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The functional relevance of the inverted repeat structure (IR/DR) in a subgroup of the Tc1/mariner superfamily of transposons has been enigmatic. In contrast to mariner transposition, where a topological filter suppresses single-ended reactions, the IR/DR orchestrates a regulatory mechanism to enforce synapsis of the transposon ends before cleavage by the transposase occurs. This ordered assembly process shepherds primary transposase binding to the inner 12DRs (where cleavage does not occur), followed by capture of the 12DR of the other transposon end. This extra layer of regulation suppresses aberrant, potentially genotoxic recombination activities, and the mobilization of internally deleted copies in the IR/DR subgroup, including Sleeping Beauty (SB). In contrast, internally deleted sequences (MITEs) are preferred substrates of mariner transposition, and this process is associated with the emergence of Hsmar1-derived miRNA genes in the human genome. Translating IR/DR regulation to in vitro evolution yielded an SB transposon version with optimized substrate recognition (pT4). The ends of SB transposons excised by a K248A excision+/integration- transposase variant are processed by hairpin resolution, representing a link between phylogenetically, and mechanistically different recombination reactions, such as V(D)J recombination and transposition. Such variants generated by random mutation might stabilize transposon-host interactions or prepare the transposon for a horizontal transfer.

Although nonviral gene therapy has great potential for use in the lung, the relative lack of cell-specific targeting has limited its applications. We have developed a new approach for cell-specific targeting based on selective nuclear import of plasmids in non-dividing cells. Using a microinjection and in situ hybridization approach, we tested several potential DNA sequences for the ability to mediate plasmid nuclear import in alveolar type II epithelial (ATII) cells. Of these, only a sequence within the human surfactant protein C (SP-C) promoter was able to mediate nuclear localization of plasmid DNA specifically in ATII cells but not in other cell types. We have mapped the minimal import sequence to the proximal 318 nucleotides of the promoter, and demonstrate that binding sites for NFI, TTF-1, and GATA-6 and the proteins themselves are required for import activity. Using intratracheal delivery of DNA followed by electroporation, we demonstrate that the SP-C promoter sequence will enhance gene expression specifically in ATII cells in mouse lung. This represents a novel activity for the SP-C promoter and thus ATII cell-specific nuclear import of DNA may prove to be a safe and effective method for targeted and enhanced gene expression in ATII cells.

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...