Factors acting on Mos1 transposition efficiency

<p>Abstract</p> <p>Background</p> <p><it>Mariner-</it>like elements (<it>MLEs</it>) are widespread DNA transposons in animal genomes. Although <it>in vitro </it>transposition reactions require only the transposase, various factors depending on the host, the physico-chemical environment and the transposon sequence can interfere with the <it>MLEs </it>transposition <it>in vivo</it>.</p> <p>Results</p> <p>The transposition of <it>Mos1</it>, first isolated from <it>drosophila mauritiana</it>, depends of both the nucleic acid sequence of the DNA stuffer (in terms of GC content), and its length. We provide the first <it>in vitro </it>experimental demonstration that MITEs of <it>MLE </it>origin, as small as 80 to 120-bp, are able to transpose. Excessive temperature down-regulates <it>Mos1 </it>transposition, yielding excision products unable to re-integrate. Finally, the super-helicity of the DNA transposon donor has a dramatic impact on the transposition efficiency.</p> <p>Conclusion</p> <p>The study highlights how experimental conditions can bias interpretation of <it>mariner </it>excision frequency and quality. <it>In vitro</it>, the auto-integration pathway markedly limits transposition efficiency to new target sites, and this phenomenon may also limit events in the natural host. We propose a model for small transposons transposition that bypasses DNA bending constraints.</p

Nuclear Importation of Mariner Transposases among Eukaryotes: Motif Requirements and Homo-Protein Interactions

Mariner-like elements (MLEs) are widespread transposable elements in animal genomes. They have been divided into at least five sub-families with differing host ranges. We investigated whether the ability of transposases encoded by Mos1, Himar1 and Mcmar1 to be actively imported into nuclei varies between host belonging to different eukaryotic taxa. Our findings demonstrate that nuclear importation could restrict the host range of some MLEs in certain eukaryotic lineages, depending on their expression level. We then focused on the nuclear localization signal (NLS) in these proteins, and showed that the first 175 N-terminal residues in the three transposases were required for nuclear importation. We found that two components are involved in the nuclear importation of the Mos1 transposase: an SV40 NLS-like motif (position: aa 168 to 174), and a dimerization sub-domain located within the first 80 residues. Sequence analyses revealed that the dimerization moiety is conserved among MLE transposases, but the Himar1 and Mcmar1 transposases do not contain any conserved NLS motif. This suggests that other NLS-like motifs must intervene in these proteins. Finally, we showed that the over-expression of the Mos1 transposase prevents its nuclear importation in HeLa cells, due to the assembly of transposase aggregates in the cytoplasm

Caractérisation des transposons à ADN Tc1-mariner chez le xénope et utilisation du transposon Sleeping Beauty comme vecteur de transgenèse germinale

LES ELEMENTS DE TYPE TC1 (TLES) ET LES ÉLÉMENTS DE TYPE MARINER APPARTIENNENT A LA SUPERFAMILLE DES TRANSPOSONS À ADN TC1-MARINER QUI SONT TRÈS LARGEMENT RÉPANDUS DANS LE RÈGNE VIVANT. GRÂCE AUX DONNÉES DE RESSOURCES GÉNOMIQUES COMBINÉES À UNE STRATÉGIE DE PCR DÉGÉNÉRÉE, NOUS AVONS IDENTIFIÉ SIX LIGNÉES DE TLES PRÉSENTS DANS LE GÉNOME DE L'AMPHIBIEN XENOPUS TROPICALIS. LA PLUPART DE CES LIGNÉES APPELÉES EAGLE, FROGGY, XEMINOS, XTTXR ET JUMPY PRÉSENTE DES CARACTÉRISTIQUES TYPIQUES DES ÉLÉMENTS TLES: UN CADRE DE LECTURE CODANT UNE TRANSPOSASE DE 340-350 ACIDES AMINÉS, ENCADRÉ PAR DES SÉQUENCES RÉPÉTÉES INVERSÉES. POUR CHACUNE DE CES LIGNÉES, NOUS AVONS IDENTIFIÉ DES COPIES CONTENANT UN CADRE DE LECTURE DE LA TRANSPOSASE INTACT SUGGÉRANT QUE CES ÉLÉMENTS POURRAIENT ÊTRE ENCORE ACTIFS. L'ANALYSE PHYLOGÉNÉTIQUE MONTRE QUE CES TLES SONT RELIÉS À DES TLES D'ACTINOPTERIGIENS ET D'AMPHIBIENS DANS UN DEUXIÈME TEMPS, NOUS AVONS IDENTIFIÉ LE PREMIER MLE, XTMAR1, ISOLÉ D'UN GÉNOME DE VERTÉBRÉ À SANG-FROID XENOPUS TROPICALIS. L'ANALYSE PHYLOGÉNÉTIQUE INCLUANT DES REPRÉSENTANTS DES DIFFÉRENTES SOUS-FAMILLES DE MLES RÉVÈLE QUE XTMAR1 EST RELIÉ AU MLE HUMAIN HSMAR2, DANS UNE DEUXIÈME LIGNÉE DE LA SOUS-FAMILLE IRRITANS. EN DERNIÈRE PARTIE, NOUS AVONS UTILISÉ LE TARNSPOSON TC1-MARINER SLEEPING BEAUTY (SB) COMME VECTEUR DE TRANSGENÈSE GERMINALE CHEZ LE XÉNOPE. CETTE TECHNIQUE REPOSE SUR LA MICROINJECTION. DANS UN EMBRYON DE XÉNOPE AU STADE UNE CELLULE, DE LA TRANSPOSASE SB ET D'UN PSEUDO-TRANSPOSON CONTENANT LE MARQUEUR FLUORESCENT GFP. DANS DES CONDITIONS OPTIMISÉES, NOUS OBTENONS UN NOMBRE IMPORTANT D'INDIVIDUS DEMI-TRANSGÉNIQUES.MARINER-LIKE ELEMENTS (MLES) AND TC1-LIKE ELEMENTS (TLES) BELONG TO THE TC1-MARINER SUPERFAMILY OF DNA TRANSPOSONS WHICH IS VERY WIDESPREAD IN ANIMALS GENOMES WE HAVE USED GENOMIC SEQUENCING DATA FROM THE FIRST ASSEMBLY OF THE AMPHIBIAN XENOPUS TROPICALIS GENOME TO IDENTIFY MULTIPLE LINEAGES OF TLES FULL-LENGTH ELEMENTS WERE ISOLATED IN EACH LINEAGE AND WERE CHARACTERIZED. MOST OF THEM EXHIBIT THE TYPICAL CHARACTERITICS OF TC1-LIKE ELEMENT THESE NEW TLES WERE NAMED EAGLE, FROGGY, JUMPY, MAYA, XEMINOS, XTTXR. PHYLOGENETICQUE STUDY INDICATE THAT THEIR CLOSEST RELATIVES ARE PRESENT IN THE GENOMES OF ACTINOPTERYGIAN AND AMPHIBIAN. WE HAVE IDENTIFIED FOR MOST OF THESE TLES, COPIES CONTAINING AN INTACT TRANSPOSASE OPEN READING FRAME SUGGESTING THAT THESE ELEMENTS MAY BE STILL ACTIVE. IN ADDITION, WE HAVE IDENTIFIED THE FIRST COMPLETE MARINER-LIKE ELEMENT, XTMAR1, WITHIN THE GENOME OF A POIKILOTHERM VERTEBRATE, XENOPUS TROPICALIS. THE PHYLOGENETIC ANALYSIS OF THE RELATIONSHIPS BETWEEN MLE TRANSPOSASES REVEALS THAT XTMAR1 IS CLOSELY RELATED TO THE HUMAN MARINER HSMAR2 AND THAT TOGTHER THEY FORM A SECOND DISTINCT LINEAGE OF THE IRRITANS SUBFAMILY. FINALLY, WE TESTED THE SLEEPING BEAUTY TRANSPOSON SYSTEM FOR ITS SUITABILITY AS A GENETIC VECTOR FOR THE GENERATION OF TRANSGENIC XENOPUS LAEVIS WE CONDUCTED A TRANSGENESIS STUDY BASED ON THE CO-INJECTION INTO ONE-CELL STAGE EMBRYOS OF THE SB TRANSPOSASE RNA AND A GFP-REPORTER PSEUDO-TRANSPOSON. THE OPTIMAL CONDITIONS LEAD US TO THE OBTENTION OF HALF TRANSGENIC ANIMALS AND A NUMBER OF SOMATIC CELL CLONES.ORSAY-PARIS 11-BU Sciences (914712101) / SudocSudocFranceF

The Ancient mariner Sails Again: Transposition of the Human Hsmar1 Element by a Reconstructed Transposase and Activities of the SETMAR Protein on Transposon Ends▿ †

Hsmar1, one of the two subfamilies of mariner transposons in humans, is an ancient element that entered the primate genome lineage ∼50 million years ago. Although Hsmar1 elements are inactive due to mutational damage, one particular copy of the transposase gene has apparently 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. A phylogenetic approach was taken to reconstruct the ancestral Hsmar1 transposase gene, which we named Hsmar1-Ra. The Hsmar1-Ra transposase efficiently mobilizes Hsmar1 transposons by a cut-and-paste mechanism in human cells and zebra fish embryos. Hsmar1-Ra can also mobilize short inverted-repeat transposable elements (MITEs) related to Hsmar1 (MiHsmar1), thereby establishing a functional relationship between an Hsmar1 transposase source and these MITEs. MiHsmar1 excision is 2 orders of magnitude more efficient than that of long elements, thus providing an explanation for their high copy numbers. We show that the SETMAR protein binds and introduces single-strand nicks into Hsmar1 inverted-repeat sequences in vitro. Pathway choices for DNA break repair were found to be characteristically different in response to transposon cleavage mediated by Hsmar1-Ra and SETMAR in vivo. Whereas nonhomologous end joining plays a dominant role in repairing excision sites generated by the Hsmar1-Ra transposase, DNA repair following cleavage by SETMAR predominantly follows a homology-dependent pathway. The novel transposon system can be a useful tool for genome manipulations in vertebrates and for investigations into the transpositional dynamics and the contributions of these elements to primate genome evolution

Survivin increased vascular development during Xenopus ontogenesis.

International audienceSurvivin is a member of the inhibitor of apoptosis proteins (IAP) family. These proteins contain one to three zinc-binding motifs termed bacculoviral IAP-binding repeats (BIRs). Survivin contains a single BIR motif. Contrary to other members that directly interact with caspases and inhibit apoptosis, Survivin is believed to have both antiapoptotic and proliferative functions. In mammals, Survivin is not detected in most adult tissues except in endothelial cells of newly formed capillaries and large blood vessels. Importantly, Survivin is highly expressed in all common human cancers. To gain a better view of Survivin expression and function during development, we used the amphibian Xenopus developmental model. We show that the genomes of X. laevis, X. tropicalis, Zebrafish, fugu pufferfish, and rainbow trout encode two different Survivin genes (Su1 and Su2), contrary to mammalian genomes, which encode a single one. In X. laevis, these two genes have a differential spatiotemporal transcription pattern. Transgenic expression of Su1 leads to an enlargement of tadpole's blood vessels with an increase in the number of endothelial cells. This effect requires a functional BIR domain and the p34/cdc2 phosphorylation site. It does not seem to rely on the antiapoptotic activity of Su1 as it is not observed in tadpoles overexpressing other antiapoptotic factors such as XIAP or BclXL. We conclude that Su1 ubiquitous gain of function leads directly or indirectly to an increase in blood vessels size via the proliferation of endothelial cells

Characterization of a novel Xenopus tropicalis cell line as a model for in vitro studies

Chantier qualité GACell lines are useful tools to facilitate in vitro studies of many biological and molecular processes. We describe a new permanent fibroblast-type cell line obtained from disaggregated Xenopus tropicalis limb bud. The cell line population doubling time was _ 24 h. Its karyotype was genetically stable with a chromosome number of 2n 5 21 and a chromosome 10 trisomy. These cells could be readily transfected and expressed transgenes faithfully. We obtained stable transformants using transposon-based gene transfer technology. These cells responded to thyroid hormone and thus can provide a complementary research tool to study thyroid hormone signaling events. In conclusion, this cell line baptized ‘‘Speedy’’ should prove useful to couple in vitro and in vivo biological studies in the X. tropicalis frog model

Dual and Opposite Effects of hRAD51 Chemical Modulation on HIV-1 Integration

The cellular DNA repair hRAD51 protein has been shown to restrict HIV-1 integration both in vitro and in vivo. To investigate its regulatory functions, we performed a pharmacological analysis of the retroviral integration modulation by hRAD51. We found that, in vitro, chemical activation of hRAD51 stimulates its integration inhibitory properties, whereas inhibition of hRAD51 decreases the integration restriction, indicating that the modulation of HIV-1 integration depends on the hRAD51 recombinase activity. Cellular analyses demonstrated that cells exhibiting high hRAD51 levels prior to de novo infection are more resistant to integration. On the other hand, when hRAD51 was activated during integration, cells were more permissive. Altogether, these data establish the functional link between hRAD51 activity and HIV-1 integration. Our results highlight the multiple and opposite effects of the recombinase during integration and provide new insights into the cellular regulation of HIV-1 replication