Uncoupling programmed DNA cleavage and repair scrambles the Paramecium somatic genome.

In the ciliate Paramecium, precise excision of numerous internal eliminated sequences (IESs) from the somatic genome is essential at each sexual cycle. DNA double-strands breaks (DSBs) introduced by the PiggyMac endonuclease are repaired in a highly concerted manner by the non-homologous end joining (NHEJ) pathway, illustrated by complete inhibition of DNA cleavage when Ku70/80 proteins are missing. We show that expression of a DNA-binding-deficient Ku70 mutant (Ku70-6E) permits DNA cleavage but leads to the accumulation of unrepaired DSBs. We uncoupled DNA cleavage and repair by co-expressing wild-type and mutant Ku70. High-throughput sequencing of the developing macronucleus genome in these conditions identifies the presence of extremities healed by de novo telomere addition and numerous translocations between IES-flanking sequences. Coupling the two steps of IES excision ensures that both extremities are held together throughout the process, suggesting that DSB repair proteins are essential for assembly of a synaptic precleavage complex.

Programmed chromosome fragmentation in ciliated protozoa: multiple means to chromosome ends.

SUMMARYCiliated protozoa undergo large-scale developmental rearrangement of their somatic genomes when forming a new transcriptionally active macronucleus during conjugation. This process includes the fragmentation of chromosomes derived from the germline, coupled with the efficient healing of the broken ends by de novo telomere addition. Here, we review what is known of developmental chromosome fragmentation in ciliates that have been well-studied at the molecular level (Tetrahymena, Paramecium, Euplotes, Stylonychia, and Oxytricha). These organisms differ substantially in the fidelity and precision of their fragmentation systems, as well as in the presence or absence of well-defined sequence elements that direct excision, suggesting that chromosome fragmentation systems have evolved multiple times and/or have been significantly altered during ciliate evolution. We propose a two-stage model for the evolution of the current ciliate systems, with both stages involving repetitive or transposable elements in the genome. The ancestral form of chromosome fragmentation is proposed to have been derived from the ciliate small RNA/chromatin modification process that removes transposons and other repetitive elements from the macronuclear genome during development. The evolution of this ancestral system is suggested to have potentiated its replacement in some ciliate lineages by subsequent fragmentation systems derived from mobile genetic elements.

Inter-generational nuclear crosstalk links the control of gene expression to programmed genome rearrangement during the Paramecium sexual cycle.

Multinucleate cells are found in many eukaryotes, but how multiple nuclei coordinate their functions is still poorly understood. In the cytoplasm of the ciliate Paramecium tetraurelia, two micronuclei (MIC) serving sexual reproduction coexist with a somatic macronucleus (MAC) dedicated to gene expression. During sexual processes, the MAC is progressively destroyed while still ensuring transcription, and new MACs develop from copies of the zygotic MIC. Several gene clusters are successively induced and switched off before vegetative growth resumes. Concomitantly, programmed genome rearrangement (PGR) removes transposons and their relics from the new MACs. Development of the new MACs is controlled by the old MAC, since the latter expresses genes involved in PGR, including the PGM gene encoding the essential PiggyMac endonuclease that cleaves the ends of eliminated sequences. Using RNA deep sequencing and transcriptome analysis, we show that impairing PGR upregulates key known PGR genes, together with ∼600 other genes possibly also involved in PGR. Among these genes, 42% are no longer induced when no new MACs are formed, including 180 genes that are co-expressed with PGM under all tested conditions. We propose that bi-directional crosstalk between the two coexisting generations of MACs links gene expression to the progression of MAC development.

Developmental timing of programmed DNA elimination in Paramecium tetraurelia recapitulates germline transposon evolutionary dynamics.

With its nuclear dualism, the ciliate Paramecium constitutes a unique model to study how host genomes cope with transposable elements (TEs). P. tetraurelia harbors two germline micronuclei (MICs) and a polyploid somatic macronucleus (MAC) that develops from one MIC at each sexual cycle. Throughout evolution, the MIC genome has been continuously colonized by TEs and related sequences that are removed from the somatic genome during MAC development. Whereas TE elimination is generally imprecise, excision of approximately 45,000 TE-derived internal eliminated sequences (IESs) is precise, allowing for functional gene assembly. Programmed DNA elimination is concomitant with genome amplification. It is guided by noncoding RNAs and repressive chromatin marks. A subset of IESs is excised independently of this epigenetic control, raising the question of how IESs are targeted for elimination. To gain insight into the determinants of IES excision, we established the developmental timing of DNA elimination genome-wide by combining fluorescence-assisted nuclear sorting with high-throughput sequencing. Essentially all IESs are excised within only one endoreplication round (32C to 64C), whereas TEs are eliminated at a later stage. We show that DNA elimination proceeds independently of replication. We defined four IES classes according to excision timing. The earliest excised IESs tend to be independent of epigenetic factors, display strong sequence signals at their ends, and originate from the most ancient integration events. We conclude that old IESs have been optimized during evolution for early and accurate excision by acquiring stronger sequence determinants and escaping epigenetic control.

GC content, but not nucleosome positioning, directly contributes to intron splicing efficiency in Paramecium.

Eukaryotic genes are interrupted by introns that must be accurately spliced from mRNA precursors. With an average length of 25 nt, the more than 90,000 introns of Paramecium tetraurelia stand among the shortest introns reported in eukaryotes. The mechanisms specifying the correct recognition of these tiny introns remain poorly understood. Splicing can occur cotranscriptionally, and it has been proposed that chromatin structure might influence splice site recognition. To investigate the roles of nucleosome positioning in intron recognition, we determined the nucleosome occupancy along the P. tetraurelia genome. We show that P. tetraurelia displays a regular nucleosome array with a nucleosome repeat length of ∼151 bp, among the smallest periodicities reported. Our analysis has revealed that introns are frequently associated with inter-nucleosomal DNA, pointing to an evolutionary constraint favoring introns at the AT-rich nucleosome edge sequences. Using accurate splicing efficiency data from cells depleted for nonsense-mediated decay effectors, we show that introns located at the edge of nucleosomes display higher splicing efficiency than those at the center. However, multiple regression analysis indicates that the low GC content of introns, rather than nucleosome positioning, is associated with high splicing efficiency. Our data reveal a complex link between GC content, nucleosome positioning, and intron evolution in Paramecium.

The unusual structure of the PiggyMac cysteine-rich domain reveals zinc finger diversity in PiggyBac-related transposases.

BACKGROUND: Transposons are mobile genetic elements that colonize genomes and drive their plasticity in all organisms. DNA transposon-encoded transposases bind to the ends of their cognate transposons and catalyze their movement. In some cases, exaptation of transposon genes has allowed novel cellular functions to emerge. The PiggyMac (Pgm) endonuclease of the ciliate Paramecium tetraurelia is a domesticated transposase from the PiggyBac family. It carries a core catalytic domain typical of PiggyBac-related transposases and a short cysteine-rich domain (CRD), flanked by N- and C-terminal extensions. During sexual processes Pgm catalyzes programmed genome rearrangements (PGR) that eliminate ~ 30% of germline DNA from the somatic genome at each generation. How Pgm recognizes its DNA cleavage sites in chromatin is unclear and the structure-function relationships of its different domains have remained elusive. RESULTS: We provide insight into Pgm structure by determining the fold adopted by its CRD, an essential domain required for PGR. Using Nuclear Magnetic Resonance, we show that the Pgm CRD binds two Zn2+ ions and forms an unusual binuclear cross-brace zinc finger, with a circularly permutated treble-clef fold flanked by two flexible arms. The Pgm CRD structure clearly differs from that of several other PiggyBac-related transposases, among which is the well-studied PB transposase from Trichoplusia ni. Instead, the arrangement of cysteines and histidines in the primary sequence of the Pgm CRD resembles that of active transposases from piggyBac-like elements found in other species and of human PiggyBac-derived domesticated transposases. We show that, unlike the PB CRD, the Pgm CRD does not bind DNA. Instead, it interacts weakly with the N-terminus of histone H3, whatever its lysine methylation state. CONCLUSIONS: The present study points to the structural diversity of the CRD among transposases from the PiggyBac family and their domesticated derivatives, and highlights the diverse interactions this domain may establish with chromatin, from sequence-specific DNA binding to contacts with histone tails. Our data suggest that the Pgm CRD fold, whose unusual arrangement of cysteines and histidines is found in all PiggyBac-related domesticated transposases from Paramecium and Tetrahymena, was already present in the ancestral active transposase that gave rise to ciliate domesticated proteins.

Functional diversification of Paramecium Ku80 paralogs safeguards genome integrity during precise programmed DNA elimination.

Gene duplication and diversification drive the emergence of novel functions during evolution. Because of whole genome duplications, ciliates from the Paramecium aurelia group constitute a remarkable system to study the evolutionary fate of duplicated genes. Paramecium species harbor two types of nuclei: a germline micronucleus (MIC) and a somatic macronucleus (MAC) that forms from the MIC at each sexual cycle. During MAC development, ~45,000 germline Internal Eliminated Sequences (IES) are excised precisely from the genome through a 'cut-and-close' mechanism. Here, we have studied the P. tetraurelia paralogs of KU80, which encode a key DNA double-strand break repair factor involved in non-homologous end joining. The three KU80 genes have different transcription patterns, KU80a and KU80b being constitutively expressed, while KU80c is specifically induced during MAC development. Immunofluorescence microscopy and high-throughput DNA sequencing revealed that Ku80c stably anchors the PiggyMac (Pgm) endonuclease in the developing MAC and is essential for IES excision genome-wide, providing a molecular explanation for the previously reported Ku-dependent licensing of DNA cleavage at IES ends. Expressing Ku80a under KU80c transcription signals failed to complement a depletion of endogenous Ku80c, indicating that the two paralogous proteins have distinct properties. Domain-swap experiments identified the α/ß domain of Ku80c as the major determinant for its specialized function, while its C-terminal part is required for excision of only a small subset of IESs located in IES-dense regions. We conclude that Ku80c has acquired the ability to license Pgm-dependent DNA cleavage, securing precise DNA elimination during programmed rearrangements. The present study thus provides novel evidence for functional diversification of genes issued from a whole-genome duplication.

Coupling DNA Damage and Repair: an Essential Safeguard during Programmed DNA Double-Strand Breaks?

DNA double-strand breaks (DSBs) are the most toxic DNA lesions given their oncogenic potential. Nevertheless, programmed DSBs (prDSBs) contribute to several biological processes. Formation of prDSBs is the 'price to pay' to achieve these essential biological functions. Generated by domesticated PiggyBac transposases, prDSBs have been integrated in the life cycle of ciliates. Created by Spo11 during meiotic recombination, they constitute a driving force of evolution and ensure balanced chromosome content for successful reproduction. Produced by the RAG1/2 recombinase, they are required for the development of the adaptive immune system in many species. The coevolution of processes that couple introduction of prDSBs to their accurate repair may constitute an effective safeguard against genomic instability.

Identification of a miniature Sae2/Ctp1/CtIP ortholog from Paramecium tetraurelia required for sexual reproduction and DNA double-strand break repair.

DNA double-strand breaks (DSBs) induced by genotoxic agents can cause cell death or contribute to chromosomal instability, a major driving force of cancer. By contrast, Spo11-dependent DSBs formed during meiosis are aimed at generating genetic diversity. In eukaryotes, CtIP and the Mre11 nuclease complex are essential for accurate processing and repair of both unscheduled and programmed DSBs by homologous recombination (HR). Here, we applied bioinformatics and genetic analysis to identify Paramecium tetraurelia CtIP (PtCtIP), the smallest known Sae2/Ctp1/CtIP ortholog, as a key factor for the completion of meiosis and the recovery of viable sexual progeny. Using in vitro assays, we find that purified recombinant PtCtIP preferentially binds to double-stranded DNA substrates but does not contain intrinsic nuclease activity. Moreover, mutation of the evolutionarily conserved C-terminal 'RHR' motif abrogates DNA binding of PtCtIP but not its ability to functionally interact with Mre11. Translating our findings into mammalian cells, we provide evidence that disruption of the 'RHR' motif abrogates accumulation of human CtIP at sites of DSBs. Consequently, cells expressing the DNA binding mutant CtIPR837A/R839A are defective in DSB resection and HR. Collectively, our work highlights minimal structural requirements for CtIP protein family members to facilitate the processing of DSBs, thereby maintaining genome stability as well as enabling sexual reproduction.

Six domesticated PiggyBac transposases together carry out programmed DNA elimination in Paramecium.

The domestication of transposable elements has repeatedly occurred during evolution and domesticated transposases have often been implicated in programmed genome rearrangements, as remarkably illustrated in ciliates. In Paramecium, PiggyMac (Pgm), a domesticated PiggyBac transposase, carries out developmentally programmed DNA elimination, including the precise excision of tens of thousands of gene-interrupting germline Internal Eliminated Sequences (IESs). Here, we report the discovery of five groups of distant Pgm-like proteins (PgmLs), all able to interact with Pgm and essential for its nuclear localization and IES excision genome-wide. Unlike Pgm, PgmLs lack a conserved catalytic site, suggesting that they rather have an architectural function within a multi-component excision complex embedding Pgm. PgmL depletion can increase erroneous targeting of residual Pgm-mediated DNA cleavage, indicating that PgmLs contribute to accurately position the complex on IES ends. DNA rearrangements in Paramecium constitute a rare example of a biological process jointly managed by six distinct domesticated transposases.

Sequence-specific DNA binding activity of the cross-brace zinc finger motif of the piggyBac transposase.

The piggyBac transposase (PB) is distinguished by its activity and utility in genome engineering, especially in humans where it has highly promising therapeutic potential. Little is known, however, about the structure-function relationships of the different domains of PB. Here, we demonstrate in vitro and in vivo that its C-terminal Cysteine-Rich Domain (CRD) is essential for DNA breakage, joining and transposition and that it binds to specific DNA sequences in the left and right transposon ends, and to an additional unexpectedly internal site at the left end. Using NMR, we show that the CRD adopts the specific fold of the cross-brace zinc finger protein family. We determine the interaction interfaces between the CRD and its target, the 5'-TGCGT-3'/3'-ACGCA-5' motifs found in the left, left internal and right transposon ends, and use NMR results to propose docking models for the complex, which are consistent with our site-directed mutagenesis data. Our results provide support for a model of the PB/DNA interactions in the context of the transpososome, which will be useful for the rational design of PB mutants with increased activity.

Improved methods and resources for paramecium genomics: transcription units, gene annotation and gene expression.

BACKGROUND: The 15 sibling species of the Paramecium aurelia cryptic species complex emerged after a whole genome duplication that occurred tens of millions of years ago. Given extensive knowledge of the genetics and epigenetics of Paramecium acquired over the last century, this species complex offers a uniquely powerful system to investigate the consequences of whole genome duplication in a unicellular eukaryote as well as the genetic and epigenetic mechanisms that drive speciation. High quality Paramecium gene models are important for research using this system. The major aim of the work reported here was to build an improved gene annotation pipeline for the Paramecium lineage. RESULTS: We generated oriented RNA-Seq transcriptome data across the sexual process of autogamy for the model species Paramecium tetraurelia. We determined, for the first time in a ciliate, candidate P. tetraurelia transcription start sites using an adapted Cap-Seq protocol. We developed TrUC, multi-threaded Perl software that in conjunction with TopHat mapping of RNA-Seq data to a reference genome, predicts transcription units for the annotation pipeline. We used EuGene software to combine annotation evidence. The high quality gene structural annotations obtained for P. tetraurelia were used as evidence to improve published annotations for 3 other Paramecium species. The RNA-Seq data were also used for differential gene expression analysis, providing a gene expression atlas that is more sensitive than the previously established microarray resource. CONCLUSIONS: We have developed a gene annotation pipeline tailored for the compact genomes and tiny introns of Paramecium species. A novel component of this pipeline, TrUC, predicts transcription units using Cap-Seq and oriented RNA-Seq data. TrUC could prove useful beyond Paramecium, especially in the case of high gene density. Accurate predictions of 3' and 5' UTR will be particularly valuable for studies of gene expression (e.g. nucleosome positioning, identification of cis regulatory motifs). The P. tetraurelia improved transcriptome resource, gene annotations for P. tetraurelia, P. biaurelia, P. sexaurelia and P. caudatum, and Paramecium-trained EuGene configuration are available through ParameciumDB ( http://paramecium.i2bc.paris-saclay.fr ). TrUC software is freely distributed under a GNU GPL v3 licence ( https://github.com/oarnaiz/TrUC ).

Multimerization properties of PiggyMac, a domesticated piggyBac transposase involved in programmed genome rearrangements.

During sexual processes, the ciliate Paramecium eliminates 25-30% of germline DNA from its somatic genome. DNA elimination includes excision of ∼45 000 short, single-copy internal eliminated sequences (IESs) and depends upon PiggyMac (Pgm), a domesticated piggyBac transposase that is essential for DNA cleavage at IES ends. Pgm carries a core transposase region with a putative catalytic domain containing three conserved aspartic acids, and a downstream cysteine-rich (CR) domain. A C-terminal extension of unknown function is predicted to adopt a coiled-coil (CC) structure. To address the role of the three domains, we designed an in vivo complementation assay by expressing wild-type or mutant Pgm-GFP fusions in cells depleted for their endogenous Pgm. The DDD triad and the CR domain are essential for Pgm activity and mutations in either domain have a dominant-negative effect in wild-type cells. A mutant lacking the CC domain is partially active in the presence of limiting Pgm amounts, but inactive when Pgm is completely absent, suggesting that presence of the mutant protein increases the overall number of active complexes. We conclude that IES excision involves multiple Pgm subunits, of which at least a fraction must contain the CC domain.

International Congress on Transposable elements (ICTE 2016) in Saint Malo: mobile elements under the sun of Brittany.

The third international conference on Transposable Elements (ICTE) was held 16-19 April 2016 in Saint Malo, France. Organized by the French Transposition Community (Research group of the CNRS: "Mobile genetic elements: from mechanism to populations, an integrative approach") and the French Society of Genetics, the conference's goal was to bring together researchers who study transposition in diverse organisms, using multiple experimental approaches. The meeting gathered 180 participants from all around the world. Most of them contributed through poster presentations, invited talks and short talks selected from poster abstracts. The talks were organized into six scientific sessions: "Taming mobile DNA: self and non-self recognition"; "Trans-generational inheritance"; "Mobile DNA genome structure and organization, from molecular mechanisms to applications"; "Remembrance of (retro)transposon past: mobile DNA in genome evolution"; and finally "The yin and the yang of mobile DNA in human health".

TFIIS-Dependent Non-coding Transcription Regulates Developmental Genome Rearrangements.

Because of their nuclear dimorphism, ciliates provide a unique opportunity to study the role of non-coding RNAs (ncRNAs) in the communication between germline and somatic lineages. In these unicellular eukaryotes, a new somatic nucleus develops at each sexual cycle from a copy of the zygotic (germline) nucleus, while the old somatic nucleus degenerates. In the ciliate Paramecium tetraurelia, the genome is massively rearranged during this process through the reproducible elimination of repeated sequences and the precise excision of over 45,000 short, single-copy Internal Eliminated Sequences (IESs). Different types of ncRNAs resulting from genome-wide transcription were shown to be involved in the epigenetic regulation of genome rearrangements. To understand how ncRNAs are produced from the entire genome, we have focused on a homolog of the TFIIS elongation factor, which regulates RNA polymerase II transcriptional pausing. Six TFIIS-paralogs, representing four distinct families, can be found in P. tetraurelia genome. Using RNA interference, we showed that TFIIS4, which encodes a development-specific TFIIS protein, is essential for the formation of a functional somatic genome. Molecular analyses and high-throughput DNA sequencing upon TFIIS4 RNAi demonstrated that TFIIS4 is involved in all kinds of genome rearrangements, including excision of ~48% of IESs. Localization of a GFP-TFIIS4 fusion revealed that TFIIS4 appears specifically in the new somatic nucleus at an early developmental stage, before IES excision. RT-PCR experiments showed that TFIIS4 is necessary for the synthesis of IES-containing non-coding transcripts. We propose that these IES+ transcripts originate from the developing somatic nucleus and serve as pairing substrates for germline-specific short RNAs that target elimination of their homologous sequences. Our study, therefore, connects the onset of zygotic non coding transcription to the control of genome plasticity in Paramecium, and establishes for the first time a specific role of TFIIS in non-coding transcription in eukaryotes.

Ku-mediated coupling of DNA cleavage and repair during programmed genome rearrangements in the ciliate Paramecium tetraurelia.

During somatic differentiation, physiological DNA double-strand breaks (DSB) can drive programmed genome rearrangements (PGR), during which DSB repair pathways are mobilized to safeguard genome integrity. Because of their unique nuclear dimorphism, ciliates are powerful unicellular eukaryotic models to study the mechanisms involved in PGR. At each sexual cycle, the germline nucleus is transmitted to the progeny, but the somatic nucleus, essential for gene expression, is destroyed and a new somatic nucleus differentiates from a copy of the germline nucleus. In Paramecium tetraurelia, the development of the somatic nucleus involves massive PGR, including the precise elimination of at least 45,000 germline sequences (Internal Eliminated Sequences, IES). IES excision proceeds through a cut-and-close mechanism: a domesticated transposase, PiggyMac, is essential for DNA cleavage, and DSB repair at excision sites involves the Ligase IV, a specific component of the non-homologous end-joining (NHEJ) pathway. At the genome-wide level, a huge number of programmed DSBs must be repaired during this process to allow the assembly of functional somatic chromosomes. To understand how DNA cleavage and DSB repair are coordinated during PGR, we have focused on Ku, the earliest actor of NHEJ-mediated repair. Two Ku70 and three Ku80 paralogs are encoded in the genome of P. tetraurelia: Ku70a and Ku80c are produced during sexual processes and localize specifically in the developing new somatic nucleus. Using RNA interference, we show that the development-specific Ku70/Ku80c heterodimer is essential for the recovery of a functional somatic nucleus. Strikingly, at the molecular level, PiggyMac-dependent DNA cleavage is abolished at IES boundaries in cells depleted for Ku80c, resulting in IES retention in the somatic genome. PiggyMac and Ku70a/Ku80c co-purify as a complex when overproduced in a heterologous system. We conclude that Ku has been integrated in the Paramecium DNA cleavage factory, enabling tight coupling between DSB introduction and repair during PGR.

Is non-homologous end-joining really an inherently error-prone process?

DNA double-strand breaks (DSBs) are harmful lesions leading to genomic instability or diversity. Non-homologous end-joining (NHEJ) is a prominent DSB repair pathway, which has long been considered to be error-prone. However, recent data have pointed to the intrinsic precision of NHEJ. Three reasons can account for the apparent fallibility of NHEJ: 1) the existence of a highly error-prone alternative end-joining process; 2) the adaptability of canonical C-NHEJ (Ku- and Xrcc4/ligase IV-dependent) to imperfect complementary ends; and 3) the requirement to first process chemically incompatible DNA ends that cannot be ligated directly. Thus, C-NHEJ is conservative but adaptable, and the accuracy of the repair is dictated by the structure of the DNA ends rather than by the C-NHEJ machinery. We present data from different organisms that describe the conservative/versatile properties of C-NHEJ. The advantages of the adaptability/versatility of C-NHEJ are discussed for the development of the immune repertoire and the resistance to ionizing radiation, especially at low doses, and for targeted genome manipulation.

Programmed Rearrangement in Ciliates: Paramecium.

Programmed genome rearrangements in the ciliate Paramecium provide a nice illustration of the impact of transposons on genome evolution and plasticity. During the sexual cycle, development of the somatic macronucleus involves elimination of ∼30% of the germline genome, including repeated DNA (e.g., transposons) and ∼45,000 single-copy internal eliminated sequences (IES). IES excision is a precise cut-and-close process, in which double-stranded DNA cleavage at IES ends depends on PiggyMac, a domesticated piggyBac transposase. Genome-wide analysis has revealed that at least a fraction of IESs originate from Tc/mariner transposons unrelated to piggyBac. Moreover, genomic sequences with no transposon origin, such as gene promoters, can be excised reproducibly as IESs, indicating that genome rearrangements contribute to the control of gene expression. How the system has evolved to allow elimination of DNA sequences with no recognizable conserved motif has been the subject of extensive research during the past two decades. Increasing evidence has accumulated for the participation of noncoding RNAs in epigenetic control of elimination for a subset of IESs, and in trans-generational inheritance of alternative rearrangement patterns. This chapter summarizes our current knowledge of the structure of the germline and somatic genomes for the model species Paramecium tetraurelia, and describes the DNA cleavage and repair factors that constitute the IES excision machinery. We present an overview of the role of specialized RNA interference machineries and their associated noncoding RNAs in the control of DNA elimination. Finally, we discuss how RNA-dependent modification and/or remodeling of chromatin may guide PiggyMac to its cognate cleavage sites.

International Congress on Transposable Elements (ICTE) 2012 in Saint Malo and the sea of TE stories.

An international conference on Transposable Elements (TEs) was held 21-24 April 2012 in Saint Malo, France. Organized by the French Transposition Community (GDR Elements Génétiques Mobiles et Génomes, CNRS) and the French Society of Genetics (SFG), the conference's goal was to bring together researchers from around the world who study transposition in diverse organisms using multiple experimental approaches. The meeting drew more than 217 attendees and most contributed through poster presentations (117), invited talks and short talks selected from poster abstracts (48 in total). The talks were organized into four scientific sessions, focused on: impact of TEs on genomes, control of transposition, evolution of TEs and mechanisms of transposition. Here, we present highlights from the talks given during the platform sessions. The conference was sponsored by Alliance pour les sciences de la vie et de la santé (Aviesan), Centre national de la recherche scientifique (CNRS), Institut national de la santé et de la recherche médicale (INSERM), Institut de recherche pour le développement (IRD), Institut national de la recherche agronomique (INRA), Université de Perpignan, Université de Rennes 1, Région Bretagne and Mobile DNA. CHAIR OF THE ORGANIZATION COMMITTEE: Jean-Marc Deragon ORGANIZERS: Abdelkader Ainouche, Mireille Bétermier, Mick Chandler, Richard Cordaux, Gaël Cristofari, Jean-Marc Deragon, Pascale Lesage, Didier Mazel, Olivier Panaud, Hadi Quesneville, Chantal Vaury, Cristina Vieira and Clémentine Vitte.

The Paramecium germline genome provides a niche for intragenic parasitic DNA: evolutionary dynamics of internal eliminated sequences.

Insertions of parasitic DNA within coding sequences are usually deleterious and are generally counter-selected during evolution. Thanks to nuclear dimorphism, ciliates provide unique models to study the fate of such insertions. Their germline genome undergoes extensive rearrangements during development of a new somatic macronucleus from the germline micronucleus following sexual events. In Paramecium, these rearrangements include precise excision of unique-copy Internal Eliminated Sequences (IES) from the somatic DNA, requiring the activity of a domesticated piggyBac transposase, PiggyMac. We have sequenced Paramecium tetraurelia germline DNA, establishing a genome-wide catalogue of -45,000 IESs, in order to gain insight into their evolutionary origin and excision mechanism. We obtained direct evidence that PiggyMac is required for excision of all IESs. Homology with known P. tetraurelia Tc1/mariner transposons, described here, indicates that at least a fraction of IESs derive from these elements. Most IES insertions occurred before a recent whole-genome duplication that preceded diversification of the P. aurelia species complex, but IES invasion of the Paramecium genome appears to be an ongoing process. Once inserted, IESs decay rapidly by accumulation of deletions and point substitutions. Over 90% of the IESs are shorter than 150 bp and present a remarkable size distribution with a -10 bp periodicity, corresponding to the helical repeat of double-stranded DNA and suggesting DNA loop formation during assembly of a transpososome-like excision complex. IESs are equally frequent within and between coding sequences; however, excision is not 100% efficient and there is selective pressure against IES insertions, in particular within highly expressed genes. We discuss the possibility that ancient domestication of a piggyBac transposase favored subsequent propagation of transposons throughout the germline by allowing insertions in coding sequences, a fraction of the genome in which parasitic DNA is not usually tolerated.