Mariner Transposons Contain a Silencer: Possible Role of the Polycomb Repressive Complex 2.

Transposable elements are driving forces for establishing genetic innovations such as transcriptional regulatory networks in eukaryotic genomes. Here, we describe a silencer situated in the last 300 bp of the Mos1 transposase open reading frame (ORF) which functions in vertebrate and arthropod cells. Functional silencers are also found at similar locations within three other animal mariner elements, i.e. IS630-Tc1-mariner (ITm) DD34D elements, Himar1, Hsmar1 and Mcmar1. These silencers are able to impact eukaryotic promoters monitoring strong, moderate or low expression as well as those of mariner elements located upstream of the transposase ORF. We report that the silencing involves at least two transcription factors (TFs) that are conserved within animal species, NFAT-5 and Alx1. These cooperatively act with YY1 to trigger the silencing activity. Four other housekeeping transcription factors (TFs), neuron restrictive silencer factor (NRSF), GAGA factor (GAF) and GTGT factor (GTF), were also found to have binding sites within mariner silencers but their impact in modulating the silencer activity remains to be further specified. Interestingly, an NRSF binding site was found to overlap a 30 bp motif coding a highly conserved PHxxYSPDLAPxD peptide in mariner transposases. We also present experimental evidence that silencing is mainly achieved by co-opting the host Polycomb Repressive Complex 2 pathway. However, we observe that when PRC2 is impaired another host silencing pathway potentially takes over to maintain weak silencer activity. Mariner silencers harbour features of Polycomb Response Elements, which are probably a way for mariner elements to self-repress their transcription and mobility in somatic and germinal cells when the required TFs are expressed. At the evolutionary scale, mariner elements, through their exaptation, might have been a source of silencers playing a role in the chromatin configuration in eukaryotic genomes.

Reliability of the nanopheres-DNA immunization technology to produce polyclonal antibodies directed against human neogenic proteins.

The molecular domestication of several DNA transposons that occurred during the evolution of the mammalian lineage, has led to the emergence of at least 43 genes, known as neogenes. To date, the limited availability of efficient commercial antibodies directed against most of their protein isoforms hampers investigation of their expression in vitro and in situ. Since immunization protocols using peptides or recombinant proteins have revealed that it is difficult to recover antibodies, we planned to produce antisera in mice using a new technique of nanopheres/DNA immunization, the ICANtibodies™ technology. Here, we investigate the possibilities of obtaining polyclonal antibodies for 24 proteins or protein domains using this immunization strategy. We successfully obtained 13 antisera that were able to detect neogenic proteins by Western blotting and ELISA in protein extracts of transiently-transfected cells and various cancer cell lines, plus another two that only detected the in ELISA and in in situ hybridizations. The features required for the production of these antibodies are analyzed and discussed, and examples are given of the advantages they offer for the study of neogenic proteins.

Characteristics of inteins in invertebrate iridoviruses and factors controlling insertion in their viral hosts.

Inteins are self-splicing proteins that occur in-frame within host-coded proteins. DNA elements coding for inteins insert specifically in highly conserved motifs of target genes. These mobile genetic elements have an uneven distribution and thus far have been found only in certain species of bacteria, archaea and fungi, a few viruses of algae and amoebozoa and in the entomopathogen, Chilo iridescent virus (CIV). Here, we report the discovery of seven new inteins parasitizing iridoviruses infecting metazoans: three within their δ DNA polymerase genes and four in genes coding for their large ribonucleotide reductase subunit. Analyses of coding sequences suggest that these inteins were acquired by ancestors shared by viruses currently classified as members of different families of viruses with large double-stranded (ds) DNA genomes and then were maintained by vertical transmission, or lost. Of significant interest is the finding that inteins present in the δ DNA polymerases of iridoviruses insert at a different location into the YGDTDS motif when compared to those found in other viruses and prokaryotes. In addition, our phylogenetic investigations suggest that inteins present in the δ DNA polymerases of these viruses might have an origin different from those found in prokaryotes. Finally, we use the sequence features of the intein insertion sites in host genes to discuss the high polymorphisms of inteins within and among viral species and the immunity of their genetic counterparts in the eukaryotic hosts of these viruses.

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 Mos1 mariner 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.

Mariner Mos1 transposase optimization by rational mutagenesis.

Mariner transposons are probably the most widespread transposable element family in animal genomes. To date, they are believed not to require species-specific host factors for transposition. Despite this, Mos1, one of the most-studied mariner elements (with Himar1), has been shown to be active in insects, but inactive in mammalian genomes. To circumvent this problem, one strategy consists of both enhancing the activity of the Mos1 transposase (MOS1), and making it insensitive to activity-altering post-translational modifications. Here, we report rational mutagenesis studies performed to obtain hyperactive and non-phosphorylable MOS1 variants. Transposition assays in bacteria have made it possible to isolate numerous hyperactive MOS1 variants. The best mutant combinations, named FETY and FET, are 60- and 800-fold more active than the wild-type MOS1 version, respectively. However, there are serious difficulties in using them, notably because they display severe cytotoxicity. On the other hand, three positions lying within the HTH motif, T88, S99, and S104 were found to be sensitive to phosphorylation. Our efforts to obtain active non-phosphorylable mutants at S99 and S104 positions were unsuccessful, as these residues, like the co-linear amino acids in their close vicinity, are critical for MOS1 activity. Even if host factors are not essential for transposition, our data demonstrate that the host machinery is essential in regulating MOS1 activity.

Modulation of Gonadotropins Activity by Antibodies.

Gonadotropins are essential for reproduction control in humans as well as in animals. They are widely used all over the world for ovarian stimulation in women, spermatogenesis stimulation in men, and ovulation induction and superovulation in animals. Despite the availability of many different preparations, all are made of the native hormones. Having different ligands with a wide activity range for a given receptor helps better understand its molecular and cellular signaling mechanisms as well as its physiological functions, and thus helps the development of more specific and adapted medicines. One way to control the gonadotropins' activity could be the use of modulating antibodies. Antibodies are powerful tools that were largely used to decipher gonadotropins' actions and they have shown their utility as therapeutics in several other indications such as cancer. In this review, we summarize the inhibitory and potentiating antibodies to gonadotropins, and their potential therapeutic applications.

Transposase concentration controls transposition activity: myth or reality?

Deciphering the mechanisms underlying the regulation of DNA transposons might be central to understanding their function and dynamics in genomes. From results obtained under artificial experimental conditions, it has been proposed that some DNA transposons self-regulate their activity via overproduction inhibition (OPI), a mechanism by which transposition activity is down-regulated when the transposase is overconcentrated in cells. However, numerous studies have given contradictory results depending on the experimental conditions. Moreover, we do not know in which cellular compartment this phenomenon takes place, or whether transposases assemble to form dense foci when they are highly expressed in cells. In the present review, we focus on investigating the data available about eukaryotic transposons to explain the mechanisms underlying OPI. Data in the literature indicate that members of the IS630-Tc1-mariner, Hobo-Ac-Tam, and piggyBac superfamilies are able to use OPI to self-regulate their transposition activity in vivo in most eukaryotic cells, and that some of them are able to assemble so as to form higher order soluble oligomers. We also investigated the localization and behavior of GFP-fused transposases belonging to the mariner, Tc1-like, and piggyBac families, investigating their ability to aggregate in cells when they are overexpressed. Transposases are able to form dense foci when they are highly expressed. Moreover, the cellular compartments in which these foci are concentrated depend on the transposase, and on its expression. The data presented here suggest that sequestration in cytoplasmic or nucleoplasmic foci, or within the nucleoli, might protect the genome against the potentially genotoxic effects of the non-specific nuclease activities of eukaryotic transposases.

Optimization of the piggyBac transposon using mRNA and insulators: toward a more reliable gene delivery system.

Integrating and expressing stably a transgene into the cellular genome remain major challenges for gene-based therapies and for bioproduction purposes. While transposon vectors mediate efficient transgene integration, expression may be limited by epigenetic silencing, and persistent transposase expression may mediate multiple transposition cycles. Here, we evaluated the delivery of the piggyBac transposase messenger RNA combined with genetically insulated transposons to isolate the transgene from neighboring regulatory elements and stabilize expression. A comparison of piggyBac transposase expression from messenger RNA and DNA vectors was carried out in terms of expression levels, transposition efficiency, transgene expression and genotoxic effects, in order to calibrate and secure the transposition-based delivery system. Messenger RNA reduced the persistence of the transposase to a narrow window, thus decreasing side effects such as superfluous genomic DNA cleavage. Both the CTF/NF1 and the D4Z4 insulators were found to mediate more efficient expression from a few transposition events. We conclude that the use of engineered piggyBac transposase mRNA and insulated transposons offer promising ways of improving the quality of the integration process and sustaining the expression of transposon vectors.

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.