The Sleeping Beauty (SB) transposable element is a promising vector for transgenesis in vertebrates and is being developed as a novel, nonviral system for gene therapeutic purposes. A mutagenesis approach was undertaken to improve various aspects of the transposon, including safety and overall efficiency of gene transfer in human cells. Deletional analysis of transposon sequences within first-generation SB vectors showed that the inverted repeats of the element are necessary and sufficient to mediate high-efficiency transposition. We constructed a "sandwich" transposon, in which the DNA to be mobilized is flanked by two complete SB elements arranged in an inverted orientation. The sandwich element has superior ability to transpose >10-kb transgenes, thereby extending the cloning capacity of SB-based vectors. We derived hyperactive versions of the SB transposase by single-amino-acid substitutions. These mutations act synergistically and result in an almost fourfold enhancement of activity compared to the wild-type transposase. When combined with hyperactive transposons and transiently overexpressed HMGB1, a cellular cofactor of SB transposition, hyperactive transposases elevate transposition by almost an order of magnitude compared to the first-generation transposon system. The improved vector system should prove useful for efficient gene transfer in vertebrates.

The Sleeping Beauty (SB) transposable element shows efficient transposition in human cells, and provides long-term transgene expression in preclinical animal models. Random chromosomal insertion of SB vectors represents a safety issue in human gene therapeutic applications, due to potential genotoxic effects associated with transposon integration. We investigated the transcriptional activities of SB in order to assess its potential to alter host gene expression upon integration. The untranslated regions (UTRs) of the transposon direct convergent, inward-directed transcription. Transcription from the 5'-UTR of SB is upregulated by the host-encoded factor high-mobility group 2-like 1 (HMG2L1), and requires a 65-base pair (bp) region not present in commonly used SB vectors. The SB transposase antagonizes the effect of HMG2L1, suggesting that natural transposase expression is under a negative feedback regulation. SB transposon vectors lacking the 65-bp region associated with HMG2L1-dependent upregulation exhibit benign transcriptional activities, at a level up to 100-times lower than that of the murine leukemia virus (MLV) long terminal repeat (LTR). Incorporation of chicken beta-globin HS4 insulator sequences in SB-based vectors reduces the transactivation of model promoters by transposon-borne enhancers, and thus may lower the risk of transcriptional activation of host genes situated close to a transposon insertion site.

We used the Sleeping Beauty (SB) transposable element as a tool to probe transposon-host cell interactions in vertebrates. The Miz-1 transcription factor was identified as an interactor of the SB transposase in a yeast two-hybrid screen. Through its association with Miz-1, the SB transposase down-regulates cyclin D1 expression in human cells, as evidenced by differential gene expression analysis using microarray hybridization. Down-regulation of cyclin D1 results in a prolonged G 1 phase of the cell cycle and retarded growth of transposase-expressing cells. G 1 slowdown is associated with a decrease of cyclin D1͞cdk4-specific phosphorylation of the retinoblastoma protein. Both cyclin D1 down-regulation and the G 1 slowdown induced by the transposase require Miz-1. A temporary G 1 arrest enhances transposition, suggesting that SB transposition is favored in the G 1 phase of the cell cycle, where the nonhomologous end-joining pathway of DNA repair is preferentially active. Because nonhomologous end-joining is required for efficient SB transposition, the transposase-induced G 1 slowdown is probably a selfish act on the transposon's part to maximize the chance for a successful transposition event.cyclin D1 ͉ protein-protein interaction ͉ transposition M obility of transposable elements is regulated by both hostand element-encoded factors, which operate by imposing constraints on transposition. Members of the Tc1͞mariner superfamily of elements are probably the most widespread DNA transposons in nature, including vertebrates (1). By far the most active Tc1͞mariner element in vertebrates is the Sleeping Beauty (SB) transposon, a reconstructed version of an ancient and extinct element in teleost fish (2). SB transposition is efficient in cells of different vertebrate classes in tissue culture (3) and in somatic (4) as well as germline tissues (5) of the mouse in vivo.SB transposition is initiated by binding of the transposase to binding sites located within the terminal inverted repeats of the element. The two ends of the element are then probably paired through interactions of transposase subunits (6), thereby forming a synaptic complex, in which excision of the transposon from the donor site likely occurs. The reaction is completed after reintegration of the element into a target site, followed by repair of transposon-induced DNA lesions by the host repair machinery.Interactions of components of the transposable element with host factors likely play important roles in the transposition process, at any of the steps described above. Indeed, we previously identified the HMGB1 protein as a cofactor of SB transposition in mammalian cells (7). HMGB1 interacts with the SB transposase in vivo and is probably involved in synaptic complex formation during transposition (7). SB transposase also interacts with the Ku protein, a component of the nonhomologous end-joining (NHEJ) pathway of double-strand DNA break repair (8), which is a limiting factor of SB transposition (8).Transposon excision sites are preferentially repaired by...

Background We have recently described a peculiar feature of the promoters in two Drosophila Tc1 -like elements, Bari1 and Bari3 . The AT-richness and the presence of weak core-promoter motifs make these promoters, that we have defined “blurry”, able to activate transcription of a reporter gene in cellular systems as diverse as fly, human, yeast and bacteria. In order to clarify whether the blurry promoter is a specific feature of the Bari transposon family, we have extended this study to promoters isolated from three additional DNA transposon and from two additional LTR retrotransposons. Results Here we show that the blurry promoter is also a feature of two vertebrate transposable elements, Sleeping Beauty and Hsmar1 , belonging to the Tc1/mariner superfamily. In contrast, this feature is not shared by the promoter of the hobo transposon, which belongs to the hAT superfamily, nor by LTR retrotransposon-derived promoters, which, in general, do not activate transcription when introduced into non-related genomes. Conclusions Our results suggest that the blurry promoter could be a shared feature of the members of the Tc1/mariner superfamily with possible evolutionary and biotechnological implications. Electronic supplementary material The online version of this article (10.1186/s13100-019-0155-6) contains supplementary material, which is available to authorized users.

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