Transposase N-terminal phosphorylation and asymmetric transposon ends inhibit piggyBac transposition in mammalian cells
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PROVIDER: PRJNA833459 | ENA |
REPOSITORIES: ENA
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Project description:DNA transposon systems are widely used in mammalian cells for genetic modification experiments, but their regulation remains poorly understood. We used biochemical and cell-based assays together with AlphaFold modeling and rational protein redesign to evaluate aspects of piggyBac transposition including the previously unexplained role of the transposase N-terminus and the need for asymmetric transposon ends for cellular activity. We found that phosphorylation at predicted casein kinase II sites in the transposase N-terminus inhibits transposition, most likely by preventing transposase-DNA interactions. Deletion of the region containing these sites releases inhibition thereby enhancing activity. We also found that the N-terminal domain promotes transposase dimerization in the absence of transposon DNA. When the N-terminus is deleted, the transposase gains the ability to carry out transposition using symmetric transposon left ends. This novel activity is also conferred by appending a second C-terminal domain. When combined, these modifications together result in a transposase that is highly active when symmetric transposon ends are used. Our results demonstrate that transposase N-terminal phosphorylation and the requirement for asymmetric transposon ends both negatively regulate piggyBac transposition in mammalian cells. These novel insights into the mechanism and structure of the piggyBac transposase expand its potential use for genomic applications.
2022-11-10 | GSE201914 | GEO
Project description:The piggyBac transposon system is widely used for biotechnology and genome engineering and is the founding member of a large superfamily of piggyBac-like elements. We investigated the role in transpositon of the nonconserved N-terminus in the piggyBac transposase, including the impact of predicted casein kinase phosphorylation sites within it.
2023-03-11 | PXD037132 | Pride
Project description:DNA transposon-based vectors have emerged as gene vehicles with a wide biomedical and therapeutic potential. So far, genomic insertion of such vectors has relied on the co-delivery of genetic material encoding the gene-inserting transposase protein, raising concerns related to persistent expression, insertional mutagenesis and cytotoxicity. This report describes potent DNA transposition achieved by direct delivery of transposase protein. By adapting integrase-deficient lentiviral particles (LPs) as carriers of the hyperactive piggyBac transposase protein (hyPBase), we demonstrate rates of DNA transposition that are comparable with the efficiency of a conventional plasmid-based strategy. Embedded in the Gag polypeptide, hyPBase is robustly incorporated into LPs and liberated from the viral proteins by the viral protease during particle maturation. We demonstrate lentiviral co-delivery of the transposase protein and vector RNA carrying the transposon sequence, allowing robust DNA transposition in a variety of cell types. Importantly, this novel delivery method facilitates a balanced cellular uptake of hyPBase, as shown by confocal microscopy, and allows high-efficiency production of clones harboring a single transposon insertion. Our findings establish engineered LPs as a new tool for transposase delivery. We believe that protein transduction methods will increase applicability and safety of DNA transposon-based vector technologies.
| S-EPMC3936723 | biostudies-literature
Project description:Continuous innovation of revolutionizing genome engineering technologies calls for an intensified focus on new delivery technologies that not only match the inventiveness of genome editors but also enable the combination of potent delivery and time-restricted action of genome-modifying bits and tools. We have previously demonstrated the use of lentivirus-derived nanoparticles (LNPs) as a protein delivery vehicle, incorporating and transferring DNA transposases, designer nucleases, or RNA-guided endonucleases fused to the N terminus of the Gag/GagPol polypeptide. Here, we establish LNP-directed transfer of the piggyBac DNA transposase protein by fusing the transposase to the integrase protein in the C-terminal end of GagPol. We show protein incorporation and proteolytic release of the DNA transposase within matured LNPs, resulting in high levels of DNA transposition activity in LNP-treated cells. Importantly, as opposed to conventional delivery methods based on transfection of plasmid DNA or in-vitro-transcribed mRNA, protein delivery by LNPs effectively results in time-restricted action of the protein (<24 hr) without compromising overall potency. Our findings refine LNP-directed piggyBac transposase delivery, at present the only available direct delivery strategy for this particular protein, and demonstrate a novel strategy for restricting and fine-tuning the exposure of the genome to DNA-modifying enzymes.
| S-EPMC5992343 | biostudies-literature
Project description:PiggyBac is a prevalent transposon system used to deliver transgenes and functionally explore the mammalian untouched genomic territory. The important features of piggyBac transposon are the relatively low insertion site preference and the ability of seamless removal from genome, which allow its potential uses in functional genomics and regenerative medicine. Efforts to increase its transposition efficiency in mammals were made through engineering the corresponding transposase (PBase) codon usage to enhance its expression level and through screening for mutant PBase variants with increased enzyme activity. To improve the safety for its potential use in regenerative medicine applications, site-specific transposition was achieved by using engineered zinc finger- and Gal4-fused PBases. An excision-prone PBase variant has also been successfully developed. Here we describe the construction of a nucleolus-predominant PBase, NP-mPB, by adding a nucleolus-predominant (NP) signal peptide from HIV-1 TAT protein to a mammalian codon-optimized PBase (mPB). Although there is a predominant fraction of the NP-mPB-tGFP fusion proteins concentrated in the nucleoli, an insertion site preference toward nucleolar organizer regions is not detected. Instead a 3-4 fold increase in piggyBac transposition efficiency is reproducibly observed in mouse and human cells.
| S-EPMC3933532 | biostudies-literature
Project description:Genomic rearrangements are a hallmark of childhood cancers, but the mutational processes underlying most of these variants remain unknown. We identified piggyBac transposable element derived 5 (PGBD5) as a highly expressed, enzymatically active domesticated human DNA transposase in a large subset of pediatric solid tumors, including rhabdoid tumors. Ectopic expression of PGBD5 in primary human cells was sufficient to induce fully penetrant cell transformation both in vitro and in immunodeficient mice in vivo. This activity required specific catalytic aspartic acid residues in the PGBD5 transposase domain as well as cellular non-homologous end-joining DNA repair, and was associated with distinct structural rearrangements defined by specific DNA sequence motifs. Similar genomic alterations, some recurrent, were found in primary human rhabdoid tumors. Thus, PGBD5 represents a new class of developmental oncogenic mutators in childhood solid tumors.
2016-12-05 | GSE81160 | GEO
Project description:The transposon piggyBac is being used increasingly for genetic studies. Here, we describe modified versions of piggyBac transposase that have potentially wide-ranging applications, such as reversible transgenesis and modified targeting of insertions. piggyBac is distinguished by its ability to excise precisely, restoring the donor site to its pretransposon state. This characteristic makes piggyBac useful for reversible transgenesis, a potentially valuable feature when generating induced pluripotent stem cells without permanent alterations to genomic sequence. To avoid further genome modification following piggyBac excision by reintegration, we generated an excision competent/integration defective (Exc(+)Int(-)) transposase. Our findings also suggest the position of a target DNA-transposase interaction. Another goal of genome engineering is to develop reagents that can guide transgenes to preferred genomic regions. Others have shown that piggyBac transposase can be active when fused to a heterologous DNA-binding domain. An Exc(+)Int(-) transposase, the intrinsic targeting of which is defective, might also be a useful intermediate in generating a transposase whose integration activity could be rescued and redirected by fusion to a site-specific DNA-binding domain. We show that fusion to two designed zinc finger proteins rescued the Int(-) phenotype. Successful guided transgene integration into genomic DNA would have broad applications to gene therapy and molecular genetics. Thus, an Exc(+)Int(-) transposase is a potentially useful reagent for genome engineering and provides insight into the mechanism of transposase-target DNA interaction.
| S-EPMC3690869 | biostudies-other
Project description:Tn5-mediated transposition of double-strand DNA has been widely utilized in various high-throughput sequencing applications. Here, we report that the Tn5 transposase is also capable of direct tagmentation of RNA/DNA hybrids in vitro. As a proof-of-concept application, we utilized this activity to replace the traditional library construction procedure of RNA sequencing, which contains many laborious and time-consuming processes. Results of Transposase assisted RNA/DNA hybrids Co-tagmEntation (termed “TRACE-seq”) are comparable to traditional RNA-seq methods in terms of detected gene number, gene body coverage, gene expression measurement, library complexity, and differential expression analysis; at the meantime, TRACE-seq enables a one-tube library construction protocol and hence is more rapid (within 6h) and convenient. We expect this tagmentation activity on RNA/DNA hybrids to have broad potentials on RNA biology and chromatin research.
2020-07-29 | GSE143422 | GEO
Project description:DNA transposons have been widely used for transgenesis and insertional mutagenesis in various organisms. Among the transposons active in mammalian cells, the moth-derived transposon piggyBac is most promising with its highly efficient transposition, large cargo capacity, and precise repair of the donor site. Here we report the generation of a hyperactive piggyBac transposase. The active transposition of piggyBac in multiple organisms allowed us to screen a transposase mutant library in yeast for hyperactive mutants and then to test candidates in mouse ES cells. We isolated 18 hyperactive mutants in yeast, among which five were also hyperactive in mammalian cells. By combining all mutations, a total of 7 aa substitutions, into a single reading frame, we generated a unique hyperactive piggyBac transposase with 17-fold and ninefold increases in excision and integration, respectively. We showed its applicability by demonstrating an increased efficiency of generation of transgene-free mouse induced pluripotent stem cells. We also analyzed whether this hyperactive piggyBac transposase affects the genomic integrity of the host cells. The frequency of footprints left by the hyperactive piggyBac transposase was as low as WT transposase (~1%) and we found no evidence that the expression of the transposase affects genomic integrity. This hyperactive piggyBac transposase expands the utility of the piggyBac transposon for applications in mammalian genetics and gene therapy.
| S-EPMC3029773 | biostudies-literature
Project description:Bacterial transposons are pervasive mobile genetic elements that exploit distinct DNA binding proteins for their horizontal spread. For example, E. coli Tn7 homes to a specific attachment site using a TniQ family protein, whereas diverse Tn7-like transposons employ Type I or Type V CRISPR-Cas systems to insert downstream of target sites specified by a guide RNA. Despite this targeting pathway diversity, transposition invariably requires TnsB, a DDE superfamily transposase that catalyses DNA excision and insertion, and TnsC, a AAA+ ATPase that is thought to communicate between the transposase and targeting proteins. How TnsC mediates this communication and thereby regulates transposition fidelity has remained elusive. Here we apply chromatin immunoprecipitation sequencing (ChIP-seq) to monitor in vivo formation of the Vibrio cholerae RNA-guided transpososome, allowing us to unambiguously resolve distinct protein recruitment events prior to integration. DNA targeting by the TniQ-Cascade complex is surprisingly promiscuous, leading to binding at hundreds of genomic off-target sites, but only a subset of those sites are licensed for TnsC and TnsB recruitment, revealing a crucial proofreading checkpoint that controls transposition fidelity. To advance the mechanistic understanding of interactions responsible for transpososome assembly, we analysed V. cholerae TnsC by cryo-EM and found that TnsC forms ATP-dependent heptameric rings, which are likely to play a critical architectural role in positioning DNA substrates for downstream integration. Collectively, our results highlight the molecular specificity imparted by consecutive binding of distinct factors to genomic target sites during RNA-guided transposition, and provide a structural roadmap to guide future engineering efforts.
2021-10-31 | GSE183114 | GEO