH1 and TRX mark transcriptionally active chromatin domains. If ASH1 or TRX function is lost or reduced, heterochromatin can spread into these domains creating a sink that diverts heterochromatic proteins from other variegating locations, which then may express a suppressed phenotype.

P elements inserted in the Telomere Associated Sequences (TAS) at the left end of the X chromosome are determiners of cytotype regulation of the entire P family of transposons. This regulation is mediated by Piwi-interacting (pi) RNAs derived from the telomeric P elements (TPs). Because these piRNAs are transmitted maternally, cytotype regulation is manifested as a maternal effect of the TPs. When a TP is combined with a transgenic P element inserted at another locus, this maternal effect is strengthened. However, when certain TPs are combined with transgenes that contain the small P element known as KP, stronger regulation arises from a zygotic effect of the KP element. This zygotic effect is observed with transgenic KP elements that are structurally intact, as well as with KP elements that are fused to an ancillary promoter from the hsp70 gene. Zygotic regulation by a KP element occurs only when a TP was present in the maternal germ line, and it is more pronounced when the TP was also present in the grand-maternal germ line. However, this regulation does not require zygotic expression of the TP. These observations can be explained if maternally transmitted piRNAs from TPs enable a polypeptide encoded by KP elements to repress P element transposition in zygotes that contain a KP element. In nature, repression by the KP polypeptide may therefore be facilitated by cytotype-mediating piRNAs.

When Drosophila mothers lacking P-element-piRNAs (i.e. ISO1) mate with certain fathers harboring active P-elements (i.e. Harwich, Har), hybrid daughters suffer from gonadal dysgenesis (GD), where ovaries collapse from uncontrolled P-element transposition. To discover the P-element driving GD, we selected hybrid D. melanogaster lines with portions of Har DNA within the ISO1 genome. We generated HISR-D and HISR-N lines retaining or losing GD induction, respectively. Despite greatly-reduced numbers of P-elements, HISR-D lines produced as many P-element piRNAs as parental Har. In these lines, we discovered a highly-truncated P-element variant: “Har-P”, which mobilized de novo in all HISR lines. Crossing P-transposase with Har-Ps in HISR-N lines restored GD and revealed a paternal P-element-piRNA-directed imprint on Har-Ps to suppress lethal somatic transposition. Har-P may weaponize P-transposase during catastrophic transposition.

Background P-element transposition in the genome causes P-M hybrid dysgenesis in Drosophila melanogaster. Maternally deposited piRNAs suppress P-element transposition in the progeny, linking them to P-M phenotypes; however, the role of zygotic piRNAs derived from paternal P elements is poorly understood. Results To elucidate the molecular basis of P-element suppression by zygotic factors, we investigated the genomic constitution and P-element piRNA production derived from fathers. As a result, we characterized males of naturally derived Q, M’ and P strains, which show different capacities for the P-element mobilizations introduced after hybridizations with M-strain females. The amounts of piRNAs produced in ovaries of F1 hybrids varied among the strains and were influenced by the characteristics of the piRNA clusters that harbored the P elements. Importantly, while both the Q- and M’-strain fathers restrict the P-element mobilization in ovaries of their daughters, the Q-strain fathers supported the production of the highest piRNA expression in the ovaries of their daughters, and the M’ strain carries KP elements in transcriptionally active regions directing the highest expression of KP elements in their daughters. Interestingly, the zygotic P-element piRNAs, but not the KP element mRNA, contributed to the variations in P transposition immunity in the granddaughters. Conclusions The piRNA-cluster-embedded P elements and the transcriptionally active KP elements from the paternal genome are both important suppressors of P element activities that are co-inherited by the progeny. Expression levels of the P-element piRNA and KP-element mRNA vary among F1 progeny due to the constitution of the paternal genome, and are involved in phenotypic variation in the subsequent generation. Electronic supplementary material The online version of this article (10.1186/s13100-018-0110-y) contains supplementary material, which is available to authorized users.

To understand how transposon landscapes (TLs) vary across animal genomes, we describe a new method called the Transposon Insertion and Depletion AnaLyzer (TIDAL) and a database of >300 TLs in Drosophila melanogaster (TIDAL-Fly). Our analysis reveals pervasive TL diversity across cell lines and fly strains, even for identically named sub-strains from different laboratories such as the ISO1 strain used for the reference genome sequence. On average, >500 novel insertions exist in every lab strain, inbred strains of the Drosophila Genetic Reference Panel (DGRP), and fly isolates in the Drosophila Genome Nexus (DGN). A minority (70%) of TL diversity across fly strains. A sharp contrast between insertion and depletion patterns indicates that many transposons are unique to the ISO1 reference genome sequence. Although TL diversity from fly strains reaches asymptotic limits with increasing sequencing depth, rampant TL diversity causes unsaturated detection of TLs in pools of flies. Finally, we show novel transposon insertions negatively correlate with Piwi-interacting RNA (piRNA) levels for most transposon families, except for the highly-abundant roo retrotransposon. Our study provides a useful resource for Drosophila geneticists to understand how transposons create extensive genomic diversity in fly cell lines and strains.

Intraspecific hybrid dysgenesis (HD) appears after some strains of D. melanogaster are crossed. The predominant idea is that the movement of transposable P elements causes HD. It is believed that P elements appeared in the D. melanogaster genome in the middle of the last century by horizontal transfer, simultaneously with the appearance of HD determinants. A subsequent simultaneous expansion of HD determinants and P elements occurred. We analyzed the current distribution of HD determinants in natural populations of D. melanogaster and found no evidence of their further spread. However, full-sized P elements were identified in the genomes of all analyzed natural D. melanogaster strains independent of their cytotypes. Thus, the expansion of P elements does not correlate with the expansion of HD determinants. We found that the ovaries of dysgenic females did not contain germ cells despite the equal number of primordial germ cells in early stages in dysgenic and non-dysgenic embryos. We propose that HD does not result from DNA damage caused by P element transposition, but it would be the disruption in the regulation of dysgenic ovarian formation that causes the dysgenic phenotypes.

Non-viral transposons have been used successfully for genetic modification of clinically relevant cells including embryonic stem, induced pluripotent stem, hematopoietic stem and primary human T cell types. However, there has been limited evaluation of undesired genomic effects when using transposons for human genome modification. The prevalence of piggyBac(PB)-like terminal repeat (TR) elements in the human genome raises concerns. We evaluated if there were undesired genomic effects of the PB transposon system to modify human cells. Expression of the transposase alone revealed no mobilization of endogenous PB-like sequences in the human genome and no increase in DNA double-strand breaks. The use of PB in a plasmid containing both transposase and transposon greatly increased the probability of transposase integration; however, using transposon and transposase from separate vectors circumvented this. Placing a eGFP transgene within transposon vector backbone allowed isolation of cells free from vector backbone DNA. We confirmed observable directional promoter activity within the 5′TR element of PB but found no significant enhancer effects from the transposon DNA sequence. Long-term culture of primary human cells modified with eGFP-transposons revealed no selective growth advantage of transposon-harboring cells. PB represents a promising vector system for genetic modification of human cells with limited undesired genomic effects.

Background Transposition of P elements in the genome causes P–M hybrid dysgenesis in Drosophila melanogaster. For the P strain, the P–M phenotypes are associated with the ability to express a class of small RNAs, called piwi-interacting small RNAs (piRNAs), that suppress the P elements in female gonads. However, little is known about the extent to which piRNAs are involved in the P–M hybrid dysgenesis in M′ and Q strains, which show different abilities to regulate the P elements from P strains. Results To elucidate the molecular basis of the suppression of paternally inherited P elements, we analyzed the mRNA and piRNA levels of P elements in the F1 progeny between males of a P strain and nine-line females of M′ or Q strains (M′ or Q progenies). M′ progenies showed the hybrid dysgenesis phenotype, while Q progenies did not. Consistently, the levels of P-element mRNA in both the ovaries and F1 embryos were higher in M′ progenies than in Q progenies, indicating that the M′ progenies have a weaker ability to suppress P-element expression. The level of P-element mRNA was inversely correlated to the level of piRNAs in F1 embryos. Importantly, the M′ progenies were characterized by a lower abundance of P-element piRNAs in both young ovaries and F1 embryonic bodies. The Q progenies showed various levels of piRNAs in both young ovaries and F1 embryonic bodies despite all of the Q progenies suppressing P-element transposition in their gonad. Conclusions Our results are consistent with an idea that the level of P-element piRNAs is a determinant for dividing strain types between M′ and Q and that the suppression mechanisms of transposable elements, including piRNAs, are varied between natural populations. Electronic supplementary material The online version of this article (10.1186/s13100-017-0096-x) contains supplementary material, which is available to authorized users.

Hybrid dysgenesis is a sterility syndrome resulting from the mobilization of certain transposable elements in theDrosophilagermline. Particularly extreme is the hybrid dysgenesis syndrome caused byP-element DNA transposons, in which dysgenic female ovaries often contain few or no germline cells. Those offspring that are produced from dysgenic germlines exhibit high rates ofde novomutation and recombination, implicating transposition-associated DNA damage as the cause of germline loss. However, how this loss occurs, in terms of the particular cellular response that is triggered (cell cycle arrest, senescence, or cell death) remains poorly understood. We demonstrate that two components of the DNA damage response, Checkpoint kinase 2 and its downstream target p53, are determine the frequency of ovarian atrophy that is associated withP-element hybrid dysgenesis. We further show that p53 is strongly induced in the germline stem cells (GSCs) of dysgenic females, and is required for their maintenance. Our observations support the critical role for p53 in conferring tolerance of transposable element activity in stem cells.

The PiggyBac (PB) transposon has emerged as a novel mutagenesis tool for understanding gene function and for phenotypic screening in eukaryotes. Successful screening of PB transposon mutants relies on efficient identification of transposon insertion site(s) (TIS) in mutant cells. However, currently available methods suffer from time-consuming steps. Here, we present the method for transposon insertion site sequencing (TIS-Seq) for high-throughput identification of TIS in transposon mutants. TIS-Seq provides qualitative and quantitative information on mutants present in a given PB transposon mutant library. TIS-Seq also facilitates identification of TIS in up to 96 individual/hand-picked mutants in a single MiniSeq/MiSeq run. TIS-Seq is a versatile method that can be easily modified to identify TIS from any kind of transposon mutant, as long as one end of the DNA sequence is known. Therefore, TIS-Seq is a promising method for transposon mutant screening. © 2017 by John Wiley & Sons, Inc. Keywords: PiggyBac; transposon insertion; transposon mutant; high throughput; mutant screening

The Drosophila melanogaster P transposable element provides one of the best cases of horizontal transfer of a mobile DNA sequence in eukaryotes. Invasion of natural populations by the P element has led to a syndrome of phenotypes known as P-M hybrid dysgenesis that emerges when strains differing in their P element composition mate and produce offspring. Despite extensive research on many aspects of P element biology, many questions remain about the genomic basis of variation in P-M dysgenesis phenotypes across populations. Here we compare estimates of genomic P element content with gonadal dysgenesis phenotypes for isofemale strains obtained from three worldwide populations of D. melanogaster to illuminate the molecular basis of natural variation in cytotype status. We show that P element abundance estimated from genome sequences of isofemale strains is highly correlated across different bioinformatics approaches, but that abundance estimates are sensitive to method and filtering strategies as well as incomplete inbreeding of isofemale strains. We find that P element content varies significantly across populations, with strains from a North American population having fewer P elements but a higher proportion of full-length elements than strains from populations sampled in Europe or Africa. Despite these geographic differences in P element abundance and structure, neither the number of P elements nor the ratio of full-length to internally-truncated copies is strongly correlated with the degree of gonadal dysgenesis exhibited by an isofemale strain. Thus, variation in P element abundance and structure across different populations does not necessarily lead to corresponding geographic differences in gonadal dysgenesis phenotypes. Finally, we confirm that population differences in the abundance and structure of P elements that are observed from isofemale lines can also be observed in pool-seq samples from the same populations. Our work supports the view that genomic P element content alone is not sufficient to explain variation in gonadal dysgenesis across strains of D. melanogaster, and informs future efforts to decode the genomic basis of geographic and temporal differences in P element induced phenotypes.

P element is a DNA transposon, known to spread in genome using transposase activity. Its activity is tissue-specific and normally observed at high temperatures within 24°C to 29°C. Here, we present a predicted RNA secondary structure domain of P element pre-mRNA which could potentially regulate the temperature sensitivity of the P element activity. In canonical P elements, the structure is a small hairpin with double-helical part interrupted by a symmetric loop and a mismatch. In M type P elements, the A.A mismatch is substituted by an A-U base pair, stabilizing the structure. The hairpin structure covers the region involving the IVS-3 5′ splice site and both pseudo-splice sites F1 and F2. While the IVS-3 and F1 binding sites of U1 snRNA are located in the double-stranded part of the structure, the F2 site is exposed in the hairpin loop. The formation of this structure may interfere with landing of U1 snRNA on IVS-3 site, while F2 is positioned for the interaction. Alignment of P element sequences supports the proposed existence of the hairpin, showing high similarity for this region. The hairpin structure, stable at low temperatures, may prevent correct IVS-3 splicing. Conversely, temperature-induced destabilization of the hairpin structure may result in the splicing at the proper IVS-3 splice site. Taking into account the increasing amount of data demonstrating the important influence of RNA folding on phenotypes determined by alternative splicing a model for possible regulation of the activity of mobile elements by pre-mRNA secondary structure seems intriguing. Предсказана вторичная структура пре-мРНК P-элемента, которая, возможно, регулирует его активность и температурную чувствительность. Структура представляет собой шпильку, более стабильную в Р-элементах М-типа, в сравнении с каноническими. Регион, образующий шпильку, находится в области 5’ сайта сплайсинга третьего интрона (IVS-3), включая оба описанных псевдо-сайта сплайсинга - F1 и F2. В то время как истинный сайт и F1 расположены, главным образом, в двухцепочечной области шпильки, F2 - экспонирован в петле. Выравнивание последовательностей Р-элементов продемонстрировало высокую степень сходства для указанного региона, что свидетельствует в пользу существования предсказанной структуры. Формирование шпильки, по нашему мнению, может препятствовать связыванию U1 snRNA с истинным сайтом сплайсинга, и, напротив, индуцированная температурой дестабилизация шпильки может приводить к корректному сплайсингу IVS-3. Таким образом, предложена гипотеза о влиянии вторичной структуры пре-мРНК мобильного элемента на его активность. Передбачена вторинна структура пре-мРНК Р-елемента, що, можливо, регулює його активність та температурну чутливість. Структура являє собою шпильку, більш стабільну у Р-елементах М-типу, порівняно з канонічними. Регіон, що утворює шпильку розташований в області 5’ сайта сплайсинга третього інтрону (IVS-3), включає обидва відомих псевдо-сайта сплайсингу - F1 та F2. При цьому 5’ сайт сплайсингу IVS-3 та F1псевдо-сайт знаходяться у дволанцюговій частині шпильки, а F2 є єкспонованим у петлі. Вирівнювання послідовностей Р-елементів продемонструвало високий рівень подібності для вказаного регіону, що свідчить на користь існування передбаченої структури. Формування шпильки, на нашу думку, може перешкоджати зв’язуванню U1 snRNA з 5’ сайтом сплайсингу IVS-3, і, навпаки, індукована температурою дестабілізація шпильки може сприяти корректному сплайсингу третього інтрону. Таким чином, запропонована гіпотеза про вплив вторинної структури пре-мРНК мобільного елементу на його активність.

The transposons of Drosophila melanogaster are regulated by small RNAs that interact with the Piwi family of proteins. These piRNAs are generated from transposons inserted in special loci such as the telomere-associated sequences at the left end of the X chromosome. Drosophila's P transposons can also be regulated by a polypeptide encoded by the KP element, a 1.15-kb-long member of the P family. Using piRNA-generating telomeric P elements (TPs) and repressor-producing transgenic KP elements, we demonstrate a functional connection between these two modes of regulation. By themselves, the TPs partially repress gonadal dysgenesis, a trait caused by rampant P-element activity in the germ line. This repression is manifested as a strictly maternal effect arising from the cytoplasmic transmission of P-specific piRNAs from mother to offspring. The repression is enhanced by genetic interactions between the TPs and other, non-telomeric P elements-a phenomenon attributable to ping-pong amplification of maternal piRNAs. KP elements, like other kinds of non-telomeric P elements, enhance regulation anchored in the TPs. However, with some TPs, the enhanced regulation is manifested as a strictly zygotic effect of the KP element. This effect is seen when the TP has few sequences in common with the KP element, a condition not conducive to ping-pong amplification of piRNAs; it can be attributed to the action of the KP repressor polypeptide. Because the effect is seen only when a TP was present in the mother's genotype, maternally generated P-element piRNAs could facilitate regulation by the KP repressor polypeptide.

Molecular mechanisms underlying the functioning of central pattern generators (CPGs) are poorly understood. Investigations using genetic approaches in the model organism Drosophila may help to identify unknown molecular players participating in the formation or control of motor patterns. Here we report Drosophila genes as candidates for involvement in the neural mechanisms responsible for motor functions, such as locomotion and courtship song. Twenty-two Drosophila lines, used for gene identification, were isolated from a previously created collection of 1064 lines, each carrying a P element insertion in one of the autosomes. The lines displayed extreme deviations in locomotor and/or courtship song parameters compared with the whole collection. The behavioral consequences of CNS-specific RNAi-mediated knockdowns for 10 identified genes were estimated. The most prominent changes in the courtship song interpulse interval (IPI) were seen in flies with Sps2 or CG15630 knockdown. Glia-specific knockdown of these genes produced no effect on the IPI. Estrogen-induced knockdown of CG15630 in adults reduced the IPI. The product of the CNS-specific gene, CG15630 (a predicted cell surface receptor), is likely to be directly involved in the functioning of the CPG generating the pulse song pattern. Future studies should ascertain its functional role in the neurons that constitute the song CPG. Other genes (Sps2, CG34460), whose CNS-specific knockdown resulted in IPI reduction, are also worthy of detailed examination.

Summary Hermes is a member of the hAT transposon superfamily that has active representatives, including McClintock's archetypal Ac mobile genetic element, in many eukaryotic species. The crystal structure of the Hermes transposase-DNA complex reveals that Hermes forms an octameric ring organized as a tetramer of dimers. Although isolated dimers are active in vitro for all the chemical steps of transposition, only octamers are active in vivo. The octamer can provide not only multiple specific DNA-binding domains to recognize repeated subterminal sequences within the transposon ends, which are important for activity, but also multiple nonspecific DNA binding surfaces for target capture. The unusual assembly explains the basis of bipartite DNA recognition at hAT transposon ends, provides a rationale for transposon end asymmetry, and suggests how the avidity provided by multiple sites of interaction could allow a transposase to locate its transposon ends amidst a sea of chromosomal DNA.

We have generated a recombinant Mos1 transposon that can insert up to 45-kb transgenes into the Caenorhabditis elegans genome. The minimal Mos1 transposon (miniMos) is 550 bp long and inserts DNA into the genome at high frequency (~60% of injected animals). Genetic and antibiotic markers can be used for selection, and the transposon is active in C. elegans isolates and Caenorhabditis briggsae. We used the miniMos transposon to generate six universal Mos1-mediated single-copy insertion (mosSCI) landing sites that allow targeted transgene insertion with a single targeting vector into permissive expression sites on all autosomes. We also generated two collections of strains: a set of bright fluorescent insertions that are useful as dominant, genetic balancers and a set of lacO insertions to track genome position.

The Sleeping Beauty (SB) transposon is the most widely used DNA transposon in genetic applications and is the only DNA transposon thus far in clinical trials for human gene therapy. In the absence of atomic level structural information, the development of SB transposon relied primarily on the biochemical and genetic homology data. While these studies were successful and have yielded hyperactive transposases, structural information is needed to gain a mechanistic understanding of transposase activity and guides to further improvement. We have initiated a structural study of SB transposase using Nuclear Magnetic Resonance (NMR) and Circular Dichroism (CD) spectroscopy to investigate the properties of the DNA-binding domain of SB transposase in solution. We show that at physiologic salt concentrations, the SB DNA-binding domain remains mostly unstructured but its N-terminal PAI subdomain forms a compact, three-helical structure with a helix-turn-helix motif at higher concentrations of NaCl. Furthermore, we show that the full-length SB DNA-binding domain associates differently with inner and outer binding sites of the transposon DNA. We also show that the PAI subdomain of SB DNA-binding domain has a dominant role in transposase's attachment to the inverted terminal repeats of the transposon DNA. Overall, our data validate several earlier predictions and provide new insights on how SB transposase recognizes transposon DNA.

Most foldback elements are defective due to the lack of coding sequences but some are associated with coding sequences and may represent the entire element. This is the case of the NOF sequences found in the FB of Drosophila melanogaster, formerly considered as an autonomous TE and currently proposed as part of the so-called FB-NOF element, the transposon that would be complete and fully functional. NOF is always associated with FB and never seen apart from the FB inverted repeats (IR). This is the reason why the FB-NOF composite element can be considered the complete element. At least one of its ORFs encodes a protein that has always been considered its transposase, but no detailed studies have been carried out to verify this. In this work we test the hypothesis that FB-NOF is an active transposon nowadays. We search for its expression product, obtaining its cDNA, and propose the ORF and the sequence of its potential protein. We found that the NOF protein is not a transposase as it lacks any of the motifs of known transposases and also shows structural homology with hydrolases, therefore FB-NOF cannot belong to the superfamily MuDR/foldback, as up to now it has been classified, and can be considered as a non-autonomous transposable element. The alignment with the published genomes of 12 Drosophila species shows that NOF presence is restricted only to the 6 Drosophila species belonging to the melanogaster group.

Transposable P elements are regulated in the germ line by piRNAs, which are small RNAs that associate with the Piwi class of proteins. This regulation, called the P cytotype, is enhanced by genetic interactions between P elements that are primary sources of these RNAs and other P elements. The enhanced regulation is thought to reflect amplification of the primary piRNAs by cleavage of mRNAs derived from the other P elements through a mechanism called the ping-pong cycle. We tested the transposase-encoding P element known as ∆2-3 for its ability to enhance cytotype regulation anchored in P elements inserted at the telomere of the left arm of the X chromosome (TP elements). The ∆2-3 P element lacks the intron between exons 2 and 3 in the structurally complete P element (CP). Unlike the CP element, it does not markedly enhance cytotype regulation anchored in TP elements, nor does it transmit transposase activity through the egg cytoplasm. However, mRNAs from both the CP and ∆2-3 elements are maternally deposited in embryos. These observations suggest that maternally transmitted CP mRNA enhances cytotype regulation by participating in the ping-pong cycle and that it encodes the P transposase in the embryonic germ line, whereas maternally transmitted ∆2-3 mRNA does not, possibly because it is not efficiently directed into the primordial embryonic germ line. Strong transposon regulation may, therefore, require ping-pong cycling with maternally inherited mRNAs in the embryo.

The hypothesis tested in this study was that single-gene mutations found previously to extend the life span of Drosophila melanogaster could do so consistently in both long-lived y w and standard w (1118) genetic backgrounds. GAL4 drivers were used to express upstream activation sequence (UAS)-responder transgenes globally or in the nervous system. Transgenes associated with oxidative damage prevention (UAS-hSOD1 and UAS-GCLc) or removal (EP-UAS-Atg8a and UAS-dTOR (FRB) ) failed to increase mean life spans in any expression pattern in either genetic background. Flies containing a UAS-EGFP-bMSRA (C) transgene associated with protein repair were found not to exhibit life extension or detectable enhanced green fluorescent protein (EGFP) activity. The presence of UAS-responder transgenes was confirmed by PCR amplification and sequencing at the 5' and 3' end of each insertion. These results cast doubt on the robustness of life extension in flies carrying single-gene mutations and suggest that the effects of all such mutations should be tested independently in multiple genetic backgrounds and laboratory environments.

Transposable elements (TEs) are both important drivers of genome evolution and genetic parasites with potentially dramatic consequences for host fitness. The recent explosion of research on regulatory RNAs reveals that small RNA-mediated silencing is a conserved genetic mechanism through which hosts repress TE activity. The invasion of the Drosophila melanogaster genome by P elements, which happened on a historical timescale, represents an incomparable opportunity to understand how small RNA-mediated silencing of TEs evolves. Repression of P-element transposition emerged almost concurrently with its invasion. Recent studies suggest that this repression is implemented in part, and perhaps predominantly, by the Piwi-interacting RNA (piRNA) pathway, a small RNA-mediated silencing pathway that regulates TE activity in many metazoan germlines. In this review, I consider the P-element invasion from both a molecular and evolutionary genetic perspective, reconciling classic studies of P-element regulation with the new mechanistic framework provided by the piRNA pathway. I further explore the utility of the P-element invasion as an exemplar of the evolution of piRNA-mediated silencing. In light of the highly-conserved role for piRNAs in regulating TEs, discoveries from this system have taxonomically broad implications for the evolution of repression.

In a classic example of the invasion of a species by a selfish genetic element, the P-element was horizontally transferred from a distantly related species into Drosophila melanogaster. Despite causing ‘hybrid dysgenesis’, a syndrome of abnormal phenotypes that include sterility, the P-element spread globally in the course of a few decades in D. melanogaster. Until recently, its sister species, including D. simulans, remained P-element free. Here, we find a hybrid dysgenesis-like phenotype in the offspring of crosses between D. simulans strains collected in different years; a survey of 181 strains shows that around 20% of strains induce hybrid dysgenesis. Using genomic and transcriptomic data, we show that this dysgenesis-inducing phenotype is associated with the invasion of the P-element. To characterize this invasion temporally and geographically, we survey 631 D. simulans strains collected on three continents and over 27 years for the presence of the P-element. We find that the D. simulans P-element invasion occurred rapidly and nearly simultaneously in the regions surveyed, with strains containing P-elements being rare in 2006 and common by 2014. Importantly, as evidenced by their resistance to the hybrid dysgenesis phenotype, strains collected from the latter phase of this invasion have adapted to suppress the worst effects of the P-element., Author Summary Some genes perform necessary organismal functions, others hijack the cellular machinery to replicate themselves, potentially harming the host in the process. These ‘selfish genes’ can spread through genomes and species; as a result, eukaryotic genomes are typically saddled with large amounts of parasitic DNA. Here, we chronicle the surprisingly rapid global spread of a selfish transposable element through a close relative of the genetic model, Drosophila melanogaster. We see that, as it spreads, the transposable element is associated with damaging effects, including sterility, but that the flies quickly adapt to the negative consequences of the transposable element.

TheDrosophila melanogaster Ptransposable element provides one of the best cases of horizontal transfer of a mobile DNA sequence in eukaryotes. Invasion of natural populations by thePelement has led to a syndrome of phenotypes known as “P-M hybrid dysgenesis” that emerges when strains differing in theirPelement composition mate and produce offspring. Despite extensive research on many aspects ofPelement biology, questions remain about the stability and genomic basis of variation in P-M dysgenesis phenotypes. Here we report the P-M status for a number of populations sampled recently from Ukraine that appear to be undergoing a shift in theirPelement composition. Gondal dysgenesis assays reveal that Ukrainian populations ofD. melanogasterare currently dominated by the P’ cytotype, a cytotype that was previously thought to be rare in nature, suggesting that a new active form of thePelement has recently spread in this region. We also compared gondal dysgenesis phenotypes and genomicPelement predictions for isofemale strains obtained from three worldwide populations ofD. melanogasterin order to guide further work on the molecular basis of differences in cytotype status across populations. We find that the number of euchromaticPelements per strain can vary significantly across populations but that totalPelement numbers are not strongly correlated with the degree of gondal dysgenesis. Our work shows that rapid changes in cytotype status can occur in natural populations ofD. melanogaster, and informs future efforts to decode the genomic basis of geographic and temporal differences inPelement induced phenotypes.

Nature Communications 7, 11126 (2016). doi:10.1038/ncomms11126, Sleeping Beauty (SB) is a prominent Tc1/mariner superfamily DNA transposon that provides a popular genome engineering tool in a broad range of organisms. It is mobilized by a transposase enzyme that catalyses DNA cleavage and integration at short specific sequences at the transposon ends. To facilitate SB’s applications, here we determine the crystal structure of the transposase catalytic domain and use it to model the SB transposase/transposon end/target DNA complex. Together with biochemical and cell-based transposition assays, our structure reveals mechanistic insights into SB transposition and rationalizes previous hyperactive transposase mutations. Moreover, our data enables us to design two additional hyperactive transposase variants. Our work provides a useful resource and proof-of-concept for structure-based engineering of tailored SB transposases., Published by Nature Publishing Group, London