Claude Universite

Bernard -Lyon

M Le Professeur

L Collet

J-F Mornex

G Annat

D Simon

M G Gay

Composantes Sante

Marie Fablet

Rita Rebollo
email: rebollo@biomserv.univ-lyon1.fr

Christian Biémont
email: biemont@biomserv.univ-lyon1.fr

Cristina Vieira
email: vieira@biomserv.univ-lyon1.fr

Batzer

Emmanuelle Lerat
email: lerat@biomserv.univ-lyon1.fr

Liliana Lopez Kleine
email: liliana.lopez@jouy.inra.fr

Eléments transposables -Transposable elements Drosophila Populations naturelles -Natural populations Délétion -Deletion Epigénétique -Epigenetics

Keywords: LTR, UTR, Sequence variability, LINEs TE, Transposable Element, UTR, UnTranslated Region, LTR, Long Terminal Repeat, ORF, Open Reading Frame, Inr, Initiator, Transposable elements, evolution, speciation, natural populations, epigenetic control

It has now been established that transposable elements (TEs) make up a variable, but significant proportion of the genomes of all organisms, from Bacteria to Vertebrates. However, in addition to their quantitative importance, there is increasing evidence that TEs also play a functional role within the genome. In particular, TE regulatory regions can be viewed as a large pool of potential promoter sequences for host genes. Studying the evolution of regulatory region of TEs in different genomic contexts is therefore a fundamental aspect of understanding how a genome works. In this paper, we first briefly describe what is currently known about the regulation of TE copy number and activity in genomes, and then focus on TE regulatory regions and their evolution. We restrict ourselves to retrotransposons, which are the most abundant class of eukaryotic TEs, and analyze their evolution and the subsequent consequences for host genomes. Particular attention is paid to much-studied representatives of the Vertebrates and Invertebrates, Homo sapiens and Drosophila melanogaster, respectively, for which high quality sequenced genomes are available.

Les éléments transposables (ET) sont une source majeure de variation génétique, ce qui leur confère un rôle essentiel dans l'évolution des génomes. Certes présents dans tous les génomes analysés à ce jour, leurs proportions sont fortement variables entre espèces et aussi entre populations, suggérant une relation unique entre génome hôte et ET. Grâce à un système modèle composé de populations naturelles de deux espèces proches (Drosophila melanogaster et D. simulans) avec des quantités différentes en ET, nous avons pu comparer les relations génome hôte/ET. Nous nous sommes particulièrement interessés à l'élément helena qui, chez D. simulans, montre une activité faible, malgré un nombre de copies élevé.

Cette activité moindre est associée à de nombreuses délétions internes des copies, suggérant un mécanisme de régulation d'ET par des délétions de l'ADN. Un autre système de régulation de l'activité des ET utilise le contrôle épigénétique, ce qui permet le maintien des copies d'ET dans le génome mais un blocage de leur activité. Le remodelage de la chromatine est un Transposable elements (TEs) are one major force of genome evolution thanks to their ability to create genetic variation. TEs are ubiquitous and their proportion is variable between species and also populations, suggesting that a tight relationship exists between genomes and TEs. The model system composed of the natural populations of the twin sisters Drosophila melanogaster and D. simulans is interesting to compare host/TE relationship, since both species harbour different amounts of TE copies. The helena element is nearly silenced in D.

simulans natural populations despite a very high copy number. Such repression is associated to abundant internally deleted copies suggesting a regulatory mechanism of TEs based on DNA deletion. Another pathway of TE regulation is through epigenetics where the host genome is able to keep intact the DNA sequences of TEs and still silence their activities.

Chromatin remodelling is well known in drosophila and specific histone modifications can be associated to specific chromatin domains. We observed an important variation on H3K27me3 and H3K9me2, two heterochromatic marks, on TE copies in D. melanogaster and D. simulans natural populations. Also, we show that derepressed lines of D. simulans exist for specific elements, have high TE transcription rates and are highly associated to non constitutive heterochromatic marks. TEs are therefore controlled by the host genome through DNA deletion and a possible chromatin remodelling mechanism. Not only genetic variability is enhanced by TEs but also epigenetic variability, allowing the host genome to be partitioned into chromatin domains. TEs are therefore mandatory to gene network regulation through their ability of "jumping epigenetics".

LONG RESUME EN FRANÇAIS

La partie codante des génomes ne constitue pas, dans beaucoup d'espèces, la majorité des séquences d'ADN présentes (Biemont and Vieira, 2006). En réalité, ces régions codantes sont souvent noyées dans une abondance de séquences répétées, qui pendant longtemps n'étaient pas associées à des « fonctions » précises et ont par conséquent été nommées « Junk DNA » [START_REF] Ohno | So much "junk DNA" in our genome[END_REF]. Aujourd'hui, multiples sont les effets décrits de ces répétitions : 1) effets directs sur le génome, telle la domestication des parties codantes des répétitions (au sein de centromères et télomères par exemple [START_REF] Sinzelle | Molecular domestication of transposable elements: from detrimental parasites to useful host genes[END_REF] ou leur utilisation dans des systèmes de régulation du génome hôte (séquences régulatrices d'une répétition permettant le contrôle des gènes [START_REF] Marino-Ramirez | Transposable elements donate lineage-specific regulatory sequences to host genomes[END_REF], ou dérivés ARN permettant la régulation de gènes par ARN interférence [START_REF] Hasler | Useful 'junk': Alu RNAs in the human transcriptome[END_REF] -2) effets indirects, tel l'apport de variabilité génétique (multiplication et recombinaisons [START_REF] Hedges | Inviting instability: Transposable elements, double-strand breaks, and the maintenance of genome integrity[END_REF] et l'augmentation de la taille des génomes hôtes [START_REF] Boulesteix | Differences in genome size between closely related species: the Drosophila melanogaster species subgroup[END_REF], deux phénomènes qui participent à l'évolution des espèces.

Les éléments transposables (ET), partie majeure du « Junk DNA », sont des séquences d'ADN capables de se multiplier à l'intérieur d'un génome, engendrant des mutations. Selon leur mode de déplacement, les ET peuvent être classés en deux grands groupes : les rétrotransposons, qui se mobilisent via une molécule d'ARN par un mécanisme de « copier/coller », et les transposons, qui se mobilisent via une molécule d'ADN par un « couper/coller » (cf. Figure 1 page 103). Les mécanismes de transposition ainsi que l'utilisation d'ET pour les réparations de cassures double brin, induisent l'augmentation du nombre de copies de ces séquences dans le génome. Les processus de délétions et recombinaisons induisent à leurs tours une diminution du nombre de copies dans le génome.

La proportion d'ET est très variable dans les espèces analysées, allant de 10% pour

Arabidopsis thaliana à 80% pour le maïs (Biemont and Vieira, 2006). De plus, des variations dans la proportion d'ET sont aussi observées entre des populations d'une même espèce (Vieira et al., 1999;[START_REF] Biemont | Genome size evolution: within-species variation in genome size[END_REF], suggérant l'existence d'une relation très étroite entre les ET et le génome. C'est exactement cette spécificité relationnelle que nous avons essayé de comprendre en étudiant un système modèle composé de deux espèces proches, D. melanogaster et D. simulans, qui ne possèdent pas la même quantité d' ET (15% et 5% environ,respectivement). Ces deux espèces sont cosmopolites, étroitement liées à l'Homme, et ont une aire de distribution assez proche, avec cependant des endroits du globe qui n'ont pas encore été colonisés par D. simulans. De plus, D. simulans, contrairement à D. melanogaster, ne possède pas un nombre constant de copies d'ET, c'est-à-dire, une faible variance du nombre de copies d'ET entre les populations naturelles ; ce qui permet une étude intraspécifique des relations ET/hôte. Aussi bien par des analyses bibliographiques que par des travaux de recherche, nous avons essayé de comprendre la relation ET/hôte chez la drosophile, avec une description des facteurs qui interviennent dans cette relation.

À travers une analyse bibliographique, nous avons constaté que les promoteurs des ET constituent un premier système de régulation car l'invasion d'un génome est dépendante des facteurs de transcription fournis par l'hôte [START_REF] Fablet | The evolution of retrotransposon regulatory regions and its consequences on the Drosophila melanogaster and Homo sapiens host genomes[END_REF]. Sachant qu'il existe plusieurs types de séquences régulatrices, spécifiques à chaque type d'ET, et qu'à ceci s'ajoute la variation de facteurs de transcription entre les espèces, nous suggérons que la relation ET/hôte est en partie une conséquence de la relation « promoteur ET / facteur de transcription de l'hôte » [START_REF] Fablet | The evolution of retrotransposon regulatory regions and its consequences on the Drosophila melanogaster and Homo sapiens host genomes[END_REF].

Parmi les systèmes de régulation qui contrent les effets mutagènes des ET et empêchent l'invasion des génomes, nous avons étudié le système de régulation par délétion avec l'exemple de l'élément helena chez la drosophile [START_REF] Rebollo | Losing helena: the extinction of a drosophila line-like element[END_REF]. Helena est un rétrotransposon de type LINE (long interspersed nuclear element), qui possède une dynamique inverse chez D. melanogaster par rapport aux autres ET car très peu de copies y sont observées (Vieira et al., 1999). L'analyse par Southern blot de cet élément dans les populations naturelles de D. melanogaster ainsi que l'analyse in silico du génome séquencé de cette espèce, montrent qu'helena est certes présent dans ces génomes mais à très faible fréquence, très détérioré et de façon fixé entre les populations, suggérant une absence d'activité de cet élément. En effet, aucune copie complète d'helena n'est détectée, ce qui nous permet d'émettre l'hypothèse d'une éventuelle extinction de cet élément chez D. melanogaster. Au contraire, dans les populations naturelles de D. simulans et dans le génome séquencé, les copies d'helena sont observées en abondance. De plus, le grand polymorphisme d'insertion et l'observation de copies complètes suggèrent que l'élément est actif. Néanmoins, l'analyse exhaustive de différentes copies d'helena dans plusieurs populations de D. simulans et dans le génome séquencé montre un mécanisme de délétions internes important, déjà décrit chez D. melanogaster [START_REF] Petrov | High rate of DNA loss in the Drosophila melanogaster and Drosophila virilis species groups[END_REF]. L'absence de transcrits observée par northern blot accentue l'idée que les copies d'helena dans le génome de D. simulans ne soient pas actives et cela probablement dû aux délétions abondantes [START_REF] Rebollo | Losing helena: the extinction of a drosophila line-like element[END_REF]. Cette analyse montre, premièrement, que l'observation d'un polymorphisme d'insertion au sein des populations de D. simulans, n'est pas corrélée à une activité forte de cet élément, mais probablement à une activité récente. Deuxièmement, nous observons beaucoup de séquences ayant des délétions internes mais qui sont très similaires entre elles (séquences nucléotidiques), suggérant la rapidité de l'apparition des délétions. L'état de l'élément helena aussi bien chez D. melanogaster que chez D. simulans semble inactif mais la présence d'une copie complète dans le génome séquencé de D. simulans suggère une possibilité d'invasion future du génome. En effet, des vagues de transpositions ont souvent été observées chez les mammifères pour des éléments de type LINE [START_REF] Adey | 3rd: Rodent L1 evolution has been driven by a single dominant lineage that has repeatedly acquired new transcriptional regulatory sequences[END_REF]Khan et al., 2006). Les mécanismes d'élimination des séquences d'ET ont déjà été décrits dans d'autres espèces chez lesquelles les recombinaisons homologues privent les éléments de type LTR (long terminal repeats) de leurs parties codantes (certaines recombinaison LTR-LTR) ou peuvent causer la délétion complète d'un élément [START_REF] Vitte | Formation of solo-LTRs through unequal homologous recombination counterbalances amplifications of LTR retrotransposons in rice Oryza sativa L[END_REF]van de Lagemaat et al., 2005). Or, le mécanisme de délétion interne décrit ici ne semble pas commun à toutes les espèces ni aux éléments de type LINE. La poursuite de ce travail par A. Granzotto et collaborateurs [START_REF] Granzotto | The evolutionary dynamics of the Helena retrotransposon revealed by sequenced Drosophila genomes[END_REF] et par E. Lerat (communication personnelle) Le deuxième système de régulation des ET que nous avons étudié est le contrôle épigénétique qui rend possible le maintien des ET dans le génome hôte tout en empêchant leur activité. Trois grandes voies épigénétiques permettent la régulation des ET et du reste du génome : la conformation de la chromatine, la méthylation de l'ADN et les processus impliquant des petits ARN [START_REF] Lisch | Epigenetic regulation of transposable elements in plants[END_REF]. Nous nous sommes premièrement concentrés sur l'analyse des structures chromatiniennes des ET dans les populations naturelles de D. melanogaster et D. simulans. L'ADN s'enroule autour d'un grand complexe protéique appelé nucléosome qui est formé de dimères d'histones. Les queues N-terminales des histones se trouvent libres du nucléosome et subissent des modifications post-traductionnelles leur conférant des états physiques précis. Les modifications de type acétylation, souvent sur les lysines N-terminales, sont observées surtout au niveau de la chromatine propice à la transcription, c'est à dire, de l'euchromatine. Au contraire, la méthylation des lysines (à l'exception de la lysine 4 sur l'histone 3 -H3K4) est plus souvent observée dans des zones où la chromatine est fermée, c'est à dire, l'hétérochromatine. Sachant qu'il existe un nombre variable de copies d'ET entre les deux espèces de drosophile analysées et aussi entre les populations naturelles de D. simulans, nous nous sommes demandé s'il existait une variation dans l'association aux marques d'histones avec ces éléments.

Notre étude porte sur quatre ET (trois éléments qui possèdent des LTR : 412, tirant et roo, et un élément de type LINE : F) dans deux populations de D. melanogaster et cinq populations de D. simulans. Premièrement, la comparaison du nombre de copies obtenu par hybridation in situ avec une sonde d'ET complète faite par Vieira et collaborateurs (Vieira et al., 1999) avec les données d'amplification par PCR quantitative d'un petit fragment d'ET (environ 400pb) suggèrent que chez D. simulans les copies d'ET sont probablement délétées, ce qui renforce l'hypothèse de mécanisme de délétion interne décrite ci-dessus. De plus, nous avons recherché les associations entre les marques d'histones permissives (H3K4me2) ou répressives (H3K27me3 et H3K9me2) et les ET, puis quantifié les transcrits de chaque ET dans les sept populations de drosophile étudiées. Il existe effectivement une variation des associations épigénétiques sur les ET non seulement entre les espèces mais aussi entre les populations naturelles. En effet, deux systèmes différents agissent chez l'espèce D. melanogaster, pour laquelle deux populations analysées présentent environ le même nombre de copies d'ET, les mêmes associations aux marques d'histones, mais une de ces populations est réprimée pour les quatre ET étudiés. Il existe probablement d'autres systèmes, tels la méthylation de l'ADN ou la RNAi, qui pourraient être différents entre ces deux populations et qui expliqueraient le comportement des ET analysés. Chez D. simulans, nous avons mis en évidence des populations dites « déréprimées » qui possèdent un taux important de transcrits d'un ET donné, beaucoup de représentations de cet élément dans le génome et des copies fortement associées à H3K27me3. Il pourrait donc exister un système de régulation épigénétique qui serait variable entre les espèces de drosophile et au sein d'une même espèce, et qui pourrait contribuer aux différences dans les quantités d'ET observées.

Il est important de comprendre que les deux systèmes étudiés (délétions et régulation épigénétique) sont complémentaires car la suppression épigénétique n'empêche pas les recombinaisons souvent délétères dues à la présence de répétitions. De plus, la suppression totale des ET d'un génome peut aussi être délétère à long terme car les ET apportent une variabilité considérée comme importante pour l'évolution des espèces [START_REF] Mcclintock | The significance of responses of the genome to challenge[END_REF]. En effet, une analyse critique de la bibliographie concernant l'impact des ET dans l'évolution des espèces ainsi que les travaux de recherche de l'équipe, nous amènent à proposer l'hypothèse selon laquelle les variations environnementales ayant un impact sur la régulation épigénétique des ET pourraient moduler la réponse du génome hôte à un quelconque stress. Un pic d'activité des ET pourrait promouvoir une augmentation rapide de la variabilité génétique et donc participer à une réponse adaptative au stress. Une répression de novo des copies récemment transposés pourrait se mettre en place dans le génome réarrangé et on observerait une stabilisation de l'activité des éléments et des systèmes de régulation. Une variation dans la taille du génome réarrangé reste possible mais pas nécessaire, permettant d'inclure les espèces à « petit génome » dans cette hypothèse d'adaptation par des bursts de transposition.

La régulation épigénétique des ET est donc, aussi bien que les effets mutagènes des ET, importante pour l'évolution des espèces. De plus, les ET créent, modulent et contrôlent des réseaux de gènes, car comme décrit plus haut, ils possèdent des séquences régulatrices capables de contrôler l'expression des gènes [START_REF] Feschotte | Transposable elements and the evolution of regulatory networks[END_REF]. A ceci s'ajoute leur caractère multi copies et ubiquitaires dans le génome, qui ne fait qu'augmenter leurs effets potentiels.

Nous proposons qu'un nouveau niveau de régulation de gènes existe, où non seulement les séquences régulatrices des ET sont nécessaires mais aussi les marques épigénétiques sur les copies d'ET. Les copies permettraient la compartimentation du génome en zones chromatiniennes définies et influenceraient aussi les gènes avoisinants par leurs marques épigénétiques.

INTRODUCTION

Apart from Greeks, with Aristotle who once proposed that human beings had "heritable characteristics" in their blood, Mendel was the first who showed the existence of "heritable factors", i.e. genes, in 1865 [START_REF] Griffiths | Modern Genetic Analysis[END_REF][START_REF] Griffiths | An introduction to genetic analysis[END_REF]. Since that time, important discoveries were made: genes are on chromosomes [START_REF] Morgan | Sex Limited Inheritance in Drosophila[END_REF], genes code for proteins [START_REF] Beadle | Genetic Control of Biochemical Reactions in Neurospora[END_REF], DNA is organized in a double helix [START_REF] Watson | Molecular structure of nucleic acids; a structure for deoxyribose nucleic acid[END_REF] etc.

In the 70's, the study of whole genomes suggested a discrepancy between genome size and "complex species": the C-value paradox (reviewed in [START_REF] Gregory | Synergy between sequence and size in large-scale genomics[END_REF]. In parallel, the discovery of jumping genes by B. McClintock in the 50's [START_REF] Mc | The origin and behavior of mutable loci in maize[END_REF][START_REF] Mcclintock | Induction of Instability at Selected Loci in Maize[END_REF] and non coding DNA repeats during the 70's, induced several hypotheses on the quality of the information handled by genomes. Jumping genes (or transposable elements -TEs) are DNA molecules capable of moving from one chromosomal location to another. Such observation was hardly accepted by the scientific community who just got used to the idea of genes fixed on chromosomes. Because of such scepticism and the lack of function for all repeats, these DNA sequences such as TEs were called "junk DNA" [START_REF] Ohno | So much "junk DNA" in our genome[END_REF]. The most complete answer from the scientific community to the C-value paradox arose from whole genome sequencing projects that began in the end of the 70's. Genome size (and gene amount) was not correlated to "species complexity" because the "junk DNA" proportion of genomes can be extremely high as it can be variable [START_REF] Gregory | Synergy between sequence and size in large-scale genomics[END_REF]. We know today that repeats can vary from 80% in some species (maize) to 10% in others (Arabidopsis) (Biemont and Vieira, 2006). It has also been shown that these repeats are essential for centromeres and telomeres integrity, they may be co-opted by the genome [START_REF] Sinzelle | Molecular domestication of transposable elements: from detrimental parasites to useful host genes[END_REF] and a high number of regulatory sequences in humans are derived from TEs [START_REF] Marino-Ramirez | Transposable elements donate lineage-specific regulatory sequences to host genomes[END_REF]. Furthermore, repeats are important recombinogenic substrates allowing for rapid genome remodelling [START_REF] Hedges | Inviting instability: Transposable elements, double-strand breaks, and the maintenance of genome integrity[END_REF]. We indeed know today that "junk DNA" is nothing but informative [START_REF] Muotri | The necessary junk: new functions for transposable elements[END_REF].

We will solely be interested in TEs because they represent the largest part of "junk DNA", are present in both euchromatin and dense heterochromatin, and have the ability to influence all the genome. TEs can be separated into two major classes given their transposition mechanisms (for a figure, please refer to "Figure 1" page 103). Class I moves through an RNA molecule and are called retrotrasposons. Class II jumps through a DNA molecule, and are called DNA transposons. Both classes can be further separated into subclasses regarding TE structure and evolutionary aspects. Finally, TE families can be distinguished regarding DNA sequence homology. TEs can be considered as genetic units bringing positive, neutral and negative effects to the genome. In order to understand the relationship that exists between TEs and genomes we studied natural regulation of TEs in Drosophila. Indeed, fruit flies are one of the most classical models for genetic studies as showed by TH Morgan researches in white-eye flies and his hypotheses on genes and chromosomes [START_REF] Morgan | Sex Limited Inheritance in Drosophila[END_REF]. Drosophila offers important experimental qualities as they are easy to keep in laboratories, breed extremely fast and can be maintained in important amounts of individuals. The existence of natural populations of Drosophila from close species, allows us to study different combinations of TEs and genomes.

The goal of my work was to understand the connexion between TE natural variation between and inside species, and genome response to TEs. A bibliographic analysis along with other members of the group, on TE regulatory regions suggests that common features exist between different types of TEs and different species [START_REF] Fablet | The evolution of retrotransposon regulatory regions and its consequences on the Drosophila melanogaster and Homo sapiens host genomes[END_REF]. However, the arm race of TEs and genomes necessarily brings specificity to each TE/genome complex, suggesting that regulatory mechanisms of TEs may vary between species and TEs analysed [START_REF] Fablet | The evolution of retrotransposon regulatory regions and its consequences on the Drosophila melanogaster and Homo sapiens host genomes[END_REF]. In order to experimentally test such suggestion, we used the system model composed of the sibling species, D. melanogaster and D. simulans along with several natural populations of both species. D. melanogaster was previously described as harbouring tree times more copies than D. simulans [16]. Such data was based on the study of natural populations allowing the observation of variation not only between species but most important, inside each species. Interestingly, all TE families are over represented in D. melanogaster natural populations compared to D. simulans ones. Few elements, as the LINElike helena element, harboured the opposite dynamic, being outnumbered in D. simulans natural populations. As a result of such observation, the first question addressed experimentally during my PhD was to understand helena opposite occurrence in our system model. Natural population variability of helena between D. simulans and D. melanogaster confirmed the specificity of TE regulation between species [START_REF] Rebollo | Losing helena: the extinction of a drosophila line-like element[END_REF]. We hypothesize on the existence of a rapid internal deletion mechanism for D. simulans TEs.

Such hypothesis was confirmed by E. Lerat in silico TE analysis and broaden to other species of Drosophila (personal communication).

The differences between D. melanogaster and D. simulans in controlling TEs and the different chromatin regions occupied by TEs in both species (Vieira et al., 1999;[START_REF] Capy | Drosophila melanogaster, Drosophila simulans: so similar yet so different[END_REF]) lead us to our second question : could epigenetic regulatory mechanisms also be variable between both species. We concentrated our analysis on chromatin remodelling processes by describing histone marks associated to TE copies. Also, we characterized all TE families analysed for copy number and expression patterns in every natural population studied. Our data suggests that D. melanogaster has more heterochromatic elements than D. simulans. Also, important variation of histone marks between all element analysed regarding natural populations or species suggest that chromatin conformation migh be specific of the context "TE/locus/genome". In D. simulans natural populations, TEs seem to be internally deleted confirming the existence of a deletion mechanism suggested for the helena element and the in silico analysis of E. Lerat. No correlation between epigenetic marks (histone post translational modifications) could however be made with TE expression or copy number. We can nevertheless propose that since expression patterns of TEs are strain-specific and often species-specific, different regulatory mechanisms might exist.

The evolutionary importance of genetic and epigenetic lineage specific regulations of TEs suggested by these experiments leads us to reflect on the impact of transposition in speciation.

We know that epigenetics is influenced by environment and is the most important regulatory mechanisms of TEs. We propose therefore, in a bibliographic survey, that changes in environment can induce changes in TE epigenetic regulation, inducing bursts of transposition.

The consequences of such bursts can reach speciation if important karyotypic modifications are observed.

Introduction

It has now been established that transposable elements (TEs) make up a significant proportion of the genomes of all organisms, ranging from more than 50% of the human genome (International Human Genome Sequencing Consortium, 2001), to about 28% of the Drosophila melanogaster genome [START_REF] Biémont | What transposable elements tell us about genome organization and evolution? The case of Drosophila[END_REF], and up to 90% of the genomes of some plants [START_REF] Bennetzen | Transposable element contributions to plant gene and genome evolution[END_REF]. The increasing data obtained from genomewide sequencing projects indicate that TEs have major functions in the organisms in which they are located. In particular, it has been shown that almost 25% of promoter regions in the human genome contain TE-derived sequences [START_REF] Jordan | Origin of a substantial fraction of human regulatory sequences from transposable elements[END_REF], and that TEs have donated transcriptional regulatory signals to many mammalian genes [START_REF] Van De Lagemaat | Transposable elements in mammals promote regulatory variation and diversification of genes with specialized functions[END_REF]. Because TEs are abundant in most genomes, they can be considered to constitute a large pool of potential promoter regions for new host regulatory sequences. Finding out how TEs are regulated is therefore important if we are to understand how the genome works.

TE regulatory regions are known to be very rapidly evolving sequences [START_REF] Arkhipova | Drosophila Retrotransposons[END_REF], a characteristic of eukaryotic regulatory regions attributed to having to cope with changing genomic environments [START_REF] Ludwig | Evidence for stabilizing selection in a eukaryotic enhancer element[END_REF]. In this article, we propose to focus on the evolution of the TE regulatory regions, and the subsequent consequences for their host genomes. We only deal with retrotransposons, which are generally the most abundant class of TEs in terms of their proportion in the genomes, and are widely distributed amongst eukaryotic genomes (Hua- Van et al., 2005). Of course, there are some exceptions, as for instance the non-autonomous Class II elements MITEs, which contributed to more than 70% of TEs in Oriza sativa, and about 50% in Arabidopsis thaliana (Hua- Van et al., 2005;[START_REF] Turcotte | Survey of transposable elements from rice genomic sequences[END_REF], or the Class II mariner element, which is very abundant in some Arthropods, such as Ceratitis capitata [START_REF] Torti | Evolution of different subfamilies of mariner elements within the medfly genome inferred from abundance and chromosomal distribution[END_REF]. Retrotransposons comprise two classes of elements, the elements bordered by long terminal repeats, known as LTR retrotransposons, and the long interspersed nuclear elements (non-LTR retrotransposons), also known as LINEs. Both these classes are mobilized via an RNA intermediate, and insert themselves into the genome by means of the reverse transcriptase that they encode in their second open reading frame (ORF). Both of these elements also encode a GAG-related, RNA binding protein in ORF1. The most important difference between LINEs and LTR elements resides in their regulatory sequences. Here, we want to show that the modalities and outcomes of the rapid evolution of the regulatory region vary according to the class of retrotransposons (LTR retrotransposons/LINEs) and the genome in which they reside. We focus particularly on two much-studied representatives of the Vertebrates and Invertebrates, Homo sapiens and D. melanogaster, respectively, for which the sequenced genomes are available.

2. Transposable elements in the genomes of H. sapiens and D. melanogaster

The major class of TEs is not the same in these two genomes

Drosophila and human genomes display quite different TE proportions (Fig. 1). LINE-1 (L1) elements are the most abundant family of autonomously-replicating retroelements in mammals, accounting for 17% of the human genome [START_REF] Bannert | Retroelements and the human genome: new perspectives on an old relation[END_REF]. Nearly 8% of human DNA consists of sequences assigned to HERVs (Human Endogenous Retroviruses) (International Human Genome Sequencing Consortium, 2001), which will be considered here to be LTR retroelements. HERVs are probably genomic traces of numerous germ-line retroviral infections [START_REF] Belshaw | Long-term reinfection of the human genome by endogenous retroviruses[END_REF], and waves of invasion are known to have occurred repeatedly during primate evolution [START_REF] Sverdlov | Retroviruses and primate evolution[END_REF]. On the contrary, LINEs and LTR retrotransposons correspond to 0.9% and 2.7% of the euchromatin of D. melanogaster, respectively [START_REF] Kaminker | The transposable elements of the Drosophila melanogaster euchromatin: a genomic perspective[END_REF]. Considering that the heterochromatin represents 30-40% of the DNA of the genome of D. melanogaster, that 50-60% of the heterochromatin exhibits sequence similarities to known TEs [START_REF] Hoskins | Heterochromatic sequences in Drosophila wholegenome shotgun assembly[END_REF]Kapitonov and Jurka, 2003), and assessing that LINEs and LTR retrotransposons display the same relative proportions in euchromatin and heterochromatin, then LINEs and LTR retrotransposons represent roughly about 3% and 10%, respectively, of the whole genome of D. melanogaster. The proportion of LINEs is significantly lower than in the human genome. Another major difference between both genomes is that the genome of D. melanogaster does not display any SINEs (Short Interspersed Nuclear Elements, usually associated with LINEs), whereas these are prevalent in the human genome (International Human Genome Sequencing Consortium, 2001).

LINEs and LTR retrotransposons do not only constitute different proportions of the genomes of D. melanogaster and H. sapiens, they also display contrasting activity patterns in both genomes. In H. sapiens, retrotransposons account for a large fraction of the genome, but have little activity (less than 0.2% of spontaneous mutations in humans are caused by L1 insertions [START_REF] Kazazian | Mobile elements and disease[END_REF]), while in D. melanogaster, in spite of their low copy number, TEs are responsible for more than 50% of naturally-occurring mutations with major morphological effects [START_REF] Eikbush | Fruit flies and humans respond differently to retrotransposons[END_REF]. In insects, sixty or so retrotransposon families are active, whereas just one LINE family, the L1 family, has been the major source of retrotranspositional activity in primates (International Human Genome Sequencing Consortium, 2001). Retrotransposons in the D. melanogaster genome seem to result from recent transposition events (Lerat et al., 2003), whereas most HERV families are now apparently extinct, even though the activity of some, such as the HERV-L family, appears to have persisted for a long time [START_REF] Benit | ERV-L elements: a family of endogenous retrovirus-like elements active throughout the evolution of mammals[END_REF], and HERV-K remained active for some time after the chimpanzee/human split [START_REF] Mayer | An almost-intact human endogenous retrovirus K on human chromosome 7[END_REF]. These HERV families consist mainly of solo LTRs, and transpositionallydeficient and transcriptionally-silent members. These differences in proportions and activity strongly suggest that TEs must be regulated differently in human and Drosophila genomes.

Overall host regulation of retrotransposon activity and copy number in the genomes of H. sapiens and D. melanogaster

Various mechanisms are involved in regulating retrotransposon activity and copy number, and they are quite different between D. melanogaster and H. sapiens. Indeed, in human cells, TEs are transcriptionally silenced due to hypermethylated CpG dinucleotides [START_REF] Lavie | CpG methylation directly regulates transcriptional activity of the human endogenous retrovirus family HERV-K (HML-2)[END_REF], which is the major way of controlling their activity. In H. sapiens, abnormalities in retrotransposon hypermethylation are frequently linked to cancers, as shown by [START_REF] Menendez | L1 and HERV-W retrotransposons are hypomethylated in human ovarian carcinomas[END_REF], who have demonstrated that L1 and HERV-W are hypomethylated in ovarian carcinomas, and that relative levels of expression of these retrotransposons are significantly higher in malignant ovarian tissues. On the contrary, until very recently, the genome of Drosophila was considered to be non-methylated [START_REF] Patel | Determination of trace amounts of 5methylcytosine in DNA by HPLC[END_REF][START_REF] Tweedie | Methylation of genomes and genes at the invertebrate-vertebrate boundary[END_REF], in contrast to most vertebrates and other insects. However, it is now realized that Drosophila does in fact methylate, mostly in young embryos [START_REF] Lyko | DNA methylation in Drosophila melanogaster[END_REF][START_REF] Lyko | DNA methylation learns to fly[END_REF], and mainly on the cytosines that are associated with the A and T nucleotides, which contrasts with the other species, in which methylation takes place on the CpG. However, the impact of methylation on the Drosophila genome remains to be clarified, since individuals with DNA methylation mutations do not display any specific dysfunction [START_REF] Kunert | A Dnmt2-like protein mediates DNA methylation in Drosophila[END_REF].

In contrast to TE regulation mechanisms in the human genome, which mainly act at the transcriptional level, the TE copy numbers are decreased by selection against insertions in the D. melanogaster genome. Selection can indeed act either directly against the deleterious effects of TE insertions or indirectly against the chromosomal rearrangements resulting from ectopic recombination between copies of elements belonging to the same family [START_REF] Charlesworth | Transposable element distribution in Drosophila[END_REF][START_REF] Biémont | Transposable element distribution in Drosophila[END_REF]. [START_REF] Eikbush | Fruit flies and humans respond differently to retrotransposons[END_REF] have suggested that one way to explain the difference in the total amount of TEs between the D. melanogaster and H. sapiens genomes could be that mammals have a much lower rate of ectopic homologous recombination than D. melanogaster [START_REF] Cooper | Factors affecting ectopic gene conversion in mice[END_REF]van de Lagemaat et al., 2005). The explanation for the differences in TE amount and composition in different genomes is not straightforward because recombination, presence or absence of heterochromatine, host effective population size and breeding system may be involved. It should thus be borne in mind that the amount of TEs may be very different in closely related species, as has been shown for the twin species D. melanogaster and D. simulans. Indeed, the total amount of TE insertion sites is three times higher in D. melanogaster than in D. simulans (Vieira et al., 1999;[START_REF] Vieira | Transposable element dynamics in two sibling species: Drosophila melanogaster and Drosophila simulans[END_REF]. Several hypotheses have been proposed to explain this difference, involving the genome properties of the species [START_REF] Kidwell | Transposable elements and host genome evolution[END_REF] or the species ecology [START_REF] Vieira | Evolution of genome size in Drosophila. Is the invader's genome being invaded by transposable elements?[END_REF], but no irrefutable evidence has been provided, and we can envisage that epigenetic mechanisms may also be responsible for controlling TEs in Drosophila.

While retrotransposon activity depends on host control mechanisms, such as methylation, recombination rate and epigenetic phenomena, they are also subject, as in the 'arms race' scenario, to their own regulatory signals, most of which are located in their 5′ regulatory regions.

Structure and variability of retrotransposon regulatory regions

Retrotransposons are transcribed via RNA polymerase II, and display a tripartite canonical eukaryotic promoter structure consisting of the TATA box, the initiator element (Inr), and the downstream promoter element (DPE) [START_REF] Arkhipova | Drosophila Retrotransposons[END_REF] (Fig. 2). The Inr and DPE motifs can act in conjunction to provide a binding site for TFIID in the absence of a TATA box, and a strict requirement for spacing between them has been demonstrated in both humans and Drosophila [START_REF] Burke | Drosophila TFIID binds to a conserved downstream basal promoter element that is present in many TATA-boxdeficient promoters[END_REF]. In some TEs, an alternative RNA start site for RNA polymerase III can be observed, as for instance 10`bp upstream of the regular RNA polymerase II start site in the mdg1 LTR retrotransposon of Drosophila [START_REF] Arkhipova | Complex patterns of transcription of a Drosophila retrotransposon in vivo and in vitro by RNA polymerases II and III[END_REF], or in the human L1 promoter [START_REF] Kurose | RNA polymerase III dependence of the human L1 promoter and possible participation of the RNA polymerase II factor YY1 in the RNA polymerase III transcription system[END_REF].

Structure of LINE element regulatory regions

Due to their retrotransposition mechanism, the regulation signals of LINEs must be located downstream from the 5′ RNA start site. Therefore, instead of TATA boxes, some internal promoters are found that consist of the Inr and the DPE [START_REF] Arkhipova | Drosophila Retrotransposons[END_REF]. The consensus 5′ UTR region of L1 copies contains such an internal promoter plus a YY1 binding site, which is necessary for the transcription to be initiated (Khan et al., 2006). Moreover, it has recently been shown that the L1 transcription start site is not always located exactly at nucleotide +1, but can range from nt -9 to + 4, so that the Inr extends into upstream sequences [START_REF] Lavie | The human L1 promoter: variable transcription initiation sites and a major impact of upstream flanking sequence on promoter activity[END_REF]. In Drosophila, even though the regulatory regions of non-LTR elements are characteristic of each element, some 5′ UTRs have the same overall structure. For the I, doc and F elements in Drosophila for example, the 5′ UTR promoter has two regions (A and B), which boost the role of the initiator. The B region can be divided into 3 regions containing DNA binding sites. The RNA machinery is recruited synergistically by all of these regions [START_REF] Minchiotti | Multiple downstream promoter modules regulate the transcription of the Drosophila melanogaster I, doc and F elements[END_REF]. In mice, the L1 5′ UTR is composed of multiple direct repeats of individual promoter elements, or monomers of about 210 bp, located upstream from a single-copy, non-monomeric sequence [START_REF] Mears | The evolution of modern lineages of mouse L1 elements[END_REF][START_REF] Goodier | A novel active L1 retrotransposon subfamily in the mouse[END_REF][START_REF] Kazazian | Mobile elements and disease[END_REF]. The monomers act synergistically to increase the promoter activity.

In addition to the internal promoter necessary for their own transcription, LINEs can also display antisense promoters (ASP). In Drosophila, the F element possesses two convergently-oriented promoters within its 5′ UTR, which overlap by approximately 100 bp [START_REF] Contursi | Functional dissection of two promoters that control sense and antisense transcription of Drosophila melanogaster F elements[END_REF]. The full-length human L1 5′ UTR also displays an antisense promoter, which can drive the transcription of adjacent cellular genes in the opposite direction [START_REF] Speek | Antisense promoter of human L1 retrotransposon drives transcription of adjacent cellular genes[END_REF]. Transcription-factor binding sites for both promoters overlap in the 5′ UTR region: two binding sites for SOX factors involved in the L1 sense transcription are located in the region from nt 400 to 600, as is an enhancer element containing binding sites for Ets and Sp1 factors, capable of functioning in either orientation [START_REF] Speek | Antisense promoter of human L1 retrotransposon drives transcription of adjacent cellular genes[END_REF]. Sequences critical for ASP activity are located in a small region extending from nt 399 to 467, and a 15-bp region located from nt 544 to 558, containing two Sp1 binding sites, may also contribute to the overall L1 ASP activity [START_REF] Speek | Antisense promoter of human L1 retrotransposon drives transcription of adjacent cellular genes[END_REF]. Chimeric transcripts derived from the L1 ASP are highly represented in expressed sequence tag (EST) datasets, from both normal and tumor cells, and the ASP can be located between 1 kb and more than 60 kb from the protein coding sequences [START_REF] Speek | Antisense promoter of human L1 retrotransposon drives transcription of adjacent cellular genes[END_REF][START_REF] Nigumann | Many human genes are transcribed from the antisense promoter of L1 retrotransposon[END_REF]. It is therefore believed that hypermethylation of L1 is a major defense mechanism, repressing the oncogenes that use this ASP as an alternative promoter, and which could be very damaging for the organism [START_REF] Roman-Gomez | Promoter hypomethylation of the LINE-1 retrotransposable elements activates sense/antisense transcription and marks the progression of chronic myeloid leukemia[END_REF].

Structure of LTR retrotransposon regulatory regions

The regulatory region of an LTR retrotransposon consists of the 5′ LTR and frequently also of a 5′ UTR, also known as the ULR (Untranslated Leader Region). The LTRs contain the promoter necessary for transcription and specify the terminator and polyadenylation signals needed for RNA processing. The LTRs consist of three discrete regions: U3 (the nearest to 5′, up to the transcription start site), R (for 'repeat', extending from the transcription start site to the polyadenylation site), and U5 (the nearest to 3′, downstream from the polyadenylation site). During reverse transcription, the R and U5 regions at the 5′ end of the RNA template are reverse transcribed first, followed by a template switch to the 3′ end of the RNA, where the reversetranscribed R region of the DNA binds to the 3′ R region of the RNA template (Varmus, 1988). The transcription start signal is located in U3, and the U5 region can have a silencing effect on transcription [START_REF] Prudhomme | A retroviral promoter and a cellular enhancer define a bipartite element which controls env ERVWE1 placental expression[END_REF]. Some LTR retrotransposons have two TATA boxes, such as the BARE-1 element in barley [START_REF] Manninen | BARE-1, a copia-like retroelement in barley (Hordeum vulgare L.)[END_REF], both of which are able to direct RNA transcription, but which do so under different conditions [START_REF] Suoniemi | The BARE-1 retrotransposon is transcribed in barley from an LTR promoter active in transient assays[END_REF]. In contrast, a substantial proportion of elements can be TATA-less, such as mdg1 and gypsy in Drosophila [START_REF] Arkhipova | Drosophila Retrotransposons[END_REF]. Bidirectional transcription is also possible for LTR retrotransposons. The Drosophila mdg1 element displays multiple start sites for the antisense direction [START_REF] Arkhipova | Properties of promoter regions of mdg1 Drosophila retrotransposons indicate that it belongs to a specific class of promoters[END_REF], and the LTRs of some families of HERVs have also been shown to be able to activate transcription in both directions, a characteristic probably linked to the presence of Sp1 binding sites [START_REF] Dunn | Transcription of two human genes from a bidirectional endogenous retrovirus promoter[END_REF][START_REF] Feuchter | Functional heterogeneity of a large family of human LTR-like promoters and enhancers[END_REF]. [START_REF] Kovalskaya | Functional human endogenous retroviral LTR transcription start sites are located between the R and U5 regions[END_REF] found transcripts promoted by the human HERV-K (HML 2) provirus and solitary LTRs, the transcriptional start point of which is located at the extreme 3′ end of the R region of the LTR. The R region is therefore excluded from transcripts initiated on LTRs, whereas a classical retroviral life-cycle model implies that the transcription is driven from between the LTR U3 and R elements. [START_REF] Kovalskaya | Functional human endogenous retroviral LTR transcription start sites are located between the R and U5 regions[END_REF] propose that a shift from the start site could be explained by the presence of at least two alternative promoters, one of which is used for viral gene expression, and the other for the transcription of retrotransposition-competent copies of the integrated provirus.

The 5′ UTR regions of LTR retrotransposons are multifunctional, and are involved in the transcriptional enhancement of the elements [START_REF] Wilson | An enhancer region within the copia untranslated leader contains binding sites for Drosophila regulatory proteins[END_REF][START_REF] Smith | The suppressor of Hairy-wing protein regulates the tissue-specific expression of the Drosophila gypsy retrotransposons[END_REF], in the isolation of transcriptional units from the neighboring regulatory elements by means of an insulator, such as in the Idefix element of Drosophila (Conte et al., 2002a), and in the interactions with the nuclear matrix via a scaffold-attachment region [START_REF] Nabirochkin | A nuclear matrix/scaffold attachment region co-localizes with the gypsy retrotransposon insulator sequence[END_REF] the Drosophila gypsy element). The 5′ UTR of Idefix in D. melanogaster has also been shown to exhibit internal ribosome entry site (IRES) activity that enables it to promote the translation of a downstream cistron in a capindependent manner [START_REF] Meignin | The 5′ untranslated region and Gag product of Idefix, a long terminal repeatretrotransposon from Drosophila melanogaster, act together to initiate a switch between translated and untranslated states of the genomic mRNA[END_REF]. Recently, an IRES upstream of each ORF has been found in the mouse L1 element [START_REF] Li | The dicistronic RNA from the mouse LINE-1 retrotransposon contains an internal ribosome entry site upstream of each ORF: implications for retrotransposition[END_REF].

Variability of the TE regulatory regions

The regulatory regions of retrotransposons are functionally very important. For instance, 10 point mutations in the U3 region of the human ERVWE1 element are enough to make 5′ and 3′ LTRs display significant different promoter activities [START_REF] Prudhomme | A retroviral promoter and a cellular enhancer define a bipartite element which controls env ERVWE1 placental expression[END_REF]. Despite this, regulatory regions of retrotransposons are often reported to be some of the most rapidly evolving regions in the sequence of the elements [START_REF] Jordan | Evidence for the role of recombination in the regulatory evolution of Saccharomyces cerevisiae Ty elements[END_REF]. In particular, the U3 sequence has been shown to be the most variable region of LTR retrotransposons, as for example in the tobacco Tnt1 element [START_REF] Casacuberta | Sequence variability within the tobacco retrotransposon Tnt1 population[END_REF]. The Retrolyc1 LTR retrotransposon from the plant Lycopersicon displays high levels of diversity in its U3 region, with a 53-bp motif repeated up to four times in tandem, the number of repeats having a significant impact on the transcription potential of the element [START_REF] Araujo | Retrolyc1 subfamilies defined by different U3 LTR regulatory regions in the Lycopersicon genus[END_REF].

In the 5′ UTR, the regulatory region of the copia LTR retrotransposon of Drosophila displays multiple copies of an 8-bp motif (TTGTGAAA), with similarity to the core sequence of the SV40 enhancer [START_REF] Mcdonald | LTR retrotransposons and the evolution of eukaryotic enhancers[END_REF]. Naturally-occurring variations in the number of these motifs are correlated with the enhancer strength of the 5′ UTR (Matyunina et al., 1996). Costas et al. (2001) PCR-amplified the 5′ LTR-UTR region of the blood LTR retrotransposon in some species of the D. melanogaster subgroup, and revealed length variability corresponding to a 49bp gap at the end of the 5′ LTR, and two gaps of 13 and 49 bp, respectively, in the UTR. Two classes of variants were subsequently identified, one of them being mainly represented by heterochromatic elements (L class), and the other corresponding to active, euchromatic elements (S class). In natural populations of D. simulans, the 412 LTR retrotransposon displays variability in sequences of its regulatory region, resulting from nucleotide substitutions, indels, and duplications of motifs, and also, in some populations, some chimerical elements, showing similarity to 412 in some regions and to mgd1 in others, were found (Mugnier et al., 2005).

All mammalian L1 LINEs have very similar coding regions, but unrelated 5′ UTRs and promoters. Five subfamilies have 87 M. Fablet et al. / Gene 390 (2007) [84][85][86][87][88][89][90][91] therefore been classified in the human genome, in which the most active copies are the most recent. In the human genome, even though L1 copies of different subfamilies all belong to the same lineage, there are striking differences in the transcription factor binding sites found in the 5′ UTRs. The active subfamily may have new binding sites that the pre-existing ones did not, such as the SRY-related transcription factor binding sites and the RUNX3 binding sites, which are essential for transcription activation and therefore allow the younger L1 copies to be active (Khan et al., 2006).

It is therefore obvious that diversity exists in the retrotransposon regulatory region, and many examples are found in the literature. We will now discuss the mechanisms that may have led to this enormous variability in regulatory region sequences, which are quite different between LTR retrotransposons and LINEs.

Evolution of the regulatory region

Retrotransposition, an error-prone mechanism

The lack of any proofreading/repair activity of RNA polymerase and reverse transcriptase means that the replication of retrotransposons (LTR and non-LTR) is a very error-prone mechanism. As a consequence, the replication of a single retrotransposon can generate a population of closely related, but not identical sequences resembling the 'quasi-species' populations of RNA viruses [START_REF] Domingo | The quasispecies (extremely heterogeneous) nature of viral RNA genome populations: biological relevancea review[END_REF][START_REF] Casacuberta | Sequence variability within the tobacco retrotransposon Tnt1 population[END_REF]. In particular, secondary structures and direct repeats, as well as runs of identical nucleotides, can considerably increase the mutation rate (Pathak andTemin, 1990, 1992). Repeated motifs are often bordered by runs of T's [START_REF] Mcdonald | LTR retrotransposons and the evolution of eukaryotic enhancers[END_REF], which are known to facilitate template slippages during reverse transcription [START_REF] Burns | High rates of frameshift mutations within homo-oligomeric runs during a single cycle of retroviral replication[END_REF]. [START_REF] Mcdonald | LTR retrotransposons and the evolution of eukaryotic enhancers[END_REF] have proposed that, since reverse transcription is prone to generating short regional duplications, which are characteristic of eukaryotic enhancers, LTR retrotransposons may play a role in the evolution of these enhancers.

Recombination in LTR retrotransposons

Recombination is a major mechanism generating variability in LTR retrotransposons. Frequent recombination events are known to occur between the two genomic RNA strands packaged within the LTR retroelement capsids [START_REF] Zhang | Retrovirus recombination depends on the length of sequence identity and is not error prone[END_REF]. [START_REF] Jordan | Evidence for the role of recombination in the regulatory evolution of Saccharomyces cerevisiae Ty elements[END_REF] found evidence for recombination by template switching between Ty1 and Ty2 elements of Saccharomyces cerevisiae. Template switching is thought to occur within the R region of the LTR, and these authors did indeed find elements containing hybrid LTRs, in which U3 regions displayed phylogenetic patterns different from those of the R and U5 regions. In natural populations of D. simulans, the regulatory region of some 412 elements has been found to result from a recombination of a 412-LTR and a mgd1-5′ UTR (Mugnier et al., 2005). Recombination has also been found in the BARE LTR retrotransposon in barley, involving BARE and Wis-2 elements [START_REF] Vicient | Variability, recombination, and mosaic evolution of the barley BARE-1 retrotransposon[END_REF]. In mice, recombination events between different ancient L1 copies have engendered new 5′ UTR active L1 copies. For instance, the G F subfamily possesses different monomers from different sequences of the F subfamily, and a new, non-monomeric region [START_REF] Goodier | A novel active L1 retrotransposon subfamily in the mouse[END_REF]. Modular evolution [START_REF] Lerat | Is the evolution of transposable elements modular?[END_REF], which results from recombination events occurring between the regulatory regions of two elements, could be a quick and efficient way to change expression patterns and the regulatory interacting system, thus allowing new variants to evade regulation by the host (Mugnier et al., 2005).

Recruitment of novel regulatory regions in LINEs

Due to their different retrotransposition mechanisms, LINEs are not affected by recombination the way LTR retrotransposons are. On the contrary, in the human L1 family, novel regulatory regions (5′ UTR) have frequently been recruited during the evolution of the family, whereas the two open reading frames have remained relatively conserved (Khan et al., 2006). The 5′ UTR of the ancestral L1 primate subfamily has been replaced at least eight times over the last 70 My. L1 families with different 5′ UTRs can coexist for a period of time without competing, because they do not rely on the same host-encoded factors for their transcription. The most powerful 5′ UTR would eventually generate a mobilization burst and would therefore give rise to a new L1 subfamily (Khan et al., 2006). Over the course of evolution, each of these subfamilies has been replaced by a new active one, and consequently all active L1 copies belong to a single lineage (Boissinot and Furano, 2005). The same life-cycle pattern has been identified in mice L1 elements, but several recombination events have complicated the phylogenetic tree [START_REF] Mears | The evolution of modern lineages of mouse L1 elements[END_REF]. In contrast to the situation in humans, active Drosophila LINE elements belonging to different lineages can be found coexisting at the same time. In the zebrafish genome, L1 elements also behave totally differently from their counterparts in the human genome: the 5′ UTRs have different sequences, and therefore the various L1 elements do not compete with each other for host transcriptional factors. The zebrafish genome thus contains more than 30 distinct L1 lineages [START_REF] Furano | L1 (LINE-1) retrotransposon diversity differs dramatically between mammals and fish[END_REF].

Some authors have also proposed that, since mammalian L1 elements have unrelated 5′ UTRs or promoters, it is possible that L1 promoters were originally derived from host genes located close to L1 insertion sites by a promoter-capturing mechanism [START_REF] Speek | Antisense promoter of human L1 retrotransposon drives transcription of adjacent cellular genes[END_REF].

Impacts on the host genome

Interference with host genome performance

Since the retrotransposon regulatory region displays promoter behavior, any insertion of a retrotransposon into the genome could affect the transcription of nearby host genes. In particular, the regulatory region could promote ectopic expression or simply modify the expression level of the host genes (Borie et al., 2000). For instance, it has been demonstrated that an LTR can bidirectionally promote the transcription of two human 88 M. Fablet et al. / Gene 390 (2007) 84-91 genes [START_REF] Dunn | Transcription of two human genes from a bidirectional endogenous retrovirus promoter[END_REF]. Ectopic transcription by L1 ASP can also be responsible for transcriptional interference and posttranscriptional silencing of cellular genes [START_REF] Nigumann | Many human genes are transcribed from the antisense promoter of L1 retrotransposon[END_REF]. van de Lagemaat et al. ( 2003) have illustrated that a major impact of TEs is their ability to induce changes in gene regulation without destroying existing gene functions. In particular, these authors showed that TE regulatory regions act as alternative promoters of many genes, therefore modifying expression patterns or tissue-specificity. LTR elements, which carry more transcription regulating signals than LINEs, are also less often found in gene promoter regions, probably because a high number of regulatory signals are more likely to alter gene expression in a greater extent and have deleterious effects [START_REF] Thornburg | Transposable elements as a significant source of transcription regulating signals[END_REF]. Most of the genes exhibiting TE regulatory regions in their promoters are involved in functions such as the stress response, immunity, and response to external stimuli, whereas genes playing a role in development and metabolism are less likely to have TE promoters inserted within their UTRs. In addition, [START_REF] Mariño-Ramírez | Transposable elements donate lineage-specific regulatory sequences to host genomes[END_REF] have shown that the regulatory sequences contributed by TEs are exceptionally lineage specific, suggesting that TEs can drive the diversification of gene regulation between evolutionary lineages. TEs can interfere with host genes, not only by promoting their transcription, but also by introducing into their 5′ UTRs novel regulatory sequences, which may act as enhancers or insulators, as in the case of the D. melanogaster elements ZAM and Idefix, respectively, which modify the proper regulation of the white gene in the eyes (Conte et al., 2002a).

Promoter competition between host genes and retrotransposons has also been shown to be a mechanism of transcriptional interference, disrupting endogenous enhancer-promoter communications (Conte et al., 2002b). The rapid evolution of the regulatory region of TEs can therefore have a wide panel of significant effects on the way the genome works.

Inter-element competition and new waves of retrotransposition

Generation of new variants of retrotransposon regulatory regions is a mechanism by which competition between elements can occur, the genome being invaded by the 'best-adapted' element. There can also be competition between different groups of copies (highly active, less active, non-autonomous) without elimination of the less active or non-autonomous elements (Fig. 3) [START_REF] Deceliere | The dynamics of transposable elements in structured populations[END_REF][START_REF] Le Rouzic | The first steps of transposable elements invasion: parasitic strategy vs. genetic drift[END_REF]. Variants of the 1731 retrotransposon in D. melanogaster, which display both extended transcriptional profiles due to changes in the LTR sequence, and altered translational strategy due to loss of frameshifting, have supplanted the more ancient forms [START_REF] Kalmykova | Selective expansion of newly evolved genomic variants of retrotransposon 1731 in the Drosophila genomes[END_REF]. Costas et al. (2001) found two classes of variants of the regulatory region in the blood LTR retrotransposon, which they suggest may compete with each other. The active blood subfamily (S) transposes at a faster rate, and therefore survives and propagates, whereas the other subfamily (L) is condemned to disappear or to remain confined within the heterochromatin. Inter-element competition has also been postulated for the two subfamilies of the tirant LTR retrotransposon in D. melanogaster and D. simulans: one subfamily, the C-type, is active and has invaded the chromosome arms, whereas transcription is impeded in another subfamily, the S-type, which is confined to the heterochromatin and has a very low copy number (Fablet et al., 2006). Host genomic constraints also interfere with inter-element competition, since, unlike D. melanogaster and D. simulans, D. mauritiana and D. sechellia do not display any expansion of the S subfamily of blood in their genomes (Costas et al., 2001). To generate new characters in this inter-element competition, inter-element recombination may, for instance, be an effective strategy by which retroelements can rapidly evolve novel regulatory sequence combinations (Jordan Fig. 3. The influence of regulatory sequences in the TE life cycle. The black curve represents the evolution of TE copy number in a given host genome. TE regulatory sequences can be subject to negative control by the host genome, leading to a decrease in TE copy number and even TE extinction. Depending on its regulatory mechanisms and the host response, a TE can display waves of increasing and decreasing copy number. The evolution of the regulatory region may allow the TE to invade a new host, in which the TE will once again be subjected to successive waves of increasing and decreasing copy number. and McDonald, 1998). What has happened to the L1 element in the human genome is an extreme form of this inter-element competition, since virtually only one lineage is active at a time (Khan et al., 2006).

The rules governing the inter-element competition are mainly determined by the host. A high degree of variability within transcriptional regulatory regions could therefore allow retrotransposons to explore transcriptional patterns in order to facilitate their coexistence with their host genome. Consistent with this hypothesis, Tnt1 elements within various plant host species tend to develop different U3 regulatory sequences that are adapted to each host genome [START_REF] Araujo | Retrolyc1 subfamilies defined by different U3 LTR regulatory regions in the Lycopersicon genus[END_REF]. Cooperation between cellular and retroviral elements is another way of creating variability in the regulation of a retrotransposon. One unusual example is that of the ERVWE1 element, the expression of which is regulated by a bipartite element consisting of a cyclic AMP-inducible LTR retroviral promoter adjacent to a cellular enhancer conferring a high level of expression and placental tropism [START_REF] Prudhomme | A retroviral promoter and a cellular enhancer define a bipartite element which controls env ERVWE1 placental expression[END_REF].

The innovation race of the regulatory region engenders a close relationship between the host factors and its transposable elements. This can result either in an adaptation between the transposable elements and their host genome, or a new burst of transposition and genome invasion. But according to the genome and type of retrotransposon considered (LTR/non-LTR), the modalities and outcomes of these evolutionary mechanisms are quite different.

CONCLUSION

In this bibliographic work, we have shown that retrotransposons differ in their regulatory sequences (from LTR elements to LINE-like elements) and, as a consequence, impact differently the genomes in which they reside. Also, we have shown that similar elements behave differently according to the species analysed. All these data suggest that a specific relationship exists between TEs and each invaded genome. Several parameters might influence the genome/TE relationship : TE families, species genomes, environment and ecological traits are described to influence TE/genome relationship [START_REF] Waterland | Transposable elements: targets for early nutritional effects on epigenetic gene regulation[END_REF]2004;Clark et al., 2007;[START_REF] Dramard | Natural epigenetic protection against the I-factor, a Drosophila LINE retrotransposon, by remnants of ancestral invasions[END_REF][START_REF] Carbone | Evolutionary breakpoints in the gibbon suggest association between cytosine methylation and karyotype evolution[END_REF]. Therefore, one way to understand such specificity is to decrease the number of different parameters involved in the equation TE/genome. The study of natural populations belonging to the same species decreases the disparities between genomic environments, since higher genetic homologies can be observed intra-species than between species, and allows for a simpler view of TE/genome interactions. D. melanogaster and D. simulans, as described above, have different amounts of repeats and TEs (~15% and ~5% of TEs respectively) [START_REF] Dowsett | Differing levels of dispersed repetitive DNA among closely related species of Drosophila[END_REF]Vieira et al., 1999). Such pattern can be observed among TE families that are often overrepresented in D. melanogaster natural populations (Vieira et al., 1999). There are a few elements that are outnumbered in natural populations of D. simulans and therefore considered as exceptions.

One of them is the LINE-like element helena. We decided to make a survey of this uncommon helena element in natural populations of both D. melanogaster and D. simulans.

The goal of this study is firstly to understand two different behaviours of one same element in two close species. Secondly, the use of natural populations allows us to test intra-specific natural variation in TE regulatory mechanisms. Surprisingly, the supposed active helena element in D. simulans natural populations is the target of an important internal deletion mechanism. Such system seems to be absent from D. melanogaster natural populations, and unpublished data by E. Lerat generalize it to most of the other D. simulans elements.

Background

Genome evolution occurs by several processes, including global genome duplications, segmental duplications and the amplification/deletion of repetitive sequences. Among the repeated sequences, transposable elements (TEs), which constitute a high proportion in many genomes, play an important role in genome evolution [1]. The transposition rates of these TEs depend on the amount and type of the TEs present in the genome; they are not constant over time, but are subject to amplification bursts in certain species and populations [2]. As a result, genomes contain widely differing amounts of TEs that are not directly correlated to their activity levels. For instance, the human genome is composed of at least 50% of TEs, but only very few are active, and they are responsible for less than 1% of mutations [3]. In contrast, in Drosophila melanogaster, only 18% of the genome is composed of TEs, but a high proportion of mutations (more than 50%) is attributable to their transposition [4].

A TE life cycle can be viewed as successive waves of transposition/loss: invasion of the host genome by TEs being followed by their progressive elimination [5,6]. For example, the LINE-1 (L1) element has colonized the entire human genome by successful waves of transposition [2,7], and today it is the most abundant TE family in this genome. However, in humans, most of the elements have been inactivated either by structural changes or by epigenetic control, such as DNA methylation [7]. In D. melanogaster, the I factor has recently reinvaded this genome after being lost from the chromosome arms [8]. TE elimination from genomes is therefore a commonly observed phenomenon, although no real-time observation of a TE extinction has ever been reported. As TEs have a considerable influence on remodeling the genome structure [9], we need to understand the dynamics of changes in their copy numbers. One way to investigate these dynamics is to analyze closely related species with differing TE amounts, such as D. simulans and D. melanogaster. These species diverged 2 to 3 million years ago [START_REF] Bie ´mont | Worldwide distribution of transposable element copy number in natural populations of Drosophila simulans[END_REF] and have differing proportions of TEs: D. melanogaster contains more than 18% of TEs, whereas D. simulans contains only 5% [START_REF] Charlesworth | The distribution of transposable elements within and between chromosomes in a population of Drosophila melanogaster. III. Element abundances in heterochromatin[END_REF].

D. simulans has fewer copies of most TEs [START_REF] Charlesworth | The distribution of transposable elements within and between chromosomes in a population of Drosophila melanogaster. III. Element abundances in heterochromatin[END_REF], but there are a few exceptions. The DNA-transposon hobo is more abundant in D. simulans, the retrovirus-like gypsy and ZAM elements have the same low number of copies in both species, and the LINE-like element helena is present in the D. simulans genome (10 insertion sites as determined by in situ hybridization), but has not been detected in the chromosome arms of D. melanogaster [START_REF] Charlesworth | The distribution of transposable elements within and between chromosomes in a population of Drosophila melanogaster. III. Element abundances in heterochromatin[END_REF][START_REF] Junakovic | Accumulation of transposable elements in the heterochromatin and on the Y chromosome of Drosophila simulans and Drosophila melanogaster[END_REF]. The striking distribution of helena in natural populations of these two species, and the fact that degenerated copies are found in the sequenced D. melanogaster genome, make this LINElike element an ideal model system to study the real time TE life cycle. Petrov and colleagues [START_REF] Vieira | Wake up of transposable elements following Drosophila simulans worldwide colonization[END_REF] proposed that deletions are common events in Drosophila, and based this suggestion on the analysis of partial helena sequences from different Drosophila species. However, it is difficult to extrapolate this to other TEs, if we take into account the fact that helena is one of the few degenerate TEs in the D. melanogaster sequenced genome [START_REF] Fablet | Ongoing loss of the tirant transposable element in natural populations of Drosophila simulans[END_REF]. Comparing closely related species with differing TE amounts, could be used to test the importance of this deletion process in regulating TE genome invasions.

We analyzed the structure and activity of helena using the sequenced genomes and of 41 natural populations of D. melanogaster and D. simulans. We show that the elimination of helena from its host genomes is a very quick process, and that it is mediated by massive internal deletions in the element [START_REF] Barolo | GFP and beta-galactosidase transformation vectors for promoter/enhancer analysis in Drosophila[END_REF]. We conclude that the process of elimination of helena is far advanced in D. melanogaster. but is still in progress in D. simulans.

Results

In silico identification of a complete copy of helena in the D. simulans genome Because no full-length copy of helena had previously been described, we performed a bioinformatic search for such a copy in the draft sequence of the D. simulans genome [16]. We found a 4912-bp copy of helena on the chromosome arm 3R (at position 1506433 -1511368 on the minus strand) (Figure 1). Helena belongs to the jockey clade [START_REF] Lerat | Sequence divergence within transposable element families in the Drosophila melanogaster genome[END_REF], has a 25-bp poly A tail, and two overlapping open reading frames (ORF1 and ORF2). The first ORF is 1737-bp and codes a 579-amino acid (aa) protein that has high similarities to the gag protein of other LINE-like elements, such as X, jockey and HeT-A [START_REF] Altschul | Gapped BLAST and PSI-BLAST: a new generation of protein database search programs[END_REF][START_REF] Felsenstein | PHYLIP-Phylogeny Inference Package (Version 3.2)[END_REF]. The gag-like protein contains the major homology region (MHR), followed by a cysteine-rich domain (CX 2 CX 4 HX 4 C, CX 2 CX 3 HX 4 C, CX 2 CX 3 HX 6 C). This region is common to all gag-like proteins, and confers an RNA or DNA single-strain binding property on these elements, as well as being essential for gag oligomerization. Helena has a coiled-coil domain located in the 5' region of the gag protein, something that had previously only been seen in L1 elements from mammals [START_REF] Bonnivard | Characterization of natural populations of Drosophila melanogaster with regard to the hobo system: a new hypothesis on the invasion[END_REF] and in some LTR retrotransposons from Drosophila [START_REF] Kaminker | The transposable elements of the Drosophila melanogaster euchromatin: a genomics perspective[END_REF]. The second ORF, which starts on the last base of ORF1, is 2721-bp, and codes a 907-aa protein corresponding to the pol gene, which is very similar to the protein of the BS and jockey elements. The pol-like protein contains all the domains necessary for its function: an apyrimidic endonuclease and an exonuclease (from amino acid 4 to 221), plus a reverse transcriptase domain (from amino acid 493 to 746). Both ORFs are intact, could produce transcripts, and are surrounded by two untranslated regions (5'UTR and 3'UTR respectively). Because the regulatory region is often defined in the 5'UTR [START_REF] Kapitonov | Molecular paleontology of transposable elements in the Drosophila melanogaster genome[END_REF], we performed a bioinformatic search for transcription factors binding sites in this region. A single region was detected containing several transcription factor binding sites, such as SP1 and upstream stimulating factor-like (USF) binding domains. This region also displays a binding site for a TATA-binding protein (TBP), and an estrogen response element (ERE).

The copies of helena in the sequenced genomes of D. melanogaster and D. simulans Using the complete sequence of helena as a query, we found 62 helena sequences in the D. simulans genome (see Additional File 1 for details). Twenty-eight of these copies were located on the chromosome arms, and the remaining 34 were in the U part of the genome, that may correspond to heterochromatin. The copies ranged in size from 107 bp to 5098 bp. However, it is difficult to determine the exact size of some copies due to the presence of numerous undetermined bases. Two copies (chr2R_13305831 and chrX_16602314) were longer than the reference sequence due to insertions. In some other cases, we may be looking at fragments of the same copy; however, the distances that separate them are too large to allow us to find out with certainty whether they come from the same copy. The estimated number of 62 copies in D. simulans may therefore be an overestimation. The average percentage identity is 96.1% for all copies, with an average of 97.4% for the copies in the euchromatin, and of 94.9% for the copies in the U part.

In the sequenced genome of D. melanogaster, we found 26 copies of helena (see Additional File 2 for details), which ranged in size from 91 bp to 4805 bp. The average percentage identity was 80.4% for all copies, with an average of 78.7% for the copies in the chromosome arms, and of 83.7% for the copies located in the U part. Most of the copies in this genome have therefore been degraded, with numerous internal deletions or insertions. All copies are truncated on the 5' side, and are DOA (Dead on arrival) copies, apart from the 3L_23487977 copy, although even this displays some internal deletions.

We analyzed in greater detail any copies that could correspond to the most recent insertions in both D. melanogaster and D. simulans. We used specific blast criteria to identify these copies: we selected matches with at least 90% identity, and a length at least 50% of that of the complete copy, with e-values of less than 10e-10 (Figure 2). In D. melanogaster, only the 3L_23487977 copy described above met all the blast criteria. It is obviously an inactive copy, since more than four deletions were detected within its sequence. In D. simulans, six copies were found that matched the blast criteria, including the complete copy on the 3R chromosome (position 1506433 -1511368 on the minus strand); the other five copies had internal deletions, and insertions were detected in four of them.

Chromatin localization of helena copies in natural populations

We used in situ hybridization to estimate the number of helena insertion sites located on the arms (euchromatin) of the polytene chromosomes from salivary glands of both D. melanogaster and D. simulans. Both species had centromeric staining, but only D. simulans from natural populations presented euchromatic bands (mean copy number 10.7 ± 2.2) with no fixed sites (see Additional File 3 for details on insertion sites per population). With this experiment we did not detect any insertions of helena in the chromosome arms, which could be explained by the short size of the elements and the divergence to the probe used.

Inter-population polymorphism

We analyzed the inter-population helena copy number polymorphism by Southern blot, using a restriction enzyme that does not cut inside the element. This method detects both heterochromatic and euchromatic sequences. As shown in Figure 3, D. melanogaster had 8 to 11 bands per population, and several bands were shared by different populations. These copies could correspond to ancient and fixed heterochromatic copies in the D. melanogaster genome. D. simulans populations contained numerous helena copies (19 to 30 copies per population) with a high level of insertion polymorphism. Since the enzyme used for the Southern blot did not cut inside the element, all bands over 4.5 kb could correspond to a complete element. Because both species harbored bands over 4.5 kb, they could have full-length helena copies.

PCR screening

Three sets of primers were used to amplify the whole ORF1 and two fragments of the ORF2. No bands corresponding to the ORF1 were observed in any of the D. melanogaster natural populations in agreement with the absence of this ORF in the sequenced genome. There was a high level of size polymorphism for the D. melanogaster ORF2, corresponding to the different internal deletions already analyzed by Petrov et al. [13,[START_REF] Eickbush | Vertical transmission of the retrotransposable elements R1 and R2 during the evolution of the Drosophila melanogaster species subgroup[END_REF]. In contrast, D. simulans displayed low size polymorphism for helena ORF1 and ORF2. Indeed, only one population out of Scheme of helena copies Figure 2 Scheme of helena copies. Representation of helena copies in the D. melanogaster and D. simulans genomes with at least 90% identity and 50% of the length of the complete copy, and with e-values of less than 10e -10 ; Triangles = insertions; Spaces = deletions.

twenty had two sets of ORF1 (Papeete). All D. simulans populations had two to three sets of ORF2.

Analysis of helena copies in D. simulans populations PCR fragments obtained from the D. simulans population screening mentioned above were cloned and sequenced. Surprisingly several common indels were detected at the same positions in different sequences from all the populations analyzed. Phylogenetic reconstructions based on the ORF1 and ORF2 (Figures 4 and5, alignments from Additional Files 4 and 5) showed that the sequences that displayed the same indels are grouped in the tree. This suggests that the deletion or insertion events were produced before the amplification of these sequences. Some copies had only a few insertions, and might be inactive since their reading frames were not preserved. However, the amplification of some of these copies could have been promoted in trans. We did not find any population that had both complete ORFs. Nevertheless, some ORFs had no internal stop codons in their sequence, suggesting that copies bearing them could be active despite the deletions.

The percentage identity between the reference copy and ORF1 ranges between 96% and 99%, meaning that these copies have not diverged much. No relationship was detected between the size and location of the deletions, and the percentage identity. For the ORF2, we found copies with a percentage identity of more than 93% that reached 100% for some copies. A common 401-bp deletion was found in copies with 93% identity with the com-plete helena copy, but no correlation was observed with the percentage identity for the other deletions or insertions. Based on the age estimation of each copy, we found that most of the oldest ORF2 fragments had the 401-bp deletion. Several young copies of both ORF1 and ORF2 displayed major internal deletions, showing that the mechanism leading to these deletions is much more powerful than copy divergence in inactivating them.

Transcript analysis in D. simulans

To test the transcriptional potential of helena in D. simulans, we performed northern blot and RT-PCR in various populations (see Materials and Methods). Since the sequenced genome was obtained from strains from North America, we added three populations from this continent (San Antonio, SW3-S2 and San Diego). No transcripts were found by Northern blot in any of the populations. However, the RT-PCR method detected transcripts of both ORF1 and ORF2 in the Valence population, an extremely low signal for ORF1 and ORF2 in three of the American populations, and ORF2 transcripts in the Amieu population. None of the other populations had any transcript for helena, implying that this element is extinct in these populations. Since the Northern blot technique is less sensitive than RT-PCR, these findings suggest that helena is transcribed at extremely low levels in the populations in which some transcripts were detected by RT-PCR.

Discussion

Our in silico and experimental analyses of the D. melanogaster genome show that helena copies are mostly DOA, devoid of ORF1, and therefore unable to transpose autonomously. All these features have been associated with elements that are in the process of inactivation [START_REF] Bonnivard | Characterization of natural populations of Drosophila melanogaster with regard to the hobo system: a new hypothesis on the invasion[END_REF]. The scenario for the helena copies in D. simulans is quite different. Analysis of the sequenced genome of this species allowed us to identify a full-length copy of helena with the structures required for an active element: two intact ORFs, a poly-A tail, and regulatory regions. The high level of insertion polymorphism detected in the natural populations suggests that helena is an active element or has been active recently. However, sequence analysis of the two ORFs of helena in the natural populations revealed two main points: first, both ORFs are intact in only very few populations; second, even though the sequences of helena are very similar at the nucleotide level, their deletion features differ.

The first point was strengthened by the almost total absence of helena transcripts in all natural populations of D. simulans, which means that very few copies were involved in transcription. Because a single master copy is enough to maintain TE transposition [START_REF] Busseau | Identification of Waldo-A and Waldo-B, two closely related non-LTR retrotransposons in Drosophila[END_REF], the putative activity of helena in this species could reside in the fulllength copy probably present in some populations such as Valence, where we did observe transcripts. Hence, we would expect sequences that are still similar at nucleotide level to differ only in the 5' end truncation size, as usually observed for LINE-like elements. However, as mentioned in the second point, we actually observed many other kinds of internal deletions that occurred throughout the length of the element. Intriguingly this deletion-promoting process appears to be quite powerful in inactivating the elements, and could be even more powerful than other mutation processes such as point mutations. This means that a real-time loss of helena is ongoing in all D. simulans populations.

The nature of the mechanisms leading to internally deleted copies is still unknown. In humans, LINE elements can be spliced [START_REF] Ebert | Histone modification and the control of heterochromatic gene silencing in Drosophila[END_REF], a process that creates internal deletions. We used bioinformatic analyses, but were unable to find splice sites in the full-length helena sequence. Also, although recombination between mRNAs can produce internal deletions [START_REF] Kim | Retroviruses in invertebrates: the gypsy retrotransposon is apparently an infectious retrovirus of Drosophila melanogaster[END_REF], helena sequences are not sufficiently divergent to allow us to infer the origin of a single copy.

Helena appears to be extinct in D. melanogaster, and this recalls the I element, which also disappeared from the D. melanogaster genome in the past, and reinvaded it only recently [START_REF] Desset | Mobilization of two retroelements, ZAM and Idefix, in a novel unstable line of Drosophila melanogaster[END_REF]. The I and helena elements are both LINE-like phylogenetic tree of the DNA sequences from the ORF2 region Figure 5 phylogenetic tree of the DNA sequences from the ORF2 region. The reconstruction was performed on the cloned DNA sequences of the ORF2 region from the different populations of D. simulans and from some sequences detected in the sequenced genome (we eliminated sequences that were too short relative to the global length of the alignment). Colored boxes represent sequences harboring common patterns of deletions and/or insertions. All the positions are given relative to the complete copy. Green box:sequences display the same 4-bp deletion (at position 547) and the same 2-bp insertion (at position 112). Dark blue box: sequences display the same 1-bp deletion (at position 521). Yellow box: sequences display the same deletions of 3 bp (at position 143), 8 bp (at position 335), 4 bp (at position 345), and an insertion of 2 bp (at position 460). Light blue box: sequences display the same 401-bp deletion (at position 179). Orange box: sequences with no deletion or insertion, very closely related to the complete copy chr3R_1506433. Black asterisks correspond to bootstrap values greater than 50%. elements, leading us to wonder whether amplification/ loss of copies could be a characteristic of this type of element. Waves of amplification/loss have been observed in humans, where only the youngest L1 subfamily is active, perhaps as a result of competition between different L1 subfamilies [2,7].

Our data show that helena has been almost entirely removed from the D. melanogaster genome, and was not subjected to the recent wave of transpositions reported for other elements [START_REF] Fablet | Ongoing loss of the tirant transposable element in natural populations of Drosophila simulans[END_REF]. In D. simulans, we did observe an insertion site polymorphism of helena, but this corresponds to copies that are being internally deleted by an efficient mechanism. This may be generalized on the basis of data on the LTR retrotransposons 412 and tirant, which have also internal deletions [START_REF] Desset | COM, a heterochromatic locus governing the control of independent endogenous retroviruses from Drosophila melanogaster[END_REF][START_REF] Prud'homme | Flamenco, a gene controlling the gypsy retrovirus of Drosophila melanogaster[END_REF]. We still do not clearly understand which mechanisms lead to a low copy number in the D. simulans genome, but a mechanism promoting internal deletions could be a major force at work [START_REF] Strohner | Recruitment of the nucleolar remodeling complex NoRC establishes ribosomal DNA silencing in chromatin[END_REF].

Conclusion

TEs are major players in genome evolution, and the way they are controlled by the host genome is one of the most fundamental questions in evolutionary genomics. Here we show that two closely related species of drosophila have a TE family at different stages in its life cycle. The mechanism by which this is achieved in D. simulans implies that a very efficient internal deletion mechanism is acting on TEs, which is more powerful than the simple neutral evolution of non-active elements. The difference in the amount of TEs between D. melanogaster and D. simulans could be explained by such a process, that doesn't seem to be very active in D. melanogaster present populations.

Methods

Natural populations

We worked on fly samples collected from several geographically-distinct, natural populations (confer each Method for natural populations investigated). These populations were maintained in the laboratory as isofemale lines or small mass cultures with around 50 pairs in each generation.

In situ hybridization

Polytene chromosomes from salivary glands of thirdinstar female larvae were prepared and treated with nicktranslated, biotinylated DNA probes, as previously described [START_REF] Tulin | The Drosophila heterochromatic gene encoding poly(ADP-ribose) polymerase (PARP) is required to modulate chromatin structure during development[END_REF]. Insertion sites were visible as brown bands resulting from a dye-coupled reaction with peroxidase substrate and diaminobenzidine. The insertion site numbers of the TE(s) were determined on all the long chromosomes arms (X, 2L, 2R, 3L, 3R), and were summed to give the total number of labeled sites per diploid genome. We did not take into account the insertions located in pericentromeric regions 20, 40, 41, 80, and 81, because TE site number estimations in these regions are difficult and not reliable for all chromosomes or all squashes. We used a probe (1278 bp) from helena of D. sechellia (AF012044). The following populations of D. melanogaster were investigated: Portugal (Chicharo), Saudi Arabia, Congo (Brazzaville), Reunion Island, Argentina (Virasoro), Bolivia, China (Canton), Vietnam, and Iso line. The D. simulans populations analyzed were from France (Valence), Russia (Moscow), Kenya (Makindu), Zimbabwe, Reunion Island, Australia (Eden, Cann River and Canberra), French Polynesia (Papeete), New Caledonia (Amieu).

Southern blot

DNA was extracted from one or five adult females by a standard phenol-chlorophorm-salt method with proteinase K digestion. The D. melanogaster populations analyzed were from Bolivia, Congo (Brazzaville), China (Canton), Portugal (Chicharo), Reunion Island, Saudi Arabia, Argentina (Virasoro), Vietnam, and ISO line. The D. simulans populations analyzed were from New Caledonia (Amieu), Australia (Eden, Cann River and Canberra), France (Valence), French Polynesia (Papeete), Russia (Moscow), Kenya (Makindu), Zimbabwe, and Reunion Island. The DNA was cut using the HindIII enzyme, which has no restriction site within the helena sequence, and therefore allowed us to estimate the number of complete helena copies. Electrophoresis of a 0.8% agarose gel containing digested DNA was carried out for 17 h. The DNA was denatured (NaOH 0.5 M), and then transferred overnight to a Hybond-N+ nylon membrane. Pre-hybridization and hybridization were carried out at 67°C using a Denhardt 5× solution. The probe used for hybridization (AF012044) was radiolabeled with 32 P, using a random procedure from Amersham.

Amplification of ORF1 and ORF2

DNA was extracted from single flies by a standard phenolchlorophorm method. The following populations of D. melanogaster were investigated: France (Valence and Saint Cyprien), Portugal (Chicharo), Saudi Arabia, Senegal, Congo (Brazzaville), Reunion Island, Guadeloupe, Argentina (Virasoro), Bolivia, and China (Canton). The D. simulans populations analyzed were from France (Valence), Russia (Moscow), Egypt (Tanta), Congo (Brazzaville), Kenya (Meru, Kwalé and Makindu), Zimbabwe, Tanzania (Arusha), Puerto Rico, Japan, Australia (Eden, Cann River and Canberra), French Polynesia (Papeete), Saint Martin, Hawaii, New Caledonia (Amieu), and Portugal (Madeira).

PCR was run using 1 Pg DNA with the two following primers -ORF1: H1for (285 5' AAC TGT AAA ATG GAT ACG AAC A 3' 306), H1rev (1808 5' GCC ACT TCA TAA ATT GTT CC 3' 1827). -ORF2: Hel2F (2325 5' CCG GGC TGG GCG ATA TGG 3' 2342), Hel2R (4548 CGT ACA TAC CAG GGG CAG TTG G 3' 4569). PCR was run in 30 cycles with annealing temperatures of 57°C (ORF1) and 56°C (ORF2). We used Euroblue taq from Eurobio. DNA amplified fragments were purified and cloned on competent bacteria (Qiagen kits). Four primers were used for sequencing: M13 forward and reverse; Seq1 (5' CTC TTC CTT CAT TTG GTA CG 3') and Seq2 (5' AAG GGG AAA CAG TGA GAA TA 3') for the complete ORF1; Seq3F (5' TTA GAC CAT GCT CTC GGT TA 3') and Seq3R (5' TGT CAA TTC CTG GAG CTT TA 3') for a fragment of ORF2. Sequencing was performed by Genome Express. Accession numbers (Genbank) from EU168807 to EU168844 correspond to ORF1 fragments. Accession numbers (Genbank) from EU170377 to EU170431 correspond to ORF2 fragments.

RT-PCR

Total RNA was extracted from four adult females, four adult males, 10 ovaries and 10 testes from D. simulans populations (France (Valence), Congo (Brazzaville), Kenya (Makindu), Zimbabwe, Australia (Canberra), New Caledonia (Amieu), Portugal (Madeira), United States (San Antonio, San Diego, Arena, SW3)) with the RNeasy protect mini kit from Qiagen. RNA extracts were treated with the Ambion's DNA-free kit. ThermoScript RT-PCR system from Invitrogen was used to synthesize four different cDNA pools (55°C for 90 min and 85°C for 5 min): a control reaction with no retrotranscriptase to test DNA contamination, a pool of total cDNA synthesized with oligo-dt primers, two specific cDNA pools obtained with H1R (ORF1) and Hel2R (ORF2), respectively, corresponding to helena transcripts. All four cDNA pools were tested for the presence of actin cDNA (house keeping gene) by PCR with Act5cfw (5'ATGTGACGAAGAAGTTG3') and Act5cRv (5'TTAGAAGCACTTGCGGTGCA3') primers. Oligo-dt and specific helena cDNA pools were analyzed by PCR using ORF1 and ORF2 specific primers (H1R/H1F and Hel2R/Hel2F).

Northern blot

Total RNA was extracted from adult females or embryos from several D. simulans populations (Valence, Makindu, Amieu, Brazzaville) with the RNeasy protect mini kit from Qiagen. Total RNA extracts were treated with the Ambion's DNA-free kit. Electrophoresis (MOPS, formaldehyde gel) was run for 3 h after RNA denaturing. After washing (water and NaOH, 75 mM) RNA was passively transferred to a nylon membrane, and cross linked for 2 hours at 64°C. Blots were pre-hybridized in hybridization buffer, then hybridized overnight at 42°C in hybridization buffer containing a 32 P-labeled helena cDNA probe. The radiolabeled cDNA probe was prepared using a Meg-aprime DNA Labeling Kit according to the manufacturer's protocol (Cat # RPN 1607; Amersham Biosciences, Little Chalfont, Buckinghamshire, England). Following hybridization, blots were washed in 2 × SSC/0.1% SDS at 42°C and then exposed to X-ray film (KODAK).

Identification of helena copies in the complete genomes

We retrieved the sequences of the chromosome arms 2L, 2R, 3L, 3R, 4, X and the unassigned part (named U) corresponding to the first release of the mosaic assembly of the genome of D. simulans available at the ftp site of the Genome Sequencing Center at the Washington University Medical School [START_REF] Pietersen | Stem cell regulation by polycomb repressors: postponing commitment[END_REF]. This mosaic assembly corresponds to different strains of D. simulans. We also used the sequenced genome of D. melanogaster [START_REF] Matyunina | Naturally occurring variation in copia expression is due to both element (cis) and host (trans) regulatory variation[END_REF]. We will refer to the helena copies found in the genomes according to the chromosome name and the start position of the copy (for example chr2L_133500 corresponds to a copy found on the 2L chromosome, and it starts at position 133500).

The helena element was only found in the databases as fragments of the reverse transcriptase (RT). We retrieved the longest sequences from D. yakuba (accession number in Genbank AF012049), D. melanogaster (AF012030) and D. virilis (U26847) to build a chimeric, 1532-bp sequence. Using this chimeric element we searched for copies in the D. simulans sequenced genome using blastn [START_REF] Costas | Amplification and phylogenetic relationships of a subfamily of blood, a retrotransposable element of Drosophila[END_REF]. The reconstructed helena sequence (ID Helena_DS) is available in Repbase [START_REF] Mugnier | New regulatory regions of Drosophila 412 retrotransposable element generated by recombination[END_REF]. Only matches with an e-value of less than 10e -10 have been retained, and any separated by distances of less than 300 bp have been merged. As the query used corresponds to a small portion of the ORF2, in order to search for longer sequences of helena, we retrieved the matches after adding 5000 bp around their positions. We then performed multiple alignments of these sequences using clustalw [START_REF] Vernhettes | The evolutionary analysis of the Tnt1 retrotransposon in Nicotiana species reveals the high variability of its regulatory sequences[END_REF] in order to detect the longest copies. By this procedure, we identified a sequence on the chromosome 3R that was the longest of the matches detected. The prediction of potentially coding parts was made using the ORF finder program available on the NCBI web site [START_REF] Charlesworth | The population genetics of Drosophila transposable elements[END_REF], and this allowed us to identify two ORFs. It was not possible to use the presence of target site duplication to determine the exact position of the beginning of the sequence, because the copy was surrounded by unidentified bases, and so we performed a blast search in the draft sequence of D. sechellia, the closest relative to D. simulans. This allowed us to find a homologous copy, and to identify the beginning of the complete copy of helena. Once this copy had been identified, it was used as a query to perform blast searches in the D. simulans and D. melanogaster genomes to determine the helena copy populations.

Sequence analysis

The computation of the percentage identity was performed using the DNADIST program in the PHYLIP package [START_REF] Daniels | The distribution of P-element sequences in Drosophila: the willistoni and saltans species groups[END_REF]. We used the sequence editor Seaview [START_REF] Hoskins | Primers used to amplify each individual insertion of tirant in the genome of natural populations. References Analysis of the genome sequence of the flowering plant Arabidopsis thaliana[END_REF] to visualize the sequences and the alignments. Splice sites and transcription binding sites were predicted by the Softberry tools [40] and Genomatix [41]; PEPcoil ([42] allowed us to find the coiled coil domain in ORF 1. Conserved domains in both ORF1 and ORF2 were predicted with the "Conserved domain search" tool from NCBI. Sequenced copies were aligned with T_coffee [43]. Phylogenetic analysis were made using maximum likelihood with HKY substitution model implemented in PhyML [44]. The reconstruction was performed on the cloned DNA sequences of the ORF1 and ORF2 region from the different populations of D. simulans, and from some sequences detected in the sequenced genome (we eliminated sequences that were too short relative to the global length of the alignment).

Age was estimated using the Bowen and McDonald method [45] with the formula Age = K/(2r), where K is the divergence between the two copies calculated from the Kimura two-parameter distance via DNAdist, and r is the synonymous substitution rate per site per million years in D. melanogaster (r = 0.016 from Li [46]). It is important to note that the age of helena copies is underestimated due to the lack of knowledge about conversion and substitution rates in D. melanogaster genome, and is also unreliable when applied to old and highly diverged copies.

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Sir Paul Nurse, Cancer Research UK Your research papers will be: ------------------------------------------------------------Eden_3 ------------------------------------------------------------Cann-River_9 ------------------------------------------------------------Amieu_2 ------------------------------------------------------------Makindu_2 ---------------------------------------------------------ATG Makindu_6 ---------------------------------------- -----------------------------------------------------------chrU_2316095 ------------------------------------------------------------chrU_4537377 ------------------------------------------------------------chrU_5384045 ------------------------------------------------------------chrU_5680622 ATGGATACGA ACAACGAAAC TTCTAACTCT CAGCTTA--G AAATAAGCCG ATTGATTATG chrU_5950518 - -----------------------------------------------------------chrU_6132643 ATGGATACGA ACAACGAAAC TTCTAACTCT CAGCTTA--G AAATAAGCCG ATTGATTATG chrX_16602314 - -----------------------------------------------------------chrU_6536133 ------------------------------------------------------------

61 chr3R_1506433
AAGAAATGCG GCAAGTAGCT GGTAAGTCTC CACTGGATAT CAGGATGGAA TTATTGAAT- Brazzaville_16 ------------------------------------------------------------Eden_3 ------------------------------------------------------------Cann-River_9 ------------------------------------------------------------Amieu_2 ----------------------------------------------------------- -----------------------------------------------------------Brazzaville_10 ----------------------------------------------------------- -----------------------------------------------------------chrU_2316095 ------------------------------------------------------------chrU_4537377 ------------------------------------------------------------chrU_5384045 ------------------------------------------------------------chrU_5680622 AAGAAATGCG GCAAGTAGCT GGTAAGTCTC CACTGGATAT CAGGATGGAG TTATTGAAT-chrU_5950518 - -----------------------------------------------------------chrU_6132643 AAGAAATGCG GCAAGTAGCT GGTAAGTCTC CACTGGATAT CAGGATGGAA TTATTGAAT-chrX_16602314 - -----------------------------------------------------------chrU_6536133 -------------------- -----------------------------------------------------------Eden_3 ------------------------------------------------------------Cann-River_9 ------------------------------------------------------------Amieu_2 ----------------------------------------------------------- ----------Cann-River_10 ------------------------------------------------CG ACGATAACAT Brazzaville_10 ------------------------------------------------ -----------------------------------------------------------chrU_2316095 ------------------------------------------------------------chrU_4537377 ------------------------------------------------------------chrU_5384045 ------------------------------------------------------------chrU_5680622 --CAACGCTT TTCATGCACT GAATCTGTTA ACCTAAGCAC AATAACTACG ACGATAACAT chrU_5950518 - -----------------------------------------------------------chrU_6132643 --CAACGCTC TTCATGCACT GAATCTGTTA ACCTAAGCAC AATAACTACG ACGATAACAT chrX_16602314 - -----------------------------------------------------------chrU_6536133 ATCAACGCTT TTCATGCACT GAATCTGGTA ACCTAAGCAC AATAACTACG ACGGCAACAT 181 chr3R_1506433 TTACAAGTGC AACTTGCAAT ACTACATCTA CAACAACTGC AGCAATCAGT AGGCCGAGCT Brazzaville_16 ------------------------------------------------------------Eden_3 ------------------------------------------------------------Cann-River_9 ------------------------------------------------------------Amieu_2 ----------------------------------------------------------- ----------------- -----------------------------------------------------------chrU_2316095 ------------------------------------------------------------chrU_4537377 ------------------------------------------------------------chrU_5384045 ------------------------------------------------------------chrU_5680622 TTACAAGTTC TACTTGCAAT ACTACATCTA CAACAACTGC AGCAATCAGT AGGCCGAGCT chrU_5950518 - -----------------------------------------------------------chrU_6132643 TTACAAGTGC AACTTGCAAT ACTACATCTA CAACAACTGC AGCAATCAGT AGGCCGAGCT chrX_16602314 - -----------------------------------------------------------chrU_6536133 TTACATGTGT TACTTGCAAT ACTACATCTA CTACA- ------------------------

241 chr3R_1506433
CCAGAGAAAA AGCTGCTACT AGATTGTCTG ATGCGCAAAA ACTTCTGACG AAAGAGAGGA Brazzaville_16 ------------------------------------------------------------Eden_3 ------------------------------------------------------------Cann-River_9 ------------------------------------------------------------Amieu_2 ----------------------------------------- -----------------------------------------------------------chrU_2316095 ------------------------------------------------------------chrU_4537377 ------------------------------------------------------------chrU_5384045 ------------------------------------------------------------chrU_5680622 CTAGAGAAAA AGCTGCTACT AGATTGTCTG ATGCGCAAAA ACTTCTGACG AAAGAGAGGA chrU_5950518 - -----------------------------------------------------------chrU_6132643 CCAGAGAAAA AGCTGCTACT AGATTGTCTG ATGCGCAAAA ACTTCTGACG AAAGAGAGGA chrX_16602314 - -----------------------------------------------------------chrU_6536133 ---------------GCAAT CAGTAGTCTG ATGCGCAAAA CCTTTTGACG AAAGAGAGGA 301 chr3R_1506433 AGAGGAAGAC CAATTTAACA CCCAACC-AT AGCTCTAAAC GAGCTGTCAG AGACGCGAAG Brazzaville_16 ------------------------------------------------------------Eden_3 ------------------------------------------------------------Cann-River_9 ----------------------------------- -----------------------------------------------------------chrU_2316095 ------------------------------------------------------------chrU_4537377 ------------------------------------------------------------chrU_5384045 ------------------------------------------------------------chrU_5680622 AGAGGAAGAC CAA--------CCAACC-AT AGCTCTAAAC GAGTTGTCAG AGACGCGAAG chrU_5950518 - -----------------------------------------------------------chrU_6132643 AGAGGAAGAC CAATTTAACA CCCAACC-AT AGCTCTAAAC GAGCTGTCAG AGACGCGAAG chrX_16602314 - -----------------------------------------------------------chrU_6536133 AGCGGGAGAC AAATTTATAA CCCAACC-TT AGCGCTAAAA GAGCTGTCAG AGACGCGAAG 361 chr3R_1506433

CTGGAGCCAT ATCCAGCCAT AAATAGTAAT TCAAACTCAA ACAATAGGTT TGCCATGCTA Brazzaville_16 -------------------------------------------AATCGTT TGGG------Eden_3 ----------------------------------------------------------- -----------------------------------------------------------chrU_2316095 ------------------------------------------------------------chrU_4537377 ------------------------------------------------------------chrU_5384045 ------------------------------------------------------------chrU_5680622 CTGGAGCCAT ATTCAGCCAC AAATAGTAAG TCAAACTCAA ACAATTGGAT GGCCATGCTA chrU_5950518 - -----------------------------------------------------------chrU_6132643 CTGGAGCCAT ATCCAGCCAT AAATAGTAAT TCAAACTCAA ACAATAGGTT TGCCATGCTA chrX_16602314 - -----------------------------------------------------------chrU_6536133 CTGGAGCCAT CCTCAGCAAC AATTAGGAAG GCAAACACAA ACAATAAGTT TGCCATGCTA 421 chr3R_1506433 -GACATGGAA TTGGACGAAA CCAGTGATGG CATAGAAACT CCACAAGTTT GTCATGATGT Brazzaville_16 ------------------------------------------------------------Eden_3 ----------------------------------------------------------- -----------------------------------------------------------chrU_2316095 ------------------------------------------------------------chrU_4537377 ------------------------------------------------------------chrU_5384045 ------------------------------------------------------------chrU_5680622 -GACATGGAA TTGGACGAAA CCAGTGATGG CATAGAAACT CCACAAGTTT GTAATGATGC chrU_5950518 - -----------------------------------------------------------chrU_6132643 -GACATGGAA TTGGACGAAA CCAGTGATGG CATAGAAACT CCACAAGTTT GTCATGATGT chrX_16602314 - --------------------------------------------------TCATGATGC chrU_6536133 -GACATGGAA TTGGACGCAA CCAGTAATGG TATAGAAATT CCACAAGTTT CTAATGATGG 481 chr3R_1506433 CGCATCCACC TCGTGTGCAT CGGCTGCTCC GAATATTCCT AATGATGACT GTGTACCAAA Brazzaville_16 ------------------------------------------------------------Eden_3 ------------------------------------------------------------Cann-River_9 CGCATCCACC TCGTGTGCAT CGGCTGCTCC GAATATTCCT AATGATGACT GTGTACCTAA Amieu_2 CGCATCCACC TCGTGTGCAT CGGCTGCTCC GAATATTCCT AATGATGACT GTGTACCAAA Makindu_2 CGCATCCACC TCGTGTGCAT CGGCTGCTCC GAAT- -------------------------Makindu_6 CGCATCCACC TCGTGTGCAT CGGCTGCTCC GAAT- -------------------------Makindu_7 CGCATCCACC TCGTGTGCAT CGGCTGCTCC GAAT- -------------------------Valence_6 CGCATCCACC TCGTGTGCAT CGGCTGCTCC GAATATTCCT AATGATGACT GTGTCCCAAA Makindu_8

CGCATCCACC TCGTGTGCAT CGGCTGCTCC GAAT--------------------------Makindu_22 CGCATCCACC TCGTGTGCAT CGGCTGCTCC GAAT--------------------------Canberra_5

CGCATCCACC TCGTGTGCAT CGGCTGCTCC GAAT- ------------------------ -------------------------------chrU_3975907 CGCATCCACC TCGTGTGCAT CGGCTGCTCC GAATATTCCT AATGATGACT GTGTACCAAA chrU_10625041 - -----------------------------------------------------------chrU_2316095 ------------------------------------------------------------chrU_4537377 ------------------------------------------------------------chrU_5384045 ------------------------------------------------------------chrU_5680622 CGCAGCCACC TCGTGTGCAT CGGCCGCTCC GAATATTCCT AATGATGACT GTGTACCAAA chrU_5950518 - ---------------------------------------------------------- ------------------------------------------------------------Eden_3 ------------------------------------------------------------Cann-River_9 TGTGACATCC AATAGCCCAC AACAGTATAC TGATAAGCAG AATTCTTATA AACAATCAAA Amieu_2 TGTGACATCC AATAACCCAC AACAGTATAC TGATAAGCAG AATTCTTATA AACAATCAAA Makindu_2 - ---------------------------------------------------CAATCAA-Makindu_6 ----------------------------------------------------CAATCAA-Makindu_7 ----------------------------------------------------CAATCAA-Valence_6 TGTGACATCC AATTGCCCAC AACAGTATAC TGATAAGCAG AATTCTTATA AACAATCAAA Makindu_8 - ---------------------------------------------------CAATCAA-Makindu_22 ----------------------------------------------------CAATCAA-Canberra_5 ----------------------------------------------------CAATCAA ---------------------------------------------------CAATCAA -----------------------------------------------------------chrU_3975907 TGTGACATCC AATAACCCAC AACAGTATAC TGATAAGCAG AATTCTTATA AACAATCAAA chrU_10625041 - -----------------------------------------------------------chrU_2316095 ------------------------------------------------------------chrU_4537377 ------------------------------------------------------------chrU_5384045 ------------------------------------------------------------chrU_5680622 TGTGACATCC ACTAGCCCAC AACAGTATAC TGATAAGCAG AATCCTTATA AACAATCAAA chrU_5950518 - ---------------------------------------------------------- ------------------------------------------------------------Eden_3 ----------------------------------------- -----------------------------------------------------------Makindu_6 ------------------------------------------------------------Makindu_7 ------------------------------------------------------------Valence_6 ACCACCGCAA ATAGTACTGA GCCTTACCAA CCTTAATGAT CTCTTTGAGC TCATTACGGA Makindu_8 ------------------------------------------------------------Makindu_22 ------------------------------------------------------------Canberra_5 ----------------------------------------------------------- ----------------------------------------------------------- -----------------------------------------------------------chrU_3975907 ACCACCGCAA ATAGTACTGA CCCTTACCAA CCTTAATGAT CTCTTTGAGC TCATTACGGA chrU_10625041 - -----------------------------------------------------------chrU_2316095 ------------------------------------------------------------chrU_4537377 ---------------------------------------------TGAGC TCATTACGGA chrU_5384045 ------------------------------------------------------------chrU_5680622 ACCACCGCAA ATAGTACTGA GCCTTACTAA CCTTAATGAA CTCTTTGAGC TCATTACG-A chrU_5950518 - -----------------------------------------------------------chrU_6132643 ACCACC----------------------------------------------------- ----------------------------------------------------------- ------------------------------ -----------------------------------------------------------chrU_3975907 GGT-CACTAG CATAAATAAT TTAACAGTTA AAGTCAATCA AGGGGA- -------------chrU_10625041 ------------------------------------------------------------chrU_2316095 ------------------------------------------------------------chrU_4537377 GGTTCATCAC CCTAGGTAAT TAACCAGTAA AGGTCAATCA GGGGGAAACA GGAGAAATTT chrU_5384045 - -----------------------------------------------------------chrU_5680622 GAT-CACTAG CCTAGATA- -----------------------------------------chrU_5950518 ------------------------------------------------------------chrU_6132643 ------------------------------------------------------------chrX_16602314 GGT-CACTAG ACTAGATAAT TTAACAGTTA AAGTCAATCA AGGAGAAACA GTGAGAATAT chrU_6536133 GGT-CACTAG CCTAGATAAT ATAA- -----------------------------GAATAT chr3R_1506433 TACCCAAA-G ACTCTGATAC TTACAGAGCT ATTGTTAATC TTTTTGATAA TTCGGGAATT Brazzaville_16 ----------------------------------------------ATAA -----------------------------------------------------------chrU_3975907 ------------------------------------------------------------chrU_10625041 ------------------------------------------------------------chrU_2316095 ------------------------------------------------------------chrU_4537377 TACCCAAAAG ATTCTGAAAT TACCAGAGTT ATTGTTATTG TTTTTGATAA TTCGGTAAGT chrU_5384045 - -----------------------------------------------------------chrU_5680622 ------------------------------------------------------------chrU_5950518 ------------------------------------------------------------chrU_6132643 ------------------------------------------------------------chrX_16602314 TACCC------------------------------------------------------ ------------------------------------------------------------chrU_3975907 ------------------------------------------------------------chrU_10625041 ------------------------------------------------------------chrU_2316095 ------------------------------------------------------------chrU_4537377 GAATTCCATA CGTACCAAAT GAAGGAAGAG AAGCCTCACA GAGTAGTTGT TAAGGGACTC chrU_5384045 - -----------------------------------------------------------chrU_5680622 ------------------------------------------------------------chrU_5950518 ------------------------------------------------------------chrU_6132643 ------------------------------------------------------------chrX_16602314 ------------------------------------------------------------chrU_6536133 GAATTCCATA CGTAC- ------------------------------------------- ----------------chr3L_18642496 ------------------------------------------------------------chrU_3975907 ------------------------------------------------------------chrU_10625041 ------------------------------------------------------------chrU_2316095 ------------------------------------------------------------chrU_4537377 CACCATAGCA CCGTAACCTA CGAAATTATC GACAACTTTA AAACATATGG -CTTTGATGT chrU_5384045 -----TAGCA CCCTAACCTA CGAAATTATC GACAACTTTA AAACATATGG GCTTTGATAT chrU_5680622 - -----------------------------------------------------------chrU_5950518 ------------------------------------------------------------chrU_6132643 ------------------------------------------------------------chrX_16602314 -

----------------------AATTATC GACAACTTTA AAACATATGG -CTTTGATGT chrU_6536133

- ---------------------------------------------------------- -----------------------------------------------------------chr3L_18642496 ------------------------------------------------------------chrU_3975907 ------------------------------------------------------------chrU_10625041 ------------------------------------------------------------chrU_2316095 ------------------------------------------------------------chrU_4537377 T-CTACAAGT ACACAACCCA AGATCCAGGA GAAATAGAGA AGAAAAACTT AATATATTCT chrU_5384045 T-CTACAAGT ACATAACCCA AGATCCAGGA GAAATAGAGA AGAAAAACTT AATATATTCT chrU_5680622 - -----------------------------------------------------------chrU_5950518 ------------------------------------------------------------chrU_6132643 ------------------------------------------------------------chrX_16602314 T-CTACAAGT ACATAACCAA AGATCCAGGA GAAATAGACA AGAAAAACTT AATATATTCT chrU_6536133 - ------------------------------------------ ------------------ -----------------------------------------------------------chr3L_18642496 ------------------------------------------------------------chrU_3975907 ------------------------------------------------------------chrU_10625041 ------------------------------------------------------------chrU_2316095 ------------------------------------------------------------chrU_4537377 TCATTAATAT AAAGACCTGT GCAAAAATTA ATGACATATA CGATATTAAC ACAATATGCC chrU_5384045

TCATTAATAT AAAGACCTAT GCAAAAATTA ATGACATATA CGATATTAAC ACAATATGCC chrU_5680622 - -----------------------------------------------------------chrU_5950518 ------------------------------------------------------------chrU_6132643 ----------------------------------------------------------- ------------------------------- -----------------------------------------------------------chr3L_18642496 ------------------------------------------------------------chrU_3975907 ------------------------------------------------------------chrU_10625041 ------------------------------------------------------------chrU_2316095 ------------------------------------------------------------chrU_4537377 GACAGAAAGT GCGGATAGAA AGAATGCGTA AATCATCTGA AATTGCACAA TGCATACGTT chrU_5384045

GACAGAAAGT GCGGATAGAA AGAATGCGTT AATCATCTGA AATAGCTCAA TGCATACGTT chrU_5680622 - -----------------------------------------------------------chrU_5950518 ------------------------------------------------------------chrU_6132643 ----------------------------------------------------------- -----------------------------------------------------------Valence_7 GTCAGGAATT CGGCCACACA GCCACGATGT GGTGAAAATC ACTTAACC- ---------- -----------------------------------------------------------chr3L_18642496 ------------------------------------------------------------chrU_3975907 -

----------------------------------AAATC ACTTAACCA-GCTATGCGTA chrU_10625041 ------------------------------------------------------------chrU_2316095 ----------------------CTCGATGT GGTGAAAATC ACTTAACCAA GCTATGCGTA chrU_4537377

GTCAGGAATT TGGCCACACA GCCTCGATGT GGTGAAAATC ACTTAACCAA GCTATGCGTA chrU_5384045 GTCAGGAATT CGGCCACAAA GCCTCGATGT GGTGAAAATC ACTTAACCAA GCTATGCGTA chrU_5680622 - -----------------------------------------------------------chrU_5950518 ------------------------------------------------------------chrU_6132643 ----------------------------------------------------------- -----------------------------------------------------------Valence_7 ----------------------------------------------------------- -----------------------------------------------------------chr3L_18642496 ------------------------------------------------------------chrU_3975907 CT

-CCC-ATG ATCA-CAGCC -------TAT CTGTATACAC TGTGGAGGAA -TCACACGGC chrU_10625041 ----------------------------------------------------CACACTGC chrU_2316095 CTTCCCAATG ATCAACAGCC -------TAT CTGTATACAC TGTGGAGGAA ATCACACGGC chrU_4537377 CTTCCCAATG ATCAACAGCC -------TAT CTGTATACAC TGTGGAGGAA ATCACACGGC chrU_5384045 CTTCCCAATG ATCAACAGCC -------TAT CTGTATACAC TGTGGAGGAA ATCACACGGC chrU_5680622

- -----------------------------------------------------------chrU_5950518 ------------------------------------------------------------chrU_6132643 ----------------------------------------------------------- -----------------------------------------------------------Valence_7 ----------------------------------------------------------- -----------------------------------------------------------chr3L_18642496 ------------------------------------------------------------chrU_3975907 AAGTTACAAG GGTTGCCAGT TTTACCAGGA GTATCTTTGA CGATCAATGG GCACTGTAAA chrU_10625041 AAGTTACAAA GGTTGCAAGT GGTACCAGGA GTATCTTCGA CGATCAATGG GCACTGTAAA chrU_2316095 AAGTTACAAG GGTTGCCAGT TTTACCAGGA GTATCTTCGA CGATCAATGG GCACTGTAAA chrU_4537377 AAGTTACAAG GGTTGCCAGT TTTACCAGGA GTATCTTCGA CGATCAATGG GCACTGTAAA chrU_5384045 AAGCTACAAA GGTTGCCAGT TTTACCAGGA GTATCTTCGA CGATCACTG---ACTGTAAA chrU_5680622 - -----------------------------------------------------------chrU_5950518 -------------------------CAGGA GTATCTTCGA CGATCAATAA GCACTGTAAA chrU_6132643 - -----------------------------------------------------------chrX_16602314 AAGCTACAAA GGCTGCCAGT TTTACCAGGA GTATCTTCGA CGATCAATGG GCACTGTAAA chrU_6536133 AAGTTACAAA CG- ---------------------CTTACA ----- -----------------------------------------------------------Valence_7 ----------------------------------------------------------- -----------------------------------------------------------chr3L_18642496 ----------------------------------------------------------- -----------------------------------------------------------chrU_5950518 GAAGCAACAA CTTAAGCAAG CCAGGTTAGA TA- ----------------G CAACACCCCA chrU_6132643 ----------------------------------------------------------- ------------------------------ -----------------------------------------------------------Valence_7 ----------------------------------------------------------- -----------------------------------------------------------chr3L_18642496 ----------------------------------------------------------- -----------------------------------------------------------chrU_5950518 GCAAAAACAG CAGCATGCAA TAGCTAGCAC TCCCAAAGAT CATACTGGAT GCTTGTCCTA chrU_6132643 - ---------------------------------------------------------- ---------------------------------------------------------- -----------------------------------------------------------Valence_7 ----------------------------------------------------------- -----------------------------------------------------------chr3L_18642496 ----------------------------------------------------------- -----------------------------------------------------------chrU_5950518 CGCAGATATT GCAAGAAATG GCAATACAAC AGCCCAGCCT CGTCTACAAA ATGTACAATT chrU_6132643 - ---------------------------------------------------------- -----------------------------------------------------------Makindu_2 AAA---------------------------------------------------------Makindu_6 AAAGGGAACT AAAATTAAGC AGCCACACCC - ---------------------------- -----------------------------------------------------------Valence_7 ----------------------------------------------------------- -------------------------- -----------------------------------------------------------chr3L_18642496 ----------------------------------------------------------- -----------------------------------------------------------chrU_5950518 AAAGGGAATT AATATTAAAC AGCAACACCC GCTTGACGTT CAGTCAATAT CGGCACAGCA chrU_6132643 - ---------------------------------------------------------- --------------Cann-River_9 ACAGGAACAA TTTATGAA------GTGG-Amieu_2 - ----------------------------Makindu_2 -----------------------------Makindu_6 ---------------------------- ----------------------------Valence_9 -----------------------------Eden_1 ACAGGAACAA TTTATGAA------GTGGC Valence_13 -----------------------------Eden_7 ACAGGAACAA TTTATGAA------GTGGC Valence_4

ACAGGAAGAA TTTATGAA------GTGGC Valence_2

ACAGGAAGAA TTTATGAA------GTGGC Valence_1 -----------------------------Valence_7 ---------------------------- -----G----Brazzaville_10 ---------------------------- ----------------------------Brazzaville_11 ACAGGAACAA TTTATGAA------GTGGC Brazzaville_12 ACAGGAACAA -------------------Brazzaville_8 ACAGGAAC---------------------chrU_963982 -----------------------------chr3L_18642496 -----------------------------chrU_3975907 ACAGGAACAA TTTATGAA------GTGGC chrU_10625041 GCAGGAACAA TTTATGAATT GTTAACTGA chrU_2316095

ACAGGAACAA TTTATGAA------GTGGC chrU_4537377

ACAGGAACAA TTTATGAA------GTGGC chrU_5384045

ACAGGAACAA TTTATGAA------TTGGC chrU_5680622 - ----------------------------chrU_5950518 GCAGGAACAA TTTATGAA------GTGGC chrU_6132643 - ----------------------------chrX_16602314 ACAGAAACAA TTTATGAA------GTGGC chrU_6536133

GCATGAACAA TATACGAA------TTGGC to acetylation of histones and methylation in H3K4, which are often observed in open chromatin structures.

Epigenetic natural variation is a common feature among species but is hardly ever studied [START_REF] Richards | Population epigenetics[END_REF]. D. melanogaster and D. simulans natural populations harbor an important TE copy number variation as described above (15% and 5% respectively). Interestingly, only 5,4% and 2,7% of the euchromatin are composed of TEs in D. melanogaster and D. simulans sequenced genomes (Clark et al., 2007). TEs seem therefore rather heterochromatic but what strikes us the most is that from the 15% of TEs present in D. melanogaster, the majority could be heterochromatic while D. simulans might harbor as much TEs in the euchromatin as in the heterochromatin. Thus, we wonder if natural variation in the histone association with TEs could explain such scenarios. We analyzed four elements in both species natural populations for TE copy number, TE expression, and TE association to histone marks. Also, we further analysed the impact of such natural variation in genome evolution. Hence, a bibliographic assay follows the histone project.

Introduction

DNA repeats increase species genetic variability and are therefore an important motor of genome evolution (Biemont and Vieira, 2006). We concentrate this study on transposable elements (TEs), which are an important component of DNA repeats. TEs are mobile sequences, that may induce mutations through their mobilisation and through copy recombination often in an individual specific manner [START_REF] Hedges | Inviting instability: Transposable elements, double-strand breaks, and the maintenance of genome integrity[END_REF]. TEs may offer new genetic features [START_REF] Muotri | The necessary junk: new functions for transposable elements[END_REF][START_REF] Sinzelle | Molecular domestication of transposable elements: from detrimental parasites to useful host genes[END_REF] and regulatory sequences [START_REF] Marino-Ramirez | Transposable elements donate lineage-specific regulatory sequences to host genomes[END_REF][START_REF] Muotri | The necessary junk: new functions for transposable elements[END_REF][START_REF] Polavarapu | Evolutionary rates and patterns for human transcription factor binding sites derived from repetitive DNA[END_REF] participating on the formation and remodelling of host gene networks [START_REF] Mcclintock | The significance of responses of the genome to challenge[END_REF][START_REF] Feschotte | Transposable elements and the evolution of regulatory networks[END_REF].

Factors that govern intra-and inter-species TE diversity are complex. They are likely a combination of intrinsic properties of TEs themselves (transposition mechanism, infectivity…), properties of host ecology (species effective size and structure…), and properties of the genome (TE regulation, gene density, genome size…). Among molecular mechanisms that could be a source of variation of TE expression/activity, is epigenetics.

Indeed, it is now well documented among the whole eukaryote taxa that regulation of TE activity involves epigenetic pathways as chromatin remodelling processes, DNA methylation and non coding small RNAs [START_REF] Lisch | Epigenetic regulation of transposable elements in plants[END_REF][START_REF] Obbard | The evolution of RNAi as a defence against viruses and transposable elements[END_REF]. TEs are maintained in the genome but are usually silenced through epigenetics, protecting the genome from negative effects of transposition/recombination, but keeping the potential for creating variability. When DNA sequences (such as genes) are positioned near or within heterochromatin, no/low expression can be detected as described for position effect variegation (PEV) models [START_REF] Girton | Chromatin structure and the regulation of gene expression: the lessons of PEV in Drosophila[END_REF]. The localization of TEs within particular chromatin environment might therefore be correlated to TE expression and further activity.

The basic unit of chromatin is the nucleosome, composed of an octamer of histones (H2A, H2B, H3 and H4, twice each) [START_REF] Ito | Role of histone modification in chromatin dynamics[END_REF]. Double stranded DNA wraps nucleosomes every 147bp and with other non histone proteins, forms the chromatin [START_REF] Ito | Role of histone modification in chromatin dynamics[END_REF]. Histones have a free N terminal tail which is the target of many post-translational modifications, as for example methylation and acetylation of lysine residues. Each histone modification is the target of chromatin remodelling factors and allow for chromatin conformation to change.

Generally, acetylation of lysines are associated to an active chromatin (euchromatin) while methylation, depending on the lysine modified, can correlate to activation (lysine 4 of histone 3) or repression (lysine 9 and 27 of histone 3) of gene expression [START_REF] Kim | Epigenetic mechanisms in mammals[END_REF]. TEs are considered as repressed elements, i.e., packaged in heterochromatic domains harbouring repressive histone marks [START_REF] Kondo | Enrichment for histone H3 lysine 9 methylation at Alu repeats in human cells[END_REF][START_REF] Ding | SDG714, a histone H3K9 methyltransferase, is involved in Tos17 DNA methylation and transposition in rice[END_REF][START_REF] Bernatavichute | Genome-wide association of histone H3 lysine nine methylation with CHG DNA methylation in Arabidopsis thaliana[END_REF]. Drosophila constitutive heterochromatin is enriched in H3K9me2 while facultative heterochromatin is labelled with H3K27me3 (Ebert et al., 2006). Furthermore heterochromatin is often enriched in TEs suggesting a role for histone modifications and chromatin related proteins in TE regulation. (Ebert et al., 2006;[START_REF] Schulze | Gene regulation by chromatin structure: paradigms established in Drosophila melanogaster[END_REF].

In this regard, several reports indicate that 1. TEs are associated with histone modifications and proteins typical of heterochromatic domains [START_REF] De Wit | Genome-wide HP1 binding in Drosophila: developmental plasticity and genomic targeting signals[END_REF][START_REF] Klenov | Repeat-associated siRNAs cause chromatin silencing of retrotransposons in the Drosophila melanogaster germline[END_REF][START_REF] Matyunina | LTR retrotransposons and the evolution of dosage compensation in Drosophila[END_REF][START_REF] Fablet | Genomic environment influences the dynamics of the tirant LTR retrotransposon in Drosophila[END_REF][START_REF] Phalke | Retrotransposon silencing and telomere integrity in somatic cells of Drosophila depends on the cytosine-5 methyltransferase DNMT2[END_REF] and 2. Distinct chromatin patterns might be observed between different TE families but also inside a TE family [START_REF] Mito | Genome-scale profiling of histone H3.3 replacement patterns[END_REF][START_REF] Fablet | Genomic environment influences the dynamics of the tirant LTR retrotransposon in Drosophila[END_REF]. The epigenetic regulation system, even if still rarely studied in natural populations, has been shown to be flexible [START_REF] Richards | Population epigenetics[END_REF]. For instance, the epigenetic polymorphism observed between twin brothers [START_REF] Ballestar | Epigenetics Lessons from Twins: Prospects for Autoimmune Disease[END_REF], or the presence of different patterns of gene methylation between Arabidopsis thaliana ecotypes [START_REF] Tanurdzic | Epigenomic consequences of immortalized plant cell suspension culture[END_REF] exemplifies epigenetic natural variation. No study on natural population has ever been made on TE histone modifications restricting our knowledge of the impact, and the dynamic of chromatin remodelling on TE diversity and stabilization in genomes. However, the need of population studies in the field of epigenetics is already claimed by many, and the importance of such experiments is no longer questionable [START_REF] Richards | Inherited epigenetic variation--revisiting soft inheritance[END_REF][START_REF] Bossdorf | Epigenetics for ecologists[END_REF][START_REF] Johannes | Epigenome dynamics: a quantitative genetics perspective[END_REF]. Drosophila natural populations are an excellent model to analyse such questions. Despite the fact that Drosophila has fewer copies of TEs when compared to other organisms such as humans (15% for Drosophila and 50% for humans), it has a high percentage of spontaneous mutations due to TE activity (Biemont and Vieira, 2006). Hence, Drosophila has putatively active elements (2004) and constitute an interesting model to the study of the impact of TEs on genetic variability and genome evolution. Studies on natural populations are approaches allowing to apprehend the genetic novelty associated with TE diversity and abundance (Fablet et al., 2006;[START_REF] Rebollo | Losing helena: the extinction of a drosophila line-like element[END_REF]. D. melanogaster and D.

simulans, for instance, contain the same TE families, with in most of the cases more than 90% of sequence identity [START_REF] Bartolome | Widespread evidence for horizontal transfer of transposable elements across Drosophila genomes[END_REF]. However, an overrepresentation of almost all TEs is observed in D. melanogaster (Vieira et al., 1999). Natural populations of D.

melanogaster are relatively homogeneous for TEs, since copies are present at high number in all strains analyzed (Vieira et al., 1999). In contrast, D. simulans natural populations are highly polymorphic since for a given element high copy number can be observed in one strain while another one is completely devoided (Vieira et al., 1999). These observations were

Results and discussion

TE occurrence in natural populations of D. melanogaster and D. simulans

All four elements analysed are retrotransposons that either have long terminal repeats (LTR) (two copia-like elements, 412 and roo, a gypsy-like element, tirant) or not (LINE-like element F) (Figure 1). They harbour open reading frames (ORFs) that may code for retrovirus like proteins as GAG, POL and ENV [START_REF] Meyerowitz | Molecular organization of a Drosophila puff site that responds to ecdysone[END_REF][START_REF] Yuki | Nucleotide sequence characterization of a Drosophila retrotransposon, 412[END_REF]Fablet et al., 2006;[START_REF] Tchurikov | Suffix-specific RNAi leads to silencing of F element in Drosophila melanogaster[END_REF]. Initial in situ hybridization experiments revealed that these elements are over-represented in D. melanogaster strains compared to D. simulans (Vieira et al., 1999). For example, Vieira and collaborators observed 18 times more F copies in D. melanogaster than in D. simulans natural populations (mean copy number between natural populations analysed). 412, tirant and F were also described as being copy number polymorphic between natural populations of D. simulans (Vieira et al., 1999;Borie et al., 2000;Fablet et al., 2006). For example, the F element varied between 37 copies in a few D.

simulans natural populations to being completely absent from others (Vieira et al., 1999). In contrast, Roo is one exception, since it was detected in high copy number in both species and all natural populations analysed (Vieira et al., 1999). All data collected by Vieira et al. was obtained through in situ hybridization of polytene chromosomes of Drosophila salivary glands using large probes corresponding to almost full length elements (Vieira et al., 1999). In situ hybridization only allowed the authors to count for copies present in the arms of polytene chromosomes (so called euchromatic). However, all four elements are present on chromosome arms and in putatively constitutive heterochromatin regions (pericentromeric staining on polytene chromosomes). In order to apprehend genome wide variation in TE copy number, we used quantitative PCR to amplify an internal region of 412, tirant, roo and F elements (Figure 2). As expected, D. melanogaster presents more TE copies than D. simulans (~4 times more, supplementary Figure 1). Interestingly, roo element, which was as highly represented in D. melanogaster as in D. simulans natural populations, has a significantly lower copy number in D. simulans (~9 fold). In addition, copy number polymorphism previously observed between natural populations of D. simulans (Vieira et al., 1999) were not reproduced. Instead, either no copy variation between D. simulans populations is observed (F Kruskal-Wallis chi p value ~0,14), or a different pattern of copy number distribution is noted among D. simulans natural populations (412 and tirant). For example, the Canberra D.

simulans strain was previously described as harbouring more than 60 copies of 412 (Vieira et al., 1999) and we observe an almost complete lack of this element in this population. The same can be described for Grand Ferrade D. simulans and the F element. We are quantifying the presence of a unique small internal region of each element (150 bp maximum) and any deleted copy in that region will not be amplified by this technique. In contrast, using a complete copy as a probe, Vieira et al were able to count every copy presented in the euchromatic regions, even importantly deleted copies. The comparison of both datasets strongly suggests that 412, tirant, roo and F are internally deleted in D. simulans natural populations. The analysis of both sequenced genomes confirmed that a large proportion of these elements present internal deletions in D. simulans genome (will be described in a separate report). These observations are consistent with previous hypothesis of an active degradation process in natural populations of D. simulans, effective on various elements such as 412 and tirant but also on other elements as the LINE-like element helena (Mugnier et al., 2005;Fablet et al., 2006;[START_REF] Rebollo | Losing helena: the extinction of a drosophila line-like element[END_REF]. Furthermore, some lines, previously described as devoid of copies of a given TE family, present copies through real time PCR. Indeed, F and tirant elements are amplified in D. simulans populations such as Grand Ferrade, Makindu or Zimbabwe initially described as empty. We interpret these differences as heterochromatic copies located in centro and telomeric domains and hence ignored in the previous in situ hybridization experiments.

We confirmed previous data showing that D. melanogaster has generally more copies of all TE families analyzed than D. simulans, and we show that intact copies in D. simulans are probably in low copy number. Are these patterns the result of differential transcriptional activity of TEs in the two Drosophila species? Full length copies of all TEs analysed can be observed in the sequenced genomes of both species [START_REF] Bartolome | Widespread evidence for horizontal transfer of transposable elements across Drosophila genomes[END_REF] and copy number variation is observed for some elements in natural populations of both species. Are these elements still producing transcripts (full length or deleted ones) and are they accounting for copy number variation of each TE?

Expression of 412, tirant, roo and F in natural populations of D. melanogaster and D. simulans

In order to test if some of the copies present in natural populations are transcriptionally active, we quantified TE expression. We extracted total RNAs from embryos and amplified total cDNA by quantitative PCR (Figure 3). The amplified region in all elements is the same as the region targeted for copy number analysis described above (Figure 1). Each TE family presented a particular expression pattern in D. melanogaster and D. simulans natural populations but globally, D. melanogaster Senegal strain harboured more transcripts than all other strains. D. melanogaster populations, Senegal and Chicharo, harboured constant patterns between all elements: Chicharo displays always lower expression level than Senegal (-3 fold, Kruskal Wallis p value 1,6 10 -3 ). Such difference can not be explained by copy number variation for all elements in D. melanogaster, except for the 412 family (Figure 2 supplementary materials).

The majority of natural populations of D. simulans harbour transcripts for at least one of the four elements. Nevertheless, no correlation between copy number and transcription level can be visualised. However, it is interesting to note that the expression of 412 in D. simulans natural populations has a tendency to follow Vieira et al. in situ hybridization quantification data [START_REF] Junakovic | Accumulation of transposable elements in the heterochromatin and on the Y chromosome of Drosophila simulans and Drosophila melanogaster[END_REF] (Figure 3 supplementary materials). One hypothesis to explain such tendency is that 412 would be transcribed at high levels from few full-length copies and that transcription may be associated to high transposition rate. In this context, each newly transposed copy would be internally deleted by the D. simulans genome, while the master copies remain intact. The second hypothesis is to think that fragments of 412 observed by in situ hybridization are producing transcripts regardless of copy quality (full-length, truncated and internally deleted copies). Such transcripts might be originated from host genes and external promoters. Further analyses on transposition rates are being held along with RACE-PCRs in order to identify external promoters. Tirant has a specific expression pattern in D. simulans, where a few natural populations, harbouring almost the same number of tirant copies (Makindu, Canberra and Zimbabwe), are either silenced or extremely transcriptionally active (700 fold between Makindu and Canberra for instance). The roo element has almost the same dynamics as tirant, since several natural populations of D. simulans harbour more transcripts than both natural populations of D. melanogaster. Both data suggests that D. simulans even thought having fewer copies than D. melanogaster, does present transcripts for TEs, comforting the idea that few copies may be responsible for high transcription rate. The F element has no copy number variation for D. simulans and no transcript variation (p values > 0,1 for Kruskal Wallis tests).

As a conclusion, expression of a TE family in a given population does not correlate with copy number and we hypothesize that different regulation systems may exist inside the same species. Moreover, populations of D. simulans harbouring the same range of TE copies can either be transcriptionally active (for example, tirant in D. simulans Makindu strain) or silenced (tirant in Canberra D. simulans strain). 412, tirant, roo and F, do not have a preferential insertion site in the host genome, they are observed in both heterochromatic and euchromatic regions. Epigenetic regulation of TEs is well known, and chromatin remodelling processes are thought to control gene expression. In D. simulans, 412 correlation between fragments present in the chromosome arms and expression suggests that the more 412 copies are present in the euchromatin regions, the higher the expression. Also, we previously showed that in D. simulans Makindu, tirant copies embedded in H3K9me2 heterochromatic domains are transcriptionally inert while copies associated with transcription are decorated with H3K27me3 [START_REF] Fablet | Genomic environment influences the dynamics of the tirant LTR retrotransposon in Drosophila[END_REF]. Do TE families present distinct chromatin status between species and between populations? Do different TE families harbour different chromatin patterns inside a population? It is important to note that the region amplified by qPCR is near or overlapping the region already described for copy number and expression assays (Figure 1).

Also, quantification of the immunoprecipitated material is relative to each TE copy number in each natural population (qPCR in the input samples). Plus, for all ChIP reactions, antibodies recognizing the histone 3 allowed us to check for nucleosome presence and lack of variation (Figure 4 supplementary materials). All elements analyzed were not associated with the histone modification typical of active regions, consistent with the common hypothesis that TEs present in the genome are globally in a repressed state (Figure 5 supplementary materials). Heterochromatin can be either constitutive (centromeres, telomeres, repeat-rich regions) or facultative (for instance, cell cycle and developmental stage dependent) [START_REF] Trojer | Facultative heterochromatin: is there a distinctive molecular signature?[END_REF]. H3K27me3 is typical from facultative heterochromatin in Drosophila (supplementary figure 6) [START_REF] Czermin | Drosophila enhancer of Zeste/ESC complexes have a histone H3 methyltransferase activity that marks chromosomal Polycomb sites[END_REF]. Patterns obtained for H3K27me3 enrichment are TE family-and population-specific (Figure 4). with H3K9me2, are observed in tirant expressed copies [START_REF] Fablet | Genomic environment influences the dynamics of the tirant LTR retrotransposon in Drosophila[END_REF].

H3K9me2 is typical from repressive constitutive heterochromatic regions (supplementary figure 6), as telomeres, centromeres, the 4 th chromosome and the Y chromosome in Drosophila [START_REF] Riddle | A lot about a little dot -lessons learned from Drosophila melanogaster chromosome 4[END_REF]. Similarly to H3K27me3, population-and TE-specific profiles are observed for H3K9me2 in embryos (Figure 4). Enrichment for H3K9me2 for TEs are weaker than compared to constitutive heterochromatic regions but are significant (supplementary figure 6). H3K9me2 patterns observed are different from the H3K27me3 ones. Roo presents a very low association with H3K9me2 in both species suggesting that roo elements are mainly outside of constitutive heterochromatin, regardless of the species analyzed and that these elements are more decorated with H3K27me3 than H3K9me2. 412

and F are more represented in the constitutive heterochromatin of D. melanogaster than D.

simulans but this does not explain both transcription profiles. Tirant is equally enriched in H3K9me2, and small variations can be observed between natural populations.

Our ChIP coupled with qPCR approach allowed us to apprehend the mean status of all individual elements within one specific TE family. This cluster analysis does not allow the detection of differences that may be present at one particular chromosomal locus (TE single copy) in each natural population. In order to have an individual picture, we observed, on polytene chromosomes, through ImmunoFish, the associations between the different copies present on the chromosome arms, and histone post-translational modifications. We analyzed H3K27me3 pattern in both natural populations of D. melanogaster and in polymorphic natural populations of D. simulans. Colocalization between elements and H3K27me3 was only observed for a few copies of the 412 element in Senegal (D. melanogaster) and Canberra (D. simulans) lines (Figure 5). All other elements present on the chromosomes arms were devoid of H3K27me3 modification. These observations suggest that if H3K27me3 is acting as a repressive mark, then expression of the elements analyzed can originate from copies on the chromosome arms devoid of any H3K27me3 modification and probably H3K9me2. One could wonder which histone marks are indeed associated to transcriptionally active copies.

TEs analysed in D. melanogaster were globally more enriched in heterochromatic marks than D. simulans TEs. Are the different histone profiles between populations a result from differential expression of enzymes in charge of chromatin modification?

b. Variation in key players of chromatin remodelling and other epigenetic pathways

In order to apprehend variation in the deposition of histone modifications, we assayed for transcriptional activity of genes involved in post-translational modification of histones and other epigenetic pathways (Figure 6). Enhancer of zeste (E(z)), responsible for methylation of H3K27me3, Su(var)3-9, responsible for H3K9me2 (Ebert et al., 2006) were significantly overexpressed in D. melanogaster compared to D. simulans natural populations. One could ask if lower expression of both enzymes could be correlated to lower association to H3K27me3 and H3K9me2 in D. simulans. We assayed for other epigenetic players, in order to check for variation between both species (supplementary figure 7). RPD3, a histone deacetylase (HDAC1 homologous), ASH1 (a multi histone methyltransferase that acts in enhancing active patterns), Su(var)4-20 (H4K20me3) and HP1 (heterochromatin protein 1) expression do not present different profiles for D. melanogaster or D. simulans. It is interesting to note that H3K9me2 is often described in association with HP1 and our data show that despite having different expression patterns for Suvar3 (9), HP1 is constant between natural populations and both species. Ash1 and Su(var)4-20 are extremely down regulated in a few D. simulans natural populations. MBD23 (methyl binding DNA protein) and DNMT2

(DNA methyl transferase, results not shown) do not present any variation between both species and natural populations. These data shows therefore that there are significant differences between the two species analyzed, D. melanogaster and D. simulans and that again, intra specific variation can be observed.

Conclusion

Our global analysis of TEs in Drosophila natural populations as illustrated in Figure 7 points to several facts. The first general conclusion is that inter and intra species variations are observed at transcriptional and epigenetic levels. TE families present distinct patterns of regulation, and variation does exist between copies of a given TE family. Although some TE expression coincides with reduced levels of repressive marks (roo in D. simulans for example), such state is not the general rule. We demonstrate that some copies of TE families are mainly devoid of repressive histone marks in the genome. One may suggest that transcripts emerge preferentially from these "nude" copies. Indeed the second conclusion is about the analysis of two natural populations of D. melanogaster (Chicharo and Senegal)

showing that each TE family presents distinct expression profiles in different populations, with transcription levels of all elements analyzed, highly different between the two populations of D. melanogaster species. Two hypotheses can be made to explain this observation: first, high transcription level in Senegal emerge from copies devoid of repressive histone modifications, while low transcription level in Chicharo results from packaging in repressive chromatin of mostly all TE copies present in the genome. However, we observed that the average enrichments for the histone methylation marks are largely similar between these two D. melanogaster populations. This suggests that an alteration of the histone methylation pattern is not the major cause of differential transcription between the two populations. If, histone post-translational modification pathways are equally effective in both D. melanogaster natural populations, repression of TEs in Chicharo might involve additional regulatory pathway. In this regard, a particular attention should be paid to the production of small interfering RNAs (siRNA) since several recent studies reveal the central role played by small RNAs in the genome, as TE silencers. The F, 412, tirant an roo elements are present in flamenco and/or 42AB regions from which emerge a high number of germinal and somatic small RNAs [START_REF] Brennecke | An epigenetic role for maternally inherited piRNAs in transposon silencing[END_REF][START_REF] Malone | Specialized piRNA pathways act in germline and somatic tissues of the Drosophila ovary[END_REF]. Chicharo line could thus be strongly silenced through small RNAs produced from 42AB and the flamenco locus. It should be noticed that no analysis has ever been performed on natural populations, and that we have no information on the variability of the insertions that can be found in the flamenco locus.

Further analysis of siRNA production will thus be helpful to enlighten silencing of TEs in natural populations. Third, some D. simulans populations present elevated levels of 412 and tirant transcripts. Interestingly, in these populations the studied TEs present more prominent association with H3K27me3 than H3K9me2. Nevertheless, since amplification of immunoprecipitated fragments of ChIP is not copy specific, one could suggest that the expressed TE copies are not associated to H3K27me3. This hypothesis is moreover in agreement with our results from ImmunoFish experiments. Copy localization of TEs in the two transcriptionally active genomes of D. simulans is mandatory to make ChIP copy specific and therefore realize the in vivo distribution of histone marks with TE copies. For example, we previously reported that an expressed tirant copy is preferentially decorated with H3K27me3 and H3K4me2 but not at all H3K9me2 compared to other copies [START_REF] Fablet | Genomic environment influences the dynamics of the tirant LTR retrotransposon in Drosophila[END_REF]. Unfortunately, TE copies are very well conserved and transcript analysis does not allow us to distinguish between copies.

The comparative analysis of TE copy number by in situ hybridization published before (Vieira et al., 1999) and the quantitative PCR assays done in this project have shown that D.

simulans strains harbour rearranged or deleted copies. Indeed, the in situ hybridization experiments performed with full length probes of the elements do not reflect the integrity of the copies detected in one genome. This has already been suggested by other works (Mugnier et al., 2005;[START_REF] Rebollo | Losing helena: the extinction of a drosophila line-like element[END_REF] in which deleted copies have been described, and definitely raises the question of TE regulation in D. simulans. Is this enigmatic TE specific deletion mechanism random or is it targeting copies present preferentially in chromosome arms (euchromatic) or in centromeric regions (heterochromatic)? We noticed that strains devoid of any copies on chromosomal arms (Vieira et al., 1999) display qPCR amplification products indicating that copies are present elsewhere in the genome. In contrast, strains harboring high number of euchromatic copies were almost not amplified by qPCR. From these observations it is tempting to hypothesize that the deletion mechanism is targeting essentially euchromatic copies.

Given the diversity of TEs and the variable degree they populate genomes, several pathways have emerged to restrict the deleterious effects of "uncontrolled" activity. From our results we propose two non-exclusive pathways. Our observations and a previous report [START_REF] Phalke | Retrotransposon silencing and telomere integrity in somatic cells of Drosophila depends on the cytosine-5 methyltransferase DNMT2[END_REF], suggest that local chromatin environment defined by histone modifications might control TE expression in D. melanogaster and D. simulans. Although the general idea is that TEs are embedded in repressive chromatin, no general rule appears from our analysis. Some copies might be packaged in constitutive repressed chromatin (H3K9me2, H3K27me3, H4K20me2) although others might be more permissive to transcriptional elongation (absence of H3K27me3 and/or H3K9me2). This pathway relies on chromatin remodellers activity such as histone methyltransferase activity and on the use of small RNAs emerging from the transposons to guide silencing. In addition, as illustrated in D. simulans, a deletion mechanism targeting all TE families tend to restrict the deleterious activity inherent to full length copies. In contrast to D. melanogaster, D. simulans populations appear as a panel of several extinction profiles of TEs with a lot of truncated copies. In a few strains we still observe high transcription but low supposed full length copy number, and in others, no transcription and only copies embedded in heterochromatic regions. Based on our observations, we hypothesize that in D. simulans, copies present in the chromosome arms are potentially active and also the preferential targets of the deletion mechanism, while copies that are conserved in heterochromatin regions, are the target of repressive histone marks (H3K9me2 and H3K27me3), and are protected from the deletion processes. Further investigations are required to determine how the deletion mechanism is set up in D. simulans natural populations. How heterochromatic copies are maintained in the genome? In the modern view of epigenetics, small RNAs have the most important role to play: they can bring specificity to the system, start the system and end the system. If, as showed above, TEs are present in the heterochromatin, are still transcribing at a low rate, they could target the deletion mechanism to copies in the chromosome arms. It is thus essential to verify if the deleted copies are indeed exclusive from euchromatin and if the heterochromatic copies are able to produce small RNAs. Natural population studies offer therefore the perfect model to study such a system.

Genetic diversity is increased by TE in natural populations. For the first time, we identified broad epigenetic variability associated to TE variation. Therefore, TEs will not only provide important sources of genetic variability for the host but also allow a second level of organization and regulation through histone modifications. It is important to note that such conclusions are only possible thanks to a populational approach where diversity can be observed directly in nature. Since TEs play a role in regulation of gene networks [START_REF] Feschotte | Transposable elements and the evolution of regulatory networks[END_REF], because they are multicopies and genome widespread, one could easily suggest that histone modifications associated to TEs are highly important to genome compartmentalization and differential regulation between populations. D. melanogaster originated in East Africa and has colonized the world in a recent past [START_REF] Lachaise | How two Afrotropical endemics made two cosmopolitan human commensals: the Drosophila melanogaster-D. simulans palaeogeographic riddle[END_REF]. The dynamics of TEs in this species is starting to be well known and it is well described that TEs are very recent and have been submitted to recent bursts of transposition and horizontal transfers (Lerat et al., 2003;[START_REF] Bartolome | Widespread evidence for horizontal transfer of transposable elements across Drosophila genomes[END_REF]. D. simulans is also an East African species but the colonization of the world started later and there are still regions where no D. simulans are found. TE dynamics in D. simulans also seems to be very different from D. melanogaster, since very few full length copies are described. Former population genetics models have proposed that the differences in the amount of TEs between the two species could be explained by differences in species effective size, with D. simulans having a more important size and a more efficient selection against TEs. Even if this statement is true, which is far from being confirmed [START_REF] Nolte | African Drosophila melanogaster and D. simulans populations have similar levels of sequence variability, suggesting comparable effective population sizes[END_REF], we shouldn't expect differences in the quality and structure of the elements. Our data show that the analysis of TE dynamics will also depend on the fine mechanism of regulation such as the epigenetic pathways, that were never included in the population genetic models, and that will allow variability in the expression levels of specific elements in specific populations. Once the system breaks down and that a TE copy is transcribed, one might observe increase in copy number. The understanding of TE impacts on genome evolution and their implications on gene networks should associate ecology, epigenetics and natural population [START_REF] Vieira | Infra-and Transspecific Clues to Understanding the Dynamics of Transposable Elements[END_REF].

Materials and methods

Natural populations

We worked on fly samples collected from several geographically distinct regions. D.

melanogaster natural populations were collected from Portugal (Chicharo) and Africa (Senegal). D. simulans natural populations are from Kenya (Makindu), Zimbabwe, Autralia (Canberra), French Polynesia (Papeete) and France (Grand Ferrade). These populations were maintained in the laboratory as isofemale lines or small mass cultures with around 50 pairs in each generation. Flies stocks were maintained at 24°C.

TE copy number estimation

DNA was extracted from 14h to 16h embryos using "DNAeasy blood and tissue kit" from Qiagen, three times for each natural population. For each sample we used DNA diluted at 0.16ng/µl. Linear real time PCR was performed using Power SYBR Green Master Mix (Applied Biosystems), on a SDS 7900 HT instrument (Applied Biosystems) with the following parameters: 50°C for two minutes, 95°C for ten minutes, and 45 cycles of 95°C 15 secondes-60°C one minute. Each genomic region was tested with specific primers designed using the program Primer Express v 2.0 (Applied Biosystems) with default parameters.

Primers sequences are available upon request. Triplicates of samples and triplicates of PCR were performed and the results obtained for each tested genes were normalized with three or four control genes treated in parallel (RP49, RNA pol II, EFG1). Raw Ct values obtained with SDS 2.2 (Applied Biosystems) were imported in Excel and normalization factor and fold changes were calculated using the GeNorm method (Vandesompele et al., , 2002). Real-time PCR and data analysis were performed at the Genomics Platform, NCCR "Frontiers in Genetics" (http://www.frontiers-in-genetics.org/genomics.htm).

TE expression quantification

RNA was isolated from 14h to 16h embryos. Extraction of RNA was done using TRIzol ® reagent (Invitrogen) according to the manufacturer's recommendation followed by chloroform /Isoamyl-alcool (24:1) purification. After DNase treatment (Ambion DNA free™), the OD260/280 (interval 1.9-2.1) of the RNA samples were determined by spectrophotometry.

The integrity of the RNA was assessed by Agilent 2100 Bioanalyser (Agilent Technologies Inc, Palo Alto CA). For each sample, one µg of total RNA was used to make cDNA using random hexamers and the Supercript II reverse transcriptase (Invitrogen). Further analysis of transcripts was done by quantitative PCR as described above (including primers).

Transcription of epigenetic factors

Total RNA was extracted from 14h to 16h embryos from all natural populations analysed (two biological replicates for each strain) with the RNeasy protect mini kit from Qiagen. 1µg of total RNA extracts were treated with the Ambion's DNA-free kit. ThermoScript RT-PCR system from Invitrogen was used to synthesize two different cDNA (55°C for 90 min and 85°C for 5 min): a control reaction with no retrotranscriptase to test DNA contamination, a pool of total cDNA synthesized with a mix of oligo-dt/random primers 1:1. The cDNA samples were diluted 80 fold, and PCR was carried out using QuantiTect SYBR Green PCR kit (Roche) on the LightCycler (Roche) using primers specific from each enzyme analysed.

Primers were chosen surrounding introns in order to amplify 150-250bp fragments of cDNA (primers available upon request). Genes analysed were chose as being part of chromatin remodelling processes, as Su(var)3-9, Ash1, E(z), HP1, HDAC3, Suvar420 and other epigenetic pathways as DNA methylation with MBD23 and dDNMT2. Quantitative PCR cycling conditions were 5 min at 95°C (1 cycle), 15 s at 95°C, followed by 10 s at 60°C and 20 s at 72°C (50 cycles). Reactions were done in duplicate, and standard curves were calculated from serial dilutions of specific amplified PCR fragments. The quantity of the transcripts was estimated relative to the RP49 and 18sRNA expression. Relative quantification was calculated as described above for TE expression.

Histone modification within TEs

Extraction of chromatin from 14h to 16h D. simulans embryos and immunoprecipitation were adapted from (Sandmann et al., 2006). Cell lysis buffer was changed to 5mMPIPES pH 8, 85 mM KCl, 0.5% Nonidet P-40 supplemented with protease inhibitors. Chromatin was shared with a Bioruptor sonicator water bath (Diagenode, Liège, Belgium) for 6 X (30-s on/30-s off) cycles at high power in order to have random fragments from 1 kb to 500 bp.

Shared chromatin was incubated overnight at 4°C with antibodies recognizing H3K9me2 (Millipore, Billerica, MA, USA; 07441), H3K27me3 (Millipore 07449), H3K4me2 (Millipore 07030), H3 (Abcam, Cambridge, UK; ab1791), and rabbit IgG (Sigma-Aldrich, St. Louis, MO, USA; I5006). The antigen-antibody complexes were washed as described before (Sandmann et al., 2006), but a second washing solution was modified as followed: TE 2X, 500 mM NaCl, 1% Triton, 0.1% SDS. To quantify each IP, real-time PCR was performed using QuantiTect SYBR Green PCR kit (Qiagen) on a MXP3000P PCR system (Stratagene, La Jolla, CA, USA). Reactions were done in duplicates, and standard curves were calculated on serial of input chromatin. To evaluate the relative enrichment of each TE after IP, we calculated the difference in cycles between the IP-enriched sample and the input DNA for TE copies and for a control (actin-CG4027). Primers were chosen in the coding region for each TE (Figure 1) and are available upon request.

Colocalisation of facultative heterochromatin and TE copies

Immunofish was adapted from [START_REF] Lavrov | Combined immunostaining and FISH analysis of polytene chromosomes[END_REF]. Fly stocks were maintained at 18°C during this experiment. Salivary glands were extracted from third-instar larvae in 1%NP40 and 0,6% NaCl. Cross linked salivary glands (2% Formaldehyde for 2 minutes) were crushed and fixated on slides for 3 minutes. Slides were conserved less than one week at 4°C in PBS.

Dried Slides were incubated in 2X SSC for 45 min at 65 °C. After being dehydrated in cold ethanol baths, polytenes were denatured for 10 min in 0,07N NaOH at room temperature.

After being washed with 2X SSC, slides were dehydrated a second time. Each TE probe was labelled during a PCR with DIG DNA labelling mix from Roche. Primers used for probe amplification target the internal region of all elements (Figure 1) and are available upon request. Slides were incubated over night at 37°C in a humid dark chamber with a denaturated mix of hybridization buffer Copy number estimation of 412, tirant, roo and F from in situ hybridization in polytene chromosomes (adapted from (Vieira et al., 1999)) and quantitative PCR.

Right graphs are adapted from (Vieira et al., 1999) and show chromosomal arm copies simulans (red bars) has very active and silenced populations for all LTR elements. Expression of all TEs is variable between both species and between all natural populations analyzed. No common pattern is observed for a specific natural population or a specific TE. Please refer to materials and methods for qPCR relative quantification and, note that reference genes are equally expressed between both species and natural populations allowing us to compare all strains together (Supplementary Figure 1). showing that both natural populations of D. melanogaster have more TE copies than D.

Figures

simulans.

Figure 2

Correlation between copy number and TE transcription for D. melanogaster natural populations.

Figure 3 Tendency of 412 transcription to be higher in high copy number natural populations of D.

simulans.

Figure 4

Histone 3 enrichment for TEs in natural populations of D. melanogaster and D. simulans.

Please, note that reference genes are equally enriched between both species and natural populations allowing us to compare all strains together (supplementary figure 1). when rp49 was analyzed and absent in a already described X region. Please, note that reference genes are equally enriched between both species and natural populations allowing us to compare all strains together (supplementary figure 1). Quantification of transcripts of genes involved chromatin post-translational modifications.

Please, note that reference genes are equally expressed between both species and natural populations allowing us to compare all strains together (supplementary Figure 1).

Introduction

Genome sequencing programs provided new clues in understanding the lack of correlation between phenotypic complexity and genome size -the so called "C value" paradox -by pointing out that genome size differs between species mostly in the non coding parts (reviewed in (Biemont and Vieira, 2006)). For instance, the human genome is composed of ~98% of non coding DNA ( 2004) while the fruit fly, Drosophila melanogaster, has a very compact genome with only ~12% of this type of sequence [START_REF] Dowsett | Differing levels of dispersed repetitive DNA among closely related species of Drosophila[END_REF]. This variable, but seemingly useless, part of the genome was named "junk DNA" and is mostly represented by repetitive sequences, such as satellite DNAs and transposable elements (TEs), which will be the focus of this short review (Figure 1 for a eukaryotic TE classification). Our work in understanding TE dynamics in natural populations led us to question the influence of TE mobilisation in speciation (Vieira et al., 1999;[START_REF] Rebollo | Losing helena: the extinction of a drosophila line-like element[END_REF][START_REF] Fablet | Genomic environment influences the dynamics of the tirant LTR retrotransposon in Drosophila[END_REF].

Speciation may be a slow process involving the fixation of genetic differences between individuals, either by changing their fitness and/or ecological specialization and/or simply inducing genic incompatibility, all followed by micro population isolation. Speciation is also thought to happen fast in the case of non-genic speciation, i.e., when important karyotypic differences are observed between individuals of the same species causing sexual isolation. We are interested in rapid evolution induced by chromosomal incompatibilities inside one species.

Chromosomal incompatibility, i.e., the impossibility of pairing for both parental homologous chromosomes, can happen with inter-specific crosses but also between individuals of the same species when chromosome rearrangements have occurred. Such incompatibility can lead to progeny lethality or sterility causing isolation of fertile survivors into micro populations. TEs are inducers of genome remodelling either through transposition, as described for lines of maize where Ac/Ds alternative transposition (from the ends of two elements) is directly responsible for major chromosomal rearrangements (translocations, duplications, inversions …) [START_REF] Zhang | Alternative Ac/Ds transposition induces major chromosomal rearrangements in maize[END_REF], or for all recombination events due to TE copies [START_REF] Hedges | Inviting instability: Transposable elements, double-strand breaks, and the maintenance of genome integrity[END_REF]. A burst of transposition might, therefore, be essential for rapid karyotypic isolation and, hence, macroevolution. TEs are often kept silenced in genomes and in order for such karyotypic changes to happen it is necessary for TEs to escape the regulation systems, which are mainly composed of epigenetic mechanisms. We propose that macroevolution and speciation could be concomitant with rapid genome remodelling induced by TE awakening from tight epigenetic repression, as indicated by many observations.

Junk and Genome: a partnership in progression

The developmental and evolutionary importance of TEs is no longer questionable. In general, all families of DNA repeats could potentially have an impact on genome organisation, either as generators of genome instability, since multicopy elements are powerful recombinogenic substrates [START_REF] Hedges | Inviting instability: Transposable elements, double-strand breaks, and the maintenance of genome integrity[END_REF], or as components of essential chromosomal domains, like centromeres and telomeres in many species [START_REF] Wong | Evolutionary dynamics of transposable elements at the centromere[END_REF][START_REF] Lamb | Plant chromosomes from end to end: telomeres, heterochromatin and centromeres[END_REF]. It is clear that TE replication might induce genetic mutations via transposition. For instance, in Drosophila a high rate of spontaneous mutations are related to transposition events (Biemont and Vieira, 2006). The immediate effects of transposition might be harmful, as illustrated by the occurrence of several diseases described in humans (reviewed in [START_REF] Callinan | Retrotransposable elements and human disease[END_REF]). Despite the damaging effects, TEs are maintained in almost all genomes either as full length or truncated copies. Full length copies have kept their ability to mutate the genome through transposition while the truncated copies have often lost the capacity to jump. However, the truncated versions might putatively be recruited by the genome. Indeed, recent reports have proposed a co-evolution of TEs and hosts, leading to the participation of TEs (often truncated copies) in complex genomic processes (as co-opted open reading frames, post transcriptional gene regulation, gene protein translation enhancement etc. as reviewed in [START_REF] Muotri | The necessary junk: new functions for transposable elements[END_REF][START_REF] Sinzelle | Molecular domestication of transposable elements: from detrimental parasites to useful host genes[END_REF]). Moreover, truncated copies may also serve as recombinogenic substrates to other truncated or full-length copies, inducing genome rearrangements. TE copies have been the source of new regulatory sequences, alternative splice sites, polyadenylation signals [START_REF] Marino-Ramirez | Transposable elements donate lineage-specific regulatory sequences to host genomes[END_REF], and new transcription factor binding sites [START_REF] Polavarapu | Evolutionary rates and patterns for human transcription factor binding sites derived from repetitive DNA[END_REF]. Also, TEs enhance genome regulation as, for example, when they are the origin of microRNAs, which are able to regulate gene expression [START_REF] Hasler | Useful 'junk': Alu RNAs in the human transcriptome[END_REF][START_REF] Piriyapongsa | Origin and evolution of human microRNAs from transposable elements[END_REF]. Since TEs are widespread in the genome and have so many influences on gene regulation, several authors have suggested a vital need for TEs in creating, remodelling and regulating gene networks [START_REF] Mcclintock | The significance of responses of the genome to challenge[END_REF][START_REF] Feschotte | Transposable elements and the evolution of regulatory networks[END_REF]. Therefore, as we hypothesize, a strong impact on genome structure and regulation could be observed after TE bursts of transposition.

Driving speciation through TE bursts of transposition

TEs have been observed in all sequenced genomes analysed to date and comparative genomics allows a broad insight into the variability in TE content between genomes of different species. For instance, TEs represent 60% of the maize genome [START_REF] Messing | Sequence composition and genome organization of maize[END_REF] and only 14% of Arabidopsis thaliana's (2000). Variations in genome size between closely related species can also be correlated to differences in the amount of TEs. For instance, in the D. melanogaster species subgroup, bigger genome sizes are in part attributed to high TE-like sequences, as estimated from the amount of reverse transcriptase related sequences by dotblot [START_REF] Boulesteix | Differences in genome size between closely related species: the Drosophila melanogaster species subgroup[END_REF]. In cotton, a significant variation in the total amount of TEs is observed between gossypium species (40% to 65%) [START_REF] Hawkins | Differential lineagespecific amplification of transposable elements is responsible for genome size variation in Gossypium[END_REF]. Such disparities depend on particular TE subfamilies, such as a massive amplification of the gypsy-like Gorge3 element in increased genome size cotton species as shown by whole genome shotgun sequencing [START_REF] Hawkins | Differential lineagespecific amplification of transposable elements is responsible for genome size variation in Gossypium[END_REF]. Variations in the relative proportion of TEs can also be intra-specific, as noted for the euchromatic copies of several TE families when counting on polytene chromosomes in natural populations of D. simulans (Vieira et al., 1999). Variation in the proportion of TEs illustrates not only the influence of a given species/population on the host/TE relationship but also suggests that TEs might play a significant role in speciation through their individual specific influence on the host genome.

TE abundance, TE-derived genomic features and chromosomic rearrangements involving TE sequences are frequently lineage specific and, therefore, suggest that variations might have either stabilized after speciation or contributed to the species evolutionary process [START_REF] Marino-Ramirez | Transposable elements donate lineage-specific regulatory sequences to host genomes[END_REF][START_REF] Bohne | Transposable elements as drivers of genomic and biological diversity in vertebrates[END_REF]. One should note that correlating TE transposition consequences and speciation is rather tricky, since an exact timing of TE bursts and natural species diversification is difficult to measure. Several researchers have tried to obtain examples of timing concordance between bursts of transposition or massive TE extinction with speciation (Table 1). Significant TE activity is observed in several species often during a period of radiation, suggesting that massive speciation and massive TE activity may be associated. The genetic distance between two organisms is calculated as a function of their genetic divergence, so every episode creating divergence, such as lineage specific transposition events, might contribute to the reproductive isolation of those organisms. Either as a cause or as a consequence of genetic differentiation, TE patterns that are different between individuals of the same species may serve not only as genetic markers for researchers but also as evidence of the speciation process occurring within the species [START_REF] Esnault | High genetic differentiation between the M and S molecular forms of Anopheles gambiae in Africa[END_REF]. For instance, significant TE insertion site polymorphism can be observed in rice for the japonica and indica cultivars, accounting for 14% of their genetic differences [START_REF] Huang | Genome-wide analysis of transposon insertion polymorphisms reveals intraspecific variation in cultivated rice[END_REF]. Since the exact evolutionary history of a species is difficult to determine, it is useful to study interspecies crosses as a model for macroevolution. Indeed, interspecies hybrids are classical examples of bursts of transposition causing important dysfunctions and potentially acting as inducers of rapid speciation (reviewed in [START_REF] Michalak | Epigenetic, transposon and small RNA determinants of hybrid dysfunctions[END_REF])) (Table 2). Three hybrids of sunflower species have a 50% larger genome than the parental lines because of a massive TE transposition and they are thought to have undergone rapid speciation (in fewer than 60 generations for one of the hybrids) [START_REF] Ungerer | Rapid hybrid speciation in wild sunflowers[END_REF][START_REF] Ungerer | Genome expansion in three hybrid sunflower species is associated with retrotransposon proliferation[END_REF]. Also, in dosage dependent crosses between A. thaliana and A. arenosa (crosses where the amount of maternal and paternal genome is variable and may be different), high expression of the paternal A. arenosa athila element in the hybrid is correlated to seed lethality [START_REF] Josefsson | Parent-dependent loss of gene silencing during interspecies hybridization[END_REF]. Such observations are essential for understanding hybrid "compatibility" since A.

arenosa and A. thaliana hybridization has succeeded at least once in nature [START_REF] Jakobsson | A unique recent origin of the allotetraploid species Arabidopsis suecica: Evidence from nuclear DNA markers[END_REF]. In insects, Drosophila buzzatii and D. koepferae are still able to interbreed and have common TE families that are maintained in each genome. Crosses between these two species highly induce the transposition of osvaldo in hybrids whilst it is repressed in both parental genomes [START_REF] Labrador | Interspecific hybridization increases transposition rates of Osvaldo[END_REF]. In wallabies, interspecific hybrids present variable centromeres composed of satellite repeats and newly replicated TE copies [START_REF] Metcalfe | Genomic instability within centromeres of interspecific marsupial hybrids[END_REF]. All these examples suggest that TE bursts of transposition occurring during hybrid speciation may induce important karyotypic changes because of the capacity of TEs to induce major chromosomal rearrangements and ectopic recombination [START_REF] Hedges | Inviting instability: Transposable elements, double-strand breaks, and the maintenance of genome integrity[END_REF][START_REF] Weil | Too many ends: aberrant transposition[END_REF]. In this way, TE mobilization will thus support novel phenotypes followed by micro population ecological isolation, the necessary components for rapid and divergent evolution. Based on hybrid crosses, we hypothesise that bursts of transposition in individuals of one species followed by intraspecific crosses could generate new phenotypes.

Persistence of TEs in most genomes, despite the initial deleterious effects, suggests a strict regulation system keeping TEs silenced. Expression of TEs is dependent on transcription factors presence as illustrated by the evolution of L1 lineage in humans. Indeed, recruitment of regulatory regions in new L1 subfamilies harbouring new transcription factor binding sites are essential for L1 expression (Khan et al., 2006). Also, cellular inhibitors may influence TE transposition post transcriptionally, as observed for some members of the APOBEC family, capable of reducing HERV-K infectivity (50 fold) [START_REF] Lee | Reconstitution of an infectious human endogenous retrovirus[END_REF] and block alu transposition in an ORF1p and ORF2p L1 independent manner [START_REF] Hulme | Selective inhibition of Alu retrotransposition by APOBEC3G[END_REF].

Moreover, transposition of mobile elements induces DNA breaks suggesting an interaction between the host DNA repair machinery and TEs. ERCC1-XPF heterodimers are implicated in DNA repair processes and limit L1 insertion [START_REF] Gasior | ERCC1/XPF limits L1 retrotransposition[END_REF]. Apart from cellular inhibitors and transcription factor dependency, TEs are also transcriptionally and posttranscriptionally regulated through epigenetic means [START_REF] Lisch | Epigenetic regulation of transposable elements in plants[END_REF]. Although regulation of TEs by the host genome might be tight it also needs to remain versatile in order to keep a "rapid genome remodelling source". Such flexibility is insured in many eukaryotic organisms via epigenetic regulatory mechanisms.

TE Epigenetic reprogramming

Epigenetic regulation of TEs involves interdependent pathways, such as chromatin remodelling factors, DNA methylation and non-coding small RNAs [START_REF] Lisch | Epigenetic regulation of transposable elements in plants[END_REF][START_REF] Obbard | The evolution of RNAi as a defence against viruses and transposable elements[END_REF] (Table 3 for a general view on TE epigenetic regulation). In rice, for instance, mutants of histone methyltransferase, specific for repressive chromatin marks, induce DNA demethylation of Tos17 (copia-like retrotransposon) and, consequently, transposition [START_REF] Ding | SDG714, a histone H3K9 methyltransferase, is involved in Tos17 DNA methylation and transposition in rice[END_REF]. Also in plants, it is possible to observe RNA dependent DNA methylation (RDDM) of TEs and genes, which is reversible since it is dependent on the presence of small interfering RNAs [START_REF] Matzke | Targets of RNA-directed DNA methylation[END_REF]. Recent investigations have highlighted the central role of RNA in controlling TE activity: such system was probably present in a common eukaryote ancestor; it is well conserved between species; and it may act as an immunological system against non self RNAs [START_REF] Obbard | The evolution of RNAi as a defence against viruses and transposable elements[END_REF]. Also, small RNAs allow for target specificity of DNA methylation or histone modification in a given sequence. For instance, epigenetic instability in long-term cultured cells of A. thaliana evolves into hypomethylation of specific TEs and subsequent activation [START_REF] Tanurdzic | Epigenomic consequences of immortalized plant cell suspension culture[END_REF]. Indeed, athila or copia elements are hypomethylated regardless of their location, but no change is observed for gypsy class elements [START_REF] Tanurdzic | Epigenomic consequences of immortalized plant cell suspension culture[END_REF]. Such specificity is possibly due to siRNAs being produced differently between TE families under stress such as this, varying from 21nt and 24nt for hypomethylated activated TEs and only 24nt for silenced gypsy class elements [START_REF] Tanurdzic | Epigenomic consequences of immortalized plant cell suspension culture[END_REF]. The epigenetic regulation system is, indeed, rather efficient; it is general because the TE families capable of invasion are multiple and divergent but, at the same time, it also appears to be specific, targeting single TE families through sequence specific small RNAs. Each pathway of the epigenetic regulation of TEs seems, therefore, to be essential and also extremely rigorous. Naturally the question arises as to how TEs could possibly invade a genome if they are being held prisoners in a perfect prison. It is well known that TEs do transpose often at a very low rate suggesting that the perfect prison is, after all, flexible. Indeed, it has recently been suggested that small RNAs can be linked with total or partial silence of elements, as observed in Drosophila hybrid dysgenesis. Indeed, intraspecific Drosophila crosses may cause hybrid dysgenesis of P and I elements showing very important deleterious effects, such as female sterility or chromosomal abnormalities [START_REF] Bucheton | The molecular basis of I-R hybrid dysgenesis in Drosophila melanogaster: identification, cloning, and properties of the I factor[END_REF][START_REF] Castro | Drosophila melanogaster P transposable elements: mechanisms of transposition and regulation[END_REF]. In these crosses individuals of the same species have different amounts of TEs since one of the parents has an empty genome. A deficit in small interfering RNA (piRNA) in the maternal gamete allows originally silenced TEs to transpose in the hybrids [START_REF] Brennecke | An epigenetic role for maternally inherited piRNAs in transposon silencing[END_REF][START_REF] Chambeyron | piRNA-mediated nuclear accumulation of retrotransposon transcripts in the Drosophila female germline[END_REF]. However, despite the associated female sterility or embryonic lethality, it should not be forgotten that a few hybrids do survive and are fertile, thus propagating newly mobilised copies and possible chromosomal changes to the population.

The study of natural populations and the observation of the natural variability that exists in epigenetic host control can lead to an understanding of TE induced macroevolution.

Epigenetic variation in hybrids, in allopolyploid species, and in single individuals could awake TEs, induce a burst of transposition and, as described above, increase karyotypic changes followed by ecological isolation. TE epigenetic regulation can be observed in somatic tissues [START_REF] Barbot | Epigenetic regulation of an IAP retrotransposon in the aging mouse: progressive demethylation and de-silencing of the element by its repetitive induction[END_REF][START_REF] Malone | Specialized piRNA pathways act in germline and somatic tissues of the Drosophila ovary[END_REF] and in germline tissues [START_REF] Malone | Specialized piRNA pathways act in germline and somatic tissues of the Drosophila ovary[END_REF].

A model has been proposed by Slotkin and co-researchers (Slotkin et al., 2009) in A. thaliana

where the naturally occurring hypomethylation of TEs in the vegetative nucleus (somatic) influences TE activity in the neighbouring sperm cell. In Drosophila, the vertical propagation of some retroviral elements (proviral amplication) depends on the inhibition, via small RNA interference, of full length retroviruses in the follicular cells adjacent to the ovocyte [START_REF] Brennecke | Discrete small RNA-generating loci as master regulators of transposon activity in Drosophila[END_REF][START_REF] Lau | Abundant primary piRNAs, endo-siRNAs, and microRNAs in a Drosophila ovary cell line[END_REF][START_REF] Malone | Specialized piRNA pathways act in germline and somatic tissues of the Drosophila ovary[END_REF]. Both types of regulation (somatic and germinal), if variable, can influence the population behaviour by creating phenotypical variations that might be inherited. Variation in TE epigenetic regulation can be observed such as in A. thaliana where the LINE-like element sadhu varies between three ecotypes showing different degrees of DNA methylation and different silencing states [START_REF] Rangwala | Meiotically stable natural epialleles of Sadhu, a novel Arabidopsis retroposon[END_REF]. Other epialleles, variation in the epigenetic regulation of a given sequence between tissues and/or individuals of the same population, were most described in plants and mice. However, improvement in population epigenetics is still necessary along with ecological epigenetic studies in order to fully understand natural population variation in epigenetic regulation [START_REF] Bossdorf | Epigenetics for ecologists[END_REF][START_REF] Johannes | Epigenome dynamics: a quantitative genetics perspective[END_REF][START_REF] Richards | Population epigenetics[END_REF]. TE epigenetic regulation is, therefore, a variable and flexible mechanism that may induce massive TE transposition in the germline and consequent chromosomal rearrangements.

The gibbon species has rapidly accumulated chromosomal rearrangements and, hence, offers an interesting model for karyotypic evolution and speciation. Carbone and collaborators recently described an example of the different epigenetic regulation of Alu elements between humans and gibbons associated with breakpoints between both species [START_REF] Carbone | Evolutionary breakpoints in the gibbon suggest association between cytosine methylation and karyotype evolution[END_REF].

They observed a CpG content higher in the gibbon alu elements near the breakpoints (typical from active elements) and such elements are undermethylated compared with humans. Alu elements present in the breakpoints are probably active and responsible, in part, for the rapid chromosomal remodelling in the gibbon. The authors propose that "the association between undermethylation and chromosomal rearrangement in gibbons suggests a correlation between epigenetic state and structural genome variation in evolution".

Conjugating two different genomes in the same organism, as is the case for hybrids or in allopolyploidization, may require significant adaptations of all the regulatory mechanisms, including TE epigenetic regulation (reviewed in [START_REF] Michalak | Epigenetic, transposon and small RNA determinants of hybrid dysfunctions[END_REF])) (Table 2). In wallabies, interspecies crosses cause a burst of transposition of a retrotransposon, together with a genome-wide hypomethylation [START_REF] O'neill | Undermethylation associated with retroelement activation and chromosome remodelling in an interspecific mammalian hybrid[END_REF]. Such a burst of transposition targeted only one parental genome and results in extended centromeres, suggesting a rapid karyotype differentiation from the parents [START_REF] O'neill | Undermethylation associated with retroelement activation and chromosome remodelling in an interspecific mammalian hybrid[END_REF]. Other natural crosses were analysed by the authors and hypomethylation of the hybrids was always observed as de novo chromosomal changes. In allopolyploidization ("condition whereby evolution is accelerated and fitness is enhanced" as suggested by Liu and Wendel), TE transposition may also be concomitant with genome-wide epigenetic changes [START_REF] Liu | Epigenetic phenomena and the evolution of plant allopolyploids[END_REF]. These examples show how genome remodelling could occur after epigenetic variation in TE copies. However, we need to establish what are the causes of genome wide epigenetic modifications and subsequent TE activation. Interspecific crosses induce genomic stress, i.e. changes in genomic stability (chromatin changes, density in repeats…) and organization (DNA recombination, TE replication, retroposed or duplicated genes…), that could indeed have an impact on epialleles and provoke TE activation. Genome-wide epigenetic changes might play a role in genome adaptation to environmental changes. One could easily hypothesize that TE awakening is due to epigenetic changes, and that those variations start at the level of one individual as a response to specific environmental changes. The ecological outcomes of TE mobilisation due to environmental changes may be numerous, such as survival of the host, increase of host fitness, micro population isolation etc. The consequent spread of these factors into populations can lead to sexual isolation and speciation.

Environment induces epigenetic reprogramming

Several investigations have demonstrated that modifications in the environment induce epigenetic modifications and, therefore, transcription state changes [START_REF] Jaenisch | Epigenetic regulation of gene expression: how the genome integrates intrinsic and environmental signals[END_REF][START_REF] Han | LINE-1 retrotransposons: modulators of quantity and quality of mammalian gene expression?[END_REF]. Such transcriptional changes are a source of phenotypic variability that may be explored by organisms through increasing the host "adaptative potential". Indeed, diet changes, temperature variation, stress etc. all have an impact on gene regulation [START_REF] Waterland | Early nutrition, epigenetic changes at transposons and imprinted genes, and enhanced susceptibility to adult chronic diseases[END_REF][START_REF] Cropley | Germ-line epigenetic modification of the murine A vy allele by nutritional supplementation[END_REF][START_REF] Gibert | Phenotypic plasticity in Drosophila pigmentation caused by temperature sensitivity of a chromatin regulator network[END_REF][START_REF] Chinnusamy | Epigenetic regulation of stress responses in plants[END_REF]. Similarly to epigenetic regulation, diet changes, temperature variations, stress etc. might affect TE transposition [START_REF] El-Sawy | Nickel stimulates L1 retrotransposition by a post-transcriptional mechanism[END_REF][START_REF] Hashida | The temperature-dependent change in methylation of the Antirrhinum transposon Tam3 is controlled by the activity of its transposase[END_REF][START_REF] Ebina | Stress management: how cells take control of their transposons[END_REF][START_REF] Cho | Endogenous retroviruses in systemic response to stress signals[END_REF]. Consequently, activation of TEs could be the result of relaxed epigenetic control induced by environmental changes. There is a huge amount of literature relating to the activation of TEs by environmental stresses, but only a few examples illustrate the link existing between environmental epigenetic instability and transposition.

Early nutrition has an impact on the epigenetic regulation of TEs, especially DNA methylation, as reviewed by [START_REF] Waterland | Early nutrition, epigenetic changes at transposons and imprinted genes, and enhanced susceptibility to adult chronic diseases[END_REF]. The aguti gene controls hair color in mice (brown in wild type). An insertion of an IAP retrotransposon in the first exon induces ectopic and variable expression of aguti. LTR from IAP elements is regulated by DNA methylation but varies between individuals. Dietary supplementation (methyl donors) shifts the phenotype to wild type brown colour, concomitant to a higher DNA methylation in the IAP element [START_REF] Waterland | Transposable elements: targets for early nutritional effects on epigenetic gene regulation[END_REF]. Heat treatment and aging induce the transcription of older heterochromatic I copies and, hence, the production of small interfering RNAs (rasiRNA) repressing active I elements in the germline [START_REF] Dramard | Natural epigenetic protection against the I-factor, a Drosophila LINE retrotransposon, by remnants of ancestral invasions[END_REF]. Individuals exposed to the pollutant benzene show decreased DNA methylation of L1 and alu1 elements [START_REF] Bollati | Changes in DNA methylation patterns in subjects exposed to low-dose benzene[END_REF] and, similarly, benzo(a)pyrene increases retrotransposition of L1 elements in HeLa cells [START_REF] Stribinskis | Activation of human long interspersed nuclear element 1 retrotransposition by benzo(a)pyrene, an ubiquitous environmental carcinogen[END_REF]. In rice, spaceflight induces transposition of several TEs, sometimes associated with hypomethylation within the element [START_REF] Long | The spaceflight environment can induce transpositional activation of multiple endogenous transposable elements in a genotype-dependent manner in rice[END_REF]. In mice, a long-term peroxisome proliferator diet induces hypomethylation of satellites, IAP and L1/L2 elements [START_REF] Pogribny | Epigenetic effects of the continuous exposure to peroxisome proliferator WY-14,643 in mouse liver are dependent upon peroxisome proliferator activated receptor alpha[END_REF].

Conclusion

Epigenetic regulation of TE copies has two main consequences: 1) the environment can have a direct influence on TE activity through epigenetic instability; 2) the presence of TE sequences in the host genome in a "harmless" state. Since bursts of transposition have been observed in several species it is tempting to suggest that the defense system has, at least temporarily, broken down. However, this failure is transient and the host may rapidly silence "de novo" TE copies. Although the benefit is not immediate, transposition might have a long term advantage. Indeed, the consequences of transposition bursts will be numerous, resulting in a renewal of genetic diversity, which is the major condition for genome evolution and the action of selection. This genetic diversity is, thus, fundamental for renewing gene networks to their "rolling circle" replication mechanism. For a recent classification of eukaryote TEs, please refer to [START_REF] Wicker | A unified classification system for eukaryotic transposable elements[END_REF] • DNA elements were extremely active during Myotis radiation. [START_REF] Pascale | Amplification of an ancestral mammalian L1 family of long interspersed repeated DNA occurred just before the murine radiation[END_REF][START_REF] Verneau | Determining and dating recent rodent speciation events by using L1 (LINE-1) retrotransposons[END_REF][START_REF] Lander | Initial sequencing and analysis of the human genome[END_REF][START_REF] Dobigny | LINE-1 amplification accompanies explosive genome repatterning in rodents[END_REF][START_REF] Ray | Multiple waves of recent DNA transposon activity in the bat, Myotis lucifugus[END_REF]

Fish

• DNA transposon bursts of transposition are concomitant with speciation events in pseudotetraploid salmonids and after genome duplication ; [START_REF] Volff | Evolution and discontinuous distribution of Rex3 retrotransposons in fish[END_REF] Note that de novo stable silencing of TEs is observed in the introgression lines. [START_REF] Ungerer | Rapid hybrid speciation in wild sunflowers[END_REF][START_REF] Kashkush | Gene loss, silencing and activation in a newly synthesized wheat allotetraploid[END_REF][START_REF] Liu | Epigenetic phenomena and the evolution of plant allopolyploids[END_REF][START_REF] Josefsson | Parent-dependent loss of gene silencing during interspecies hybridization[END_REF][START_REF] Ungerer | Genome expansion in three hybrid sunflower species is associated with retrotransposon proliferation[END_REF] sequencing programs in order to locate each copy and to make ChIP copy specific, but also bioinformatic analysis.

Apart from the chromatin remodelling factors analysed in this work, DNA methylation and small interfering RNAs are also part of the epigenetic TE regulation. It has been proposed that the original role of epigenetic defence, especially small RNAs, is to protect the genome against foreign DNA, as TEs, and act as an extrapolated immunological system [START_REF] Obbard | The evolution of RNAi as a defence against viruses and transposable elements[END_REF]. Therefore, TEs and epigenetic regulation factors have coexisted and coevolved for a long time. One could wonder if epigenetic regulation system was not adapted for genes while specific for parasite DNAs. The ability of TEs to insert near genes and to be genome widespread would perhaps generalise the flexible epigenetic mechanisms to its neighbourhood, as already observed for heterochromatin spreading. Genes would thus be epigenetically regulated thanks to TEs. Chromatin territories would be created, and specific regulatory elements would be targeted to each specific region. Also, as suggested by our data, if epigenetic regulation is actually element-specific, invasion of genomes by TEs become possible if they are unknown for the system. However, de novo silencing of TEs through epigenetic learning would avoid TE activity to be maintained. Hence, TEs that are new for a genome may have the opportunity to engage invasion until the moment epigenetic mechanisms start to act and then suppress TE activity. In that case, epigenetic flexibility allows a renouveau of genetic material to the first contact with TEs. If any of these hypotheses is true, it means that efficiency of the epigenetic system to control the first invasion is essential. If no "epigenetic immunological system" is engaged then the genome will be invaded and the TEs will cause important chromosomal changes, and sometimes even macroevolution.

The FASEB Journal Transposable elements (TEs), which are DNA sequences that can move and multiply along the chromosomes, are now considered to be full-fledged components of genomes, able to play various and numerous functional roles (1,2). However, their dynamics within a given genome and natural populations is far from being fully understood (3,4), since TE amount and genomic distribution vary considerably between different species and populations. In most organisms, a high proportion of the heterochromatic genomic compartment is composed of TEs, which are usually organized in clusters (5,6) and mainly correspond to deleted elements. However, an increasing amount of data shows that heterochromatin also harbors complete copies of TEs, which may constitute a "reservoir" of new elements that may be reactivated (7)(8)(9) and thus are potentially able to invade gene-rich regions like the euchromatin, which may have a considerable evolutionary impact. This idea is supported by data showing that the distribution of TEs between euchromatin and heterochromatin is variable and depends on the populations or strains analyzed (10 -13). However, we still do not have a refined analysis of the structure and activity of TEs located in different chromatin conformations within a given genome and between genomes. From this perspective, tirant, an endogenous retrovirus from Dro-sophila belonging to the gypsy-like long terminal repeat (LTR) retrotransposon subclass, is an interesting model, since its genomic copy number varies considerably in different natural populations of Drosophila simulans, ranging from 0 euchromatic insertion in most worldwide populations, to 2 to 5 in East African populations [START_REF] Bie ´mont | Worldwide distribution of transposable element copy number in natural populations of Drosophila simulans[END_REF][START_REF] Vieira | Wake up of transposable elements following Drosophila simulans worldwide colonization[END_REF]. A previous study of the tirant regulatory region in natural populations of Drosophila melanogaster and D. simulans revealed two subfamilies of tirant regarding the 5Ј LTRuntranslated region (UTR) [START_REF] Fablet | Ongoing loss of the tirant transposable element in natural populations of Drosophila simulans[END_REF]. One subfamily, called C type, corresponds to the euchromatic insertions in African populations, and is found in the heterochromatin of all populations worldwide. This C type has been shown to be expressed when located in euchromatin. The other subfamily, called S type, is found in all populations, at very low copy numbers [START_REF] Fablet | Ongoing loss of the tirant transposable element in natural populations of Drosophila simulans[END_REF], and despite its high levels of sequence conservation between populations, is not found to be actively transcribed.

In an attempt to understand the dynamics of tirant and the influence of genomic localization on its activity, we relate data on the in vitro expression of tirant and on genome walking and sequence analysis. We show that the chromatin environment is a hallmark for expression and that heterochromatin harbors full-length elements that have invasive features. We tested whether the different 5Ј LTR-UTR variants were able to promote expression using the reporter gene technique and correlated this to the genomic location of each tirant insertion and local chromatin structure. We then identified the genomic context of the insertions in the D. simulans sequenced genome, and described the copies of tirant.

MATERIALS AND METHODS

Drosophila natural populations

We worked on fly samples collected from several geographically distinct natural populations of D. melanogaster and D. simulans. A list of the populations analyzed is provided in Supplemental Table 1. These populations were maintained in the laboratory at 17°C as isofemale lines or small mass cultures with around 50 pairs in each generation.

Reporter gene assays

Constructions

We used 3 variants of the tirant 5Ј LTR-UTR region previously amplified by polymerase chain reaction (PCR) from natural population samples ( 14) (GenBank accession numbers: AY756122, AY756118, and AY756121; see Fig. 1 for a detailed diagram). We refer to these sequences as S/C-i,j, where S or C is the type, i is the number of repeats of a 19-bp motif in the LTR, and j is the number of repeats of a 102-bp motif in the 5Ј UTR: S-1,5 (AY756122), C-2,2 (AY756121), and C-2,4 (AY756118) (Fig. 1). The 5Ј LTR-UTR regions were cloned separately upstream of a lacZ reporter gene into the SphI-PstI (for S-1,5) and SphI-XbaI (for C-2,2 and C-2,4) sites of the pPelican plasmid [START_REF] Barolo | GFP and beta-galactosidase transformation vectors for promoter/enhancer analysis in Drosophila[END_REF]. Plasmids were amplified in TOP 10 competent cells from Invitrogen (Carlsbad, CA, USA) and purified with Plasmid Mini/Midi kits from Qiagen (Courtaboeuf, France) (final elution with water). The presence of the 5Ј LTR-UTR regions in the plasmids were confirmed by PCR and restriction profiles.

Transfection into S2 cells

Transfections were done using Celfectin (Invitrogen). Cells were then incubated for 72 h at 22°C. S2 cells were cotransfected with tirant constructs and the pGL3-Control vector (Promega, Madison, WI, USA), consisting of a luciferase gene downstream of an SV40 promoter. ␤-Galactosidase activity was normalized vs. luciferase activity.

The ratios of the reported ␤-galactosidase activity to the luciferase activity were compared for the various constructions. After cell lysis, the ␤-galactosidase and luciferase activities were measured using the ␤-gal reporter gene assay, chemiluminescence kit (Roche Diagnostics, Mannheim, Germany), and the Luciferase Assay System kit (Promega), respectively.

Each transfection was repeated six times. Three wells were transfected with pPelican alone, as a negative control, and three others were transfected with a vector carrying a lacZ gene downstream of an SV40 promoter, as a positive control.

Genome-walking analysis

We determined the insertion site sequences for each copy of tirant with the Universal Genome Walker kit (Clontech, Mountain View, CA, USA) in three genomes from natural populations of D. simulans: the Makindu population from Kenya (5 euchromatic copies), the Chicharo population from Portugal (0 euchromatic copies), and the Zimbabwe population (2 euchromatic copies). Genomic DNA was extracted from 20 female adults from each population. For each population, 4 genomic libraries were obtained using the restriction enzymes supplied by the provider: DraI, EcoRV, PvuII, and StuI, with 9, 5, 0, and 2 restriction sites, respectively, along the tirant reference sequence. Adaptors were ligated according to the supplier's instructions, and two nested PCRs were subsequently done, with primers specific to tirant LTR and to the adaptors. The external tirant-specific primer can amplify both C and S types: 5Ј GTT TAG AGG CGT GGG GGT TTA GAA TC 3Ј. The internal tirant-specific primers specifically used for the C and S types are 5Ј TGT AAG CAT AAT GAA CAT GCC GAC TC 3Ј and 5Ј TGT AAA CAT AAT TTC CAT GCC ACT TC 3Ј, respectively. PCRs were done in 2 steps using the Advantage Genomic PCR kit (Clontech).

PCR amplification of tirant entire copies

To amplify each copy of tirant, we designed specific primers from the flanking sequences of each insertion (Supplemental Table 2). We realized 3-step PCRs with the Expand Long Template PCR System (Roche) with an annealing temperature of 60°C and the System 1 buffer, provided by the supplier.

Populational screening of tirant insertions

For some of the insertion sites determined by genome walking in the Makindu population that corresponded to potentially full-length copies of tirant, we designed a pair of primers with the forward primer in the env gene, and the reverse primer in the 3Ј flanking region. We looked for the presence of PCR products in a pool of 3 flies for each natural population, which would indicate that the corresponding insertion of tirant was shared by several populations. The Makindu population was systematically used as a positive control for the PCR assays. The screened populations and the list of the primers are presented in Supplemental Table 3. We used the EuroBlueTaq enzyme from Eurobio (Les Ullis, France). The PCR was a 3-step reaction run in 30 cycles with an annealing temperature of 57°C. Amplified products were migrated on a 1% agarose gel.

Local chromatin structure analysis

Chromatin immunoprecipitation (ChIP)

Extraction of chromatin from 16-h D. simulans Makindu embryos and immunoprecipitation (IP) were adapted from Sandmann et al. (16). Cell lysis buffer was changed to 5 mM PIPES pH 8, 85 mM KCl, 0.5% Nonidet P-40 supplemented with protease inhibitors. Chromatin was shared with a Bioruptor sonicator water bath (Diagenode, Lie `ge, Belgium) for 6 ϫ 30-s on/30-s off cycles at high power in order to have random fragments from 1 kb to 500 bp. Shared chromatin was incubated overnight at 4°C with antibodies recognizing H3K9me2 (Millipore, Billerica, MA, USA; 07441), H3K27me3 (Millipore 07449), H3K4me2 (Millipore 07030), H3 (Abcam, Cambridge, UK; ab1791), and rabbit IgG (Sigma-Aldrich, St. Louis, MO, USA; I5006). The antigenantibody complexes were washed as described before ( 16), but a second washing solution was modified as followed: TE 2ϫ, 500 mM NaCl, 1% Triton, 0.1% SDS.

Real-time PCR

To quantify each IP, real-time PCR was performed using QuantiTect SYBR Green PCR kit (Qiagen) on a MXP3000P PCR system (Stratagene, La Jolla, CA, USA). Reactions were done in duplicates, and standard curves were calculated on serial of input chromatin. To evaluate the relative enrichment of C and S copies after IP, we calculated the difference in cycles between the IP-enriched sample and the input DNA for tirant copies and for a control (actin-CG4027). C 2 and S 5 copies (see Table 1, Makindu population) were amplified using primers in the flanking region and in the tirant LTR (C 2 specific primers: forward, 5Ј GTG TTC CAG TTG CCG TCT TC 3Ј; reverse, 5Ј TTT TCT GGG GTG TTG TAC GC 3Ј; S 5 specific primers: forward, 5Ј TGC TCT CAA CTG CGC GCG AGT TAC 3Ј; reverse, 5Ј GTA AA ATA ATT TCC ATG CCA CTT C 3Ј).

Analysis of tirant copies from the D. simulans sequenced genome

We retrieved the sequences of the chromosome arms 2L, 2R, 3L, 3R, 4, X, and the unassigned part (named U), of the first release of the mosaic assembly of the genome of D. simulans available at the ftp site of the Genome Sequencing Center at the Washington University Medical School (http://hgdownload. cse.ucsc.edu/downloads.html#droSim). This mosaic assembly corresponds to different D. simulans strains. In the following, we refer to the tirant copies using the chromosome name and the start position of the copy (e.g., chr2L_21040234 corresponds to a copy found on chromosome 2L and that starts at position 21040234).

To search for any kind of copy of tirant of the two variant types in the D. simulans sequenced genome, we used the sequences of the S type (GenBank AY756122) and the C type (GenBank AY756123) cloned by Fablet et al. (14). As these sequences corresponded only to the 5Ј LTR-UTR and 5Ј region of the first open reading frame (ORF) gag, we also used the complete sequenced tirant from D. melanogaster (GenBank AY928610) [START_REF] Lerat | Sequence divergence within transposable element families in the Drosophila melanogaster genome[END_REF], which is a C type, to identify sequences corresponding to internal parts and to potentially complete copies of tirant in D. simulans. With all these sequences, we searched for tirant copies in the D. simulans sequenced genome using Blastn [START_REF] Altschul | Gapped BLAST and PSI-BLAST: a new generation of protein database search programs[END_REF]. Matches with an e_value Ͻ 10 Ϫ10 were retained, and those with distances Ͻ 300 bp were merged. The detected copies were compared pairwise with the three query sequences using the program matcher (EMBOSS website; http://emboss.bioinformatics. nl/cgi-bin/emboss/matcher), and the corresponding percentage identity was computed with the dnadist module from the PHYLIP package [START_REF] Felsenstein | PHYLIP-Phylogeny Inference Package (Version 3.2)[END_REF]. The detection of the ORFs was performed using the ORF Finder program at the National Center for Biotechnology Information website (www.ncbi. nlm.nih.gov/projects/gorf/).

RESULTS

Reporter gene assays

Three types of tirant 5Ј LTR-UTR regions were tested for the promotion of lacZ expression in S2 cells: S-1,5, which is an S type, and C-2,2 and C-2,4, which are C types with two and four 102-bp motifs in the 5Ј UTR, respectively (see Fig. 1). Note that the number of repeats of the 102-bp motif is always 5 for all S types previously identified in natural populations [START_REF] Fablet | Ongoing loss of the tirant transposable element in natural populations of Drosophila simulans[END_REF]. As shown in Fig. 2, the activity ratios for S-1,5, C-2,2, and C-2,4 were significantly different from both the positive and negative controls (see Materials and Methods for It is not possible to determine the position on the chromosomes of some sites, especially those corresponding to insertions into other transposable elements. NA, electrophoresis of the PCR product shows multiple bands, possibly indicating embedded insertions of tirant. details). One-tailed pairwise t tests revealed a significant difference between C-2,2 and C-2,4 (Pϭ0.012), and between S-1,5 and C-2,4 (Pϭ0.006), whereas C-2,2 and S-1,5 were not significantly different (Pϭ0.216). This shows that both the C and S types are able to promote the expression of the reporter gene and are therefore theoretically able to promote tirant expression in an endogenous context.

Analysis of the genomic context of each copy of tirant

The reporter gene assay results cannot be directly extrapolated to the endogenous copies of tirant without taking into account the genomic localization of each copy (i.e., in euchromatin or heterochromatin), which could influence transcription. We therefore determined the genomic localization of each insertion of tirant by the genome-walking technique.

The genome-walking protocol allowed us to identify 10, 7, and 4 tirant insertion sites in the genomes of the Makindu, Zimbabwe, and Chicharo populations, respectively. The results are presented in Table 1. The number of insertions was slightly higher than that obtained by in situ hybridization [START_REF] Bie ´mont | Worldwide distribution of transposable element copy number in natural populations of Drosophila simulans[END_REF]. This is due to the following facts: 1) only euchromatic copies on the chromosome arms were detected by in situ hybridization, 2) full-length copies as well as solo LTRs could be detected by genome walking, whereas the detection size threshold for in situ hybridization excluded that for solo LTRs, and 3) the genome walking was done on DNA extracted from a pool of 20 individuals, thus revealing the sum of the insertions resulting from individual variability, whereas insertion sites were estimated for single individuals by the in situ hybridization technique.

Since the sequenced genome of D. simulans was very poorly annotated at the time of the study, we used the annotations of the corresponding regions in the D. melanogaster sequenced genome. The only shared site among the genomes of the 3 natural populations analyzed in the present study corresponded to an insertion into the Maupiti Islands centromeric DNA, which is an A ϩ T-rich region displaying many insertions of TEs, mainly non-LTR retrotransposons, which showed a high degree of similarity with the F and doc elements (9). Of the other 18 insertions of tirant identified in the 3 genomes, 7 were found within other TEs, such as hobo [START_REF] Bonnivard | Characterization of natural populations of Drosophila melanogaster with regard to the hobo system: a new hypothesis on the invasion[END_REF], frogger [START_REF] Kaminker | The transposable elements of the Drosophila melanogaster euchromatin: a genomics perspective[END_REF], MAX [START_REF] Kaminker | The transposable elements of the Drosophila melanogaster euchromatin: a genomics perspective[END_REF], diver2 [START_REF] Kapitonov | Molecular paleontology of transposable elements in the Drosophila melanogaster genome[END_REF], R1 [START_REF] Eickbush | Vertical transmission of the retrotransposable elements R1 and R2 during the evolution of the Drosophila melanogaster species subgroup[END_REF], and Rt1b [START_REF] Busseau | Identification of Waldo-A and Waldo-B, two closely related non-LTR retrotransposons in Drosophila[END_REF]. Five of the 18 insertions were heterochromatic, and the remaining 6 were found in unannotated DNA which probably corresponds to noncoding DNA. With the data of the exact insertion site for each copy of tirant, we were able to design primers specific to the flanking region of each individual copy of tirant, in order to amplify it by long PCR in the different populations. As expected from previous Southern blot results [START_REF] Fablet | Ongoing loss of the tirant transposable element in natural populations of Drosophila simulans[END_REF], we obtained full-length amplicons, as well as shorter ones, for each of the 3 populations tested. The detailed results and the correspondence with insertion sites are shown in Table 1. Five of the insertions in the Makindu population were of the expected size for a full-length tirant, i.e., 8.5 kb. In the other cases, the amplicons corresponding to short fragments were assumed to be solo LTRs (the amplicons were sequenced for insertions Makindu 7 and Zimbabwe 4). We used a PCR test to check whether the potentially full-length insertions were present in other populations than Makindu, and we found that these insertions were specific to the Makindu population and were not found in the other populations.

To determine whether local chromatin environment could vary between C and S copies, we identified histone modification marks on two different tirant full-length copies: C 2 and S 5 (see Table 1). ChIP followed by copy-specific real-time PCR analysis was performed using antisera against permissive (H3K4me2) and repressive (H3K9me2 and H3K27me3) histone marks [START_REF] Ebert | Histone modification and the control of heterochromatic gene silencing in Drosophila[END_REF]. We observed that the S-type copy of tirant is strongly associated with the repressive histone marks H3K9me2 and H3K27me3. In contrast, the C-type copy of tirant is characterized by the coenrichment of the repressive mark H3K27me3 and the permissive H3K4me2 (Fig. 3). These observations suggest that the only full-length S-type copy present in the Makindu genome is embedded in a repressive chromatin environment, while the C-type copy can be in a bivalent chromatin domain.

D. simulans sequenced genome analysis

Among the 20 copies of tirant identified in the D. simulans sequenced genome, 5 are of the S type and 15 of the C type (Table 2 and Fig. 4A,B). For each type, we found one almost-complete sequence. The full-length S-type copy U_8880559 is located in the U part of the genome and corresponds to a sequence of 8508 bp. It is flanked by two LTRs of 427 bp sharing 99.30% identity, which indicates that this is a recent insertion. Two ORFs correspond to potentially complete gag (positions 2028 to 2978) and env (positions 6847 to 8118) genes. The pol ORF (positions 3218 to 6455) displays some frameshifts that result in inframe stop codons. The full-length C-type copy U_10384153 is also located in the U part of the genome, and corresponds to a sequence of 6803 bp, surrounded by undetermined bases, which made it impossible for us to determine the presence of LTRs at the extremities. Two ORFs correspond to potentially complete gag (positions 911 to 2044) and env (positions 5693 to 6801) genes; the pol ORF (positions 2284 to 5515) displays a large internal deletion that creates a frameshift.

Comparison of the cloned flanking regions with those of the copies identified in silico showed that the 2Lrandom-887610 copy (Table 1; Fig. 4B) is present in both the Makindu and Zimbabwe populations (insertions Ma-kindu 7 and Zimbabwe 4). The sequencing of PCR products from populations shows that this insertion is a solo LTR. However, it is not possible to conclude about the corresponding insertion found in the sequenced genome, since it is flanked downstream by undetermined bases. The other insertions detected in the 3 populations correspond to unique insertions that are not present in the sequenced genome of D. simulans.

DISCUSSION

The S and C types of tirant, previously characterized in D. melanogaster and D. simulans [START_REF] Fablet | Ongoing loss of the tirant transposable element in natural populations of Drosophila simulans[END_REF], appear to behave quite differently in the genome of D. simulans. It had previously been shown that the C type was polymorphic, presenting varying numbers of a 102-bp motif in its UTR, whereas the S type was monomorphic, with always 5 repeats of this motif in its UTR. In addition, the S-type copy number was low and homogeneous among D. simulans natural populations, while the Ctype copy number was high in East African populations, but quite low in the surrounding populations [START_REF] Fablet | Ongoing loss of the tirant transposable element in natural populations of Drosophila simulans[END_REF].

Tirant activity

Reporter gene assays in S2 cells suggest that both the S and C types are able to promote the expression of the reporter gene, with significant differences in the expression strength between the C-type sequences tested. However, as revealed by previous RT-PCR experiments on gonads, only the C-type sequences are expressed when located in the euchromatin [START_REF] Fablet | Ongoing loss of the tirant transposable element in natural populations of Drosophila simulans[END_REF]. The S type, which was localized in the heterochromatin, was not expressed in any of the natural populations analyzed ( 14), but we show in this study, by the in vitro experiments, that it is potentially able to drive expression. Two hypotheses can be proposed to explain this discrepancy: either the S type is inhibited in its endogenous natural context (e.g., by a repressive genomic environment, such as heterochromatin), or it is able to promote expression in a D. melanogaster genetic context (e.g., S2 cells), but is repressed in a D. simulans genetic context. An alternative hypothesis would be that tirant expression is allowed in S2 cells, which are derived from embryonic cells, whereas it could be specifically repressed in some tissues, such as ovaries.

The observed situation for tirant in D. simulans is comparable to cases reported in D. melanogaster, in which one line displays genetic instability-also known as the permissive line-with an elevated copy number and a high rate of activity for a particular TE, such as the endogenous retroviruses gypsy [START_REF] Kim | Retroviruses in invertebrates: the gypsy retrotransposon is apparently an infectious retrovirus of Drosophila melanogaster[END_REF], ZAM, and Idefix [START_REF] Desset | Mobilization of two retroelements, ZAM and Idefix, in a novel unstable line of Drosophila melanogaster[END_REF]. Stable lines-also known as restrictive lines-are not totally devoid of these elements but have few or no copies in the chromosome arms [START_REF] Desset | COM, a heterochromatic locus governing the control of independent endogenous retroviruses from Drosophila melanogaster[END_REF][START_REF] Prud'homme | Flamenco, a gene controlling the gypsy retrovirus of Drosophila melanogaster[END_REF]. The Makindu population, which has full-length copies of tirant in the euchromatin, behaves like a permissive line in which the control of tirant is partly or totally impaired, which would explain its activity and relative high copy number in this population. In contrast, the other populations analyzed in this study only have a few inactive heterochromatic copies of tirant.

Tirant copies genomic contexts

S type

The genome-walking protocol showed the presence of one or two S-type insertions per genome (i.e., per population), which is consistent with what was previously found by Southern blot analysis [START_REF] Fablet | Ongoing loss of the tirant transposable element in natural populations of Drosophila simulans[END_REF]. One of these insertions is heterochromatic (Makindu 8), and the other three are located in copies of either the R1 non-LTR retrotransposon (Makindu 5 and Zimbabwe 1) or the Rt1b non-LTR retrotransposon (Chicharo 4). Rt1b, also known as Waldo-A, belongs to the R1 clade [START_REF] Busseau | Identification of Waldo-A and Waldo-B, two closely related non-LTR retrotransposons in Drosophila[END_REF]. We cannot precisely locate the insertions of these elements, but we know that a high proportion of R1 copies is located in centromeric heterochromatin [START_REF] Eickbush | Vertical transmission of the retrotransposable elements R1 and R2 during the evolution of the Drosophila melanogaster species subgroup[END_REF], or in ribosomal genes that are often inactivated by local heterochromatin formation [START_REF] Strohner | Recruitment of the nucleolar remodeling complex NoRC establishes ribosomal DNA silencing in chromatin[END_REF]. In this regard, we found that the unique full-length S-type copy of tirant in the Makindu genome is associated with H3K9me2 and H3K27me3, two specific repressive histone marks [START_REF] Ebert | Histone modification and the control of heterochromatic gene silencing in Drosophila[END_REF]. This confirms the assumption that S-type copies of tirant are inserted into inactivated genomic regions, mainly heterochromatin, which could explain the ab- sence of S-type transcripts in endogenous conditions revealed by RT-PCR, even though the S-type 5Ј LTR-UTR region was shown to drive expression in the S2 cells.

In the sequenced genome of D. simulans, 5 S-type copies were detected, only one of which corresponded to a full-length copy. The shorter ones displayed highpercentage identities with the full-length S-type copy, except the U_13736243 copy (94.58% identity, Table 2), which is the shortest sequence for this type. Moreover, the shorter copies were surrounded by undetermined bases, indicating that they could be longer, and that we are probably underestimating the copy number of the S type. We were unable to identify the exact location of the S-type insertions, since the D. simulans sequenced genome was not annotated yet.

C type

Unlike the S type, which appears to be exclusively heterochromatic, insertions of the C type are both euchromatic and heterochromatic. In the Chicharo genome, in which no tirant copy can be detected using in situ hybridization, 3 C-type copies were found when analyzing the genomic sequence, one in the heterochromatin, another in centromeric DNA (Maupiti Islands), which is also heterochromatic, and the last one in the LTR retrotransposon diver2. Diver2 insertions in the D. melanogaster genome are either centromeric or telomeric [START_REF] Kapitonov | Molecular paleontology of transposable elements in the Drosophila melanogaster genome[END_REF] and so correspond to heterochromatin. Therefore, all tirant insertions found by genome walking in the Chicharo genome can be assumed to be heterochromatic.

The tirant C-type insertion 7 from Zimbabwe is of particular interest since its flanking region displays blast matches with the heterochromatic gene parp [START_REF] Tulin | The Drosophila heterochromatic gene encoding poly(ADP-ribose) polymerase (PARP) is required to modulate chromatin structure during development[END_REF]. Tulin et al. (31) showed that the genomic region where parp is located in D. melanogaster is rich in TEs, especially gypsy elements that have lost their LTRs and insulators. These authors also showed that a mutation in the regulatory region of the gene parp deregulates the LTR-retrotransposon copia, the transcription level of which increases 50-fold. The idea that the insertion of tirant near parp in the Zimbabwe genome could regulate other TEs, or promote changes in chromatin structure warrants further investigation. While it was previously assumed from Southern blot data that the Zimbabwe genome had no full-length copy of tirant, the long PCR experiments suggest that insertion 1 is potentially full length. This discrepancy could be explained by the presence of restriction polymorphism in this sequence.

In the Makindu genome, the size of PCR products obtained for half of the detected insertions indicates that the copies are (potentially) full length. Insertion sites are varied for these copies: intron of a gene, Maupiti Islands, R1 transposable element, and unannotated DNA. Interestingly, we observed that one of these full-length C-type tirant copies, insertion C 2, harbors simultaneously repressive H3K27me3 and permissive H3K4me2 histone marks. In the past few years, several studies in mouse and human ES cells reported the occurrence of such "bivalent domains" predominantly on key developmental regulators (reviewed in Pietersen and van Lohuizen;ref. 32). It was proposed that this unusual combination of marks keeps genes repressed or expressed at very low level but poised for later activation or complete repression [START_REF] Pietersen | Stem cell regulation by polycomb repressors: postponing commitment[END_REF]. In this regard, we can propose that the Makindu genome contains C-type copies of tirant susceptible to be expressed in response to appropriate developmental or cellular cues. Full-length insertions are thus not associated specifically with euchromatin but are also present in heterochromatic regions.

Internal dynamics of tirant in the D. simulans genome: the cohabitation of two subfamilies Subfamilies with differing regulatory regions have been reported for various TEs, such as copia [START_REF] Matyunina | Naturally occurring variation in copia expression is due to both element (cis) and host (trans) regulatory variation[END_REF], blood [START_REF] Costas | Amplification and phylogenetic relationships of a subfamily of blood, a retrotransposable element of Drosophila[END_REF], and 412 [START_REF] Mugnier | New regulatory regions of Drosophila 412 retrotransposable element generated by recombination[END_REF] in Drosophila, and Tnt1 in plants [START_REF] Vernhettes | The evolutionary analysis of the Tnt1 retrotransposon in Nicotiana species reveals the high variability of its regulatory sequences[END_REF]. In some cases, such as copia, subfamilies are associated with different levels of expression (4,[START_REF] Pietersen | Stem cell regulation by polycomb repressors: postponing commitment[END_REF]. The situation observed for blood in the D. melanogaster subgroup of species [START_REF] Costas | Amplification and phylogenetic relationships of a subfamily of blood, a retrotransposable element of Drosophila[END_REF], is very similar to what we found for tirant. One of the blood subfamilies, long (L), is mainly heterochromatic, and its insertion sites are shared by most populations. The other subfamily, short (S), is potentially active and euchromatic. One particular population of D. simulans has more blood insertions on the chromosome arms than the other populations of the species ( 13), which is interpreted as an invasion of euchromatin by the S subfamily [START_REF] Costas | Amplification and phylogenetic relationships of a subfamily of blood, a retrotransposable element of Drosophila[END_REF]. The authors, therefore, assume the existence of competition between the subfamilies, leading to the elimination from euchromatin of the L subfamily, which is replaced by the S subfamily. This situation is quite similar to what we observe for tirant, with the S type restricted to the heterochromatin and untranscribed, and the C type that has invaded euchromatin in the East African populations of D. simulans.

The dynamics of the tirant types is not incompatible with an overall loss of the element from the genome of most D. simulans populations worldwide, as was previously proposed ( 14) but indicates that one population (Makindu), which can be considered as permissive for tirant, has been subjected to some deregulation, leading to transposistions, and an increase in copy number. Our results also suggest that the S and C types of tirant are subjected to several different mechanisms of regulation, which include chromatin conformation and therefore the epigenetic regulation machinery.

For a long time, heterochromatin has been considered to be the graveyard of TEs, since the absence of recombination would lead to the accumulation of these sequences [START_REF] Charlesworth | The population genetics of Drosophila transposable elements[END_REF]. This model is consistent with data showing that TEs are organized in clusters in the heterochromatin and that most of them are highly rearranged and deleted (6,[START_REF] Daniels | The distribution of P-element sequences in Drosophila: the willistoni and saltans species groups[END_REF]. However, analyses of the Drosophila heterochromatic sequences (8,[START_REF] Hoskins | Primers used to amplify each individual insertion of tirant in the genome of natural populations. References Analysis of the genome sequence of the flowering plant Arabidopsis thaliana[END_REF] and several experimental studies have shown that fulllength elements persist in this part of the genome, and in some cases could be active (7,9). The identification of full-length S-type copies of tirant in the heterochromatin of the D. simulans natural populations and sequenced genome, associated with the data obtained for the in vitro expression of these type of elements, suggest that the heterochromatin harbors potentially invasive elements, which may be reactivated under particular conditions that have not yet been determined and that warrant further studies.

The data obtained in Fablet et al comes from somatic tissues analysis since embryos were used in all experiments [START_REF] Fablet | Genomic environment influences the dynamics of the tirant LTR retrotransposon in Drosophila[END_REF]. Transposition events in somatic tissues, a already discussed above, may have deleterious effects for the genome but also may have an impact in germline regulation. Germline regulation of TEs is necessary for genome integrity so inheritance of proper genetic material can be done. Difference between somatic and germinal regulation of TEs has already been described but again, no variation in natural populations has ever been researched. In order to understand TE regulation in germinal tissues of natural populations, I assayed for tirant transcription (gag, pol and env) in ovaries of both D. melanogaster and D. simulans natural populations (Figure 1 hereafter). Again, different expression levels are observed among natural populations of D. simulans where only one strain is active. Moreover, contrarily to the expression test made in embryos and presented above, D. melanogaster natural populations are silenced for tirant. Therefore, a silencing mechanism is present in the germline but is absent in the somatic tissues. An alternative hypothesis is that enhancers of tirant expression are present in the somatic tissues but absent in the germinal line. Further experiments are being held in the laboratory in order to understand tirant regulation in germinal tissues.

_________________________________________________________________

Les éléments transposables (ET) sont une source majeure de variation génétique, ce qui leur confère un rôle essentiel dans l'évolution des génomes. Certes présents dans tous les génomes analysés à ce jour, leurs proportions sont fortement variables entre espèces et aussi entre populations, suggérant une relation unique entre génome hôte et ET. Grâce à un système modèle composé de populations naturelles de deux espèces proches (Drosophila melanogaster et D. simulans) avec des quantités différentes en ET, nous avons pu comparer les relations génome hôte/ET. Nous avons pu montré que les ET sont contrôlés par le génome hôte par des délétions internes et probablement par un système épigénétique variable. De plus, dans certaines populations analysées, des copies peuvent échapper à ce contrôle et envahir le génome. Les ET sont donc des grands créateurs de variabilité génétique mais permettent aussi une territorialisation chromatinienne du génome car ils portent des modifications épigénétiques précises et sont capables de les éttendre à leurs environnements génomiques. Ceci leur confére la fonction "d'épigénétique mobile". ___________________________________________________________________________ TE variation in natural populations of Drosophila : copy number, transcription and chromatin state _______________________________________________________________________ Transposable elements (TEs) are one major force of genome evolution thanks to their ability to create genetic variation. TEs are ubiquitous and their proportion is variable between species and also populations, suggesting that a tight relationship exists between genomes and TEs. The model system composed of the natural populations of the twin sisters Drosophila melanogaster and D. simulans is interesting to compare host/TE relationship, since both species harbour different amounts of TE copies. The helena element is nearly silenced in D. simulans natural populations despite a very high copy number. Such repression is associated to abundant internally deleted copies suggesting a regulatory mechanism of TEs based on DNA deletion. Another pathway of TE regulation is through epigenetics where the host genome is able to keep intact the DNA sequences of TEs and still silence their activities. Chromatin remodelling is well known in drosophila and specific histone modifications can be associated to specific chromatin domains. We observed an important variation on H3K27me3 and H3K9me2, two heterochromatic marks, on TE copies in D. melanogaster and D. simulans natural populations. Also, we show that derepressed lines of D. simulans exist for specific elements, have high TE transcription rates and are highly associated to non constitutive heterochromatic marks. TEs are therefore controlled by the host genome through DNA deletion and a possible chromatin remodelling mechanism. Not only genetic variability is enhanced by TEs but also epigenetic variability, allowing the host genome to be partitioned into chromatin domains. TEs are therefore mandatory to gene network regulation through their ability of "jumping epigenetics".

  Universitaire de Technologie B Institut de Science Financière et d'Assurance Institut Universitaire de Formation des Maîtres Directeur : M. Le Professeur F. Gieres Directeur : M. C. Collignon Directeur : M. B. Guiderdoni Directeur : M. le Professeur J. Lieto Directeur : M. le Professeur C. Coulet Directeur : M. le Professeur R. Lamartine Directeur : M. le Professeur J-C. Augros Directeur : M R. Bernard VARIATION DES ELEMENTS TRANSPOSABLES DANS LES POPULATIONS NATURELLES DE DROSOPHILA : NOMBRE DE COPIES, TRANSCRIPTION ET ETAT DE LA CHROMATINE.

  système épigénétique bien décrit chez la drosophile. Les régions chromatiniennes des génomes sont associées à différents types de modifications d'histone. Nous avons mis en évidence, dans des populations de D. melanogaster et D. simulans, une variation conséquente de modifications d'histones de type hétérochromatique, H3K27me3 et H3K9me2, associées à des copies de différents ET. De plus, nous avons décrit des populations chez D. simulans dites déréprimées, chez lesquelles certains éléments sont surexprimés et présentent des localisations probablement hétéchromatiques. Les ET sont donc contrôlés par le génome hôte par des délétions internes et probablement par un système épigénétique variable. De plus, dans certaines populations, des copies peuvent échapper à ce contrôle et envahir le génome. Les ET sont donc des grands créateurs de variabilité génétique mais permettent aussi une territorialisation chromatinienne du génome car ils portent des modifications épigénétiques précises et sont capables de les éttendre à leurs environnements génomiques. Ceci leur confére la fonction "d'épigénétique mobile". TE VARIATION IN NATURAL POPULATIONS OF DROSOPHILA : COPY NUMBER, TRANSCRIPTION AND CHROMATIN STATE

  montre que le système de délétions internes est commun à d'autres drosophilidae et agit sur tous les rétrotransposons analysés chez D. simulans. De plus, D. melanogaster semble être la seule à échapper à ce système même si l'élément helena s'y trouve très dégradé. On observe donc : 1) une relation spécifique entre l'élément helena et le génome de D. melanogaster par rapport aux autres éléments de cette espèce, 2) un comportement différent entre les ET et le génome de D. melanogaster par rapport aux autres espèces de drosophiles. Les systèmes d'élimination des séquences d'ET sont donc variables entre les espèces et entre les éléments et participent à la spécificité de la relation ET/hôte.

Fig. 1 .

 1 Fig. 1. Relative proportions of the various TE classes in the sequenced genomes of Homo sapiens and Drosophila melanogaster.

Fig. 2 .

 2 Fig. 2. Structure of typical LTR retrotransposon (A) and LINE (B) regulatory regions. Only the most common features are shown. The black elements are mandatory and found in every retrotransposon, whereas the gray elements are not essential. However, for LTR retrotransposons, the presence of either the TATA box or the downstream promoter element (DPE) is required. TATA: TATA box, Inr: Initiator.

helena structure Figure 1

 1 helena structure. Full-length copy of helena in the D. simulans genome (3R: 1506433 -15011368). DNA sequence: UTR, untranslated region; ORF, open reading frame; AAA, polyA tail. 5'UTR: ERE, estrogen response element; GATA, SP1, stimulating protein 1; USF-like, upstream stimulating factor-like; Protein sequences: MHR, major homology region; CCHC, cysteine rich domain; AP, apyrimidic. See Materials and Methods for prediction information.

  Southern blot analysis of D. melanogaster and D. simulans populations Figure 3 Southern blot analysis of D. melanogaster and D. simulans populations. Lanes 1 to 9 are D. melanogaster populations (Bolivia, Brazzaville, Canton, Chicharo, Reunion Island, Arabia, Virasoro, Vietnam, and ISO for the 9 th and 20 th lane). Lanes 1 to 19 are D. simulans populations (Amieu, Eden, Valence, Canberra, Papeete, Moscow, Makindu, Zimbabwe, Cann River and Reunion Island). For both Southern blots, the DNA size is estimated in base pairs. phylogenetic tree of the DNA sequences from the ORF1 region Figure 4 phylogenetic tree of the DNA sequences from the ORF1 region. The reconstruction was performed on the cloned DNA sequences of the ORF1 region from the different populations of D. simulans, and from some sequences detected in the sequenced genome (we eliminated sequences that were too short relative to the global length of the alignment). Colored boxes identify sequences harboring common patterns of deletions and/or insertions. All the positions are given by reference to the complete copy. Green box: sequences display the same deletions of 118 bp (at position 45), 3 bp (at position 839), 6 bp (at position 846), 1 bp (at position 854) and a 1-bp insertion (at position 415). Blue box: sequences display the same 28-bp deletion (at position 1092) -those with a red star also have a 77-bp deletion (at position 508), and a 91-bp deletion (at position 593). Yellow box: sequences display the same deletions of 1 bp (at position 160), 28 bp (at position 165), 19 bp (at position 954), 2 bp (at position 989), and 37 bp (at position 1006), and an insertion of 1 bp (at position 322). Orange box: sequences with no deletion or insertion, very closely related to the complete copy chr3R_1506433. Black asterisks correspond to bootstrap values greater than 50%.

  available free of charge to the entire biomedical community peer reviewed and published immediately upon acceptance cited in PubMed and archived on PubMed Central yours -you keep the copyright Submit your manuscript here: http://www.biomedcentral.com/info/publishing_adv.asp BioMedcentral 10. Li YJ, Satta Y, Takahata N: Paleo-demography of the Drosophila melanogaster subgroup: application of the maximum likelihood method. Genes & genetic systems 1999, 74(4):117-127. 11. Vieira C, Lepetit D, Dumont S, Biemont C: Wake up of transposable elements following Drosophila simulans worldwide colonization. Mol Biol Evol 1999, 16(9):1251-1255. 12. Biemont C, Nardon C, Deceliere G, Lepetit D, Loevenbruck C, Vieira C: Worldwide distribution of transposable element copy number in natural populations of Drosophila simulans. Evolution Int J Org Evolution 2003, 57(1):159-167. 13. Petrov DA, Hartl DL: High rate of DNA loss in the Drosophila melanogaster and Drosophila virilis species groups. Molecular Biology and Evolution 1998, 15(3):293-302. 14. Lerat E, Rizzon C, Biemont C: Sequence divergence within transposable element families in the Drosophila melanogaster genome. Genome Res 2003, 13(8):1889-1896. 15. Arkhipova IR, Lyubomirskaya NV, Ilyn YV: Drosophila Retrotransposons. Berlin: Springer; 1995. 16. Assembly/Alignement/Annotation of 12 relatedDrosophila species [http://rana.lbl.gov/drosophila/index.html] 17. Malik HS, Burke WD, Eickbush TH: The age and evolution of non-LTR retrotransposable elements. Molecular biology and evolution 1999, 16(6):793-805. 18. Hohjoh H, Singer MF: Cytoplasmic ribonucleoprotein complexes containing human LINE-1 protein and RNA. Embo J 1996, 15(3):630-639.19. Pardue ML, Danilevskaya ON, Lowenhaupt K, Wong J, Erby K: The gag coding region of the Drosophila telomeric retrotransposon, HeT-A, has an internal frame shift and a length polymorphic region. J Mol Evol 1996, 43(6):572-583. 20. Casavant NC, Scott L, Cantrell MA, Wiggins LE, Baker RJ, Wichman HA: The end of the LINE?: lack of recent L1 activity in a group of South American rodents. Genetics 2000, 154(4):1809-1817. 21. Rohrmann GF, Karplus PA: Relatedness of baculovirus and gypsy retrotransposon envelope proteins. BMC Evol Biol 2001, 1:1. 22. Fablet M, Rebollo R, Biemont C, Vieira C: The evolution of retrotransposon regulatory regions and its consequences on the Drosophila melanogaster and Homo sapiens host genomes. Gene 2007, 390(1-2):84-91. 23. Lozovskaya ER, Nurminsky DI, Petrov DA, Hartl DL: Genome size as a mutation-selection-drift process. Genes Genet Syst 1999, 74(5):201-207. 24. Brookfield JF: Evolutionary forces generating sequence homogeneity and heterogeneity within retrotransposon families. Cytogenet Genome Res 2005, 110(1-4):383-391. 25. Belancio VP, Hedges DJ, Deininger P: LINE-1 RNA splicing and influences on mammalian gene expression. Nucleic Acids Res 2006, 34(5):1512-1521. 26. Roy SW, Penny D: Widespread intron loss suggests retrotransposon activity in ancient apicomplexans. Molecular biology and evolution 2007, 24(9):1926-1933. 27. Bucheton A, Vaury C, Chaboissier MC, Abad P, Pelisson A, Simonelig M: I elements and the Drosophila genome. Genetica 1992, 86(1-3):175-190. 28. Fablet M, McDonald JF, Biemont C, Vieira C: Ongoing loss of the tirant transposable element in natural populations of Drosophila simulans. Gene 2006, 375:54-62. 29. Mugnier N, Biemont C, Vieira C: New regulatory regions of Drosophila 412 retrotransposable element generated by recombination. Molecular biology and evolution 2005, 22(3):747-757. 30. Vieira C, Biemont C: Transposable element dynamics in two sibling species: Drosophila melanogaster and Drosophila simulans. Genetica 2004, 120(1-3):115-123. 31. Biemont C, Monti-Dedieu L, Lemeunier F: Detection of transposable elements in Drosophila salivary gland polytene chromosomes by in situ hybridization. Methods Mol Biol 2004, 260:21-28.

  TTCATGCACT GAATCTGTTA ACCTAAGCAC AATA-CTAC-

3 .

 3 Histone modifications within 412, tirant, roo and F elements in natural populations of D. melanogaster and D. simulans a. Chromatin immunoprecipitation of histone marks associated to TEs ChIP analysis coupled with qPCR allowed us to observe permissive (H3K4me2) and repressive marks (H3K9me2 and H3K27me3) of histones on TE copies during embryogenesis.

  412 and tirant are globally more enriched in H3K27me3 in D. melanogaster natural populations than in D. simulans ones while no difference is observed between both species for roo and F. H3K27me3 enrichment does not correlate with transcript variation of the four elements analysed. Indeed, D. melanogaster populations, although displaying different amount of transcripts, are both associated to H3K27me3. D. simulans natural populations harbour distinct patterns of H3K27me3 on different TE families including populations as enriched as D. melanogaster, and others completely devoid of such histone mark. Strikingly, the most transcriptionally active 412 population of D. simulans, Canberra, has the highest association of H3K27me3 among all natural populations. Similarly, tirant transcriptionally active line, Makindu, also harbors the highest H3K27me3 association. It is important to note that, as described by Fablet et al, bivalent marks as H3K27me3 and small association to H3K4me2, along with low association

Figure 1 TE

 1 Figure 1 TE schematic representation TEs analysed are LTR elements (412, tirant and roo) or LINE-like elements (F). Boxes represent open reading frames (ORF), a part from the LTR (long terminal repeats) box. ORF may code for retrovirus-like proteins GAG, POL and ENV. Primers used for quantitative PCR amplification (estimation of copy number, TE expression and enrichment in specific histone marks) are showed in black arrows. The probe used to observe colocalization of histone marks and TE single copy is represented as a green line.

Figure 2

 2 Figure 2

(

  euchromatic) for the four elements analyzed. Note that D. melanogaster has more elements than D. simulans and intra specific variation is observed for D. simulans. Left graphs show quantification of copy internal regions through quantitative PCR. Absence or different patterns of variation are observed for D. simulans natural populations. Blue bars represent D.melanogaster natural populations and red bars D. simulans. Please refer to materials and methods for qPCR relative quantification and, note that reference genes are equally represented between both species and natural populations allowing us to compare all strains together (Supplementary Figure1).

Figure 3 Transcription

 3 Figure 3 Transcription of TEs in natural populations of D. melanogaster and D. simulans D. melanogaster (bleu bars) harbors transcription for the four elements analyzed. D.

Figure

  Figure 3

3

 3 Figure 3

Figure 4 Figure 5 Figure 6 Figure 7

 4567 Figure 4

Figure 1 Ct

 1 Figure 1 Ct comparison between D. melanogaster and D. simulans for reference genes. For all experiments (copy number, transcription estimation and ChIP) reference genes used for both species, D. melanogaster and D. simulans are either equally represented, equally transcribed or equally associated to histone post translational modifications. Such data allow us to compare natural populations of D. simulans with populations of D. melanogaster. Also, D. melanogaster TE copy number present earlier Cts than D. simulans natural populations

Figure 5 H3K4me2

 5 Figure 5 H3K4me2 ChIP data A. H3K4me2 lack f enrichment for TEs in natural populations of D. melanogaster and D. simulans. B. Enrichment of H3K4me2 was nevertheless observed in absolute quantification of the reference gene (actin) compared to TEs. C. H3K4me2 was also relative enriched to actin

Figure 6 Specific

 6 Figure 6 Specific H3K9me2 association to constitutive heterochromatin and comparison with TEs A. Satellite 1688 located in constitutive heterochromatin of D. melanogaster Chicharo strain is enriched in H3K9me2 but not H3K27me3. B. Higher enrichment of satellite 1688 compared to TEs in D. melanogaster Chicharo strain.

Figure 7

 7 Figure 7

Figure 3 Figure 4 Figure 5 Figure 6 Figure

 3456 Figure 3

FiguresFigure 1

 1 Figures

••

  Acquisition and consequent transposition of an endogenous retrovirus element in Entamoeba histolytica and lineage specific enrichment in TEs might affect speciation Mus musculus & M. caroli crosses induce retroelements hypomethylation on chromosome 10, the substrate of double minute chromosome formation in interspecies hybrids. (Brown et al., 2008) Insects • D. melanogaster intraspecific crosses can result in hybrid dysgenesis, associated with P or I elements mobilisation, dependent on rasiRNA production in the germinal cell line and causing several abnormalities (such as female sterility); • Crosses between D. buzzatti & D. koepferae awake osvaldo copies in the hybrid. (Labrador et al., 1999; Brennecke et al., 2008) Marsupials • Interspecific macropodid hybrids (Macropus rufogriseus & M. agilis) present centromeric instability due to TEs and satellite replication, probably inducing karyotypic isolation relative to the parental species; • Genome-wide hypomethylation and centromeric expansion, due to TE activation, are observed in M. eugenii or Wallabia bicolor hybrids. (O'Neill et al., 1998; Metcalfe et al., 2007) Plants • In A. thaliana & A. arenosa dosage-dependent crosses, the usually silenced paternal athila elements are activated concomitantly with the deregulation of polycomb complexdependent gene regulation; • Wheat allotetraploid formation is accompanied by TE activation, DNA methylation and gene expression alterations; • Helianthus annuus & H. petiolari hybrids have a 50% larger genome than parental individuals due to TE amplification ; • DNA introgression in Zizania latifolia causes TE activation through modifications in DNA methylation and morphological novelties compared to the primordial line.

  , we show that some full-length copies of tirant are present in heterochromatic regions in Drosophila simulans and that when tested in vitro, these copies have a functional promoter. However, when inserted in heterochromatic regions, tirant copies are inactive in vivo, and only transcription of euchromatic copies can be detected. Thus, our data indicate that the localization of the element is a hallmark of its activity in vivo and raise the question of genomic invasions by transposable elements and the importance of their genomic integration sites.chromatin ⅐ endogenous retrovirus ⅐ natural populations

Figure 1 .

 1 Figure 1. Structure of the variants of the 5ЈLTR-UTR region tested. Rectangles and ovals stand for the different tandem repeats. Variants are named as follows: S/C-i,j, where S or C is the subfamily, i is the number of 19-bp tandem repeats in the LTR, and j is the number of 102-bp repeats in the 5ЈUTR.

Figure 2 .

 2 Figure 2. Reporter gene assays, transfection into S2 cells results. Activity ratio: ratio of the ␤-galactosidase and luciferase activities measured. Negative control corresponds to transfection of an empty pPelican vector, and positive control to transfection of a vector carrying the lacZ gene downstream of an SV40 promoter. No sd could be calculated for the positive control because 2 of 3 measurements were out of range for the machine used.

Figure 3 .

 3 Figure 3. Histone marks association with full-length C-and S-type copies of tirant. ChIP analysis of C-and S-type copies of tirant in D. simulans embryos from the population of Makindu. See Materials and Methods for fold enrichment computation. H3K9me2, H3K27me3, and H3K4me2 are epigenetic marks characteristic of constitutive heterochromatin, nonconstitutive heterochromatin, and euchromatin, respectively.

Figure 4 .

 4 Figure 4. Structure of the tirant copies found in the D. simulans sequenced genome. A) S-type copies. B) C-type copies. Black boxes, LTR regions; blue boxes, gag genes, red boxes, pol genes, green boxes, env genes; hatched boxes, undetermined bases (N bases).

Figure 1 .

 1 Figure 1. Relative quantification of tirant gag, pol and env genes in natural populations of D. melanogaster (blue) and D. simulans (red). High expression of tirant in ovaries of D. simulans Makindu strain is observed. For materials and methods please refer to article page 88. These results are part of Hubert et al. in preparation.

  Eléments transposables, Drosophila, Populations naturelles, Délétion, Epigénétique ___________________________________________________________________________ LBBE UMR CNRS 5558 43 Bd du 11 novembre 1918 -69622 cedex Villeurbanne

32. Genome Sequencing Center at the Washington University

	Medical School	[http://hgdownload.cse.ucsc.edu/down
	loads.html#droSim]
	33. Ensembl Genome Browser	[http://www.ensembl.org/
	index.html]

34. Karlin S, Altschul SF: Methods for assessing the statistical significance of molecular sequence features by using general scoring schemes. Proc Natl Acad Sci USA 1990, 87(6):2264-2268. 35. Repbase [http://www.girinst.org/] 36. Thompson JD, Higgins DG, Gibson TJ: CLUSTAL

W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice.

  ORF Finder [http://www.ncbi.nlm.nih.gov/gorf/gorf.html] 38. Felsenstein J:

	Nucleic Acids Res 1994,
	22(22):4673-4680.
	37.

An alternating least squares approach to infer- ring phylogenies from pairwise distances

. Syst Biol 1997, 46(1):101-111. 39. Galtier N, Gouy M, Gautier C: SEAVIEW

and PHYLO_WIN: two graphic tools for sequence alignment and molecular phylogeny

  Softberry tools [http://www.softberry.com/berry.phtml] 41. Genomatix [http://www.genomatix.de] 42. Lupas A, Van Dyke M, Stock J:

. Comput Appl Biosci 1996, 12(6):543-548. 40.

Predicting coiled coils from pro- tein

	43. Notredame C, Higgins DG, Heringa J: T-Coffee: A novel method
	for fast and accurate multiple sequence alignment. J Mol Biol
	2000, 302(1):205-217.
	44. Guindon S, Gascuel O: A

sequences. Science 1991, 252(5010):1162-1164.

simple, fast, and accurate algorithm to estimate large phylogenies by maximum likelihood

. Systematic biology 2003, 52(5):696-704. 45. Bowen NJ, McDonald JF: Drosophila

euchromatic LTR retro- transposons are much younger than the host species in which

they reside. Genome research 2001, 11(9):1527-1540. 46. Li W: Molecular Evolution. Sunderland, MA: Sinauer Associates; 1997.

Supplementary

Table 1 :

 1 helena copies in the Drosophila simulans sequenced genome

	chromosome	strand	start	stop	length (bp)	%identity
						with the
						complete
						helena
	2L £	+	1592410	1592944		99.25
	2L ¥	+	1674490	1674844		99.21
	2L *	-	15248615	15252309	3680	99.75
	2L £	+	17606797	17606986		100.00
	2L £¥	+	21324887	21324994		90.74
	2L £	-	21614564	21615205		99.22
	2L £	+	21940141	21940640		96.59
	2R £	-	1707808	1707920		97.32
	2R £	-	11963548	11964592	1045	99.81
	2R £	-	13305831	13310157	4327	99.22
	2R £	-	14875310	14876458	1149	99.91
	2R ¥	-	14892514	14892896		98.35
	2R £	-	15489919	15490045		96.06
	3L £	+	6812601	6816422	3821	99.63
	3L £	-	13781457	13782139		99.56
	3L £	-	14741536	14741739		99.51
	3L £	-	18635059	18635811		99.07
	3L ¥	-	18642496	18643290		100.00
	3L £	+	22376004	22376483		100.00
	3R £¥	-	30161	30267		88.79
	3R £	+	5983652	5983777		95.20
	3R £	-	1504547	1505432		99.77
	3R §	-	1506433	1511368	4912	/
	3R £	+	17102251	17102891		99.53
	3R £	+	18466755	18467294		99.81
	X £	+	4098908	4099039		96.92
	X £¥	-	16594484	16595282		93.59
	X £*	+	16602314	16610995	8682	96.94
	X £	-	16622045	16623141	1096	95.62
	U £	+	233264	234099		95.08
	U ¥$	-	963982	966622	2641	97.01
	U £	+	1677297	1679692	2396	96.16
	U ¥	-	1817942	1818183		96.28
	U £$	+	2316095	2319355	3260	94.32
	U £¥	+	3850078	3850295		98.17
	U $	+	3975907	3980988	5098	97.12

Table 2 :

 2 helena copies in the Drosophila melanogaster sequenced genome

	chromosome	strand	start	stop	length (bp)	%identity
						with the
						complete
						helena
	2L $	+	20553860	20554967	1108	94.00
	2L	+	20502277	20502573	297	76.00
	2L *	-	21790993	21793986	2994	61.00
	2R *	-	483468	488273	4805	83.00
	2R *	+	1630552	1632697	2146	64.66
	2R *	-	1841627	1843766	1907	68.81
	3L #	+	16603717	16607074	3358	65.00
	3L	+	18688097	18688680	583	70.05
	3L $	-	23487977	23490595	2619	93.00
	3R	-	484629	484719	91	79.00
	3R	-	401148	401432	285	92.00
	3R *	+	3313732	3314009	278	95.00
	3R *	-	3884273	3886085	1813	70.21
	3R *	-	7777247	7779392	2146	64.65
	X	-	18697277	18697732	458	84.00
	X *	-	22519248	22521013	1765	90.00
	X $	-	2255945	2256468	524	88.00
	U	+	1548034	1548124	91	86.00
	U	+	1572147	1572700	554	90.00
	U	-	3163038	3163543	506	72.00
	U	-	3164132	3164716	585	70.00
	U $	-	390489	392808	2320	87.00
	U $	+	4397920	4400343	2423	94.00
	U *	+	5254588	5256562	1975	67.34
	U	+	5813553	5813690	138	92.00
	U	+	5814272	5814990	719	95.00

* sequences with internal deletions and insertions $ sequences with internal deletions # sequence with insertions

Table 3 :

 3 Helena copy number in D. melanogaster and D. simulans populations by in situ hybridization in polytene chromosoms

Alignment: /home/lerat/MANIP/simulans/HELENA/ARTICLE/revision/

alignment_ORF1.ali Seaview [blocks=10 fontsize=10 A4] on Fri Dec 21 10:42:44 2007 1 chr3R_1506433 ATGGATACGA ACAACGAAAC TTCTAACTCT CAGCTTA--G AAATAAGCCG ATTGATTATG Brazzaville_16

CAACAACTGC AGCAATCAGT AGGCCGAGCT Makindu_22 TTACAAGTGC AACTTGCAAT ACTACATCTA CAACAACTGC AGCAATCAGT AGGCCGAGCT Canberra_5 TTACAAGTGC TACTTGCAAT ACTACATCTA CAACAACTGC AGCAATCAGT AGGCCGAGCT Cann-River_2 TTACAAGTGC TACTTGCAAT ACTACATCTA CAACAACTGC AGCAATCAGT AGGCCGAGCT Cann-River_6 TTACAAGTGC TACTTGCAAT ACTACATCTA CAACAACTGC AGCAATCAGT AGGCCGAGCT Cann-River_7 TTACAAGTGC TACTTGCAAT ACTACATCTA CAACAACTGC AGCAATCAGT AGGCCGAGCT Cann_River_5 TTACAAGTGC TACTTGCAAT ACTACATCTA CAACAACTGC AGCAATCAGT AGGCCGAGCT Cann-River_8 TTACAAGTGC AACTTGCAAT ACTACATCTA CAACAACTGC AGCAATCAGT AGGCCGAGCT Valence_5 TTACAAGTGC TACTTGCAAT ACTACATCTA CAACAACTGC AGCAATCAGT AGGCCGAGCT Valence_9 TTACAAGTGC TACTTGCAAT ACTACATCTA CAACAACTGC AGCAATCAGT AGGCCGAGCT Eden_1 TTACAAGTGC TACTTGCAAT ACTACATCTA CAACAACTGC AGCAATCAGT AGGCCGAGCT Valence_13 TTACAAGTGC TACTTGCAAT ACTACATCTA CAACAACTGC AGCAATCAGT AGGCCGAGCT Eden_7 TTACAAGTGC TACTTGCAAT ACTACATCTA CAACAACTGC AGCAATCAGT AGGCCGAGCT Valence_4 TTACAAGTGC TACTTGCAAT ACTACATCTA CAACAACTGC AGCAATCAGT AGGCCGAGCT Valence_2 TTACAAGTGC TACTTGCAAT ACTACATCTA CAACAACTGC AGCAATCAGT AGGCCGAGCT Valence_1 TTACAAGTGC TACTTGCAAT ACTACATCTA CAACAACTGC AGCAATCAGT AGGCCGAGCT Valence_7 TTACAAGTGC TACTTGCAAT ACTACATCTA CAACAACTGC AGCAATCAGT AGGCCGAGCT Makindu_cM1 TTACAAGTGC TACTTGCAAT ACTACATCTA CAACAACTGC AGCAATCAGT AGGCCGAGCT Makindu_cM6 TTACAAGTGC TACTTGCAAT ACTACATCTA CAACAACTGC AGCAATCAGT AGGCCGAGCT Makindu_1 TTACAAGTGC TACTTGCAAT ACTACATCTA CAACAACTGC AGCAATCAGT AGGCCGAGCT Amieu_5 TTACAAGTGC AACTTGCAAT ACTACATCTA CAACAACTGC AGCAATCAGT AGGCCGAGCT Eden_11

		-
	Makindu_2	TTACAAGTGC TACTTGCAAT ACTACATCTA CAACAACTGC AGCAATCAGT AGGCCGAGCT
	Makindu_6	TTACAAGTGC TACTTGCAAT ACTACATCTA CAACAACTGC AGCAATCAGT AGGCCGAGCT
	Makindu_7	TTACAAGTGC TACTTGCAAT ACTACATCTA CAACAACTGC AGCAATCAGT AGGCCGAGCT
	Valence_6	TTACAAGTGC TACTTGCAAT ACTACATCTA CAACAACTGC AGCAATCAGT AGGCCGAGCT
	Makindu_8	TTACAAGTGC TACTTGCAAT ACTACATCTA

TCAAACTCAA ACAATAGGTT TGCCATGCTA Makindu_22 CTGGAGCCAT ATCCAGCCAC AAATAGTAAT TCAAACTCAA ACAATAGGTT TGCCATGCTA Canberra_5 CTGGAGCCAT ATCCAGCCAC AAATAGTAAT TCAAACTCAA ACAATAGGTT TGCCATGCTA Cann-River_2 CTGGAGCCAT ATCCAGCCAC AAATAGTAAT TCAAACTCAA ACAATAGGTT TGCCATGCTA Cann-River_6 CTGGAGCCAT ATCCAGCCAC AAATAGTAAT TCAAACTCAA ACAATAGGTT TGCCATGCTA Cann-River_7 CTGGAGCCAT ATCCAGCCAC AAATAGTAAT TCAAACTCAA ACAATAGGTT TGCCATGCTA Cann_River_5 CTGGAGCCAT ATCCAGCCAC AAATAGTAAT TCAAACTCAA ACAATAGGTT TGCCATGCTA Cann-River_8 CTGGAGCCAT ATCCAGCCAT AAATAGTAAT TCAAACTCAA ACAATAGGTT TGCCATGCTA Valence_5 CTGGAGCCAT ATCCAGCCAC AAATAGTAAT TCAAACTCAA ACAATAGGTT TGCCATGCTG Valence_9 CTGGAGCCAT ATCCAGCCAC AAATAGTAAT TCAAACTCAA ACAATAGGTT TGCCATGCTG Eden_1 CTGGAGCCAT ATCCAGCCAC AAATAGTAAT TCAAACTCAA ACAATAGGTT TGCCATGCTA Valence_13 CTGGAGCCAT ATCCAGCCAC AAATAGTAAT TCAAACTCAA ACAATAGGTT TGCCATGCTG Eden_7 CTGGAGCCAT ATCCAGCCAC AAATAGTAAT TCAAACTCAA ACAATAGGTT TGCCATGCTA Valence_4 CTGGAGCCAT ATCCAGCCAC AAATAGTAAT TCAAACTCAA ACAATAGGTT TGCCATGCTG Valence_2 CTGGAGCCAT ATCCAGCCAC AAATAGTAAT TCAAACTCAA ACAATAGGTT TGCCATGCTG Valence_1 CTGGAGCCAT ATCCAGCCAC AAATAGTAAT TCAAACTCAA ACAATAGGTT TGCCATGCTG

		-
	Cann-River_9	CTGGAGCCAT ATCCAGCCAC AAATAGTAAT TCAAACTCAA ACAATAGGTT TGCCATGCTA
	Amieu_2	CTGGAGCCAT GTCCAGCCAC AAATAGTAAT TCAAACTCAA ACAATAGGTT TGCCATGCCA
	Makindu_2	CTGGAGCCAT ATCCAGCCAC AAATAGTAAT TCAAACTCAA ACAATAGGTT TGCCATGCTA
	Makindu_6	CTGGAGCCAT ATCCAGCCAC AAATAGTAAT TCAAACTCAA ACAATAGGTT TGCCATGCTA
	Makindu_7	CTGGAGCCAT ATCCAGCCAC AAATAGTAAT TCAAACTCAA ACAATAGGTT TGCCATGCTA
	Valence_6	CTGGAGCCAT ATCCAGCCAC AAATAGTAAT TCAAACTCAA ACAATAGGTT TGCCATGCTG
	Makindu_8 CTGGAGCCAT ATCCAGCCAC AAATAGTAAT Valence_7 CTGGAGCCAT ATCCAGCCAC AAATAGTAAT TCAAACTCAA ACAATAGGTT TGCCATGCTG
	Makindu_cM1	CTGGAGCCAT ATCCAGCCAC AAATAGTAAT TCAAACTCAA ACAATAGGTT TGCCATGCTG
	Makindu_cM6	CTGGAGCCAT ATCCAGCCAC AAATAGTAAT TCAAACTCAA ACAATAGGTT TGCCATGCTG
	Makindu_1	CTGGAGCCAT ATCCAGCCAC AAATAGTAAT TCAAACTCAA ACAATAGGTT TGCCATGCTA
	Amieu_5	CTGGAGCCAT ATCCAGCCAT AAATAGTAAT TCAAACTCAA ACAATAGGTT TGCCATGCTA
	Eden_11	CTGGAGCCAT ATCCAGCCAC AAATAGTAAT TCAAACTCAA ACAATAGGTT TGCCATGCTA
	Cann-River_10	CTGGAGCCAT ATCCAGCCAC AAATAGTAAT TCAAACTCAA ACAATAGGTT TGCCATGCCA
	Brazzaville_10 CTGGAGCCAT ATCCAGCCAC AAATAGTAAT TCAAACTCAA ACAATAGGTT TGCCATGCCA
	Cann-River_3	CTGGAGCCAT ATCCAGCCAT AAATAGTAAT TCAAACTCAA ACAATAGGTT TGCCGTGCTA
	Cann-River_13	CTGGAGCCAT ATCCAGCCAC AAATAGTAAT TCAAACTCAA ACAATAGGTT TGCCATGCTA
	Eden_4	CTGGAGCCAT ATCCAGCCAC AAATAGTAAT TCAAACTCAA ACAATAGGTT TGCCATGCTA
	Makindu_10	CTGGAGCCAT ATCCAGCCAC AAATAGTAAT TCAAACTCAA ACAATAGGTT TGCCATGCTA
	Makindu_11	CTGGAGCCAT ATCCAGCCAC AAATAGTAAT TCAAACTCAA ACAATAGGTT TGCCATGCTA
	Brazzaville_11 CTGGAGCCAT ATCCAGCCAC AAATAGTAAT TCAAACTCAA ACAATAGGTT TGCCATGCTA
	Brazzaville_12 CTGGAGCCAT ATCCAGCCAC AAATAGTAAT TCAAACTCAA ACAATAGGTT TGCCATGCTA
	Brazzaville_8	CTGGAGCCAT ATCCAGCCAC AAATAGTAAT TCAAACTCAA ACAATAGGTT TGCCATGCTA
	chrU_963982	CTGGAGCCAT ATCCAGCCAC AAATAGTAAT TCAAACTCAA ACAATAGGCT TGCCATGCTA
	chr3L_18642496 CTGGAGCCAT ATCCAGCCAT AAATAGTAAT TCAAACTCAA ACAATAGGTT TGCCATGCTA
	chrU_3975907	CTGGAGCCAT ATCCAGCCAC AAATAGTAAT TCAAACTCAA ACAATAGGTT TGCCATGCTA
	chrU_10625041	-

- Cann-River_2 ACCACCGCAA ATAGTACTGA GCCTTACCAA CCTTAATGAT CTCTTTGAGC TCATTACGGA Cann-River_6 ACCACCGCAA ATAGTACTGA GCCTTACCAA CCTTAATGAT CTCTTTGAGC TCATTACGGA Cann-River_7 ACCACCGCAA ATAGTACTGA GCCTTACCAA CCTTAATGAT CTCTTTGAGC TCATTACGGA Cann_River_5 ACCACCGCAA ATAGTACTGA GCCTTACCAA CCTTAATGAT CTCTTTGAGC TCATTACGGA Cann-River_8 ACCACCGCAA ATAGTACTGA GCCTTACCAA TCTTAATGAT CTCTATGAGC TCATTACGGA Valence_5 ACCACCGCAA ATAGTACTGA GCCTTACCAA CCTTAATGAT CTCTTTGAGC TCATTACGGA Valence_9 ACCACCGCAA ATAGTACTGA GCCTTACCAA CCTTAATGAT CTCTTTGAGC TCATTACGGA Eden_1 ACCACCGCAA ATAGTACTGA GCCTTACCAA CCTTAATGAT CTCTTTGAGC TCATTACGGA Valence_13 ACCACCGCAA ATAGTACTGA GCCTTACCAA CCTTAATGAT CTCTTTGAGC TCATTACGGA Eden_7 ACCACCGCAA ATAGTACTGA GCCTTACCAA CCTTAATGAT CTCTTTGAGC TCATTACGGA Valence_4 ACCACCGCAA ATAGTACTGA GCCTTACCAA CCTTAATGAT CTCTTTGAGC TCATTACGGA Valence_2 ACCACCGCAA ATAGTACTGA GCCTTACCAA CCTTAATGAT CTCTTTGAGC TCATTACGGA Valence_1 ACCACCGCAA ATAGTACTGA GCCTTACCAA CCTTAATGAT CTCTTTGAGC TCATTACGGA Valence_7 ACCACCGCAA ATAGTACTGA GCCTTACCAA CCTTAATGAT CTCTTTGAGC TCATTACGGA Makindu_cM1 ACCACCGCAA ATAGTACTGA GCCTTACCAA CCTTAATGAT CTCTTTGAGC TCATTACGGA Makindu_cM6 ACCACCGCAA ATAGTACTGA GCCTTACCAA CCTTAATGAT CTCTTTGAGC TCATTACGGA Makindu_1

GGTGAAAATC ACTTAACCAA GCTATGCGTA Makindu_22 GTCAGGAATT CGGCCACACA GCCACGATGT GGTGAAAATC ACTTAACCAA GCTATGCGTA Canberra_5 GTCAGGAATT CGGCCACACA GCCACGATGT GGTGAAAATC ACTTAACCAA GCTATGCGTA Cann-River_2 GTCAGGAATT CGGCCACACA GCCACGATGT GGTGAAAATC ACTTAACCAA GCTATGCGTA Cann-River_6 GTCAGGAATT CGGCCACACA GCCACGATGT GGTGAAAATC ACTTAACCAA GCTATGCGTA Cann-River_7 GTCAGGAATT CGGCCACACA GCCACGATGT GGTGAAAATC ACTTAACCAA GCTATGCGTA Cann_River_5 GTCAGGAATT CGGCCACACA GCCACGATGT GGTGAAAATC ACTTAACCAA GCTATGCGTA Cann-River_8 GTCAGGAATT CGGCCACACA GCCACGATGT GGTGAAAATC ACTTAACCAA GCTATGCGTA Valence_5 GTCAGGAATT CGGCCACACA GCCACGATGT GGTGAAAATC ACTTAACCAA GCTATGCGTA Valence_9 GTCAGGAATT CGGCCACACA GCCACGATGT GGTGAAAATC ACTTAACCAA GCTATGCGTA Eden_1 GTCAGGAATT CGGCCACACA GCCACGATGT GGTGAAAATC ACTTAACCCA GCTATGCGTA Valence_13 GTCAGGAATT CGGCCACACA GCCACGATGT GGTGAAAATC ACTTAACCAA GCTATGCGTA Eden_7 GTCAGGAATT CGGCCACACA GCCACGATGT GGTGAAAATC ACTTAACCAA GCTATGCGTA Valence_4 GTCAGGAATT CGGCCACACA GCCACGATGT GGTGAAAATC ACTTAACCAA GCTATGCGTA Valence_2 GTCAGGAATT CGGCCACACA GCCACGATGT GGTGAAAATC ACTTAACCAA GCTATGCGTA Valence_1 -

			-
		chrX_16602314	GACAGAAAGT GCGGATAGAA AGAATGCGTA AATCATCTGA AATTGCTCAA TGCATACGTT
		chrU_6536133	GACAGAAAAT GCGGATGGAA AAAATGCGTA AGTCTTCGGA AATTGCTCAA TGCATACGTT
	1081
	chr3R_1506433	GTCAGGAATT CGGCCACACA GCCTCGATGT GGTGAAAATC ACTTAACCAA GCTATGCGTA
	Brazzaville_16 GTCAGGAATT CGGCCACACA GCCACGATGT GGTGAAAATC ACTTAACCAA GCTATGCGTA
	Eden_3	GTCAGGAATT CGGCCACAAA GCCTCGATGT GGTGAAAATC ACTCAACCAA GCTATGCGTA
	Cann-River_9	GTCAGGAATT CGGCCACAAA GCCTCGATGT GGTGAAAATC ACTTAACCAA GCTATGCGTA
	Amieu_2	GTCAGGAATT CGGCCACACA GCCTCGATGT GGTGAAAATC ACTTAACCAA GCTATGCGTA
	Makindu_2	GTCAGGAATT CGGCCACACA GCCACGATGT GGTGAAAATC ACTTAACCAA GCTATGCGTA
	Makindu_6	GTCAGGAATT CGGCCACACA GCCACGATGT GGTGAAAATC ACTTAACCAA GCTATGCGTA
	Makindu_7	GTCAGGAATT CGGCCACACA GCCACGGTGT GGTGAAAATC ACTTAACCAA GCTATGCGTA
	Valence_6	GTCAGGAATT CGGCCACACA GCCACGATGT GGTGAAAATC ACTTAACCAA GCTATGCGTA
	Makindu_8	GTCAGGAATT CGGCCACACA GCCACGATGT

AGCCCAGCCT CGTCTACATA ATGTACAATT Makindu_22 CGCAGATATT GCAAGAAATG GCAATACAAC AGCCCAGCCT CGTCTACATA ATGTACAATT Canberra_5 CGCAGATATT GCAAGAAATG GCAATACAAC AGCCCAGCCT CGTCTACATA ATGTACAATT Cann-River_2 CGCAGATATT GCAAGAAATG GCAATACAAC AGCCCAGCCT CGTCTACATA ATGTACAATT Cann-River_6 CGCAGATATT GCAAGAAATG GCAATACAAC AGCCCAGCCT CGTCTACATA ATGTACAATT Cann-River_7 CGCAGATATT GCAAGAAATG GCAATACAAC AGCCCAGCCT CGTCTACATA ATGTACAATT Cann_River_5 CGCAGATATT GCAAGAAATG GCAATACAAC AGCCCAGCCT CGTCTACATA ATGTACAATT Cann-River_8 CGCAGATATT GCAAGAAATG GCAATACAAC AGCCCAGCCT CGTCTACATA ATGTACAATT Valence_5 CGCAGATATT GCAAGAAATG GCAATACAAC AGCCCAGCCT CGTCTACATA ATGTACAATT Valence_9 CGCAGATATT GCAAGAAATG GCAATACAAC AGCCCAGCCT CGTCTACATA ATGTACAATT Eden_1 CGCAGATATT GCAAGAAATG GCAATACAAC AGCCCAGCCT CGTCTACATA ATGTACAATT Valence_13 CGCAGATATT GCAAGAAATG GCAATACAAC AGCCCAGCCT CGTCTACATA ATGTACAATT Eden_7 CGCAGATATT GCAAGAAATG GCAATACAAC AGCCCAGCCT CGTCTACATA ATGTACAATT Valence_4 CGCAGATATT GCAAGAAATG GCAATACAAC AGCCCAGCCT CGTCTACATA ATGTACAATT Valence_2 CGCAGATATT GCAAGAAATG GCAATACAAC AGCCCAGCCT CGTCTACATA ATGTACAATT Valence_1 -

		-
	Makindu_2	CGCAGATATT GCAAGAAATG GCAATACAAC AGCCCAGCCT CGTCTACATA ATGTACAATT
	Makindu_6	CGCAGATATT GCAAGAAATG GCAATACAAC AGCCCAGCCT CGTCTACATA ATGTACAATT
	Makindu_7	CGCAGATATT GCAAGAAATG GCAATACAAC AGCCCAGCCT CGTCTACATA ATGTACAATT
	Valence_6	CGCAGATATT GCAAGAAATG GCAATACAAC AGCCCAGCCT CGTCTACATA ATGTACAATT
	Makindu_8	CGCAGATATT GCAAGAAATG GCAATACAAC

  Deep washing of slides once again was carried and followed by PBS washing. DAPI staining was carried with Vectashield Hard Set, AbCys mounting medium for fluorescence from Vector laboratories (1:3 with DAPI Vectashield Hard Set, AbCys). The slides were observed with AxioImager Z1 fluorescent microscope (Zeiss). The image obtained was treated with ImageJ software.

	(Formamide + 50% Dextran sulfate sodium salt + 20xSSC), hareng sperm and
	DIG labelled probe. The next morning, slides were washed in 2xSSC, four times at 42°C and
	15min at room temperature in PBS. Slides were than incubated for one hour with blocking
	solution (PBS, 5% BSA, 0,1% Triton ) at room temperature. Histone modification H3K27me3
	was targeted using Millipore 07449 antibody over night at 4°C, diluted 100 times in blocking
	solution. PBS washing steps the next morning were followed by deep washing (300mM of
	NaCl, 0,2% NP40, 0,2% Tween and PBS, second wash gas 400mM NaCl) in a shaking table
	for 15min each. Polytenes were incubated sequentially with Anti Dig monoclonal from Roche
	(1/25), AlexaFluor 555 Donkey Anti mouse IgG (1/100) and AlexaFluor 488 Donkey anti
	rabbit IgG (1/100) for 2h each.

Table 1 :

 1 . Boxes represent open reading frames, triangles are either inverted repeats (IR), in blue, or long terminal repeats (LTR), in green, and small blue arrows are duplicated TE burst of transposition concomitant with radiation periods

	insertion site representations. DDE elements : transposases carrying aspartate (D), aspartate (D,)
	glutamate (E) motif. MITE : miniature inverted repeated elements ; ERV : endogenous retrovirus ;
	LINE : long interspersed nuclear element ; SVA : composite element composed of parts of SINE,
	VNTR (variable number of tandem repeats) and alu repeats -firt box represents CCCTCT hexamere
	repeats ; SINE : short interspersed nuclear element -red box represents diagnostic features ; Gag, Pol,
	Env : retroviral-like proteins coded by TE open reading frames.

Table 2 :

 2 Hybrid analysis : epigenetic remodelling and TE activation

	General view of TE epigenetic regulation
		Position effect variegation (PEV) is the mechanism describing transcription
		variation of a given gene correlated to its chromatin localization. Mutations in Su(var)
		genes responsible for such variegation are often accompanied by TE amplification.
		The major function of this gene family is to post-translationally modify histone N
	Histone modifications	terminal ends. Usually histone methylation in lysine residues (H3K9m, H3K27m, H4K20m) is typically from a closed chromatin conformation, in contrast to acetylation of histones and methylation in H3K4 which are often observed in open chromatin
		structures. TEs are highly associated with repressive marks as in general H3K9me3 in
		humans, H4K20me3 in drosophila and H3K9me2 in plants. Regarding chromatin
		malleability, histone variants and non histone proteins (as HP1) are also involved in
		TE regulation.
	DNA methylation	In plants and mammals, DNA methylation plays an important role in silencing TEs. In insects, DNA methylation is observed as a silencing process in genes and TEs.
		Post translational gene silencing (PTGS) through small interfering RNAs (siRNA)
		processed by the AGO/DICER/RISC complex is also a mechanism that can be used to
		silence TEs. Indeed siRNAs derived from TE copy transcripts can target full length
		and putatively active TE transcripts, thus preventing TE transposition. Piwi related
		RNAs (piRNAs or rasiRNAs for repeated associated small interfering RNAs) in
	Non coding RNAs	Drosophila are processed by the piwi/Aub/AGO3 pathway, are 24-30 nt and are known to silence TEs in the germline whereas endo-siRNA (endogenous small interfering RNAs) processed by DICER2/AGO2 are 21 nt and are capable of somatic silencing
		TEs. Germinal and somatic silencing are therefore possible thanks to non coding
		RNAs. However, the presence and the transcription of a TE copy in the genome is
		essential to engage PTTES (post translational transposable element silencing). The
		idea of the immune system is, hence, appropriate since having non coding RNAs of a
		given TE family will preserve the genome from further invasions.

Table 3 :

 3 General view of TE epigenetic variationGENERAL CONCLUSIONSTEs harbor an intimate relationship with the genomes in which they are present. As a consequence of the immediate deleterious results of some transpositions, several mechanisms have been selected that allow genome to counteract negative effects. Elimination of TE sequences as observed with helena is also observed with LTR elements, SINEs, and almost all elements in D. simulans, D. sechelia and D. yakuba[START_REF] Rebollo | Losing helena: the extinction of a drosophila line-like element[END_REF][START_REF] Granzotto | The evolutionary dynamics of the Helena retrotransposon revealed by sequenced Drosophila genomes[END_REF], E. Lerat personal communication). Interestingly, the D. simulans twin sister, D. melanogaster, does not harbour the same deletion mechanism, showing again how specific the arm race for invasion/protection might be. TE expression can also be suppressed without elimination of their sequences. Indeed, through epigenetic regulation, silencing of TEs is possible while they are still maintained in the genome. Chromatin data obtained in this work shows the existence of epigenetic variability in TE families between natural populations and species of Drosophila. Our data do not show a natural variation in the epigenetic regulation of TEs since no correlation can be observed between TE expression and local chromatin structure. It is important to note that further data is needed on TE copy specific analysis in order to correlate histone marks and TE dynamics. Such kind of project needs not only deep

• Research Communication Genomic environment influences the dynamics of the tirant LTR retrotransposon in Drosophila Marie Fablet,* Emmanuelle Lerat,* Rita Rebollo,* Be ´atrice Horard, † Nelly Burlet,* Sonia Martinez,* E ´milie Brasset, ‡ Eric Gilson, † Chantal Vaury, ‡ and Cristina Vieira* ,1

  CNRS, UMR 5558, Laboratoire de Biome ´trie et Biologie Evolutive, Universite ´Lyon 1, Villeurbanne, France; † CNRS UMR 5161/ENS Lyon, Faculte ´de me ´decine Lyon Sud, Laboratoire de Biologie Mole ´culaire de la Cellule, Universite ´Lyon 1/HCL, Pierre Be ´nite, France; and ‡ UMR/CNRS 6247, INSERM, U931, Faculte ´de Me ´decine, BP38, Clermont Universite ´, Clermont-Ferrand, France

*

TABLE 1 .

 1 Tirant insertion sites determined by genome walking

	Insertion	Type	Location	Comments	Length
	Makindu population
	1	C	X	Unannotated DNA	Full length
	2	C	2L	tkv gene intron	Full length
	3	C	3R	Unannotated DNA	Full length
	4	C	Centromeric DNA	Maupiti islands	Full length
	5	S		R1 non-LTR retrotransposon	Full length
	6	C	2L	hobo transposon	Short fragment
	7	C	2L	frogger LTR retrotransposon	Short fragment
	8	S	2R	Heterochromatin	Short fragment
	9	C		MAX LTR retrotransposon	Short fragment
	10	C	3R	Unannotated DNA	NA
	Zimbabwe population
	1	S		R1 non-LTR retrotransposon	Full length
	2	C	3	Heterochromatin	Long fragment
	3	C	2L	3Ј from CG13786 gene	Short fragment
	4	C	2L	Heterochromatin	Short fragment
	5	C	3R	Unannotated DNA	Short fragment
	6	C	Centromeric DNA	Maupiti islands	Short fragment
	7	C	3	Heterochromatin	NA
	Chicharo population
	1	C	Centromeric DNA	Maupiti islands	Full length
	2	C		Heterochromatin	Long fragment
	3	C		diver2 LTR retrotransposon	Short fragment
	4	S		Rt1b non-LTR retrotransposon	NA

TABLE 2 .

 2 Positions of the tirant copies in the D. simulans sequenced genome

								Percentage identity
							tirant Dm	C type	S type
	Chromosome	Strand	Start	Stop	Type	Length (bp)	AY928610	AY756123	AY756122	chrU_8880559
	U	Ϫ	13736532	13737472	S	942	79.10	72.97	88.03	94.58
	U	Ϫ	5979828	5980937	S	1110	85.57	NA	NA	99.28
	U	Ϫ	8880559	8889066	S	8508	80.06	73.35	99.47	/
	U	ϩ	3039715	3042707	S	2993	78.43	NA	NA	98.47
	U	Ϫ	2603202	2604990	S	1791	76.16	NA	NA	99.44
	U	ϩ	1610739	1611425	C	687	89.70	89.10	58.48	60.00
	3L	Ϫ	22549046	22549538	C	493	85.39	NA	NA	82.62
	U	Ϫ	1396504	1396898	C	422	96.68	99.26	89.01	88.99
	U	ϩ	28708	29409	C	702	94.14	NA	NA	86.55
	U	ϩ	9615110	9615888	C	780	93.93	NA	NA	85.19
	U	ϩ	10384153	10390953	C	6803	98.63	99.26	78.16	80.81
	U	Ϫ	15708778	15709918	C	1142	95.97	NA	NA	74.82
	U	ϩ	3501254	3503485	C	2232	97.17	NA	NA	75.21
	U	ϩ	6605489	6606440	C	952	92.30	94.11	79.87	79.38
	U	ϩ	7436025	7437064	C	1040	95.76	NA	NA	82.19
	2L-random	ϩ	887610	888048	C	440	90.38	90.76	86.11	87.78
	2L	ϩ	21040234	21040766	C	533	83.86	NA	NA	74.56
	2L	ϩ	21042156	21045168	C	2845	82.96	67.84	65.90	80.52
	2L	ϩ	21052705	21053256	C	552	86.79	NA	NA	84.04
	2L	ϩ	21205201	21205440	C	240	91.25	NA	NA	86.67

NA, value cannot be computed.

M.Fablet et al. / Gene 390 (2007) [84][85][86][87][88][89][90][91]

(page number not for citation purposes)

Correspondence: Universite ´de Lyon, F-69000, Lyon, Universite ´Lyon 1, CNRS, UMR5558, Laboratoire de Biome ´trie et Biologie Evolutive, F-69622, Villeurbanne, France. E-mail: vieira@biomserv.univ-lyon1.fr doi: 10.1096/fj.08-123513

Acknowledgements

We thank Monika Ghosh for reviewing the English text. This work was funded by the Centre National de la Recherche Scientifique (UMR 5558 and GDR 2157 on Transposable Elements).

Acknowledgements

We thank Monika Ghosh for reviewing the English text. This work was funded by the Centre National de la Recherche Scientifique (UMR 5558) and the IMPBio program.

Acknowledgements

We thank Nelly Burlet, Olaf Nickel, Dr Sameer Phalke, Professor Dr Gunter Reuter, Dr Jamy Peng and Dr Gary Karpen for their valuable help and Dr Marie Fablet for all the comments on previous versions of this manuscript. This work was funded by CNRS ANR, Rhone Alpes region project, FINOVI and Fapesp. Pr Eric Gilson is an associate member of European Union 6 th framework program The Epigenome Network of Excellence (NoE) and his laboratory was supported by European Union 6 th framework program grant RISCRAD and ARECA framework program from Canceropole Lyon Auvergne Rhône Alpes.

Acknowledgments

Owing to space limitations, we sincerely regret the omission of many outstanding publications in this field. We thank Dr. Marie Fablet and Dr. Daphne Reiss for their valuable advice. We thank Valerie James for the English corrections. This work was supported by ANR Genemobile, FINOVI and CIBLE2008 (Région Rhône Alpes).

We thank Christian Bie ´mont for useful comments, and Monika Ghosh for reviewing the English text. The work in C. Vieira's laboratory was funded by the Centre National de la Recherche Scientifique (UMR 5558 and GDR 2157 on TEs). The work in E.G.'s laboratory was funded by ARC (ARECA program on epigenetic profiling).

Madeira_3'p'1 CC-AATAAGG ATACAAGAA-

 -------------Madeira__3'10 CC-AATAAGG ATACAAGAA--------------Canberra_810 CC-AATAAGG ATACAAGAAA TCCT---------Eden_4'9 CC-AATAAGG ATACAAGAA--------------Eden_4'4 CC-AATAAGG ATACAAGAA--------------Eden_4'3 CC-AATAAGG ATACAAGAA--------------Eden_4'11 CC-AATAAGG ATACAAGAA--------------Eden_4'12 CC-AATAAGG ATACAAGAAA TC-----------Amieu_1 CC-AATAAGG ATACAAGAA--------------chrU_5384045 CC-AATAAGG ATACAAGAAA TCCTTGAAAT AAT Zimbabwe_72 CC-AATAAGG ATAGAAGAAA TCCTTGAAAT A--chrU_3975907 CC-AATAAGG ATAGAAGAAA TCCTTGAAAT AAT Canberra_87 CC-AATAAGG ATAGAAGAAA TCCT---------Canberra_88 CC-AATAAGG ATAGAAGAAA TCCT---------Canberra_83 CC-AATAAGG ATAGAAGAAA TCCT---------Valence_13 CC-AATAAGG ATAGAAGAA--------------Papeete_12 CC-AATAAGG ATAGAAGAA--------------Zimbabwe_710 CC-AATAAGG ATAGAAGAAA TCCT---------Papeete_6 CC-AATAAGG ATAGAAGAA--------------Canberra_815 CC-AATAAGG ATAGAAGAAA TCCT---------Papeete_2 CC-AATAAGG ATAGAAGAA- -------------Brazzaville_1 CC-AATAAGG ATAGAAGAA- -------------Madeira_3'9

CC-AATAAGG ATAGAAGAA--------------Madeira_3'8 CC-AATAAGG ATAGAAGAA--------------Madeira_3'7 CC-AATAAGG ATAGAAGAAA -------------Eden_4'13 CC-AATAAGG ATAGAAGAA--------------Madeira_4'10 CC-AATAAGG ATAGAAGAA--------------Amieu_68

CC-AATAAGG ATAGAAGAA- -------------Amieu_69 CC-AATAAGG ATAGAAGAA- -------------Amieu_4 CC-AATAAGG ATAGTAGAA- -------------Makindu_5'4 ---------------------------------Papeete_10 CC-AATAAGG ATAGAAG----------------Canberra_84 CC-AATAAGG ATAGAAGAAA TCC----------Cann CC-AATAATG ATAGAAGAA--------------Amieu_61 CC-AATAAGG ATAGAAGAAA TCCT---------Amieu_62 CC-A-----------------------------Amieu_65 CC-AATAAGG ATAGAAGAA-CC-AATAAGG ATAGAAGAAT TATCTGAAAT AAT chrU_5679045 CC-AATA------GAAGAAA TCCTTGAAAT AAT Amieu_8 CC-AATAAGG ATAGAAGAAA -------------Valence_14 CC-AATAAGG ATAGAAGAA--------------Valence_2 ---------------------------------Valence_4 CC-AATAAGG ATAGAAGAAA -------------Zimbabwe_76 CC-AATAAGG ATAGAAGAAA TCC----------Zimbabwe_712 CC-AATAATG ATAGAAGAAA TCCTTGAAAT AAT chrU_5950518 --CC-AATAAGG ATAGAAGAAA TCCTTGAAAT AAT

Abbreviations

DOA, dead on arrival; LINE, long interspersed nuclear element; L1, LINE 1; LTR, long terminal repeat; ORF, open reading frame; TE, transposable element

Authors' contributions

RR and LL carried out the molecular and genetic studies, RR and EL did the bioinformatic analysis, CV and CB contributed to the design of the study, CV designed and coordinated the study. All the authors contributed to writing of the paper. All the authors have read and approved the final manuscript.

Additional material

Additional File 1

Additional File 4

Alignment of helena ORF1. The data provided the alignement used to construct the tree on Figure 4 Click here for file [http://www.biomedcentral.com/content/supplementary/1471-2164-9-149-S4.pdf]

Additional File 5

Alignment of helena ORF2. The data provided the alignement used to construct the tree on Figure 5 Click here for file [http://www.biomedcentral.com/content/supplementary/1471-2164-9-149-S5.pdf] -----------------------------------------------------------Canberra_815 --------------------CATCTGGAAA ACTCTCTCCA ATTAAATACG GTTCTGAGCT Papeete_2 - ---------------------------------------ATTAAATACG GTTCTGAGCT Brazzaville_1 ---------------------------------------- ---------------------------------------------------------- ---------------------------------------------------------- ----------------------------------------------------------Zimbabwe_71 A-----------------------------------------------------------Valence_6 A-----------------------------------------------------------Makindu_51 A-----------------------------------------------------------Zimbabwe_79 A-----------------------------------------------------------Zimbabwe_77 A-----------------------------------------------------------Zimbabwe_714 A-----------------------------------------------------------Zimbabwe_715 A-----------------------------------------------------------Papeete_5 ------------------------------------------------------------Papeete_1 ------------------------------------------------------------Eden_4'14 A-----------------------------------------------------------Valence_5 A-----------------------------------------------------------Madeira_3'5 A-----------------------------------------------------------Madeira_3'p'1 A-----------------------------------------------------------Madeira__3'10 A-----------------------------------------------------------Canberra_810 A-----------------------------------------------------------Eden_4'9 A-----------------------------------------------------------Eden_4'4 A----------------------------------------------------------

---------------------------------------ATTAAATACG CTTCTGAGCT Papeete_1 ----------------------------------------ATTAAATACG CTTCTGAGCT Eden_4'14 -------------------A CaTCTGGAAA ACTCTTTCCa ATTAAATACG CTTCTGAGCT Valence_5 -------------------A CATCTGGAAA ACTCTTTCCA ATTAAATACG CTTCTGAGCT Madeira_3'5 ----------------ACaA CaTCTGGAAA ACTCTTTCCa ATTAAATACG CTTCTGAGCT Madeira_3'p'1 ----------------ACaA CaTCTGGAAA ACTCTTTCCa ATTAAATACG CTTCTGAGCT Madeira__3'10 ---------------gacaa caTCTGGAAA ACTCTTTCCa ATTAAATACG CTTCTGAGCT Canberra_810 ----------------------TCTGGAAA ACTCTTTCCA ATTAAATACG CTTCTGAGCT Eden_4'9 ---------------gACAA CaTCTGGAAA ACTCTTTCCa ATTAAATACG CTTCTGAGCT Eden_4'4 -------------------A CaTCTGGAAA ACTCTTTCCa ATTAAATACG CTTCTGAGCT Eden_4'3 ----------------ACaA

-Eden_4'3 A-----------------------------------------------------------Eden_4'11 A-----------------------------------------------------------Eden_4'12

A- ----------------------------------------------------------Amieu_1 A-----------------------------------------------------------chrU_5384045 A---------------------------------------------------------- -----------------------------------------------------------Zimbabwe_71 ------------------------------------------------------------Valence_6 ------------------------------------------------------------Makindu_51 ------------------------------------------------------------Zimbabwe_79 ------------------------------------------------------------Zimbabwe_77 ------------------------------------------------------------Zimbabwe_714 ------------------------------------------------------------Zimbabwe_715 ------------------------------------------------------------Papeete_5 ------------------------------------------------------------Papeete_1 -

-----------------------------------------------------------Eden_4'14 ------------------------------------------------------------Valence_5 ------------------------------------------------------------Madeira_3'5 ------------------------------------------------------------Madeira_3'p'1 ------------------------------------------------------------Madeira__3'10 ------------------------------------------------------------Canberra_810 ------------------------------------------------------------Eden_4'9 ------------------------------------------------------------Eden_4'4 ------------------------------------------------------------Eden_4'3 ------------------------------------------------------------Eden_4'11 ------------------------------------------------------------Eden_4'12 ------------------------------------------------------------Amieu_1

- -----------------------------------------------------------chrU_5384045 ----------------------------------------------------------- -----------------------------------------------------------Zimbabwe_71 ------------------------------------------------------------Valence_6 ------------------------------------------------------------Makindu_51 ------------------------------------------------------------Zimbabwe_79 ------------------------------------------------------------Zimbabwe_77 ------------------------------------------------------------Zimbabwe_714 ------------------------------------------------------------Zimbabwe_715 ------------------------------------------------------------Papeete_5 ------------------------------------------------------------Papeete_1 ------------------------------------------------------------Eden_4'14 ------------------------------------------------------------Valence_5 ------------------------------------------------------------Madeira_3'5 ------------------------------------------------------------Madeira_3'p'1 ------------------------------------------------------------Madeira__3'10 ------------------------------------------------------------Canberra_810 ------------------------------------------------------------Eden_4'9 ------------------------------------------------------------Eden_4'4 ------------------------------------------------------------Eden_4'3 ------------------------------------------------------------Eden_4'11 ------------------------------------------------------------Eden_4'12 ------------------------------------------------------------Amieu_1 ------------------------------------------------------------chrU_5384045 ----------------------------------------------------------- ---------------------------------------------------------- ------------------------------------------------------ -----------------------------------------------------------Zimbabwe_71 ------------------------------------------------------------Valence_6 ------------------------------------------------------------Makindu_51 ------------------------------------------------------------Zimbabwe_79 ------------------------------------------------------------Zimbabwe_77 ------------------------------------------------------------Zimbabwe_714 ------------------------------------------------------------Zimbabwe_715 ------------------------------------------------------------Papeete_5 ------------------------------------------------------------Papeete_1 ------------------------------------------------------------Eden_4'14 ------------------------------------------------------------Valence_5 ------------------------------------------------------------Madeira_3'5 ------------------------------------------------------------Madeira_3'p'1 ------------------------------------------------------------Madeira__3'10 ------------------------------------------------------------Canberra_810 ------------------------------------------------------------Eden_4'9 ------------------------------------------------------------Eden_4'4 ------------------------------------------------------------Eden_4'3 ------------------------------------------------------------Eden_4'11 ------------------------------------------------------------Eden_4'12 ------------------------------------------------------------Amieu_1 ------------------------------------------------------------chrU_5384045 ----------------------------------------------------------- -----------------------------------------------------------Zimbabwe_71 ------------------------------------------------------------Valence_6 ------------------------------------------------------------Makindu_51 ------------------------------------------------------------Zimbabwe_79 ------------------------------------------------------------Zimbabwe_77 ------------------------------------------------------------Zimbabwe_714 ------------------------------------------------------------Zimbabwe_715 ------------------------------------------------------------Papeete_5 ------------------------------------------------------------Papeete_1 -----------------------------------------------------------

-Eden_4'14 ------------------------------------------------------------Valence_5 ------------------------------------------------------------Madeira_3'5 ------------------------------------------------------------Madeira_3'p'1 ------------------------------------------------------------Madeira__3'10 ------------------------------------------------------------Canberra_810 ------------------------------------------------------------Eden_4'9 ------------------------------------------------------------Eden_4'4 ------------------------------------------------------------Eden_4'3 ------------------------------------------------------------Eden_4'11 ------------------------------------------------------------Eden_4'12

- -----------------------------------------------------------Amieu_1 ------------------------------------------------------------chrU_5384045 ----------------------------------------------------------- -----------------------------------------------------------Zimbabwe_71 ------------------------------------------------------------Valence_6 ------------------------------------------------------------Makindu_51 ------------------------------------------------------------Zimbabwe_79 ------------------------------------------------------------Zimbabwe_77 ------------------------------------------------------------Zimbabwe_714 ------------------------------------------------------------Zimbabwe_715 ------------------------------------------------------------Papeete_5 ------------------------------------------------------------Papeete_1 ------------------------------------------------------------Eden_4'14 ------------------------------------------------------------Valence_5 ------------------------------------------------------------Madeira_3'5 ------------------------------------------------------------Madeira_3'p'1 ------------------------------------------------------------Madeira__3'10 ------------------------------------------------------------Canberra_810 ------------------------------------------------------------Eden_4'9 ------------------------------------------------------------Eden_4'4 ------------------------------------------------------------Eden_4'3 ------------------------------------------------------------Eden_4'11 ------------------------------------------------------------Eden_4'12 ------------------------------------------------------------Amieu_1 ------------------------------------------------------------chrU_5384045 ----------------------------------------------------------- ------------------------------------------------G CC--------Zimbabwe_71 -------------------------------------------------G CC--------Valence_6 -------------------------------------------------G CC-TATTAAG Makindu_51 -------------------------------------------------G CC-TATTAAG Zimbabwe_79 -------------------------------------------------G CCCTATTAAG Zimbabwe_77 -------------------------------------------------G CC-TATTAAG Zimbabwe_714 -------------------------------------------------G CC--------Zimbabwe_715 -------------------------------------------------G CC-TATTAAG Papeete_5 ------------------------------------------------------------Papeete_1 -----------------------------------------------------------

-Eden_4'14 -------------------------------------------------G CC-TATTAAG Valence_5 -------------------------------------------------G CC-TATTAAG Madeira_3'5 -------------------------------------------------G CC-TATTAAG Madeira_3'p'1 -------------------------------------------------G CC-TATTAAG Madeira__3'10 -------------------------------------------------G CC-TATTAAG Canberra_810 -------------------------------------------------G CC-TATTAAG Eden_4'9 -------------------------------------------------G CC-TATTAAG Eden_4'4 -------------------------------------------------G CC-TATTAAG Eden_4'3 -------------------------------------------------G CC-TATTAAG Eden_4'11 -------------------------------------------------G CC-TATTAAG Eden_4'12 -------------------------------------------------G CC-TATTAAG Amieu_1 -------------------------------------------------G CC-TATTAAG chrU_5384045

- ------------------------------------------------G --------------------------------------------Valence_4 AAATAAGAGA GACTCACCAG TACCTACAAA TGCCATTGCA GATGTCTTGG CC-TATTAAG Zimbabwe_76 AAATAAGAGA GACTCACCAG TACCTACAAA TGCCATTGCA GATGTCTTGG CC-TATTAAG Zimbabwe_712 AAATAAGAGA GAC----CAG TACCTACAAA TGACATTGCA GATGTCTTGG CC-TATTAAG chrU_5950518 AAATAAAAAA GACTCACCA- ---------------------------------------

CONCLUSION

The helena element is common in D. melanogaster and D. simulans natural populations.

However, helena is extinct in D. melanogaster since it is no longer able to transpose due to the lack of complete open reading frames. In D. simulans, few natural populations harbour complete copies of helena but all of these strains have numerous internally deleted copies of this element. Given the repetitive nature of TEs, recombination is often the cause of copy internal deletion [START_REF] Bennetzen | Mechanisms and rates of genome expansion and contraction in flowering plants[END_REF]. More specifically, solo LTR formation allows the genome to keep the regulatory parts of the elements and throw the coding parts away. In barley, TE conversion into solo LTRs is faster than element insertion, and therefore counteracts with TE invasion [START_REF] Shirasu | A contiguous 66-kb barley DNA sequence provides evidence for reversible genome expansion[END_REF]. In rice, 190Mb of LTR elements were deleted in the last 8 million years [START_REF] Ma | Analyses of LTR-retrotransposon structures reveal recent and rapid genomic DNA loss in rice[END_REF]. Important deletions can also induce TE extinction and have severe consequences on the host genome as described in rodents where massive radiation seems to concord in time with TE extinction [START_REF] Casavant | The end of the LINE?: lack of recent L1 activity in a group of South American rodents[END_REF]. Thanks

to comparative genomics, we know that precise TE deletion can also occur when target sites duplications (created upon retrotransposon insertions) are the objects of recombination (van de Lagemaat et al., 2005). The host genome is therefore able to eliminate TE sequences.

Nevertheless, one should not forget that recombination events can also be harmful for the host genome when unequal homologous recombination occurs [START_REF] Hedges | Inviting instability: Transposable elements, double-strand breaks, and the maintenance of genome integrity[END_REF].

Indeed 0.3% of human genetic diseases are probably related to Alu unequal homologous recombination [START_REF] Deininger | Alu repeats and human disease[END_REF]. As a conclusion, our data show that deletion occurs inside the TE sequence, which is not a common described phenomenon especially in the case of LINE elements, such as helena, in which only 5' truncations are expected from the transposition mechanism.

Sequence deletion is therefore a very effective system for suppressing the transposition activity of TEs, but the consequences can also be deleterious for the host. Genome regulation system is also capable of avoiding transposition by controlling the epigenetic environment of TE copies while keeping their sequences unmodified. Full-length TEs might therefore be protected from deletion by epigenetic means but also might be harmless for the host genome.

Epigenetic regulation of TEs involves chromatin remodeling factors, DNA methylation and non coding small RNAs [START_REF] Lippman | The role of RNA interference in heterochromatic silencing[END_REF] simulans. Our data demonstrate that although TEs are mainly decorated with repressive histone marks we observed intra and inter specific variations suggesting that association between TEs and epigenetic marks is dependent on the TE family/Genome analyzed.

Author Summary

Transposable elements (TEs) are DNA sequences able to move inside the genome.

Transposition and recombination events of TEs may induce mutations and spread copies in the genome. Activity of TE copies is dependent on chromatin localization and since the major part of transposition events is lineage specific, TE copy localization is variable between species, and inside species. We use the model system composed of the fruit flies, D.

melanogaster and D. simulans, along with natural populations from both species in order to compare histone post translational modifications and TE dynamics. Our data suggests that different associations of histone marks exists between these very close species. Also, natural populations of both D. melanogaster and D. simulans do not behave equally to TE presence since TE expression varies between populations analyzed. This research shows how population studies in epigenetics are mandatory to understand how TEs are contributing to genome evolution.

established from the estimation of TE copy number through in situ hybridization experiments by which centromeric, telomeric and dense heterochromatic regions can not be individually counted (Vieira et al., 1999). Therefore, variation in copy number between D. melanogaster and D. simulans natural populations reflects only euchromatic copies. Also, euchromatic TE representation is different between both species sequenced genomes (~5% and 2% for D.

melanogaster and D. simulans respectively (Clark et al., 2007)) suggesting a role for TE chromatin localization in host response.

We propose to investigate somatic TE association with a subset of histone marks in regard of TE copy number variation and TE transcription in natural populations of both D.

melanogaster and D. simulans. The TEs chosen for this assay were copy number polymorphic between natural populations of D. simulans (Vieira et al., 1999) and chosen as being LTR elements (412, tirant and roo) and LINE-like elements (F). No DNA transposons are copy number polymorphic between natural populations of D. simulans and were therefore excluded from our analysis. For each TE family, we observed transcriptionally derepressed populations in both species of Drosophila. Transcription profiles are specific for each natural population and also each TE analysed. Histone patterns are polymorphic between natural populations and distinct between TE families. We confirm that different associations to previously described regulatory marks exist between both species. Also, and more importantly, single natural populations can act as transcriptionally active lines for specific TEs, showing that mobile elements can counteract global host regulation system, and that natural populations may be used as natural mutants. These findings enlighten the nuanced world of natural variation of TE epigenetic marks and show that even with a highly described epigenetic regulatory system, genomes can be invaded and TEs can colonize populations.

H3K27me3 and H3K9me2 enrichment on TEs in natural populations of D. melanogaster and D. simulans.

Variation of H3K27me3 and H3K9me2 is observed between D. melanogaster (blue) and D.

simulans (red) and between all natural populations analysed. 412 and tirant are polymorphic for H3K27me3 but roo and F harbour lower fold changes. The roo element has the highest enrichment rate for H3K9me2 while the F element has the lowest. Please, note that reference genes are equally enriched between both species and natural populations allowing us to compare all strains together (supplementary figure 1). Quantification of transcripts of genes involved in H3K27me3 and H3K9me2 chromatin post-translational modifications. Please, note that reference genes are equally expressed between both species and natural populations allowing us to compare all strains together (supplementary Figure 1).

COMPLEMENTARY WORK

As a member of the « Transposable element, evolution and populations » team, I had the opportunity to participate to other members' projects. I will succinctly discuss them below, outlining when I conceived the experiment.

The tirant element regulation Tirant, as described above, is an LTR retrotransposon present in both species of D. melanogaster and D. simulans [START_REF] Fablet | The evolution of retrotransposon regulatory regions and its consequences on the Drosophila melanogaster and Homo sapiens host genomes[END_REF]. Copy number polymorphism of this element is observed in D. simulans in which most natural populations are empty (polytene chromosomal arms) of tirant copies (Vieira et al., 1999). However, through Southern blot, Fablet et al. (2000) have showed that these strains of D. simulans presented tirant copies, henceforth located in heterochromatic regions, that are not polytenized [START_REF] Fablet | Genomic environment influences the dynamics of the tirant LTR retrotransposon in Drosophila[END_REF]. In addition, characterization of tirant copies in both species revealed two different subfamilies: a common type present in both species (C-type) and a D. simulans type, specific to this species (S-type). Interestingly the S-type was specific to heterochromatic regions as showed by genome walking and Southern blot deductions [START_REF] Fablet | Genomic environment influences the dynamics of the tirant LTR retrotransposon in Drosophila[END_REF]. The C-type on the other hand, could be observed in both heterochromatic and euchromatic regions (Fablet et al., 2006). In order to verify that both types of elements were indeed present in a specific local chromatin conformation, I ran a ChIP assay targeting euchromatic (H3K4me2) and heterochromatic (H3K9me2, H3K27me3) histone marks associated with locus-specific insertions of tirant. Indeed, tirant S-type was highly enriched in H3K9me2, suggesting its presence in dense and constitutive heterochromatin, whereas the C-type presents a weaker enrichment in H3K9me2 and H3K27me3, but a higher one in H3K4me2. The S-type is indeed present in a heterochromatic region as observed through genome walking. Since only C-type transcripts were found (Fablet et al., 2006), Fablet et al wanted to verify that the lack of expression of tirant S-type was indeed due to a heterochromatic silencing. In order to test this hypothesis, we transfected promoters specific to the S-type or the C-type along with a reporter gene in S2 cells of Drosophila [START_REF] Fablet | Genomic environment influences the dynamics of the tirant LTR retrotransposon in Drosophila[END_REF]. Both promoters were able to induce expression of the reporter gene suggesting that it is only the heterochromatic localisation of the S-type that is responsible for its silencing. Here follows the scientific article published on these data.

SUPPLEMENTARY TABLE S1.

Populations tested for the presence of Makindu-specific insertions of tirant.

In brackets are the number of isofemale lines tested for each population.

SUPPLEMENTARY TABLE S2.

SUPPLEMENTARY TABLE S3.

Primers used to test for the presence of Makindu-specific insertions of tirant in natural populations of D. simulans.

For each of the following PCR reactions, the forward primer is located within the ORF envelope, and the reverse primer is designed in the 3' flanking region of tirant.