Sleeping Beauty transposase modulates cell-cycle progression through interaction with Miz-1

Abstract

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...

A flk‐1 promoter/enhancer reporter transgenic Xenopus laevis generated using the Sleeping Beauty transposon system: An in vivo model for vascular studies

“…The chromatin binding protein HMGB1 was identified as a host cofactor for the SB transposase and was shown to increase transposition in mammalian cell lines (Zayed et al, 2003). In addition, the transcription factor Miz-1 was shown to interact with the SB transposase (Walisko et al, 2006). These, and potentially other host factors, may influence the efficiency of SB transposition and should be considered when using SB in different cells and organisms.…”

Interference with Cell-Cycle Progression by Parasitic Genetic Elements: Sleeping Beauty Joins the Club

“…2). We have shown that downregulation of cyclin D1 by the SB transposase is associated with a decrease of cyclin D1/cdk4-specific phosphorylation of Rb, thereby potentiating a delay in the G 1 /S transition 25 (Fig. 2).…”

“…In a combined approach of genome-wide expression analysis by gene chip hybridization and yeast two-hybrid cloning, we found that the SB transposase downregulates expression of the cyclin D1 gene through physical interaction with the host-encoded Miz-1 protein 25 ( Fig. 2).…”

“…Miz-1 is a zinc finger transcription factor of cell cycle regulatory genes such as cyclin D1 and p15 INK4B . 26,27 In contrast to Myc, that antagonizes transactivation of these promoters by Miz-1, 26,27 the SB transposase only affects cyclin D1 expression 25 ( Fig. 2).…”

Excision Efficiency Is Not Strongly Coupled to Transgenic Rate: Cell Type-Dependent Transposition Efficiency ofSleeping BeautyandpiggyBacDNA Transposons

“…There are a number of proteins described to interact with the SB transposase, most of which are involved in DNA repair processes or in cell cycle regulation (Zayed et al, 2003;Izsvák et al, 2004;Walisko et al, 2006). As these proteins could differ in their cell type-specific distribution and activity, we measured the mRNA expression levels of four SBinteracting factors (DNAPKcs, HMGB1, Ku70, Ku80) in the HeLa and HEK-293 cell lines.…”