This thesis is an investigation of the concepts and tools required for the successful application of genome ecology on real genomic transposable element (TE) data. Beginning with the formation of an interdisciplinary working group to examine the distinction between ecology and evolution within genome ecology. By establishing these definitions it was possible to account for the relative effect of proxies for these processes in explaining the variation with the TE communities in a group of genomes. This resulted in the finding that ecological processes were could only explain variation in closely related groups of genomes. Thus ecological methods, developed for examining species distributions along a linear transect, were used to analyze the 30 B.taurus chromosomes. This resulted in the identification of 8 TE species responsible for explaining the spatial distribution in the B.taurus chromosomes. This successful application of ecological methods on TE data promises to inspire many other promising interdisciplinary studies. / OGS

Els elements transposables (TEs) són seqüències repetitives amb el tret definitori de canviar la seva posició al genoma. Ocupen fraccions importants dels genomes eucariotes, y, tot i que solen considerar-se paràsits genètics, també s'especula amb la possibilitat de que tinguessin alguna funció cel·lular que encara ens és desconeguda. Tot i així, sembla evident que tenen un paper important com facilitadors de l'evolució, ja que generen variabilitat al genoma de l'hoste. El TE Galileo està implicat en la generació de reordenacions cromosòmiques adaptatives naturals a l'espècie Drosophila buzzatii, en la que hauria generat variabilitat amb valor adaptatiu per a l'hoste. A més, tots els elements Galileo trobats en treballs anteriors eren defectius – composats bàsicament d'estructures similars a la dels elements Foldback – i no es van poder establir relacions d'homologia amb ninguna seqüència coneguda. Amb aquest rerefons, en aquesta tesi es va plantejar caracteritzar l'element genètic mòbil Galileo en diferents espècies de Drosophila i analitzar la seva dinàmica evolutiva. D'aquesta forma, en una primera fase es van buscar elements Galileo complets en diferents espècies del gènere Drosophila: D. buzzatii, D. mojavensis, D. virilis, D. willitoni, D. ananassae, D. pseudoobscura i D. persimilis, fent servir tant mètodes bioinformàtics com experimentals (depenent de si el genoma analitzat estava seqüenciat o no). Les còpies trobades presenten llargues Repeticions Invertides Terminals (TIR) de fins a 1,2 Kb, una elevada identitat amb seqüències de Galileo descrites anteriorment i, a més, contenen una zona codificant que ha permès classificar Galileo com a membre de la superfamília de l'element P. Posteriorment, mitjançant anàlisis filogenètiques, hem trobat l'existència de subfamílies de Galileo en tres espècies (D. buzzatii, D. mojavensis, D. virilis) i evidència d'activitat transposicional recent (D. willitoni, D. ananassae, D. pseudoobscura, D. persimilis i D. mojavensis). En una segona fase de la tesi, hem dut a terme experiments amb part de la proteïna que es codifica a Galileo i hem comprovat que interacciona amb les TIR de Galileo, confirmant que aquesta seqüència és la responsable de la reacció de transposició. Finalment, hem analitzat en detall la diversitat de Galileo al genoma de D. mojavensis i hem detectat una diversitat estructural molt important, on l'intercanvi de seqüències entre elements pareix força freqüent per l'evolució dels TEs. / Los elementos transponibles (TEs) son secuencias repetitivas cuya característica definitoria es la capacidad de cambiar de posición en el genoma. Ocupan fracciones muy importantes de los genomas de eucariotas, y aunque se suelen considerar parásitos genéticos, también se especula con la posibilidad de que pudieran tener alguna función celular que aún nos es desconocida. No obstante, parece evidente que tienen un papel importante como facilitadores de la evolución, al generar variabilidad en el genoma del huésped. El TE Galileo está implicado en la generación de reordenaciones cromosómicas adaptativas naturales en la especie Drosophila buzzatii, con lo que habría generado variabilidad adaptativa para el huésped. Además, todos los elementos Galileo encontrados en trabajos anteriores eran defectivos – compuestos básicamente de estructuras similares a las de los elementos Foldback – y no se pudieron establecer relaciones de homología con ninguna secuencia conocida. Con este trasfondo, en esta tesis se planteó caracterizar el elemento genético móvil Galileo en diferentes especies de Drosophila y analizar su dinámica evolutiva. De esta manera, en una primera fase se buscaron elementos Galileo completos en en diferentes especies del género Drosophila: D. buzzatii, D. mojavensis, D. virilis, D. willitoni, D. ananassae, D. pseudoobscura y D. persimilis, utilizando métodos tanto bioinformáticos como experimentales (dependiendo de si el genoma analizado estaba secuenciado o no). Las copias encontradas presentan largas Repeticiones Invertidas Terminals (TIR) de hasta 1,2 Kb, una elevada identidad con secuencias de Galileo descritas con anterioridad y, además, contienen una zona codificante que ha permitido clasificar Galileo como miembro de la superfamilia del elemento P. Posteriormente, mediante análisis filogenéticos, hemos encontrado la existencia de subfamilias de Galileo en tres especies (D. buzzatii, D. mojavensis, D. virilis) y evidencias de actividad transposicional reciente (D. willitoni, D. ananassae, D. pseudoobscura, D. persimilis y D. mojavensis). En una segunda fase de la tesis, hemos llevado a cabo experimentos con parte de la proteína que codifica Galileo y hemos comprobado que interacciona con las TIR de Galileo, confirmando que esta secuencia es la responsable de la reacción de transposición. Finalmente, hemos analizado en detalle la diversidad de Galileo en el genoma de D. mojavensis y hemos detectado una diversidad estructural muy importante, lo que sugiere que el intercambio de secuencias entre elementos podría ser bastante frecuente para la evolución de los TEs. / Transposable elements (TE) are repetitive sequences whose ability to change their location in the genome defines them. They made up a important proportion of the eukaryotic genomes, and although they are often considered as genetic parasites, it has been also argued that they might have some still unknown cellular function. Nevertheless, it is clear that they play a role as drivers of their host evolution, due to the fact that TEs generate genetic variability. The TE Galileo is involved in the generation of adaptive chromosomal rearrangements in natural populations of Drosophila buzzatii, indicating that it would be a driver of adaptation in its host. Moreover, all Galileo elements found in previous works were incomplete – mainly composed by Foldback-like structures – and homology relationships could not be established with any known sequence. With this background, this thesis was proposed to characterise the mobile genetic element Galileo in different Drosophila species and analyse its evolutionary dynamics. Thus, in a first phase we searched for complete copies of Galileo in different species of the Drosophila genus: D. buzzatii, D. mojavensis, D. virilis, D. willitoni, D. ananassae, D. pseudoobscura and D. persimilis, using both bioinformatic and experimental methods (depending on whether the analysed genome was available or not). The copies found present long TIR (up to 1.2 Kb), high sequence identity with previously found Galileo sequences and, moreover, they harbour coding sequences that have allowed the classification of Galileo as a member of the P-element superfamily. Subsequently, by means of phylogenetic analyses, we have found that there are Galileo subfamilies in three different species (D. buzzatii, D. mojavensis, D. virilis) and evidence of recent transpositional activity (in D. willitoni, D. ananassae, D. pseudoobscura, D. persimilis and D. mojavensis). In a second phase of the thesis, we have conducted experiments with part of the Galileo protein and detected specific binding to the Galileo TIR, confirming that this sequence is responsible for the transposition reaction. Finally, we have thoroughly studied the Galileo variability in the D. mojavensis genome and found a striking structural variation, suggesting that the exchange of sequences among different Galileo copies might be quite common and important for TEs evolution.

Repetitive sequences, including transposable elements, represent approximately half of the mammalian genome. Epigenetic mechanisms evolved to repress these potentially deleterious mobile elements. However, such elements can be variably silenced between individuals – so called ‘metastable epialleles’. The best known example is the Avy locus where an endogenous retrovirus (ERV) of the intracisternal A-particle (IAP) class was spontaneously inserted upstream of the agouti coat colour gene, resulting in variable IAP promoter DNA methylation, variable expressivity of coat phenotype, and environmentally modulated transgenerational epigenetic inheritance within genetically identical individuals. It is not known whether the behaviour exhibited by the ERV at Avy represents a common occurrence throughout the genome or is unusual. Taking a genetic approach in purified cell populations, I have conducted a systematic genome-wide screen of murine metastable epialleles. I have identified over 100 murine IAPs with properties of metastable epialleles. Like Avy, each exhibits a stable epigenetic state within an individual but epigenetic variability between individuals. Methylation levels are locus-specific within an individual, suggesting cis-acting control. The same screening strategy was applied for identification of metastable epialleles associated with other types of LTR-retroelements. However, many of identified candidates showed no inter-individual methylation variation upon experimental validation. These results suggest that IAPs are the dominant class of ERVs capable of acquiring epigenetic states that are variable between genetically identical individuals. I have conducted an analysis of IAP induced initiation and termination of transcription events using de novo assembled transcriptomes generated for B and T cells. 143 IAPs have been identified to overlap de novo assembled transcripts. 33 IAPs are metastable epialleles. Several of them show an inverse correlation between LTR promoter methylation and adjacent gene expression. In addition, I have shown that metastable epialleles have a characteristic pattern of histone modification and are flanked by the methylation sensitive binding factor CTCF, providing testable hypotheses concerning the establishment and/or maintenance of the variable methylation state. My findings indicate that metastability is, in general, specific to the IAP class of ERVs, that only around 1% of these elements have this unusual epigenetic property and that the ability to impact transcription, such as at agouti in Avy, is not a ubiquitous feature of these loci.

The evolution of a gene’s expression profile is commonly assumed to be independent of its genomic neighborhood. This is, however, in contrast to what we know about the lack of autonomy between expression of neighboring genes in extant taxa. Indeed, in all eukaryotic genomes, genes of similar expression-profile tend to cluster, reflecting chromatin level dynamics. Does it follow that if a gene increases expression in a particular lineage then the genomic neighbors will also increase in their expression or is gene expression evolution autonomous? To address this, I consider evolution of human gene expression since the human-chimp common ancestor, allowing for both variation in estimation of current expression level and error in Bayesian estimation of the ancestral state. I find that in all tissues and both sexes, the change in gene expression of a focal gene on average predicts the change in gene expression of neighbors. The effect is highly pronounced in the immediate vicinity but extends much further. Sex-specific expression change is also genomically clustered. As genes increasing their expression in humans tend to avoid nuclear lamina domains and be enriched for the gene activator 5-hydroxymethylcytosine, chromatin level mechanisms are likely regulators of this phenomenon. Firstly established in Primates and then expanded to compacted genome of yeasts, the phenomenon of correlation in change in gene expression of the neighbouring genes I describe as “expression piggy-backing”, an analog of hitchhiking. Extending the same principle to non-coding genes I find a possible role of lincRNAs in regulating expression of their neighbours, mediated by a coupling between splicing and chromatin modification. Finally I employ insertions of human endogenous retroviruses (HERVs), as a naturally occurring transgene experiment, to find out how randomly scattered sequences would affect the expression profile of their neighboring genes. I show these retroviruses to be the focus of transcription in human ES cells and define a transcription factor, LBP9, as a novel pluripotency-associated agent. Transcription results in neighbourhood modification including the generation of chimaeric transcripts. Predictions were confirmed experimentally by collaborators.

MicroRNAs (miRNAs) are an abundant and diverse class of small, non-protein coding RNAs that guide the post-transcriptional repression of messenger RNA (mRNA) targets in a sequence-specific manner. Hundreds, if not thousands of distinct miRNA sequences have been described, each of which has the potential to regulate a large number of mRNAs. Over the last decade, miRNAs have been ascribed roles in nearly all biological processes in which they have been tested. More recently, interest has grown in understanding how individual miRNAs evolved, and how they are regulated. In this work, we demonstrate that Transposable Elements are a source for novel miRNA genes and miRNA target sites. We find that primate-specific miRNA binding sites were gained through the transposition of Alu elements. We also find that remnants of Mammalian Interspersed Repeat transposition, which occurred early in mammalian evolution, provide highly conserved functional miRNA binding sites in the human genome. We also provide data to support that long non-coding RNAs (lncRNAs) can provide a novel miRNA binding substrate which, rather than inhibiting the miRNA target, inhibits the miRNA. As such, lncRNAs are proposed to function as endogenous miRNA "sponges," competing for miRNA binding and reducing miRNA-mediated repression of protein-coding mRNA targets. We also explored how dynamic changes to miRNA binding sites can occur by A-to-I editing of the 3 `UTRs of mRNA targets. These works, together with knowledge gained from the regulatory activity of endogenous and exogenously added miRNAs, provided a platform for algorithm development that can be used in the rational design of artificial RNAi triggers with improved target specificity. The cumulative results from our studies identify and in some cases clarify important mechanisms for the emergence of miRNAs and miRNA binding sites on large (over eons) and small (developmental) time scales, and help in translating these gene silencing processes into practical application.

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第18526号 / 農博第2083号 / 新制||農||1026(附属図書館) / 学位論文||H26||N4870(農学部図書室) / 31412 / 京都大学大学院農学研究科農学専攻 / (主査)教授 奥本 裕, 教授 米森 敬三, 教授 冨永 達 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

Whole genome sequencing (WGS) projects for model mammalian organisms exposed the magnitude to which transposable elements (TEs) have contributed to DNA content, but led to inferences on repeat composition and activity patterns which do not capture the true diversity within Mammalia. Understanding of the evolutionary importance of TEs and the development of TEs for biological applications are hindered when considering only the data gathered from a limited sampling of model organisms. The research presented here begins with analysis of a mouse lemur WGS dataset, revealing an exception in the primate lineage to the generalizations garnered from model organisms. A recently active TE was uncovered with evidence of horizontal transfer involving a primate and another mammal; this discovery may lead to a useful transfection vector with known efficacy in mammalian cells. Furthermore, an opportunity to observe atypical patterns of TE diversification within a mammalian family was achieved through survey sequencing and comparative analyses of five vespertilionid taxa and a phyllostomid outgroup. A potential inverse relationship between the two classes of TEs was tentatively described, and further explored by an in-depth analysis of the dominant TE family from each class in a species with WGS data available. The interplay of the two classes could not previously be investigated in mammals, due to ~40 million years of Class II TE inactivity in model organisms. Through exploration of activity patterns of both classes, this study provides insight on the relationship between TEs and their host.

Les éléments transposables (ET) sont des séquences d‟ADN capables de se déplacer dans les génomes qui les hébergent. Les ET de type mariner (mariner-like element : MLE) ont été caractérisés chez les animaux et chez les plantes à fleurs terrestres mais pas chez les microalgues marines. Dans le présent travail, les MLE ont été recherchés chez les diatomées marines (Bacillariophycées) qui sont des microalgues possédant une enveloppe externe siliceuse. Elles ont colonisé tous les milieux aquatiques et constituent une part majeure du phytoplancton; de ce fait, elles jouent un rôle écologique clé dans le milieu marin.La caractérisation des MLE des diatomées a été réalisée au moyen d‟approches moléculaire et bio-informatique. La présence de MLE dans le génome de 10 espèces de diatomées a été mise en évidence grâce à l‟amplification de fragments d‟environ 380 pb. Ces fragments correspondent à une partie de la séquence conservée codant l‟enzyme responsable de la transposition des MLE : la transposase. L‟analyse des séquences obtenues, par des méthodes phylogénétiques, ainsi que la classification des MLE de diatomées mettent en évidence leur appartenance au groupe des MLE végétaux de la superfamille Tc1-mariner. Néanmoins, les séquences MLE de diatomées divergent considérablement par rapport aux MLE des plantes terrestres. Afin de déterminer si les diatomées ont la capacité de produire la transposase, l‟expression des MLE a été recherchée chez des diatomées soumises à des stress thermiques de courte durée (5 h). Nos travaux montrent que les MLE sont exprimés chez les trois espèces testées incluant la diatomée modèle Phaeodactylum tricornutum dont le génome a été séquencé récemment. L‟expression des MLE des diatomées est variable selon les conditions thermiques et selon les espèces.L‟ensemble de nos résultats suggère que les MLE sont ubiquistes dans les génomes de diatomées et qu‟ils sont présents de manière ancestrale dans la lignée végétale. Les MLE des diatomées forment trois nouvelles sousfamilles de la superfamille Tc1-mariner, la plus répandue des superfamilles d‟ET. De plus, leur expression suggère qu‟il existe des MLE capables de se déplacer dans le génome des diatomées. Si la transposition était vérifiée, ils pourraient alors être développés comme outils de mutagenèse. / Transposable elements (TE) are sequences able to move between two loci in the host genomes. Mariner-like element (MLE) are well characterized in animal and land plant genomes but not in marine microalgae. In this work, we have looked for MLE in marine diatoms (Bacillariophyceae) that are microalgae having a siliceous wall. They have colonized all aquatic environments and are a major component of the phytoplankton, so they play a major role in the ecology of marine environments.To characterize the diatom MLE, molecular and bio-informatics approaches were used. The presence of MLE was detected in ten diatom species which exhibited sequence fragments of about 380 pb. These fragments were identified as a section of the conserved sequence which encodes the enzyme responsible for MLE transposition, the transposase. Phylogeny and classification analysis of these fragments revealed that diatom MLE belong to the Tc1-mariner superfamily, and more precisely to the vegetal MLE group. Nevertheless, diatom MLE sequences diverged from the flowering plant MLE. In order to determine if MLE transposase is produced in diatoms, MLE expression was looked for in diatoms under thermal stresses. Our results showed that diatom MLE were expressed in the three species tested, including the model diatom Phaeodactylum tricornutum which was completely sequenced recently. Diatom MLE expression was dependent on the thermal conditions and on the species.Our results suggest that MLE are widespread in diatom genomes and that they have an ancestral presence in the green lineage. Diatom MLE cluster in three subfamilies in the huge ET Tc1-mariner superfamilly. Finally, the diatom MLE expression detected could reflect the existence of active MLE in diatom genomes. If this hypothesis were verified, it could lead to the development of mutagenesis tools.

Les cellules, et tout particulièrement les cellules germinales, maintiennent l'intégrité de leur génome en prévenant d'éventuelles mutations comme celles dues à la mobilité des éléments transposables (ET). Dans la lignée germinale des animaux, une classe particulière de petits ARN régulateurs, les PIWI-interacting RNA (piRNA), sont les acteurs majeurs du contrôle des ET. Chez Drosophila melanogaster, il existe des souches dites réactives, dépourvues de copies actives de l'élément I, ET exprimé dans la lignée germinale femelle. Les femelles de ces souches voient leur capacité à réprimer l'invasion de leur génome par l'élément I augmenter avec l'âge. Des données antérieures ont montré qu'une fois acquise, la capacité à réprimer l'élément I est transmise maternellement au travers des générations. Mes travaux de thèse ont permis de montrer que la transmission de la capacité à réprimer l'élément I n'est pas corrélée à des modifications de l'activité transcriptionnelle des loci producteurs de piRNA, mais semble uniquement véhiculée par les piRNA. En effet, les piRNA de l'élément I déposés dans l'embryon vont amorcer la production de piRNA complémentaires dans les ovaires de la descendance, ce qui induit une forte accumulation de piRNA antisens à l'élément I. Ainsi, les piRNA maternellement déposés assurent la transmission de la capacité à réprimer l'élément I, acquise suite au vieillissement des ascendants maternels. Mes résultats mettent en évidence le rôle des piRNA comme support moléculaire d'une composante non génétique de l'information héritable, indépendante de la chromatine et déterminante pour le maintien de l'intégrité du génome. / Cells, especially germinal stem cells, maintain genomic integrity by averting the propagation of mutations, generated as a consequence of DNA damage. In particular, they must avoid the deleterious activity of transposable elements (TEs). In animal germlines, one of the key players of the TE repression involves a specific class of small regulatory RNAs, the PIWI-interacting RNAs (piRNAs). In Drosophila melanogaster, there are reactive strains that are devoid of functional copies of the I element, a TE specifically expressed in the female germ line. When they get older, females of these strains can acquire a strong capacity to repress the I element invasion. Anterior works have shown that once acquired, this capacity to repress the I element is maternally transmitted over generations. The results obtained during my thesis revealed that the transmission of the capacity to repress the I element is not correlated with increased transcriptional activity of piRNA producer loci but seems only mediated by the piRNAs. Indeed, I element piRNAs deposition in the embryo after aging treatment correlates with the production of complementary piRNAs in the ovaries of the progeny. This results in a strong accumulation of antisense I element piRNAs. The maternally deposited piRNAs ensure the transmission of the capacity to repress the I element acquired after ancestor aging. My results highlight the molecular support of a DNA- and chromatin-independant component of heritable information essential for the maintenance of genome integrity.

Les éléments transposables (ET) ont un impact majeur sur le fonctionnement etla dynamique des génomes, à l’échelle de l’individu et de l’espèce. Le cilié Parameciumest un modèle original pour l’étude des ET. Chaque individu unicellulaire a un génomegerminal qui subit, lors des processus sexuels, des réarrangements massifs, comprenantl’élimination des ET et de leurs vestiges à copie unique, pour former un génome somatiqueoptimisé pour l’expression des gènes. La programmation épigénétique de cesréarrangements implique des petits ARN dans un processus complexe de soustractiongénomique.Au cours de ma thèse, j’ai effectué des analyses bioinformatiques et biostatistiques dedonnées hétérogènes à l’échelle du génome pour : (i) Identifier et analyser des propriétésintrinsèques, de dizaines de milliers de vestiges d’ET à copie unique, appelés "InternalEliminated Sequences" (IES). (ii) Comprendre le rôle de déterminants génétiques et dedifférents facteurs épigénétiques dans le ciblage et l’élimination des IES.L’ensemble de ces analyses met en lumière la co-Évolution des ET et des mécan-Ismes de défense de l’hôte. / Transposable elements (TE) have major impact on the function and dynamicsof genomes, both at the level of the individual and of the species. The ciliate Parameciumprovides an original model for studies of TE. Each individual unicell has a germlinegenome that undergoes massive rearrangements at each sexual generation including thephysical elimination of TE and their single copy remnants, yielding a somatic genomestreamlined for gene expression. The epigenetic programming of the rearrangementsinvolves small RNAs in a complex process of genomic subtraction.During my thesis, I carried out bioinformatic and biostatistical analyses of heteroge-Neous, genome-Scale datasets in order to : (i) Identifiy and study the intrinsic propertiesof tens of thousands of TE remnants know as "Internal Eliminated Sequences" (IES).(ii) Explore the roles of genetic determinants and epigenetic factors in the targeting andelimination of the IESs.Taken together, the studies illustrate the co-Evolution of TE and host defense mecha-Nisms.