Transposase Activity Research Articles

Abstract A64: Functional genomics of metastatic Ewing sarcoma

Abstract Ewing sarcoma (ES) is a poorly differentiated bone and soft tissue tumor of high metastatic potential. ES mainly affects children, adolescents, and young adults (AYAs) at a frequency of ~1.5 cases per million globally. Ewing neoplasms strikingly converge on a single recurrent initiating event, which is a chromosomal translocation that generates a fusion transcript between the EWSR1 gene and a gene of the ETS family of transcription factors, most commonly FLi1 (85%). After 20 years since the discovery of the EWS-FLi1 fusion, ES remains a clinical challenge with unacceptably low survival rates, primarily due to metastasis. The bulk of research conducted to date (>95%) being focused on the primary tumor has resulted in a critical knowledge gap. To address this issue and unravel the dysregulated pathways in ES tumor evolution and metastatic dissemination, we harnessed the genome-wide insertional mechanism of transposons and the transduction efficiency of lentiviruses to engineer ES cell models. Human mesenchymal stem cells (hMSC), the putative cells of origin of ES, were engineered to constitutively express EWS-Fli1 accompanied by the inducible expression of a highly active transposase. Upon activation of the former, transposon-mediated mutagenesis will activate oncogenes and inactivate tumor suppressor genes, to mediate transformation of the transduced that can now engraft when implanted in recipient mice. Xenografted tumors are resected and the mice observed for development of distant metastases. Matching primary and metastatic tumors are sequenced to uncover the genes commonly affected by transposition in the two compartments. Pathway-oriented bioinformatic analysis will further reveal candidate metastatic driver genes. Matching of the targeted pathways with drugs will be used to validate the candidates by in vitro and in vivo metastasis assays. Citation Format: Wajih Jawhar, Paul Waterhouse, Rama Khokha, Takeaki Ishii, Robert Turcotte, Nada Jabado, Livia Garzia. Functional genomics of metastatic Ewing sarcoma [abstract]. In: Proceedings of the AACR Special Conference on the Advances in Pediatric Cancer Research; 2019 Sep 17-20; Montreal, QC, Canada. Philadelphia (PA): AACR; Cancer Res 2020;80(14 Suppl):Abstract nr A64.

Identification of RAG-like transposons in protostomes suggests their ancient bilaterian origin

BackgroundV(D) J recombination is essential for adaptive immunity in jawed vertebrates and is initiated by the RAG1-RAG2 endonuclease. The RAG1 and RAG2 genes are thought to have evolved from a RAGL (RAG-like) transposon containing convergently-oriented RAG1-like (RAG1L) and RAG2-like (RAG2L) genes. Elements resembling this presumptive evolutionary precursor have thus far only been detected convincingly in deuterostomes, leading to the model that the RAGL transposon first appeared in an early deuterostome.ResultsWe have identified numerous RAGL transposons in the genomes of protostomes, including oysters and mussels (phylum Mollusca) and a ribbon worm (phylum Nemertea), and in the genomes of several cnidarians. Phylogenetic analyses are consistent with vertical evolution of RAGL transposons within the Bilateria clade and with its presence in the bilaterian ancestor. Many of the RAGL transposons identified in protostomes are intact elements containing convergently oriented RAG1L and RAG2L genes flanked by terminal inverted repeats (TIRs) and target site duplications with striking similarities with the corresponding elements in deuterostomes. In addition, protostome genomes contain numerous intact RAG1L-RAG2L adjacent gene pairs that lack detectable flanking TIRs. Domains and critical active site and structural amino acids needed for endonuclease and transposase activity are present and conserved in many of the predicted RAG1L and RAG2L proteins encoded in protostome genomes.ConclusionsActive RAGL transposons were present in multiple protostome lineages and many were likely transmitted vertically during protostome evolution. It appears that RAGL transposons were broadly active during bilaterian evolution, undergoing multiple duplication and loss/fossilization events, with the RAGL genes that persist in present day protostomes perhaps constituting both active RAGL transposons and domesticated RAGL genes. Our findings raise the possibility that the RAGL transposon arose earlier in evolution than previously thought, either in an early bilaterian or prior to the divergence of bilaterians and non-bilaterians, and alter our understanding of the evolutionary history of this important group of transposons.

Evolution and domestication of Tc1/mariner transposons in the genome of African coelacanth (Latimeria chalumnae).

Here, we comprehensively analysed the abundance, diversity, and activity of Tc1/mariner transposons in African coelacanth (Latimeria chalumnae). Fifteen Tc1/mariner autonomous transposons were identified and grouped into six clades: DD34E/Tc1, DD34D/mariner, DD35D/Fot, DD31D/pogo, DD30-31D/pogo-like, and DD32-36D/Tigger, belonging to three known families: DD34E/Tc1, DD34D/mariner, and DD×D/pogo (DD35D/Fot, DD31D/pogo, DD30-31D/pogo-like, and DD32-36D/Tigger). Thirty-one miniature inverted-repeat transposable element (MITE) transposons of Tc1/mariner were also identified, and 20 of them display similarity to the identified autonomous transposons. The structural organization of these full Tc1/mariner elements includes a transposase gene flanked by terminal inverted repeats (TIRs) with TA dinucleotides. The transposases contain N-terminal DNA binding domain and a C-terminal catalytic domain characterized by the presence of a conservative D(Asp)DE(Glu)/D triad that is essential for transposase activity. The Tc1/mariner superfamily in coelacanth exhibited very low genome coverage (0.3%), but it experienced an extraordinary difference of proliferation dynamics among the six clades identified; moreover, most of them exhibited a very recent and current proliferation, suggesting that some copies of these transposons are putatively active. Additionally, at least four functional genes derived from Tc1/mariner transposons were found. We provide an up-to-date overview of Tc1/mariner in coelacanth, which may be helpful in determining genome and gene evolution in this living fossil.

Complex Evolutionary History of Mboumar, a Mariner Element Widely Represented in Ant Genomes

Mboumar-9 is an active mariner-transposable element previously isolated in the ant Messor bouvieri. In this work, a mariner-like element, Mboumar, isolated from 22 species of ants, is analyzed. These species belong to nine different subfamilies, including Leptanillinae, the most primitive ant subfamily, and Myrmicinae and Formicidae, the most derived ones. Consequently, Mboumar-like elements seem to be well-represented in ant genomes. The phylogenetic tree drawn for mariner elements is highly inconsistent with the phylogeny of host ants, with almost identical elements found in clearly distant species and, on the contrary, more variable elements in closely related species. The inconsistency between the two phylogenetic trees indicates that these transposable elements have evolved independently from the speciation events of the ants that host them. Besides, we found closer genetic relationships among elements than among their host ants. We also found potential coding copies with an uninterrupted open reading frame of 345 aa in 11 species. The putative transposase codified by them showed a high sequence identity with the active Mboumar-9 transposase. The results of selection tests suggest the intervention of purifying selection in the evolution of these elements. Overall, our study suggests a complex evolutionary history of the Mboumar-like mariner in ants, with important participation of horizontal transfer events. We also suggest that the evolutionary dynamics of Mboumar-like elements can be influenced by the genetic system of their host ants, which are eusocial insects with a haplodiploid genetic system.

Assaying Chromatin Accessibility Using ATAC-Seq in Invertebrate Chordate Embryos.

Cis-regulatory elements (CREs) are non-coding DNA regions involved in the spatio-temporal regulation of gene expression. Gene regulatory changes drive animal development and play major roles during evolution of animal body plans. Therefore, we believe that determining CREs at different developmental stages and across animal lineages is critical to understand how evolution operates through development. The Assay for Transposase-Accessible Chromatin followed by high-throughput sequencing (ATAC-seq) is a powerful technique for the study of CREs that takes advantage of Tn5 transposase activity. Starting from fewer than 105 cells, in a 1-day procedure, it is possible to detect, at a genome-wide level, CREs located in open chromatin regions with high resolution. Here, we describe a detailed step-by-step ATAC-seq protocol for invertebrate chordate marine embryos. We have successfully applied this technique to amphioxus and two species of tunicate embryos. We also show an easy workflow to analyze data generated with this technique. Moreover, we point out that this method and our bioinformatic pipeline are efficient to detect CREs associated with Wnt signaling pathway by simply using embryos treated with a drug that perturbs this pathway. This approach can be extended to other signaling pathways and also to embryo mutants for critical genes. Our results therefore demonstrate the power of ATAC-seq for the identification of CREs that play essential functions during animal development in a wide range of invertebrate or vertebrate animals.

A highly soluble Sleeping Beauty transposase improves control of gene insertion.

The Sleeping Beauty (SB) transposon system is an efficient non-viral gene transfer tool in mammalian cells but its broad use has been hampered by uncontrolled transposase gene activity from DNA vectors, posing a risk for genome instability, and by the inability to use transposase protein directly. Here, we used rational protein design based on the crystal structure of the hyperactive SB100X variant to create an SB transposase (hsSB) with enhanced solubility and stability. We demonstrate that hsSB can be delivered with transposon DNA to genetically modify cell lines and embryonic, hematopoietic and induced pluripotent stem cells (iPSCs), overcoming uncontrolled transposase activity. We used hsSB to generate chimeric antigen receptor (CAR) T-cells, which exhibit potent anti-tumor activity in vitro and in xenograft mice. We found that hsSB spontaneously penetrates cells, enabling modification of iPSCs and generation of CAR-T cells without the use of transfection reagents. Titration of hsSB to modulate genomic integration frequency achieved as few as two integrations per genome.

Biochemical analysis of nucleosome targeting by Tn5 transposase.

Tn5 transposase is a bacterial enzyme that integrates a DNA fragment into genomic DNA, and is used as a tool for detecting nucleosome-free regions of genomic DNA in eukaryotes. However, in chromatin, the DNA targeting by Tn5 transposase has remained unclear. In the present study, we reconstituted well-positioned 601 dinucleosomes, in which two nucleosomes are connected with a linker DNA, and studied the DNA integration sites in the dinucleosomes by Tn5 transposase in vitro. We found that Tn5 transposase preferentially targets near the entry–exit DNA regions within the nucleosome. Tn5 transposase minimally cleaved the dinucleosome without a linker DNA, indicating that the linker DNA between two nucleosomes is important for the Tn5 transposase activity. In the presence of a 30 base-pair linker DNA, Tn5 transposase targets the middle of the linker DNA, in addition to the entry–exit sites of the nucleosome. Intriguingly, this Tn5-targeting characteristic is conserved in a dinucleosome substrate with a different DNA sequence from the 601 sequence. Therefore, the Tn5-targeting preference in the nucleosomal templates reported here provides important information for the interpretation of Tn5 transposase-based genomics methods, such as ATAC-seq.

Development of a metabolic pathway transfer and genomic integration system for the syngas-fermenting bacterium Clostridium ljungdahlii

BackgroundClostridium spp. can synthesize valuable chemicals and fuels by utilizing diverse waste-stream substrates, including starchy biomass, lignocellulose, and industrial waste gases. However, metabolic engineering in Clostridium spp. is challenging due to the low efficiency of gene transfer and genomic integration of entire biosynthetic pathways.ResultsWe have developed a reliable gene transfer and genomic integration system for the syngas-fermenting bacterium Clostridium ljungdahlii based on the conjugal transfer of donor plasmids containing large transgene cassettes (> 5 kb) followed by the inducible activation of Himar1 transposase to promote integration. We established a conjugation protocol for the efficient generation of transconjugants using the Gram-positive origins of replication repL and repH. We also investigated the impact of DNA methylation on conjugation efficiency by testing donor constructs with all possible combinations of Dam and Dcm methylation patterns, and used bisulfite conversion and PacBio sequencing to determine the DNA methylation profile of the C. ljungdahlii genome, resulting in the detection of four sequence motifs with N6-methyladenosine. As proof of concept, we demonstrated the transfer and genomic integration of a heterologous acetone biosynthesis pathway using a Himar1 transposase system regulated by a xylose-inducible promoter. The functionality of the integrated pathway was confirmed by detecting enzyme proteotypic peptides and the formation of acetone and isopropanol by C. ljungdahlii cultures utilizing syngas as a carbon and energy source.ConclusionsThe developed multi-gene delivery system offers a versatile tool to integrate and stably express large biosynthetic pathways in the industrial promising syngas-fermenting microorganism C. ljungdahlii. The simple transfer and stable integration of large gene clusters (like entire biosynthetic pathways) is expanding the range of possible fermentation products of heterologously expressing recombinant strains. We also believe that the developed gene delivery system can be adapted to other clostridial strains as well.

Piggybat transposase for the generation of CD19 specific chimeric antigen receptor T cells

Background & Aim Introduction DNA transposon systems offer an alternative to viral vectors for genetic modification of eukaryotic cells. The Piggybac system is being used in CAR T-cell clinical trials presently, but its widespread use may be limited by licensing restrictions. Transposon mining of the little brown bat (Myotis lucifugus) genome has revealed the presence of an active DNA transposase named Piggybat. Aims To optimise manufacturing conditions and evaluate the genomic integration profile of the Piggybat transposase in the generation of CD19 specific CAR T-cells (CAR19-T) compared to Piggybac transposase. Methods, Results & Conclusion Methods CAR19-T were generated from healthy donors (n=3) by co-electroporation of two plasmids encoding the transposase and a CAR19 transposon. T-cells were co-cultured with irradiated feeder cells and cytokines. CAR19-T expansion, CAR expression, memory phenotype, CD19 specific activation and cytotoxicity were assessed after a 2-week culture period. CAR gene integration copy number was determined by droplet digital polymerase chain reaction and integration site analysis was conducted by next generation sequencing of PCR amplicons spanning genomic DNA/CAR gene intersection sites. Results Over 2 weeks, Piggybat CAR19-T expanded a mean of 26 fold with 52% CAR expression, and Piggybac CAR19-T expanded a mean of 11 fold with 67% CAR expression. Piggybat and Piggybac CAR19-T had a mean 59% and 48% CD8+ T-cells respectively, with a similar CAR19-T memory phenotype. Using 50ug/ml concentration of each transposon and transposase plasmids, Piggybat had fewer genomic integrants than Piggybac (mean 3.4 and 10.1 copies per cell respectively). Integrant copy number could be titrated by altering transposon plasmid concentration. Peak CAR19-T expansion, expression and MFI were achieved using 1ug Piggybat transposase plasmid and 5ug transposon plasmid. Piggybat and Piggybac CAR19-T demonstrated similar CD19 specificity and cytotoxicity. Piggybat CAR19-T showed 1920 unique genomic integration sites, 11.13% within 2.5kb of transcription start sites (TSS) and 43.72% being intragenic, compared to Piggybac with 2697 unique integration sites, 20.33% TSS localised and 47.77% intragenic. Conclusions Compared to Piggybac, Piggybat transposase can generate CD19 specific CAR T-cells with similar CAR expression, function and insertion site profile, but fewer integrants per cell. Thus, Piggybat is a new DNA transposon that may allow cost-effective CAR T-cell production for clinical use.

An Efficient System for Ds Transposon Tagging in Brachypodium distachyon.

Transposon tagging is a powerful tool that has been widely applied in several species for insertional mutagenesis in plants. Several efforts have aimed to create transfer-DNA (T-DNA) insertional mutant populations in Brachypodium distachyon, a monocot plant used as a model system to study temperate cereals, but there has been a lack of research aimed at using transposon strategies. Here, we describe the application of a maize (Zea mays) Dissociation (Ds) transposon tagging system in B distachyon The 35S::AcTPase cassette and Ds element were constructed within the same T-DNA and transformed into B distachyon plants. The Ds element was readily transposed to other chromosomes or to the same chromosome under the function of Activator (Ac) transposase. Through homologous chromosome synapsis, recombination, and segregation, the Ds element separated from the Ac element. We selected stable Ds-only plants using G418 and GFP assays and analyzed 241 T0 lines, some of which were highly efficient at producing Ds-only progeny. Through thermal asymmetric interlaced PCR, we isolated 710 independent Ds flanking sequences from Ds-only plants. Furthermore, we identified a large collection of mutants with visible developmental phenotypes via this transposon tagging system. The system is relatively simple and rapid in comparison to traditional T-DNA insertion strategies, because once efficiency lines are obtained they can be reused to generate more lines from nontransposed plants without the use of time-consuming tissue culture steps.

Dimerization through the RING-Finger Domain Attenuates Excision Activity of the piggyBac Transposase.

The movement of the piggyBac transposon is mediated through its cognate transposase. The piggyBac transposase binds to the terminal repeats present at the ends of the transposon. This is followed by excision of the transposon and release of the nucleoprotein complex. The complex translocates, followed by integration of the transposon at the target site. Here, we show that the RING-finger domain (RFD) present toward the C-terminus of the transposase is vital for dimerization of this enzyme. The deletion of the RFD or the last seven residues of the RFD results in a monomeric protein that binds the terminal end of the transposon with nearly the same affinity as wild type piggyBac transposase. Surprisingly, the monomeric constructs exhibit >2-fold enhancement in the excision activity of the enzyme. Overall, our studies suggest that dimerization attenuates the excision activity of the piggyBac transposase. This attribute of the piggyBac transposase may serve to prevent excessive transposition of the piggyBac transposon that might be catastrophic for the host cell.

New insights into the transposition mechanisms of IS6110 and its dynamic distribution between Mycobacterium tuberculosis Complex lineages.

The insertion Sequence IS6110, only present in the pathogens of the Mycobacterium tuberculosis Complex (MTBC), has been the gold-standard epidemiological marker for TB for more than 25 years, but biological implications of IS6110 transposition during MTBC adaptation to humans remain elusive. By studying 2,236 clinical isolates typed by IS6110-RFLP and covering the MTBC, we remarked a lineage-specific content of IS6110 being higher in modern globally distributed strains. Once observed the IS6110 distribution in the MTBC, we selected representative isolates and found a correlation between the normalized expression of IS6110 and its abundance in MTBC chromosomes. We also studied the molecular regulation of IS6110 transposition and we found a synergistic action of two post-transcriptional mechanisms: a -1 ribosomal frameshift and a RNA pseudoknot which interferes translation. The construction of a transcriptionally active transposase resulted in 20-fold increase of the transposition frequency. Finally, we examined transposition in M. bovis and M. tuberculosis during laboratory starvation and in a mouse infection model of TB. Our results shown a higher transposition in M. tuberculosis, that preferably happens during TB infection in mice and after one year of laboratory culture, suggesting that IS6110 transposition is dynamically adapted to the host and to adverse growth conditions.

Abstract 3002: Therapeutic targeting of PGBD5-induced DNA repair dependence in pediatric solid tumors

Abstract Despite intense efforts, the cure rates of childhood solid tumors are not satisfactory. Resistance to intensive chemotherapy is common, and targets for molecular therapies are largely undefined, particularly for tumors caused by aberrant developmental and epigenetic factors. We have now found that the majority of childhood solid tumors, rhabdoid tumors, neuroblastoma, medulloblastoma and Ewing sarcoma, express an active DNA transposase PGBD5. Using functional genetic approaches, we observed that mouse and human cells deficient in non-homologous end joining (NHEJ) DNA repair cannot tolerate the expression of PGBD5. In a chemical screen of DNA repair inhibitors, we identified the ATR/ATM inhibitor AZD6738 as a specific sensitizer of PGBD5-dependent DNA damage and apoptosis. Ectopic expression of PGBD5 was sufficient to induce hypersensitivity to AZD6738, and endogenous PGBD5 was necessary for its anti-tumor effects. We found that PGBD5-expressing tumor cells accumulate unrepaired DNA damage in response to AZD6738 treatment, and undergo apoptosis in both dividing and G1 phase cells in the absence of measureable DNA replication stress. Accordingly, AZD6738 exhibits nanomolar potency against the majority of neuroblastoma, medulloblastoma, Ewing sarcoma and rhabdoid tumor cells tested, while sparing non-transformed human and mouse embryonic fibroblasts in vitro. Finally, AZD6738 exhibits excellent single-agent activity against high-risk neuroblastoma and medulloblastoma xenografts, with induction of apoptosis and tumor regression in vivo. These findings delineate a therapeutically actionable synthetic lethality for the majority of childhood solid tumors. Citation Format: Anton Henssen, Casie Reed, Eileen Jiang, Wong Wai, Elisa de Stanchina, Yasumichi Kuwahara, Hajime Hosoi, Johannes H. Schulte, Neil Ganem, John Petrini, Alex Kentsis. Therapeutic targeting of PGBD5-induced DNA repair dependence in pediatric solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3002. doi:10.1158/1538-7445.AM2017-3002

Abstract 4888: Human PGBD5 DNA transposase promotes site-specific oncogenic mutations in rhabdoid tumors

Abstract Genomic rearrangements are a hallmark of childhood solid tumors, but their mutational causes remain poorly understood. Here, we identify the piggyBac transposable element derived 5 (PGBD5) gene as an enzymatically active human DNA transposase expressed in the majority of rhabdoid tumors, a lethal childhood cancer. Using assembly-based whole-genome DNA sequencing, we observed previously unknown somatic genomic rearrangements in primary human rhabdoid tumors. These rearrangements were characterized by deletions and inversions involving PGBD5-specific signal (PSS) sequences at their breakpoints, with some recurrently targeting tumor suppressor genes, leading to their inactivation. PGBD5 was found to be physically associated with human genomic PSS sequences that were also sufficient to mediate PGBD5-induced DNA rearrangements in rhabdoid tumor cells. We found that ectopic expression of PGBD5 in primary human cells was sufficient to promote penetrant cell transformation in vitro and in immunodeficient mice in vivo. This activity required specific catalytic residues in the PGBD5 transposase domain, as well as end-joining DNA repair, and induced distinct structural rearrangements, involving PSS-associated breakpoints, similar to those found in primary human rhabdoid tumors. Thus, PGBD5 defines a distinct class of oncogenic mutators and induces site-specific somatic DNA rearrangements in human cancer. Citation Format: Anton G. Henssen, Richard Koche, Jiali Zhuang, Eileen Jiang, Casie Reed, Amy Eisenberg, Eric Still, Elias Rodríguez-Fos, Santiago Gonzalez, Montserrat Puiggròs, Andrew N. Blackford, Christopher E. Mason, Elisa de Stanchina, Mithat Gönen, Anne-Katrin Emde, Minita Shah, Kanika Arora, Catherine Reeves, Nicholas D. Socci, Elizabeth Perlman, Cristina R. Antonescu, Charles W. Roberts, Hanno Steen, Elizabeth Mullen, Stephen P. Jackson, David Torrents, Zhiping Weng, Scott A. Armstrong, Alex Kentsis. Human PGBD5 DNA transposase promotes site-specific oncogenic mutations in rhabdoid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4888. doi:10.1158/1538-7445.AM2017-4888

Transposase-driven rearrangements in human tumors.

A new study shows that aberrant DNA transposase activity promotes structural alterations that are clonally selected to drive tumor development. This discovery uncovers novel mechanisms of tumor-suppressor gene inactivation and highlights a new approach to cancer gene identification.

PGBD5 promotes site-specific oncogenic mutations in human tumors.

Genomic rearrangements are a hallmark of human cancers. Here, we identify the piggyBac transposable element derived 5 (PGBD5) gene as an active DNA transposase expressed in the majority of childhood solid tumors, including lethal rhabdoid tumors. Using assembly-based whole-genome DNA sequencing, we found previously undefined genomic rearrangements in human rhabdoid tumors. These rearrangements involved PGBD5-specific signal (PSS) sequences at their breakpoints, recurrently inactivating tumor suppressor genes. PGBD5 was physically associated with genomic PSS sequences that were also sufficient to mediate PGBD5-induced DNA rearrangements in rhabdoid tumor cells. Ectopic expression of PGBD5 in primary immortalized human cells was sufficient to promote cell transformation in vivo. This activity required specific catalytic residues in the PGBD5 transposase domain, as well as end-joining DNA repair, and induced structural rearrangements with PSS breakpoints. This defines PGBD5 as an oncogenic mutator and provides a plausible mechanism for site-specific DNA rearrangements in childhood and adult solid tumors.

Transposition of the bamboo Mariner-like element Ppmar1 in yeast

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.

Unlocking Tn3-family transposase activity in vitro unveils an asymetric pathway for transposome assembly

The Tn3 family is a widespread group of replicative transposons that are notorious for their contribution to the dissemination of antibiotic resistance and the emergence of multiresistant pathogens worldwide. The TnpA transposase of these elements catalyzes DNA breakage and rejoining reactions required for transposition. It also is responsible for target immunity, a phenomenon that prevents multiple insertions of the transposon into the same genomic region. However, the molecular mechanisms whereby TnpA acts in both processes remain unknown. Here, we have developed sensitive biochemical assays for the TnpA transposase of the Tn3-family transposon Tn4430 and used these assays to characterize previously isolated TnpA mutants that are selectively affected in immunity. Compared with wild-type TnpA, these mutants exhibit deregulated activities. They spontaneously assemble a unique asymmetric synaptic complex in which one TnpA molecule simultaneously binds two transposon ends. In this complex, TnpA is in an activated state competent for DNA cleavage and strand transfer. Wild-type TnpA can form this complex only on precleaved ends mimicking the initial step of transposition. The data suggest that transposition is controlled at an early stage of transpososome assembly, before DNA cleavage, and that mutations affecting immunity have unlocked TnpA by stabilizing the protein in a monomeric activated synaptic configuration. We propose an asymmetric pathway for coupling active transpososome assembly with proper target recruitment and discuss this model with respect to possible immunity mechanisms.

Forward genetic screen of human transposase genomic rearrangements.

BackgroundNumerous human genes encode potentially active DNA transposases or recombinases, but our understanding of their functions remains limited due to shortage of methods to profile their activities on endogenous genomic substrates.ResultsTo enable functional analysis of human transposase-derived genes, we combined forward chemical genetic hypoxanthine-guanine phosphoribosyltransferase 1 (HPRT1) screening with massively parallel paired-end DNA sequencing and structural variant genome assembly and analysis. Here, we report the HPRT1 mutational spectrum induced by the human transposase PGBD5, including PGBD5-specific signal sequences (PSS) that serve as potential genomic rearrangement substrates.ConclusionsThe discovered PSS motifs and high-throughput forward chemical genomic screening approach should prove useful for the elucidation of endogenous genome remodeling activities of PGBD5 and other domesticated human DNA transposases and recombinases.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-2877-x) contains supplementary material, which is available to authorized users.

Abstract 1576: Insertional mutagenesis to identify molecular mechanisms of breast cancer dormancy and metastasis

Abstract The PiggyBac transposon insertional mutagenesis system (PBTS) is a powerful genomic screen previously demonstrated to identify cancer-causing genes and which we have adapted to identify genes regulating breast cancer cell dormancy and metastases. Transposition takes places through a cut and paste mechanism catalyzed by the PiggyBac transposase, which excises and re-integrates the transposon into TTAA chromosomal sites. We have adapted the PBTS system using the well characterized D2.0R in vitro and in vivo models of breast cancer cell dormancy. D2.0R cells grow as primary tumors in the mammary fat pad, disseminate to the lungs, but remain dormant. The PBTS system induces random genome-wide gene activations or inactivations that we expect will induce dormant cells to proliferate and form metastases in vivo. Identification of these genes is performed using high throughput sequencing and existing computational algorithms. Candidate dormancy and metastasis genes will be functionally validated for their roles in dormancy and metastases. We have validated the performance of the PBTS in our model system. D2.0R cells were transfected with 20 copies of the PiggyBac transposon ATP-1. Single clones were obtained from the pooled population and expanded under selection. Pooled D2.0R cells and D2.0R single clones were subsequently transduced with lenti-hyPBase transposase and double-selected for the stable integration of the ATP-1 transposon and the lenti-hyPBase transposase. gDNA was extracted from these cells, sheared and amplified by splinkerette-PCR and sequenced using an Illumina platform. Our data show that there is a significantly higher variability in the integration sites of the ATP-1 transposon in D2.0R cells stably expressing hy-PBase as compared with non-hy-PBase expressing D2.0R cells carrying the ATP-1 transposons. This demonstrates that cells from each clone acquire new insertions sites upon ATP-1 re-mobilization due to hyPBase transposase activity. Likewise, we found a significantly higher variability in the ATP-1 insertions in cells from the pooled cultures than in the cells from individual cellular clones. We also observed that reads from both the 5’ and 3’ ends of the ATP-1 insertion sites of the transposon cluster together, showing a good correlation between reads from both ends. A complimentary approach will be performed by introducing the PBTS system into the poorly metastatic MMTV-Myc transgenic mammary cancer model to identify genes and pathways that induce metastases. Since Myc is overexpressed in ∼30% of human breast cancers, this is a relevant model to employ. These results demonstrate that our experimental protocols are able to induce transposon mobilization which should identify genes that regulate tumor dormancy and metastases. Identifying genes that promote dormancy and metastases will lead to novel strategies that could prevent disease recurrence or treat metastatic breast cancer. Citation Format: Laura Vera Ramirez, Sven Bilke, Robert Walker, Paul Meltzer, Tinghu Qiu, Roland Rad, Jeffrey E. Green. Insertional mutagenesis to identify molecular mechanisms of breast cancer dormancy and metastasis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1576.