Corinne Augé-Gouillou

Human and other mammalian genomes contain transposons of the mariner family

Internal fragments of the putative transposase gene of mariner‐like elements (MLEs) were amplified from human, mouse, rat, chinese hamster, sheep and bovine genomic DNAs by polymerase chain reaction (PCR). The sequences identified in human, ovine and bovine genomes correspond to ancient degenerate transposons. Screening mammalian sequence libraries identified a truncated element in the human ABL gene and the sequence of its 5′‐ITR was determined. This ITR sequences were used in PCR experiments with DNA from six mammalian species and detected full‐sized and deleted MLEs. The presence of MLE in mammalian genomes demonstrates that they are ubiquitous mobile elements found from fungi to man. This observation strongly raises the possibility that MLE could constitute tools for the modification of eucaryotic genomes.

Assembly of the mariner Mos1 Synaptic Complex

ABSTRACT The mobility of transposable elements via a cut-and-paste mechanism depends on the elaboration of a nucleoprotein complex known as the synaptic complex. We show here that the Mos1 synaptic complex consists of the two inverted terminal repeats of the element brought together by a transposase tetramer and is designated paired-end complex 2 (PEC2). The assembly of PEC2 requires the formation of a simpler complex, containing one terminal repeat and two transposase molecules and designated single-end complex 2 (SEC2). In light of the formation of SEC2 and PEC2, we demonstrate the presence of two binding sites for the transposase within a single terminal repeat. We have found that the sequence of the Mos1 inverted terminal repeats contains overlapping palindromic and mirror motifs, which could account for the binding of two transposase molecules “side by side” on the same inverted terminal repeat. We provide data indicating that the Mos1 transposase dimer is formed within a single terminal repeat through a cooperative pathway. Finally, the concept of a tetrameric synaptic complex may simply account for the inability of a single mariner transposase molecule to interact at the same time with two kinds of DNA: the inverted repeat and the target DNA.

COMPUTER ANALYSES REVEAL A HOBO-LIKE ELEMENT IN THE NEMATODE CAENORHABDITIS ELEGANS, WHICH PRESENTS A CONSERVED TRANSPOSASE DOMAIN COMMON WITH THE TC1 -MARINER TRANSPOSON FAMILY

The present report describes the use of computer analyses to reveal a hobo-like element in the genome of Caenorhabditis elegans. This hobo-like sequence is 3039 bp long, contains two inverted terminal repeats of 25-27 bp and probably does not encoded a functional transposase. Sequence comparisons suggest that each transposase of hobo elements probably has a D(D/S)E motif. Thus the transposases of the hAT superfamily of transposons appear to be close to the other transposases and intregrases.

Liver-enriched HNF-3α and ubiquitous factors interact with the human transferrin gene enhancer

The human transferrin gene enhancer is organized in two domains. Domain A contains a single enhanson designated 1a. Domain B contains four enhansons named Ib, II, III and IV. We demonstrate here that the liver‐enriched transcription factor HNF‐3α interacts with enhanson Ia and that enhansons Ib and IV are binding sites for members of the NF1 family. In addition, enhansons II and III seem to be respectively the targets for the AP4 protein and for EIII, a factor not yet completely identified. Analysis of mutated enhancer regions establishes that each enhanson is required for full enhancer activity and that the proteins binding to enhansons II, III and IV may interact within a multiprotein complex. This enhancer region presents no activity in the Sertoli cells of testis, where transferrin is also synthesized. We demonstrate that in Sertoli cells, the members of the HNF‐3 family are not expressed; this fact may account for the inactivity of the enhancer in these cells.

Factors acting on Mos1 transposition efficiency

BackgroundMariner- like elements (MLEs) are widespread DNA transposons in animal genomes. Although in vitro transposition reactions require only the transposase, various factors depending on the host, the physico-chemical environment and the transposon sequence can interfere with the MLEs transposition in vivo.ResultsThe transposition of Mos1, first isolated from drosophila mauritiana, depends of both the nucleic acid sequence of the DNA stuffer (in terms of GC content), and its length. We provide the first in vitro experimental demonstration that MITEs of MLE origin, as small as 80 to 120-bp, are able to transpose. Excessive temperature down-regulates Mos1 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.ConclusionThe study highlights how experimental conditions can bias interpretation of mariner excision frequency and quality. In vitro, 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.

Physical properties of DNA components affecting the transposition efficiency of the mariner Mos1 element

Previous studies have shown that the transposase and the inverted terminal repeat (ITR) of the Mos1mariner elements are suboptimal for transposition; and that hyperactive transposases and transposon with more efficient ITR configurations can be obtained by rational molecular engineering. In an attempt to determine the extent to which this element is suboptimal for transposition, we investigate here the impact of the three main DNA components on its transposition efficiency in bacteria and in vitro. We found that combinations of natural and synthetic ITRs obtained by systematic evolution of ligands by exponential enrichment did increase the transposition rate. We observed that when untranslated terminal regions were associated with their respective natural ITRs, they acted as transposition enhancers, probably via the early transposition steps. Finally, we demonstrated that the integrity of the Mos1 inner region was essential for transposition. These findings allowed us to propose prototypes of optimized Mos1 vectors, and to define the best sequence features of their associated marker cassettes. These vector prototypes were assayed in HeLa cells, in which Mos1 vectors had so far been found to be inactive. The results obtained revealed that using these prototypes does not circumvent this problem. However, such vectors can be expected to provide new tools for the use in genome engineering in systems such as Caenorhabditis elegans in which Mos1 is very active.

In vitro recombination and inverted terminal repeat binding activities of the Mcmar1 transposase.

The Mcmar1 mariner element (MLE) presents some intriguing features with two large, perfectly conserved, 355 bp inverted terminal repeats (ITRs) containing two 28 bp direct repeats (DRs). The presence of a complete ORF in Mcmar1 makes it possible to explore the transposition of this unusual MLE. Mcmar1 transposase (MCMAR1) was purified, and in vitro transposition assays showed that it is able to promote ITR-dependent DNA cleavages and recombination events, which correspond to plasmid fusions and transpositions with imprecise ends. Further analyses indicated that MCMAR1 is able to interact with the 355 bp ITR through two DRs: the EDR (external DR) is a high-affinity binding site for MCMAR1, whereas the IDR (internal DR) is a low-affinity binding site. The main complex detected within the EDR contained a transposase dimer and only one DNA molecule. We hypothesize that the inability of MCMAR1 to promote precise in vitro transposition events could be due to mutations in its ORF sequence or to the specific features of transposase binding to the ITR. Indeed, the ITR region spanning from EDR to IDR resembles a MITE and could be bent by specific host factors. This suggests that the assembly of the transposition complex is more complex than that of those involved in the mobility of the Mos1 and Himar1 mariner elements.

SETMAR isoforms in glioblastoma: A matter of protein stability

Glioblastomas (GBMs) are the most frequent and the most aggressive brain tumors, known for their chemo- and radio-resistance, making them often incurable. We also know that SETMAR is a protein involved in chromatin dynamics and genome plasticity, of which overexpression confers chemo- and radio-resistance to some tumors. The relationships between SETMAR and GBM have never been explored. To fill this gap, we define the SETMAR status of 44 resected tumors and of GBM derived cells, at both the mRNA and the protein levels. We identify a new, small SETMAR protein (so called SETMAR-1200), enriched in GBMs and GBM stem cells as compared to the regular enzyme (SETMAR-2100). We show that SETMAR-1200 is able to increase DNA repair by non-homologous end-joining, albeit with a lower efficiency than the regular SETMAR protein. Interestingly, the regular/small ratio of SETMAR in GBM cells changes depending on cell type, providing evidence that SETMAR expression is regulated by alternative splicing. We also demonstrate that SETMAR expression can be regulated by the use of an alternative ATG. In conclusion, various SETMAR proteins can be synthesized in human GBM that may each have specific biophysical and/or biochemical properties and characteristics. Among them, the small SETMAR may play a role in GBMs biogenesis. On this basis, we would like to consider SETMAR-1200 as a new potential therapeutic target to investigate, in addition to the regular SETMAR protein already considered by others.

Expression and Purification of the Eukaryotic MBP-MOS1 Transposase from Sf21 Insect Cells

[Abstract] Here, we present the full-length protocol for purifying the recombinant MOS1 transposase from insect cells used in our recent publication (Pflieger et al., 2014), which involved a N-terminal MBP-tag and maltose-affinity chromatography. Due to their overall basic properties, transposases are often difficult to purify, especially because they tend to aggregate. Since the 90s, we chose a method of purification without a denaturation step. Our first priority was to preserve the 3D structure of the protein in order to maintain its biochemical activities with the highest specific activity. Nevertheless, our production/purification made from bacteria regularly contain truncated products (or degradation products) and their levels increase with concentration of purified transposase. In contrast, production/purification made from eukaryotic cells do not contain such degradation product. We thus developed a protocol involving the pVL1392 baculovirus transfer vector and the BaculoGold TM baculovirus expression system, allowing the expression of recombinant MOS1 from baculovirus-infected Sf21 cells.