Research

Transposable elements are “jumping genes” with an ability to change their genomic positions (Fig. 1). Transposons make up significant fractions of genomes; for example, about 45% of the human genome is derived from transposon DNA. Transposons are best viewed as molecular parasites that propagate themselves using resources of the host cell. Despite their parasitic nature, there is increasing evidence that transposable elements are a powerful force in genome evolution. Transposons are natural gene delivery vehicles that are being developed as genetic tools. We follow two major lines of research: 1) molecular biology and cellular regulation of DNA transposition in vertebrate cells using the Sleeping Beauty (SB) element as a research tool, and 2) development of transposons as gene vectors for insertional mutagenesis in vertebrate models and for human gene therapy.

 

 

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Figure 1. The Sleeping Beauty transposon system. (a) Components and structure of a two-component gene transfer system based on Sleeping Beauty. A gene of interest (orange box) to be mobilized is cloned between the terminal inverted repeats (IR/DR, black arrows) that contain binding sites for the transposase (white arrows). The transposase gene (purple box) is physically separated from the IR/DRs, and is expressed in cells from a suitable promoter (black arrow). The transposase consists of an N-terminal DNA-binding domain, a nuclear localization signal (NLS) and a catalytic domain characterized by the DDE signature. (b) Mechanism of Sleeping Beauty transposition. The transposable element carrying a gene of interest (GOI, orange box) is maintained and delivered as part of a DNA vector (blue DNA). The transposase (purple circle) binds to its sites within the transposon inverted repeats (black arrows). Excision takes place in a synaptic complex. Excision separates the transposon from the donor DNA, and the double-strand DNA breaks that are generated during this process are repaired by host factors. The excised element integrates into a TA site in the target DNA (green DNA) that will be duplicated and will be flanking the newly integrated transposon.

 

The ancient mariner sails again

Csaba Miskey

The human Hsmar1 elements are inactive due to mutational damage, but one particular copy of the transposase gene has been under selection. This transposase coding region is part of the SETMAR gene, in which a histone methylatransferase SET domain is fused to an Hsmar1 transposase domain. We took a phylogenetic approach to reconstruct the ancestral Hsmar1 transposon that efficiently mobilizes by a cut-and-paste mechanism in human cells and zebrafish embryos. The SETMAR protein binds, and introduces single-strand nicks into Hsmar1 inverted repeat sequences. Pathway choice for DNA break repair is different in response to transposon cleavage mediated by the Hsmar1 transposase and SETMAR in vivo. The novel transposon system can be a useful tool for investigations into the transpositional dynamics and contribution of these elements to primate genome evolution.

 

 

Domesticated, transposon-derived cellular genes

Ludivine Sinzelle

We reconstructed the functional components of a Harbinger element in zebrafish, including a transposase and a second protein of unknown function that has a Myb-like trihelix domain. The reconstructed transposon preferentially inserts into a 15-bp consensus target sequence in human cells. The Myb-like protein is required for transposition, interacts with the transposase, and enables transposition in part by promoting nuclear import of the transposase and by binding to the transposon ends. We investigated the functions of two, transposon-derived human proteins: HARBI1, a domesticated transposase-derived protein and NAIF1 that contains a trihelix motif similar to that described in the Myb-like protein. Physical interaction, subcellular localization and DNA-binding activities of HARBI1 and NAIF1 suggest strong functional homologies between the Harbinger system and their related, host-encoded counterparts.

 

 

RNA interference and epigenetic regulation of transposition

Tobias Jursch, Andrea Schorn

We are looking at the possible involvement of RNA interference in transposon silencing in vertebrates, and at the effect of chromatin structure of both donor and target sites on transposition. RNA interference is involved in transposon regulation in C. elegans and Drosophila, and it has been implicated to play similar roles in vertebrates. Andrea is investigating transposon regulation by RNA interference in zebrafish. Transposition of SB is enhanced by CpG methylation of transposon donor DNA, and Tobias is currently testing models for the enhancing effect of DNA methylation.

 

 

Loss-of-function insertional mutagenesis

Ivana Grabundzija

Transposons can be applied as useful research tools for gene discovery, thereby contributing to our understanding of gene function in vertebrates. We are taking advantage of local hopping for regional saturation mutagenesis in mice, where the primary transposon donor locus can be determined by targeting the transposon to a chromosomal region of interest. We began to work on regional transposon mutagenesis of the Williams-Beuren syndrome locus (in collaboration with Thomas Floss, GSF), with the goal to uncover the genetic basis of this disease.

 

 

Transposons as non-viral vectors for gene therapeutic approaches

Ismahen Ammar, Csaba Miskey, Katrin Voigt

DNA-based transposons are natural gene delivery vehicles, and molecular reconstruction of SB represents a cornerstone in applying transposition-mediated gene delivery in vertebrate species, including humans. We coordinate a research project within the framework of EU FP6 with the goal of developing novel, non-viral gene delivery technologies for ex vivo gene-based therapies.

SB transposition occurs into chromosomes in a random manner, which is clearly undesired for human applications due to potential genotoxic effects associated with transposon integration. We succeeded in targeting SB transposition into predetermined chromosomal loci. We employed modular targeting fusion proteins (Fig. 2), in which the module responsible for target binding can be a natural DNA-binding protein or domain, or an artificial protein such as a designer zinc finger. Targeted transposition could be a powerful method for safe transgene integration in human applications. 
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Figure 2. Experimental strategies for targeting Sleeping Beauty transposition. The common components of the targeting systems include a transposable element that contains the IRs (arrowheads) and a gene of interest equipped with a suitable promoter. The transposase (purple circle) binds to the IRs and catalyzes transposition. A DNA-binding protein domain (red oval) recognizes a specific sequence (turquoise box) in the target DNA (parallel lines). (a) Targeting with transposase fusion proteins. Targeting is achieved by fusing a specific DNA-binding protein domain to the transposase. (b) Targeting with fusion proteins that bind the transposon DNA. Targeting is achieved by fusing a specific DNA-binding protein domain to another protein (white oval) that binds to a specific DNA sequence within the transposable element (yellow box). In this strategy, the transposase is not modified. (c) Targeting with fusion proteins that interact with the transposase. Targeting is achieved by fusing a specific DNA-binding protein domain to another protein (light green oval) that interacts with the transposase. In this strategy, neither the transposase nor the transposon is modified.

 

 

Checkpoint controls in Sleeping Beauty element (SB)  transposition

Diana Pryputniewicz

Our understanding the way of how eukaryotic recombinases are working is still mostly based on assuming analogies to bacterial transposons. Besides the basic chemical reaction, the different elements have a variety of “built-in” regulatory mechanisms, often involving host factors, to provide specificity to the transposition reaction. The main function of a regulation is to impose “quality control” on transposition in the form of regulatory checkpoints, at which certain molecular requirements have to be fulfilled for the transpositional reaction to proceed. The role of these regulatory checkpoints is to avoid accumulation of incorrect reaction products in genomes, possessing a threat of genome instability associated by transposition. Our ultimate goal is to reconstruct the entire transposition process of the SB in vitro, and to decipher the checkpoint controls of the reaction. A fascinating question is the differential regulation of transposition and the transposition derived V(D)J recombination.

 

 

Recombination and DNA repair

Yongming Wang

Cellular mechanisms that are directly involved in repairing transposition-inflicted DNA lesions or can attenuate DNA damage should have crucial role in establishing stable host-transposon co-existence. Our results suggest that DNA damage repair of lesions generated by transposition are differently regulated from any other repair process.  This differential regulation manifests in actively influencing the accessibility of host repair factors to the DNA lesions generated by transposition. SB transposon takes advantage of the cellular repair machinery and/or during DNA replication to amplify their own genome. This process is active in germinal, but strongly inhibited in somatic cells.

 

 

Transposition and stress/developmental signaling

Dawid Grzela, Anantharam Deveraj

Transposons occupy a significant portion of our genomes. However, the vast majority of transposons remain silent due to accumulated mutations in their genomes. The transposition of the few, active copies is strongly regulated, but this control is sensitive to environmental stress. Our preliminary results show that transposons might exist in a “latent” form in the genome and are able to sense developmental and environmental changes and manipulate stress signaling.

 

 

Finding the active copy of RAT-IAP endogenous retrovirus

Yongming Wang

The endogenous retrovirus, Rat-IAP was repeatedly demonstrated to influence the expression of rat genes, without knowing the active copy. A phenotype of hypodactyly in rat was associated with a recent retrotransposition event. We have identified an “active” copy of an IAP-type endogenous retroelement in the rat genome, and showed that the element is active in retrotransposition. The transpositionally active copy has an intact env gene, so element might be capable of infection.

 

 

Isolating hyperactive transposase versions by directed evolution.

Lajos Mátés

 It is widely believed that naturally occurring transposons have not been selected for the highest possible activity, and are strongly downregulated to avoid insertional inactivation of essential genes. Using DNA shuffling technology combined with molecular evolutionary approaches, we were able to find aspecial combination of synergistic mutations that resulted in a significant, ~100-fold increase in transposase activity in SB. The 100-fold hyperactive SB system approaches integration rates of viral vectors opening new avenues for gene therapeutic approaches (INTHER-FP6 coordination) as well as for genome manipulation techniques in vivo.

 

 

Transposon mutagenesis in rat spermatogonial stem cells

Lajos Mátés, Janine Fröhlich

Transposons can be harnessed as vehicles for introducing genetic mutations into genomes. The genes inactivated by transposon insertion are "tagged" by the transposable element, which can be used for subsequent cloning of the mutated allele. The SB system is active in all vertebrates, including rats. While embryonic stem cell technology is not established in the rat, the technology of maintaining and expanding spermatogonial stem cells became available. Thus, we have extended the utilization of the SB tranposon to rats, with the goal of knocking out genes implicated in disease development by transposon mutagenesis in vivo. The project has enormous potential to develop powerful genomic tools for rat that is the preferred model organism of cardiovascular, behavioral studies.

Rat_Spermatogonial400

 

Deciphering the genetic background of hormone induced breast cancer

Andrea Schmitt

The SB transposon is suitable for somatic mutagenesis and emerged as a new tool in cancer research an alternative to retroviral mutagenesis. Transposon based insertional mutagenesis screen is able to identifiy both oncogenes and tumor-suppressor genes that normally protect against cancer. My laboratory is engaged in a project using a rat model to study the genetics of the estrogen-induced mammary cancer. Unlike the situation in mouse, the development of mammary cancer is similar to human as it is also estrogen-dependent. The susceptibility to estrogen-induced mammary cancer behaves as a complex trait controlled by a QTL and multiple gene-gene interactions. The transposon mutagenesis approach is expected to be a powerful tool to decipher the regulatory network.