Physical properties of DNA components affecting the transposition efficiency of the <Emphasis Type="Italic">mariner Mos1</Emphasis> element

Previous studies have shown that the transposase and the inverted terminal repeat (ITR) of the Mos1 mariner elements are suboptimal for transposition; and that hyperactive transposases and transposon with more efficient ITR configurations can be obtained by rational molecular engineering. In an attempt to determine the extent to which this element is suboptimal for transposition, we investigate here the impact of the three main DNA components on its transposition efficiency in bacteria and in vitro. We found that combinations of natural and synthetic ITRs obtained by systematic evolution of ligands by exponential enrichment did increase the transposition rate. We observed that when untranslated terminal regions were associated with their respective natural ITRs, they acted as transposition enhancers, probably via the early transposition steps. Finally, we demonstrated that the integrity of the Mos1 inner region was essential for transposition. These findings allowed us to propose prototypes of optimized Mos1 vectors, and to define the best sequence features of their associated marker cassettes. These vector prototypes were assayed in HeLa cells, in which Mos1 vectors had so far been found to be inactive. The results obtained revealed that using these prototypes does not circumvent this problem. However, such vectors can be expected to provide new tools for the use in genome engineering in systems such as Caenorhabditis elegans in which Mos1 is very active.     

cAMP protein kinase phosphorylates the Mos1 transposase and regulates its activity: evidences from mass spectrometry and biochemical analyses

Genomic plasticity mediated by transposable elements can have a dramatic impact on genome integrity. To minimize its genotoxic effects, it is tightly regulated either by intrinsic mechanisms (linked to the element itself) or by host-mediated mechanisms. Using mass spectrometry, we show here for the first time that MOS1, the transposase driving the mobility of the mariner Mos1 element, is phosphorylated. We also show that the transposition activity of MOS1 is downregulated by protein kinase AMP cyclic-dependent phosphorylation at S170, which renders the transposase unable to promote Mos1 transposition. One step in the transposition cycle, the assembly of the paired-end complex, is specifically inhibited. At the cellular level, we provide evidence that phosphorylation at S170 prevents the active transport of the transposase into the nucleus. Our data suggest that protein kinase AMP cyclic-dependent phosphorylation may play a double role in the early stages of genome invasion by mariner elements.     

The mariner Mos1 transposase produced in tobacco is active in vitro

The mariner-like transposon Mos1 is used for insertional mutagenesis and transgenesis in different animals (insects, nematodes), but has never been used in plants. In this paper, the transposition activity of Mos1 was tested in Nicotiana tabacum, but no transposition event was detected. In an attempt to understand the absence of in planta transposition, Mos1 transposase (MOS1) was produced and purified from transgenic tobacco (HMNtMOS1). HMNtMOS1 was able to perform all transposition reaction steps in vitro: binding to ITR, excision and integration of the same pseudo-transposon used in in planta transposition assays. The in vitro transposition reaction was not inhibited by tobacco nuclear proteins, and did not depend on the temperature used for plant growth. Several hypotheses are proposed that could explain the inhibition of HMNtMOS1 activity in planta.     

Manipulating the <Emphasis Type="Italic">Caenorhabditis elegans</Emphasis> genome using <Emphasis Type="Italic">mariner</Emphasis> transposons

Tc1, one of the founding members of the Tc1/mariner transposon superfamily, was identified in the nematode Caenorhabditis elegans more than 25 years ago. Over the years, Tc1 and other endogenous mariner transposons became valuable tools for mutagenesis and targeted gene inactivation in C. elegans. However, transposition is naturally repressed in the C. elegans germline by an RNAi-like mechanism, necessitating the use of mutant strains in which transposition was globally derepressed, which causes drawbacks such as uncontrolled proliferation of the transposons in the genome and accumulation of background mutations. The more recent mobilization of the Drosophila mariner transposon Mos1 in the C. elegans germline circumvented the problems inherent to endogenous transposons. Mos1 transposition strictly depends on the expression of the Mos transposase, which can be controlled in the germline using inducible promoters. First, Mos1 can be used for insertional mutagenesis. The mobilization of Mos1 copies present on an extrachromosomal array results in the generation of a small number of Mos1 genomic insertions that can be rapidly cloned by inverse PCR. Second, Mos1 insertions can be used for genome engineering. Triggering the excision of a genomic Mos1 insertion causes a chromosomal break, which can be repaired by transgene-instructed gene conversion. This process is used to introduce specific changes in a given gene, such as point mutations, deletions or insertions of a tag, and to create single-copy transgenes.     

Regulation of mariner transposition: the peculiar case of mos1

Background

Mariner elements represent the most successful family of autonomous DNA transposons, being present in various plant and animal genomes, including humans. The introduction and co-evolution of mariners within host genomes imply a strict regulation of the transposon activity. Biochemical data accumulated during the past decade have led to a convergent picture of the transposition cycle of mariner elements, suggesting that mariner transposition does not rely on host-specific factors. This model does not account for differences of transposition efficiency in human cells between mariners. We thus wondered whether apparent similarities in transposition cycle could hide differences in the intrinsic parameters that control mariner transposition.

Principal Findings

We find that Mos1 transposase concentrations in excess to the Mos1 ends prevent the paired-end complex assembly. However, we observe that Mos1 transposition is not impaired by transposase high concentration, dismissing the idea that transposase over production plays an obligatory role in the down-regulation of mariner transposition. Our main finding is that the paired-end complex is formed in a cooperative way, regardless of the transposase concentration. We also show that an element framed by two identical ITRs (Inverted Terminal Repeats) is more efficient in driving transposition than an element framed by two different ITRs (i.e. the natural Mos1 copy), the latter being more sensitive to transposase concentration variations. Finally, we show that the current Mos1 ITRs correspond to the ancestral ones.

Conclusions

We provide new insights on intrinsic properties supporting the self-regulation of the Mos1 element. These properties (transposase specific activity, aggregation, ITR sequences, transposase concentration/transposon copy number ratio…) could have played a role in the dynamics of host-genomes invasion by Mos1, accounting (at least in part) for the current low copy number of Mos1 within host genomes.     

The bacterial Tn<Emphasis Type="Italic">9</Emphasis> chloramphenicol resistance gene: an attractive DNA segment for <Emphasis Type="Italic">Mos1 mariner</Emphasis> insertions

The eukaryotic mariner transposons are currently thought to have no sequence specificity for integration other than to insert within a TA contained in a degenerated [TA]_1–4 tract, either in vitro or in vivo. We have investigated the properties of a suspected hotspot for the integration of the mariner Mos1 element, namely the Tn9 cat gene that encodes a chloramphenicol acetyl transferase. Using in vitro and bacterial transposition assays, we confirmed that the cat gene is a preferential target for MOS1 integration, whatever its sequence environment, copy number or chromosomal locus. We also observed that its presence increases transposition rates both in vitro and in bacterial assays. The structural and sequence features that constitute the attractiveness of cat were also investigated. We first demonstrated that supercoiling is essential for the cat gene to be a hot spot. In contrast to the situation for Tc1-like elements, DNA curvature and bendability were not found to affect integration target preferences. We found that Mos1 integrations do not occur randomly along the cat gene. All TA dinucleotides that are preferred for integration were found within either TATA or TA×TA motifs. However, these motifs are not sufficient to constitute an attractive dinucleotide, since four TATA and TA×TA sites are cold spots. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Hvmar1, a mariner‐like element from the tobacco budworm Heliothis virescens,can transpose in Drosophila melanogaster

Transposable elements of the mariner family are widespread and have been found in the genome of plants, animals and insects. However, most of these elements contain multiple inactivating mutations and so far, only three naturally occurring mariner elements are known to be functional. In a previous study, a mariner‐like element called Hvmar1 was discovered in the genome of the tobacco budworm Heliothis virescens. Further analysis of the Hvmar1 nucleotide sequence revealed the presence of 30‐bp imperfect inverted terminal repeats and an intact open reading frame, which is considered to encode a functional transposase. In the present study, we show that the Hvmar1 element is active using interplasmid transposition assays in Drosophila melanogaster embryos. When injected into Drosophila embryos, the helper plasmid produced a transposase that was able to mediate transposition of the Hvmar1 element from a donor to a target plasmid. The transposition efficiency of Hvmar1 in D. melanogaster is approximately 11‐fold lower than that of the well‐known Mos1 mariner transposon. However, this efficiency is comparable to those observed previously with Mos1 in non‐Drosophila insects. We identified 10 independent interplasmid transposition events, albeit the recovery of these events was rare. In each case the Hvmar1 element transposed in a precise manner, with the characteristic TA dinucleotides being duplicated on insertion. Furthermore, two of the target sites identified have been used previously by Mos1 for insertion. The active transposition of Hvmar1 in D. melanogaster provides a basis for examining the mobility of this element in its natural host as well as a starting point for comparative studies with Mos1 and other functional mariner transposons.     

Factors acting on <Emphasis Type="Italic">Mos1</Emphasis> transposition efficiency

Background  

Mariner-like elements (MLEs) are widespread DNA transposons in animal genomes. Although in vitro transposition reactions require only the transposase, various factors depending on the host, the physico-chemical environment and the transposon sequence can interfere with the MLEs transposition in vivo.     

Analyses of<Emphasis Type="Italic"> cis</Emphasis> -acting elements that affect the transposition of<Emphasis Type="Italic"> Mos1 mariner</Emphasis> transposons in vivo

The left (5) inverted terminal repeat (ITR) of the Mos1 mariner transposable element was altered by site-directed mutagenesis so that it exactly matched the nucleotide sequence of the right (3) ITR. The effects on the transposition frequency resulting from the use of two 3 ITRs, as well as those caused by the deletion of internal portions of the Mos1 element, were evaluated using plasmid-based transposition assays in Escherichia coli and Aedes aegypti. Donor constructs that utilized two 3 ITRs transposed with greater frequency in E. coli than did donor constructs with the wild-type ITR configuration. The lack of all but 10 bp of the internal sequence of Mos1 did not significantly affect the transposition frequency of a wild-type ITR donor. However, the lack of these internal sequences in a donor construct that utilized two 3 ITRs resulted in a further increase in transposition frequency. Conversely, the use of a donor construct with two 3 ITRs did not result in a significant increase in transposition in Ae. aegypti. Furthermore, deletion of a large portion of the internal Mos1 sequence resulted in the loss of transposition activity in the mosquito. The results of this study indicate the possible presence of a negative regulator of transposition located within the internal sequence, and suggest that the putative negative regulatory element may act to inhibit binding of the transposase to the left ITR. The results also indicate that host factors which are absent in E. coli, influence Mos1 transposition in Ae. aegypti.Communicated by G. P. Georgiev     

Transposase-transposase interactions in MOS1 complexes: a biochemical approach

Transposases are proteins that have assumed the mobility of class II transposable elements. In order to map the interfaces involved in transposase-transposase interactions, we have taken advantage of 12 transposase mutants that impair mariner transposase-transposase interactions taking place during transposition. Our data indicate that transposase-transposase interactions regulating Mos1 transposition are sophisticated and result from (i) active MOS1 dimerization through the first HTH of the N-terminal domain, which leads to inverted terminal repeat (ITR) binding; (ii) inactive dimerization carried by part of the C-terminal domain, which prevents ITR binding; and (iii) oligomerization. Inactive dimers are nonpermissive in organizing complexes that produce ITR binding, but the interfaces (or interactions) supplied in this state could play a role in the various rearrangements needed during transposition. Oligomerization is probably not due to a specific MOS1 domain, but rather the result of nonspecific interactions resulting from incorrect folding of the protein. Our data also suggest that the MOS1 catalytic domain is a main actor in the overall organization of MOS1, thus playing a role in MOS1 oligomerization. Finally, we propose that MOS1 behaves as predicted by the pre-equilibrium existing model, whereby proteins are found to exist simultaneously in populations with diverse conformations, monomers and active and inactive dimers for MOS1. We were able to identify several MOS1 mutants that modify this pre-existing equilibrium. According to their properties, some of these mutants will be useful tools to break down the remaining gaps in our understanding of mariner transposition.     

Endogenous Transposases Affect Differently <Emphasis Type="BoldItalic">Sleeping Beauty</Emphasis> and <Emphasis Type="BoldItalic">Frog Prince</Emphasis> Transposons in Fish Cells

Fish cells stably expressing exogenous genes have potential applications in the production of fish recombinant proteins, gene-function studies, gene-trapping, and the production of transgenic fish. However, expression of a gene of interest after random integration may be difficult to predict or control. In the past decade, major contributions have been made in vertebrate-gene transfer, by using tools derived from DNA transposons. Among them, the Sleeping Beauty (SB) and Frog Prince (FP) transposons, derived, respectively, from fish and frog genomes, mediate transposition in a large variety of cells, although with different efficiency. This study was aimed at assessing the activities of the SB and the FP transposases in fish cell lines from genetically distant species (CHSE-214, RTG-2, BF-2, EPC, and SAF-1). Their transpositional ability was evaluated by the plasmid-based excision assay, the colony formation assay, and the footprint patterns. The results reveal that while both transposases are active in all cell lines, the transposition rates and the precision of the transposition are overall higher with FP than SB. Our results also indicated a key role of cell-specific host factors in transposition, which was associated with the presence of Tc1-like endogenous transposases; this effect was more accentuated in the two salmonid cell lines transfected with SB. This result agrees with previous studies supporting the use of transposons in heterologous organisms to prevent from genomic instability and from impeding the precise activity of the exogenous transposase.     

Target Capture during Mos1 Transposition

DNA transposition contributes to genomic plasticity. Target capture is a key step in the transposition process, because it contributes to the selection of new insertion sites. Nothing or little is known about how eukaryotic mariner DNA transposons trigger this step. In the case of Mos1, biochemistry and crystallography have deciphered several inverted terminal repeat-transposase complexes that are intermediates during transposition. However, the target capture complex is still unknown. Here, we show that the preintegration complex (i.e., the excised transposon) is the only complex able to capture a target DNA. Mos1 transposase does not support target commitment, which has been proposed to explain Mos1 random genomic integrations within host genomes. We demonstrate that the TA dinucleotide used as the target is crucial both to target recognition and in the chemistry of the strand transfer reaction. Bent DNA molecules are better targets for the capture when the target DNA is nicked two nucleotides apart from the TA. They improve strand transfer when the target DNA contains a mismatch near the TA dinucleotide.     

In vivo Tn<Emphasis Type="Italic">5</Emphasis>-based transposon mutagenesis of Streptomycetes

This paper reports the in vivo expression of the synthetic transposase gene tnp(a) from a hyperactive Tn5 tnp gene mutant in Streptomyces coelicolor. Using the synthetic tnp(a) gene adapted for Streptomyces codon usage, we showed random insertion of the transposon into the Streptomycetes genome. The insertion frequency for the hyperactive Tn5 derivative is 98% of transformed S. coelicolor cells. The random transposition has been confirmed by the recovery of ~1.1% of auxotrophs. The Tn5 insertions are stably inherited in the absence of apramycin selection. The transposon contains an apramycin resistance selection marker and an R6Kγ origin of replication for transposon rescue. We identified the transposon insertion loci by random sequencing of 14 rescue plasmids. The majority of insertions (12 of 14) were mapped to putative open-reading frames on the S. coelicolor chromosome. These included two new regulatory genes affecting S. coelicolor growth and actinorhodin biosynthesis.     

Target site selection by the <Emphasis Type="Italic">mariner</Emphasis>-like element, <Emphasis Type="Italic">Mos1</Emphasis>

The eukaryotic transposon Mos1 is a class-II transposable element that moves using a “cut-and-paste” mechanism in which the transposase is the only protein factor required. The formation of the excision complex is well documented, but the integration step has so far received less investigation. Like all mariner-like elements, Mos1 was thought to integrate into a TA dinucleotide without displaying any other target selection preferences. We set out to synthesize what is currently known about Mos1 insertion sites, and to define the characteristics of Mos1 insertion sequences in vitro and in vivo. Statistical analysis can be used to identify the TA dinucleotides that are non-randomly targeted for transposon integration. In vitro, no specific feature determining target choice other than the requirement for a TA dinucleotide has been identified. In vivo, data were obtained from two previously reported integration hotspots: the bacterial cat gene and the Caenorhabditis elegans rDNA locus. Analysis of these insertion sites revealed a preference for TA dinucleotides that are included in TATA or TA × TA motifs, or located within AT-rich regions. Analysis of the physical properties of sequences obtained in vitro and in vivo do not help to explain Mos1 integration preferences, suggesting that other characteristics must be involved in Mos1 target choice.     

Hyperactive mariner transposons are created by mutations that disrupt allosterism and increase the rate of transposon end synapsis

New applications for transposons in vertebrate genetics have spurred efforts to develop hyperactive variants. Typically, a genetic screen is used to identify several hyperactive point mutations, which are then incorporated in a single transposase gene. However, the mechanisms responsible for the increased activity are unknown. Here we show that several point mutations in the mariner transposase increase their activities by disrupting the allostery that normally serves to downregulate transposition by slowing synapsis of the transposon ends. We focused on the conserved WVPHEL amino acid motif, which forms part of the mariner transposase dimer interface. We generated almost all possible single substitutions of the W, V, E and L residues and found that the majority are hyperactive. Biochemical analysis of the mutations revealed that they disrupt signals that pass between opposite sides of the developing transpososome in response to transposon end binding. In addition to their role in allostery, the signals control the initiation of catalysis, thereby preventing non-productive double-strand breaks. Finally, we note that such breaks may explain the puzzling ‘self-inflicted wounds’ at the ends of the Mos1 transposon in Drosophila.     

cis and trans factors affecting Mos1 mariner evolution and transposition in vitro, and its potential for functional genomics

Mos1 and other mariner/Tc1 transposons move horizontally during evolution, and when transplanted into heterologous species can transpose in organisms ranging from prokaryotes to protozoans and vertebrates. To further develop the Drosophila Mos1 mariner system as a genetic tool and to probe mechanisms affecting the regulation of transposition activity, we developed an in vitro system for Mos1 transposition using purified transposase and selectable Mos1 derivatives. Transposition frequencies of nearly 10^–3/target DNA molecule were obtained, and insertions occurred at TA dinucleotides with little other sequence specificity. Mos1 elements containing only the 28 bp terminal inverted repeats were inactive in vitro, while elements containing a few additional internal bases were fully active, establishing the minimal cis-acting requirements for transposition. With increasing transposase the transposition frequency increased to a plateau value, in contrast to the predictions of the protein overexpression inhibition model and to that found recently with a reconstructed Himar1 transposase. This difference between the ‘natural’ Mos1 and ‘reconstructed’ Himar1 transposases suggests an evolutionary path for down-regulation of mariner transposition following its introduction into a naïve population. The establishment of the cis and trans requirements for optimal mariner transposition in vitro provides key data for the creation of vectors for in vitro mutagenesis, and will facilitate the development of in vivo systems for mariner transposition.     

IPB7 transposase behavior in Drosophila melanogaster and Aedes aegypti

Transposons are used in insect science as genetic tools that enable the transformation of insects and the identification and isolation of genes though their ability to insert in or near to them. Four transposons, piggyBac, Mos1, Hermes and Minos are commonly used in insects beyond Drosophila melanogaster with piggyBac, due to its wide host range and frequency of transposition, being the most commonly chosen. The utility of these transposons as genetic tools is directly proportional to their activity since higher transposition rates would be expected to lead to higher transformation frequencies and higher frequencies of insertion throughout the genome. As a consequence there is an ongoing need for hyperactive transposases for use in insect genetics, however these have proven difficult to obtain. IPB7 is a hyperactive mutant of the piggyBac transposase that was identified by a genetic screen performed in yeast, a mammalian codon optimized version of which was then found to be highly active in rodent embryonic stem cells with no apparent deleterious effects. Here we report the activity of IPB7 in D. melanogaster and the mosquito, Aedes aegypti. Somatic transposition assays revealed an increase in IPB7's transposition rate from wild-type piggyBac transposase in D. melanogaster but not Ae. aegypti. However the use of IPB7 in D. melanogaster genetic transformations produced a high rate of sterility and a low transformation rate compared to wild-type transposase. This high rate of sterility was accompanied by significant gonadal atrophy that was also observed in the absence of the piggyBac vector transposon. We conclude that IPB7 has increased activity in the D. melanogaster germ-line but that a component of the sterility associated with its activity is independent of the presence of the piggyBac transposon.     

DNA-binding activity and subunit interaction of the mariner transposase

Mos1 is a member of the mariner/Tc1 family of transposable elements originally identified in Drosophila mauritiana. It has 28 bp terminal inverted repeats and like other elements of this type it transposes by a cut and paste mechanism, inserts at TA dinucleotides and codes for a transposase. This is the only protein required for transposition in vitro. We have investigated the DNA binding properties of Mos1 transposase and the role of transposase–transposase interactions in transposition. Purified transposase recognises the terminal inverted repeats of Mos1 due to a DNA-binding domain in the N-terminal 120 amino acids. This requires a putative helix–turn–helix motif between residues 88 and 108. Binding is preferentially to the right hand end, which differs at four positions from the repeat at the left end. Cleavage of Mos1 by transposase is also preferentially at the right hand end. Wild-type transposase monomers interact with each other in a yeast two-hybrid assay and we have used this to isolate mutations resulting in reduced interaction. These mutations lie along the length of the protein, indicating that transposase–transposase interactions are not due to a single interaction domain. One such mutation which retains both DNA-binding and catalytic activity has greatly reduced ability to excise Mos1 from plasmid DNA through coordinate cleavage of the two ends and transposition in vitro is lowered to a level 20-fold below that of the wild-type. This suggests that transposase–transposase interaction is required to form a synaptic complex necessary for coordinate cleavage at the ends of Mos1 during transposition. This mutant enzyme allows insertion at dinucleotides other than TA, including sequences with GC base pairs. This is the first example of a mariner/Tc1 transposase with altered target specificity.     

New Mos1 mariner transposons suitable for the recovery of gene fusions in vivo and in vitro

The Drosophila Mos1 element can be mobilized in species ranging from prokaryotes to protozoans and vertebrates, and the purified transposase can be used for in vitro transposition assays. In this report we developed a ‘mini-Mos1’ element and describe a number of useful derivatives suitable for transposon mutagenesis in vivo or in vitro. Several of these allow the creation and/or selection of tripartite protein fusions to a green fluorescent protein–phleomycin resistance (GFP-PHLEO) reporter/selectable marker. Such X-GFP-PHLEO-X fusions have the advantage of retaining 5′ and 3′ regulatory information and N- and C-terminal protein targeting domains. A Mos1 derivative suitable for use in transposon-insertion mediated linker insertion (TIMLI) mutagenesis is described, and transposons bearing selectable markers suitable for use in the protozoan parasite Leishmania were made and tested. A novel ‘negative selection’ approach was developed which permits in vitro assays of transposons lacking bacterial selectable markers. Application of this assay to several Mos1 elements developed for use in insects suggests that the large mariner pM[cn] element used previously in vivo is poorly active in vitro, while the Mos1-Act-EGFP transposon is highly active.     

Structural Basis for the Inverted Repeat Preferences of mariner Transposases

The inverted repeat (IR) sequences delimiting the left and right ends of many naturally active mariner DNA transposons are non-identical and have different affinities for their transposase. We have compared the preferences of two active mariner transposases, Mos1 and Mboumar-9, for their imperfect transposon IRs in each step of transposition: DNA binding, DNA cleavage, and DNA strand transfer. A 3.1 Å resolution crystal structure of the Mos1 paired-end complex containing the pre-cleaved left IR sequences reveals the molecular basis for the reduced affinity of the Mos1 transposase DNA-binding domain for the left IR as compared with the right IR. For both Mos1 and Mboumar-9, in vitro DNA transposition is most efficient when the preferred IR sequence is present at both transposon ends. We find that this is due to the higher efficiency of cleavage and strand transfer of the preferred transposon end. We show that the efficiency of Mboumar-9 transposition is improved almost 4-fold by changing the 3′ base of the preferred Mboumar-9 IR from guanine to adenine. This preference for adenine at the reactive 3′ end for both Mos1 and Mboumar-9 may be a general feature of mariner transposition.