Postintegrative Gene Silencing within the Sleeping Beauty Transposition System

Abstract

The Sleeping Beauty (SB) transposon represents an important vehicle for in vivo gene delivery because it can efficiently and stably integrate into mammalian genomes. In this report, we examined transposon expression in human cells using a novel nonselective fluorescence-activated cell sorter-based method and discovered that SB integrates ϳ20 times more frequently than previously reported within systems that were dependent on transgene expression and likely subject to postintegrative gene silencing. Over time, phenotypic analysis of clonal integrants demonstrated that SB undergoes additional postintegrative gene silencing, which varied based on the promoter used for transgene expression. Molecular and biochemical studies suggested that transposon silencing was influenced by DNA methylation and histone deacetylation because both 5-aza-2-deoxycytidine and trichostatin A partially rescued transgene silencing in clonal cell lines. Collectively, these data reveal the existence of a multicomponent postintegrative gene silencing network that efficiently targets invading transposon sequences for transcriptional silencing in mammalian cells.Transposable elements play an important role in evolution which is demonstrated by an estimated 50 genes within the human genome that are transposon derived, including genes vital to our adaptive immunity (16) and DNA repair (33). Amazingly, sequences recognized as derived from transposable elements account for approximately 45% of the overall human genome (31). Transposable elements can be broken down into two main groups: those which rely on an RNA intermediate for remobilization (retro-elements) and those which are remobilized directly as DNA (DNA transposons). Fossils of one group of DNA transposons, the Tc1/mariner type, are found throughout nature. Remnants, which have been deactivated through the accumulation of mutations over time (21,22), have been isolated from a variety of organisms, including fish (22), Xenopus laevis (30, 51), insects (49), Caenorhabditis elegans (48), Drosophila melanogaster (47), and humans (43, 52). Recognizing the many potential uses of an active and efficient DNA transposon system, Ivics et al. reconstructed a functional Tc1/ mariner-like element from inactive transposon remnants found in fish and named it Sleeping Beauty (SB) (21). Shortly following its description, our group established the possible utility of this system as a means to genetically modify somatic tissues in adult mammals to treat animal models of human disease (40,57,59).DNA transposons of the Tc1/mariner type contain a simple structure in which the only components required for transposition are inverted repeats (IRs), which flank the DNA to be transposed, and Sleeping Beauty transposase. As with other DNA transposons, SB transposition occurs through a "cut and paste" mechanism mediated by binding to the IRs of Sleeping Beauty transposase, which can be supplied either in cis from an autonomous element or in trans from a nonautonomous element. The excision and integration steps are me...

Nonviral Gene Delivery with the Sleeping Beauty Transposon System

“…An additional factor that may provoke transgene silencing is the cargo DNA, particularly the type of promoter used to drive expression of the gene of interest. Indeed, it was previously shown that transgene constructs delivered into mouse cells by SB transposition can be subject to epigenetic regulation by CpG methylation and that a determinant of epigenetic modifications of the integrating transposon vector is the cargo transgene construct, with the promoter playing a major role (Garrison et al, 2007). However, with careful promoter choice, several studies have established that SB-mediated transposition provides long-term expression in vivo, as discussed previously.…”

Gene duplication in the epigenomic era: Roles of chromatin modifications

“…17,21 The 18 new data sets were obtained with antibodies against H2AK5ac, H2AK9ac, H2BK120ac, H2BK12ac, H2BK20ac, H2BK5ac, H3K14ac, H3K18ac, H3K23ac, H3K27ac, H3K36ac, H3K4ac, H3K9ac, H4K12ac, H4K16ac, H4K5ac, H4K8ac and H4K91ac. Characterizations of the distributions of these histone Gene duplication and chromatin modification DNA methylation has been suggested to implicate in the silencing of the Sleeping Beauty transposon, 25,26 and H3K9 methylations also seem to be important for transposon silencing. 24 It is possible that similar mechanisms or biological processes exist for selectively repressing newly duplicated (or retrotransposed) sequences.…”

Use of Transposon-Transposase Systems for Stable Genetic Modification of Embryonic Stem Cells

“…Embryonic stem cells, for instance, have clear molecular defense mechanisms against viral promoter sequences (Meilinger et al, 2009;Rowe et al, 2010). When addressing this question for the SB transposon, it was revealed that the effect of silencing depends rather on the cargo sequence and not on the vector itself (Garrison et al, 2007;Zhu et al, 2010). All these encouraging characteristics further supported the use of SB as a tool for gene therapy and provided the basis for the first clinical trial initiated by a non-viral vector: SB is used in the treatment of a B-lymphoid malignancy by ex vivo genetically modified autologous T-cells (Williams, 2008).…”