HMGXB4 Targets Sleeping Beauty Transposition to Germinal Stem Cells.

Transposons are parasitic genetic elements that frequently hijack vital cellular processes of their host. HMGXB4 is a known Wnt signaling-regulating HMG-box protein, previously identified as a host-encoded factor of Sleeping Beauty (SB) transposition. Here, we show that HMGXB4 is predominantly maternally expressed, and marks both germinal progenitor and somatic stem cells. SB piggybacks HMGXB4 to activate transposase expression and target transposition to germinal stem cells, thereby potentiating heritable transposon insertions. The HMGXB4 promoter is located within an active chromatin domain, offering multiple looping possibilities with neighboring genomic regions. HMGXB4 is activated by ERK2/MAPK1, ELK1 transcription factors, coordinating pluripotency and self-renewal pathways, but suppressed by the KRAB-ZNF/TRIM28 epigenetic repression machinery, also known to regulate transposable elements. At the post-translational level, SUMOylation regulates HMGXB4, which modulates binding affinity to its protein interaction partners and controls its transcriptional activator function via nucleolar compartmentalization. When expressed, HMGXB4 can participate in nuclear-remodeling protein complexes and transactivate target gene expression in vertebrates. Our study highlights HMGXB4 as an evolutionarily conserved host-encoded factor that assists Tc1/Mariner transposons to target the germline, which was necessary for their fixation and may explain their abundance in vertebrate genomes.

Transcriptionally promiscuous "blurry" promoters in Tc1/mariner transposons allow transcription in distantly related genomes.

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.

Transcriptional activities of the Sleeping Beauty transposon and shielding its genetic cargo with insulators.

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.

Interference with cell cycle progression by parasitic genetic elements: sleeping beauty joins the club.

Transposable elements are discrete segments of DNA that have the distinctive ability to move and replicate within genomes. Similar to viruses, transposons are best viewed as molecular parasites that propagate themselves using resources of the host cell. Many viruses have developed strategies to modulate the host cell cycle machinery and cellular self-destruct mechanisms to maximize the chance for successful infection and the production of virus progeny. Recent evidence shows that transposable elements have also evolved mechanisms to modulate cell cycle progression for their own benefit. Thus, interference with the cell cycle seems to be a shared strategy of parasitic selfish genetic elements.

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

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 D1/cdk4-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.

Development of hyperactive sleeping beauty transposon vectors by mutational analysis.

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 transposable element: evolution, regulation and genetic applications.

Members of the Tc1/mariner superfamily of transposable elements isolated from vertebrate species are inactive due to the accumulation of mutations. A representative of a subfamily of fish elements estimated to be last active > 10 million years ago has been reconstructed, and named Sleeping Beauty(SB). This element opened up new avenues for studies on DNA transposition in vertebrates, and for the development of transposon tools for genetic manipulation in important model species and in humans. Multiple transposase binding sites within the terminal inverted repeats, a transpositional enhancer sequence, unequal affinity of the transposase to the binding sites and the activity of the cellular HMGB1 protein all contribute to a highly regulated assembly of SB synaptic complexes, which is likely a requirement for the subsequent catalytic steps. Host proteins involved in double-strand DNA break repair are limiting factors of SB transposition in mammalian cells, underscoring evolutionary, structural and functional links between DNA transposition, retroviral integration and V(D)J recombination. SB catalyzes efficient cut-and-paste transposition in a wide range of vertebrate cells in tissue culture, and in somatic tissues as well as the germline of the mouse and zebrafish in vivo, indicating its usefulness as a vector for transgenesis and insertional mutagenesis.