Made available in DSpace on 2015-03-26T13:51:51Z (GMT). No. of bitstreams: 1 texto completo.pdf: 482532 bytes, checksum: 1c1dce279e9ef945c0bd479992a99516 (MD5) Previous issue date: 2009-02-20 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The hemibiothrophic basidiomycete Moniliophthora perniciosa, the causal agent of witche's broom disease in cocoa (Theobroma cacao), presents different transposable elements in its genome. A transposable element of Class II belonging to the super-family PIF/Harbinger, named Boto, had been partially characterized in previous studies. The element Boto, as well as other elements PIF/Harbinger, presents two ORFs (ORF 1 and ORF 2). OFR 2 codifies the transposase while ORF 1 codifies one protein which function is unknown. The aim of this work was to prove the Boto element activity and characterize the ORF 1. For assure the activity of the transposable element Boto in the genome of M pernicious was carried out the evaluation of the integration profile in isolates resultants of sexual cycle. Three of such isolates showed variation in the copy number of transposable element Boto, proving Boto activity. Furthermore, PCR reactions were carried out for detection of insertion sites of this element in different isolates. Among the 28 isolates used in this experiment, 14 showed amplification products with expected size. The products of amplification of two isolates, randomly chosen, were cloned and sequenced. The analysis of the obtained sequences showed that in both isolated sequenced the element Boto did not transpose for the insertion site analyzed in the M. perniciosa genome. It proves the activity of this element in the genome of M. perciciosa, because, in the isolated used as reference for this study, the Boto element was inserted in that position. In the in silico analysis of the ORF 1 sequence, the presence of two possible introns was verified, one containing 55 pb and another 48 pb. This sequence codifies a protein of 422 amino acids residues. To demonstrate the presence of the two introns, experiments of RT-PCR were carried out using two oligonucleotides groups. Amplified products obtained, when cDNA was used as template of PCR reaction, were cloned and sequenced, being confirmed the position and the size of the two predicted introns. Analysis of RT-PCR also showed that this ORF is expressed even in normal conditions of cultivation of the fungi, what is important for activity of this element, since other studies showed that the expression of this ORF is fundamental to the transposition of elements PIF/Harbinger. The results of this work showed that Boto is active in the M. perniciosa genome and it can have an important role as generating agent of genetic variability in this phytopathogen. It is worth to emphasize that this is the first description of an element of the superfamily PIF/Harbinger that presents two introns in ORF 1. / O basidiomiceto hemibiotrófico Moniliophthora perniciosa, agente causal da vassoura-de-bruxa no cacaueiro (Theobroma cacao) apresenta diferentes elementos transponíveis em seu genoma. Um elemento transponível da Classe II pertencente à superfamília PIF/Harbinger, denominado Boto, foi parcialmente caracterizado em estudos anteriores. O elemento Boto, assim como os demais elementos PIF/Harbinger, apresenta duas ORFs (Open Reading Frame), uma que codifica a transposase e outra, denominada ORF 1, cuja seqüência de aminoácidos deduzida representa uma proteína de função é desconhecida. Este trabalho foi conduzido com o objetivo de comprovar a atividade de Boto e caracterizar a ORF 1. Para a confirmação da atividade do elemento transponível Boto no genoma de M. perniciosa realizou-se a avaliação do perfil de integração em isolados resultantes do ciclo sexual, isto é, obtidos a partir da germinação de basidiósporos. Três isolados apresentaram variação no número de cópias, demonstrando a atividade de Boto. Foi realizado PCR para detecção dos sítios de inserção deste elemento em diferentes isolados. Dentre os 28 isolados utilizados no experimento, 14 apresentaram produtos de amplificação com o tamanho esperado. Os produtos de amplificação de dois isolados, escolhidos aleatoriamente, foram clonados e seqüenciados. Na análise das seqüências obtidas, verificou-se que, em ambos os isolados sequenciados, o elemento Boto provavelmente não sofreu transposição para os sítios analisados no genoma de M. perniciosa. Isto comprova a atividade deste elemento, pois, no isolado usado como referência para este estudo, o elemento Boto estava inserido naquela posição. Na análise da seqüência da ORF 1, verificou-se a presença de dois possíveis íntrons, um contendo 55 pb e outro de 48 pb. Esta seqüência codifica uma proteína de 422 resíduos de aminoácidos. Para comprovar a presença dos íntrons, experimentos de RT-PCR foram realizados com a utilização de dois conjuntos de oligonucleotídeos. Os amplificados obtidos, quando cDNA foi utilizado como molde da reação de PCR, foram clonados e sequenciados, sendo confirmada a posição e o tamanho dos dois íntrons preditos. Análise de RT-PCR mostrou que esta ORF é expressa mesmo em condições normais de cultivo do fungo, o que é importante para atividade deste elemento, visto que outros estudos mostram que a expressão desta ORF é fundamental à transposição de elementos PIF/Harbinger. Os resultados deste trabalho mostram que Boto é ativo no genoma de M. perniciosa e pode ter um importante papel como agente gerador de variabilidade genética neste fitopatógeno. Vale ressaltar que esta é a primeira descrição de um elemento da superfamília PIF/Harbinger que apresenta dois íntrons na ORF 1.

Os elementos de transposição são seqüências discretas que são capazes de mover- se de um lócus para outro, constituindo uma parte significante do genoma de eucariotos. São agrupados em duas classes principais, os elementos da Classe I, que se transpõem via um intermediário de RNA (retrotransposon), e os elementos da Classe II, que se transpõem via mecanismo de DNA do tipo corta e cola (transposon). A análise das seqüências de um banco de cDNA construído com RNA mensageiro da glândula salivar de Rhynchosciara americana mostrou a presença de representantes das duas classes de elementos. Nesse trabalho caracteriza-se quarto elementos de transposição tipo mariner, onde as seqüências consenso de nucleotídeos foram derivadas de múltiplas cópias defectivas contendo deleções, mudança no quadro de leitura e códons de terminação. Ramar1, um elemento full-length e Ramar2 um elemento defectivo que contém uma deleção na região interna da ORF da transposase, mas mantém e as extremidades intactas. Ramar3 e Ramar4 são elementos defectivos que apresentam muitas deleções no interior da ORF. As seqüências preditas das transposases demostraram que Ramar1 e Ramar2 estão filogeneticamente muito próximos dos elementos mariner da subfamília mauritiana. Enquanto, Ramar3 e Ramar4 pertencem às subfamílias mellifera e irritans, respectivamente. Hibridização in situ mostrou que Ramar1 localiza-se em muitas regiões do cromossomo, principalmente na heterocromatina pericentromérica, enquanto Ramar2 aparece em uma única banda no cromossomo A. Resultado ainda mais curioso foi a caracterização molecular de um elemento de retrotransposição, denominado RaTART, que provavelmente é o responsável pela reconstituição telomérica em R.americana, assim como os elementos TART, HeT-A e TAHRE de Drosophila. Experimentos de Southern Blots do retroelemento RaTART indicam que este está representado por seqüências repetidas no genoma de R.americana, enquanto que Northern Blots mostraram uma expressão em diferentes estágios do desenvolvimento e o transcrito de alto peso molecular detectado representa o retrotransposon non-LTR inteiro. Enquanto a localização cromossômica de RaTART por hibridização in situ mostrou uma marcação predominante nas extremidades dos cromossomos, indicando possivelmente o primeiro elemento de transposição descrito em R.americana com função definida na estrutura do cromossomo. O último retrotransposon, identificado nesse projeto, presente no genoma de R.americana, denominado R2Ra, foi isolado a partir de uma varredura em um banco genômico construído no bacteriófago lambda dash usando como sonda o recombinante pRa1.4 que contém a unidade de repetição do rDNA. A análise da seqüência mostrou a presença de regiões conservadas, como o domínio de transcriptase reversa e o motivo zinc finger na região amino-terminal. O sítio de inserção no gene 28S do rDNA é altamente conservado em R.americana e a análise filogenética mostrou que este elemento pertence ao grupo R2. A localização cromossômica confirma que o elemento móvel R2Ra se insere em um sítio específico no gene rDNA. / Transposable elements are discrete sequences that are able to move from one locus to another within the genome, constituting a significant part of eukaryotic genome. They are grouped into two main types, Class I elements transpose via an RNA intermediate (retrotransposon), and Class II elements transpose via a DNA \"cut-and-paste\" mechanism (transposons). The analysis of sequences of a cDNA bank constructed from mRNA of the salivary glands of Rhynchosciara americana showed the presence of putative types of two classes elements. In the present thesis we describe four mariner elements, where the nucleotides consensus sequences were derived from multiple defective copies containing deletions, frame shifts and stop codons. Ramar1, a full-length element and Ramar2 is a defective mariner element that contains a deletion overlapping most of the internal region of the transposase ORF and the extremities of the element maintain intact. Ramar3 e Ramar4 are defective mariner element that were impossible to predict a complete ORF. Predicted transposase sequences demonstrated that Ramar1 and Ramar2 are phylogenetically very close to mariner-like elements of mauritiana subfamily. However, Ramar3 and Ramar4 belong to mellifera and irritans subfamilies, respectively. In situ hybridisations showed Ramar1 localized in several chromosome regions, mainly in pericentromeric heterochromatin and their boundaries, while Ramar2 appeared as a single band in chromosome A. More interesting data were the molecular characterization of the non-LTR retrotransposon element, called as RaTART, that probably is the responsible by telomeric reconstruction in R.americana, as well as the telomeric retrotransposable elements TART, Het-A and TAHRE of Drosophila. Southern blot analysis indicated that this transposable element is represented by repeat sequences in the genome of R. americana, and Northern blot analysis showed a expression in different developmental stages and the transcript of high molecular mass detected represents the full-length non-LTR retrotransposon. However, the chromosomal localization of the retroelement by in situ hybridisation showed a labelling predominant on chromosome ends, indicating possibly the first transposable element described in R.americana with a defined role in chromosome structure. The last retrotransposon, identified in this project, present in the genome of Rhynchosciara americana, called R2Ra, was isolated from screening of a lambda dash genomic library using as probe the recombinant pRa1.4 of rDNA. The analysis of sequence showed the presence of conserved regions, like transcriptase reverse domain and zinc finger motif in the amino terminal region. The insertion site is high conserved in R.americana and a phylogenetic analysis showed that this element belongs to the R2 clade. The chromosomal localization confirm that the R2Ra mobile element insert into the site specific in rDNA gene.

Les génomes nucléaires eucaryotes sont empaquetés au sein d’une structure nucléoprotéique appelée chromatine et dont l’unité fondamentale est le nucléosome. Celui-ci est composé d’un octamère d’histones, contenant deux molécules de chacune des histones H2A, H2B, H3 et H4, autour duquel 147 pb d’ADN sont enroulées. Les modifications post-traductionnelles (PTMs) des histones et de l’ADN (méthylation des cytosines) constituent des marqueurs épigénomiques primaires qui participent à la régulation et au contrôle de l’accessibilité des différentes régions du génome. Ainsi, la chromatine forme une structure dynamique influencée par les changements environnementaux et développementaux et contribue à orchestrer diverses fonctions du génome. L’objectif principal de ma thèse était de caractériser l’organisation spatiale et la dynamique temporelle des états chromatiniens chez Arabidopsis par des approches à l’échelle du génome permettant l’étude des profils de méthylation de l’ADN et d’un ensemble de modifications post-traductionnelles des histones. La méthylation de l’ADN, une marque caractéristique de l’inactivation épigénétique et de l’hétérochromatine chez les plantes et les mammifères, est largement confinée aux séquences répétées, dont les éléments transposables (TEs). Par mon travail de thèse, j’ai montré que chez Arabidopsis les séquences de TEs faiblement méthylées ou non associées à des petits ARN interférents (siRNAs), donc potentiellement non régulées par la machinerie de RNA-directed DNA methylation (RdDM), peuvent acquérir une méthylation de l’ADN par diffusion à partir de séquences adjacentes ciblées par les siRNAs. Cette diffusion de la méthylation de l’ADN sur des régions promotrices pourrait expliquer, au moins en partie, l’impact négatif des TEs associés à des siRNAs sur l’expression des gènes à proximité immédiate. Dans une seconde partie de ma thèse, j’ai contribué à l’analyse intégrée de la méthylation de l’ADN et de onze modifications des histones. L’utilisation d’analyses combinatoires et en cluster m’a permis de montrer que l’épigénome d’Arabidopsis présente des principes simples d’organisation. En effet, ces analyses nous ont conduit à distinguer quatre états fondamentaux de la chromatine chez Arabidopsis, préférentiellement associés aux gènes actifs, aux gènes inactifs, aux TEs et aux régions intergéniques. Dans une troisième partie, j’ai intégré des données épigénomiques et transcriptomiques obtenues à différents temps afin d’étudier les dynamiques temporelles des états chromatiniens en réponse à un stimulus externe, la première exposition à la lumière des plantules suite à la germination. Ces travaux nous ont permis de montrer que la monoubiquitination de l’histone H2B participe à la modulation fine et sélective des changements rapides de l’expression de gènes. L’ensemble du travail présenté contribue à une meilleure compréhension de l’organisation de la chromatine le long du génome des plantes et de la dynamique des états chromatiniens en réponse aux changements de l’environnement. / Eukaryotic genomes are packed into the confines of the nucleus through a nucleoproteic structure called chromatin. Chromatin is a dynamic structure that can respond to developmental or environmental cues to regulate and orchestrate the functions of the genome. The fundamental unit of chromatin, the nucleosome, consists of a protein octamer, which contains two molecules of each of the core histone proteins (H2A, H2B, H3, H4), around which 147 bp of DNA is wrapped. The post-translational modifications (PTMs) of histones and methylation of the cytosine residues in DNA (DNA methylation) constitute primary epigenomic markers that dynamically alter the interaction of DNA with nucleosomes and participate in the regulation and control access to the underlying DNA. The main objective of my thesis was to understand the spatial and temporal dynamics of chromatin states in Arabidopsis by investigating on a genome-wide scale, patterns of DNA methylation and a set of well-characterized histone post-translational modifications. DNA methylation, a hallmark of epigenetic inactivation and heterochromatin in both plants and mammals, is largely confined to transposable elements and other repeat sequences. I show in this thesis that in Arabidopsis, methylated TE sequences having no or few matching siRNAs, and therefore unlikely to be targeted by the RNA-directed DNA methylation (RdDM) machinery, acquire DNA methylation through spreading from adjacent siRNA-targeted regions. Further, I propose that this spreading of DNA methylation through promoter regions can explain, at least in part, the negative impact of siRNA-targeted TE sequences on neighbouring gene expression. In a second part, I have contributed to integrative analysis of DNA methylation and eleven histone PTMs. I have shown through combinatorial and cluster analysis that the Arabidopsis epigenome shows simple principles of organisation and can be distinguished into four primary types of chromatin that preferentially index active genes, repressed genes, TEs, and intergenic regions. Finally, in a third part, I integrated epigenomics with transcriptome data at three different time points in a developmental window to investigate the temporal dynamics of chromatin states in response to an external stimulus. This used the light-induced transcriptional response as a paradigm to assess the impact of histone H2B monoubiquitination (H2Bub), and showed that this PTM is associated with active transcription and implicated in the selective fine-tuning of gene expression. Taken together, the work presented here contributes significantly to our understanding of the spatial organisation of chromatin states in plants, its dynamic nature and how it can contribute to allow plants to respond to a signal from the environment.

The retrotransposon Ylt1 was described previously from the genome of the dimorphic fungus Yarrowia lipolytica. Remarkably, Ylt1 is currently the largest LTR retrotransposon reported from fungal genomes. However, little was known about its biology and its interactions with host genome. So, the aim of this work was the characterization of properties of Ylt1.Analysis of proteins encoded by Ylt1 (Gag protein and integrase) was carried out during this work. To enable their detection, both proteins were tagged with HA epitopes. The sizes of Gag protein and putative precursors of Gag protein and integrase were estimated, and a model for the proteolytic processing of the polyprotein of Ylt1 was proposed. It was shown that Gag protein of Ylt1 is about 2-fold larger than Gag proteins of other studied yeast retrotransposons. An analysis of Ylt1 expression was also performed. Production of the Ylt1 Gag protein under different conditions was analyzed by Western blotting. Expression of Ylt1 occurred on all tested carbon sources. The amount of Ylt1 decreased rapidly upon transition to stationary growth phase, in the presence of copper sulfate and under heat shock conditions. It is suggested that Ylt1 is expressed in actively growing cells, whereas stress conditions have a negative impact on its expression. Such expression pattern was not previously reported for other yeast retrotransposons. Activity of Ylt1 in vivo was characterized using an Ylt1 elements tagged with SUC2 gene of Saccharomyces cerevisiae. Mobilization of the marked Ylt1 element and its transposition from autonomous plasmid into host genome was observed in performed experiments. Obtained results strongly support the idea that Ylt1 is transpositionally active. Formation of tandem repeats by newly inserted Ylt1 elements was observed in several cases. It is suggested that integrase function was affected in this case, and that the integration was mediated by homologous recombination instead. Analysis of the Ylt1 insertion specificity and of the Ylt1 distribution in the genome of Y. lipolytica E150 was done. The remarkable sequence specificity of Ylt1 insertions, which is unusual for LTR retrotransposons, was revealed during this analysis. Also, it was shown that Ylt1 insertions are found mainly in intergenic regions, often at a significant distance (>500 bp) from the next reading frame. No association of Ylt1 insertions with tRNA genes was observed. Searches for Ylt1-related elements in the Y. lipolytica genome database were performed. The novel Ty3/gypsy element Tyl6 was found in the genome of Y. lipolytica E150. The sequence analysis of this element was carried out. It was shown that structural properties of Tyl6 resemble the properties of the Ty3 element of S. cerevisiae. However, two reading frames of Tyl6 (gag and pol) are separated by -1 frame-shift, which was not previously reported for retrotransposons of hemiascomycetous yeasts. Phylogenetic analysis placed Tyl6 within chromoviruses, and the Tse3 element of S. exiguus was shown to be the closest relative of Tyl6. The distribution of Tyl6 among Y. lipolytica strains was analyzed. Interestingly, the novel element was found only in strains derived from the strain YB423-12. The strains of independent origin included in the analysis were shown to be Tyl6-free. The same distribution was previously reported for the retrotransposon Ylt1 and for the DNA transposon Mutyl. Two models of the evolution of transposable elements in Y. lipolytica genome were proposed based on these results.

The following contains two projects focused on the fungal pathogen, Fusarium oxysporum. The first project was purely computational in the examination of transposable elements (TEs), which are mobile sequences with the ability to multiply and move in their host genome. In F. oxysporum, TEs such as miniature impala elements are associated with the secreted in xylem gene that are related to its virulence over its host. The F. oxysporum species complex can be utilized as a model system for the examination of TE content and TE expression during the infection cycle. To find whether TEs play a role in the infection process and if their expression changes when fungi are in planta, a comparison was made using RNA-seq data from a pathogenic (Fo5176) and a non-pathogenic strain (Fo47) of F. oxysporum interacting with the model plant Arabidopsis thaliana. Complementary to this, the copy numbers of the same TEs were calculated in the two aforementioned strains and in F. oxysporum f.sp. lycopersici 4287 (Fo4287) to find if there was a correlation between expression and copy number. Using these two different datasets together showed that TE expression and copy number are lower in the non-pathogenic strain and unlinked in the infection course. The second project examined the growth inhibition of Fusarium oxysporum isolates Fo32931 (the isolate pathogenic to immunocompromised humans) and Fo4287 with the use of extracts from chilies of Capsicum chinense. Pepper plants were grown from seed and the peppers were harvested for an ethanol (100%) extraction. After preparation, the optical density of growth of the F. oxysporum isolates was measured for a 48-hour period with 96-well plate containing varying concentrations of the extracts and controls. Growth curves were analyzed and normalized to a growth control. After doing High Performance Liquid Chromatography, an estimated concentration of capsaicin (the causal agent of the burning sensation from hot chilis) was established. A correlation between the amount of growth inhibition and the concentration of capsaicin was made. Taken together, the data suggests that an increase of capsaicin concentration in extracts is correlated with reduced growth for the two tested isolates of F. oxysporum.

Cílem práce je seznámení se s problematikou uchovávání informace v DNA, provést rešerši na téma transpozony, bioinformatické nástroje a algoritmy, které jsou používány k jejich detekci v nasekvenovaných genomech a vytvořit tak stručný úvod do obsáhle problematiky, včetně jejího zasazení do kontextu současně probíhajícího výzkumu v dané oblasti. Na základě přehledu stávajících algoritmů a nástrojů pro detekci transpozonů je navržen a implementován nástroj pro hledání tzv. LTR transpozonů.

L’alternance de périodes glaciaires et interglaciaires durant les 20 derniers Ma a mené à des changements environnementaux répétés au niveau du plateau continental antarctique. C’est dans ce contexte que les téléostéens de la famille des Nototheniidae se sont adaptés et diversifiés à travers plusieurs vagues de radiations (dont les Trematominae), dominant l’Ichtyofaune australe. Parmi les Nototheniidae, le groupe « Trematomus » (genres Cryothenia, Pagothenia, Trematomus et Indonotothenia) est celui où l’on observe la plus grande diversité chromosomique, avec des nombres diploïdes de chromosomes allant de 24 à 58, impliquant de nombreux réarrangements ayant accompagné les spéciations. Nous avons cherché à caractériser ces remaniements chromosomiques. Avec un caryotype ancestral inféré de 2n = 48, une conservation des unités chromosomiques entre espèces, et une constance des tailles de génome, l’hypothèse de réarrangements structuraux sans polyploïdisation préalable est la plus probable. Afin de reconstruire l’histoire évolutive de ces événements, nous avons recherché les homologies chromosomiques interspécifiques. Ceci nous a permis de reconstituer les remaniements (majoritairement des fusions) que nous avons repositionnés sur la phylogénie résolue des « Trematomus ». Contrairement à ce qui a été publié pour le genre Notothenia, nos résultats suggèrent des acquisitions multiples et indépendantes. Les éléments transposables (ETs) peuvent être impliqués dans les remaniements chromosomiques par le biais de recombinaisons ectopiques. Ils participent alors à la diversification des lignées au cours de l’évolution. En raison de leur régulation épigénétique, leur mobilisation massive peut être induite en cas de variations environnementales importantes. Nous nous sommes intéressés à trois super-familles d’ETs (DIRS, Gypsy and Copia) dans ces génomes. Les DIRS1 ont montré des patrons d’insertions en points chauds dans les régions centromériques et péricentromériques. Etant donné leur mode de transposition décrit et leur propension à s’insérer dans des copies préexistantes, nous proposons un rôle des éléments DIRS1 comme facilitateurs des fusions observées lors de la diversification des « Trematomus ». / In the last 20 My, multiple glacial-interglacial cycles led to strong and repeated environmental changes on the Antarctic continental shelf. In this changing environment, nototheniid fishes diversified through several rounds of species radiation (one of which within Trematominae), and now constitute the dominant group in Antarctic teleosts. Among Nototheniidae, the group « Trematomus » (genera Cryothenia, Pagothenia, Trematomus and Indonotothenia) exhibits the highest chromosomal diversity, with diploid chromosome numbers ranging between 24 and 58, involving many rearrangements probably linked to speciation. We characterized the nature of these chromosomal repatternings. With an inferred ancestral state of 2n = 48 acrocentric chromosomes, a conserved number of chromosomal structural units, and a constancy of the genomes sizes we measured; the hypothesis of structural modifications is favored rather than a whole genome duplication associated to drastic reductions. In order to reconstruct an evolutionary scenario of such chromosomal rearrangements accompanying the trematomine diversification, we identified interspecific chromosomal homologies. This allowed us to reconstruct the rearrangements events (mostly centric and tandem fusions). We plotted them on a phylogeny we reconstructed based on our own ddRAD-seq data. Contrary to what was reported for the Notothenia, our results are in favor of independent acquisitions. Transposable elements (TEs) can lead to chromosomal rearrangements through ectopic recombination events, hinting at a role as drivers of specific-lineage diversification. Moreover, due to their epigenetic regulation, TEs can be mobilized when thermic changes occur. We focused on three retrotransposon superfamilies (DIRS, Gypsy and Copia) in nototheniid genomes. The DIRS1 showed unexpected accumulation patterns of insertion in the centromeric and pericentromeric regions. Given the mechanism of DIRS1 transposition and their tendency to sometimes insert on pre-existing copies (homing), we suggest a role of DIRS1 elements as facilitators of the fusions that occurred during the trematomine radiation.

L'abondance d'éléments génétiques mobiles dans le génome humain a un impact critique sur son évolution et son fonctionnement. Même si la plupart des éléments transposables sont inactifs en raison de l'accumulation de mutations, le rétrotransposon LINE-1 (Long Interspersed Element-1 ; ou L1) continue de se mobiliser et d'influer sur notre génome. Il a ainsi contribué à l'évolution de l'homme moderne, mais aussi à l'apparition de maladies génétiques. Les séquences du rétrotransposon L1 correspondent à 17% de la masse totale de l’ADN humain. Une copie active de L1 est capable de se mobiliser de manière autonome par un mécanisme de type «copier-coller» qui met en jeu un intermédiaire ARN et une étape de transcription inverse. Cependant, peu de choses sont connues sur les voies cellulaires impliquées dans la mobilité de L1. Notre laboratoire a découvert, par des cribles double-hybride, une interaction entre la protéine ORF2p de L1 et le récepteur α associé aux œstrogènes (ERRα), un membre de la famille des récepteurs nucléaires. Ici, nous avons confirmé et étendu cette observation à plusieurs autres membres de la superfamille des récepteurs de stéroïdes en utilisant un test de double-hybride fluorescent (F2H) en culture cellulaire. Pour mieux comprendre le rôle potentiel d’ERRα dans le cycle de rétrotransposition de L1, nous avons effectué des expériences de suppression et de surexpression qui suggèrent qu’ERRα est un régulateur positif de la rétrotransposition. Collectivement, ces données relient les voies de signalisation des stéroïdes avec la régulation post-traductionnelle de la rétrotransposition de L1, ce qui suggère un modèle par lequel ERRα et probablement autres récepteurs nucléaires peuvent recruter le RNP L1 vers des emplacements chromosomiques spécifiques. / The abundance of genetic mobile elements in our DNA has a critical impact on the evolution and function of the human genome. Even if most transposable elements are inactive due to the accumulation of mutational events, the Long INterspersed Element-1 (LINE-1 or L1) retrotransposon continues to diversify and impact our genome, being involved in the evolution of modern humans and in the appearance of genetic diseases or in tumorigenesis. L1 forms 17% of human DNA. It is autonomously active being replicated through an RNA-mediated ‘copy-and-paste’ mechanism. The L1 element encodes two proteins, ORF1p and ORF2p, which associate with the L1 mRNA to form L1 ribonucleoprotein particles, the core of the retrotransposition machinery. However, little is known about the cellular pathways involved in L1 replication. Our laboratory has discovered by yeast 2-hybrid screens an interaction between L1 ORF2p and the estrogen-related receptor α (ERRα), a member of the nuclear receptor family. Here, we confirmed and extended this observation to several other members of the steroid receptor superfamily using a fluorescent two-hybrid assay (F2H) in human cultured cells. To get further insight into the potential role of ERR in L1 replication cycle, we performed ERR siRNA-mediated knock-down and overexpression experiments, which suggest that ERR is a positive regulator of retrotransposition. Moreover, the artificial tethering and concentration of ERR to a large and repetitive genomic array inhibits retrotransposition. Collectively, these data link steroid signaling pathways with the post-translational regulation of L1 retrotransposition, suggesting a model by which ERRα, and probably several other nuclear receptors, can recruit the L1 RNP to specific chromosomal locations, acting as tethering factors.

Upon fertilization, the early embryo sustains most of the cellular processes using the maternally deposited reserves in the egg itself until the zygotic gene expression takes charge. Among the plethora of essential components provided by the mother are small non-coding RNAs called PIWI-interacting RNAs (piRNAs), which provide immunity to the zygote against transposon challenge. In this thesis, I have presented three different functions of piRNAs in Drosophila melanogaster- in maintenance of genomic integrity, telomere protection and their role as an adaptive immune system against genomic parasites. In Chapter 2, I have described the phenotypic effects of the loss of piRNA function in early embryos. The mutations affecting the piRNA pathway are known to cause embryonic lethality. To describe this lethality in detail, I have shown that all the characterized piRNA mutants show compromised zygotic genomic integrity during early embryogenesis. In addition, two piRNA pathway components, Aubergine (Aub) and Armitage (Armi) are also required for telomere resolution during early embryogenesis. Aub and Armi recruit telomeric protection complex proteins, HOAP and HP1, to the telomeric ends and thus avoid activation of the Non-homologous end joining (NHEJ) DNA repair pathway at the telomeres. There are about 120 transposon families in Drosophila melanogaster and piRNA pathway mutations cause activation of many of the resident transposons in the genome. In Chapter 3, I have described the effects of infection by a single transposon, P-element, in naïve strains by introduction through the zygote. Activation of the P-element leads to desilencing of unrelated transposons, causing accumulation of germline DNA damage which is linked to severely reduced fertility in the hybrid females. However, there is partial restoration of fertility as the hybrid progeny age, which correlates with P-element piRNA production and thus P-element silencing. Additionally, a number of transposons mobilize into piRNA generating heterochromatic clusters in the genome, and these insertions are stably inherited in the progeny. Collectively our data shows that piRNA production can be triggered in the adults in an absence of maternal contribution and that piRNAs serve as an adaptive immune system which helps resolve an internal genetic conflict between the host and the parasite. In an effort to understand the phenotypic effects of piRNA dysfunction in Drosophila, we have uncovered new exciting roles for piRNAs in development and presented evidence how transposons can act as architects in restructuring the host genome.

Les deux sous-espèces annuelles de téosinte qui sont les plus proches parents sauvages du maïs sont d’excellents systèmes pour étudier l’adaptation locale car leur distribution couvre un large éventail de conditions environnementales. Zea mays ssp. parviglumis est distribuée dans un habitat chaud et mésique en dessous de 1800 m d’altitude, tandis que Zea mays ssp. mexicana prospère dans des conditions sèches et fraîches à des altitudes plus élevées. Nous avons combiné des approches d’écologie inverse et de génétique association afin d’identifier les déterminants de l'adaptation locale chez ces téosintes. A partir de données de séquençage haut débit (HTS) de six populations comprenant des populations de basses et hautes altitudes, une étude précédente a identifié un sous-ensemble de 171 polymorphismes nucléotidiques (SNP candidats) présentant des signaux de sélection. Nous avons utilisé ces SNP candidats pour tester l'association entre la variation génotypique et phénotypique de 18 caractères. Notre panel d’association était constitué de 1663 plantes provenant de graines de 11 populations échantillonnées le long de deux gradients d’altitude. Il a été évalué deux années consécutives dans deux jardins communs. Nous avons contrôlé sa structure neutre en utilisant 18 marqueurs microsatellites. La variation phénotypique a révélé l’existence d'un syndrome altitudinal composé de dix caractères. Nous avons ainsi observé une augmentation de la précocité de floraison, une diminution de la production de talles et de la densité en stomates des feuilles ainsi qu’une augmentation de la taille, de la longueur et du poids des grains avec l’élévation croissante du site de collecte des populations. Ce syndrome a évolué malgré des flux de gènes détectables entre populations. Nous avons montré que le pourcentage de SNP candidats associés aux différents caractères dépend de la prise en compte de la structure neutre soit en cinq groupes génétiques (71,7%), soit en onze populations (11,5%), indiquant une stratification complexe. Nous avons testé les corrélations entre les variables environnementales et les fréquences alléliques des SNP candidats sur 28 populations. Nous avons trouvé un enrichissement à la fois pour les SNP présentant des associations phénotypiques et les SNP présentant des corrélations environnementales dans trois larges inversions chromosomiques, confirmant leur rôle dans l'adaptation locale. Pour explorer la contribution de la variation structurale à l'évolution adaptative, nous nous sommes concentrés sur le contenu en éléments transposables (ET) des six populations séquencées (HTS). Ces éléments constituent environ 85% du génome du maïs et contribuent à sa variabilité fonctionnelle. Nous avons effectué la première description populationnelle des ET chez les téosintes pour deux catégories d'insertions, celles présentes et celles absentes du génome de référence du maïs. Nous avons ensuite recherché des polymorphismes liés aux ET présentant des fréquences alléliques contrastées entre populations de basse et de haute altitude. Nous avons identifié un sous-ensemble d'insertions candidates. Enfin, nous avons génotypé, dans un panel d'association, des insertions d’ET connues pour avoir contribué à l'évolution phénotypique du maïs. Contrairement à ce qui a été observé chez le maïs, certaines de ces insertions n'ont montré aucun effet phénotypique chez les téosintes, ce qui suggère que leur effet dépend du fond génétique. Notre étude apporte de nouvelles connaissances sur l’adaptation altitudinale chez les plantes. Elle ouvre la discussion sur les défis soulevés par l'utilisation (1) d'outils de génomique des populations pour identifier la variation adaptative, (2) de populations naturelles en génétique d’association, et (1) de ressources génétiques sauvages pour l'amélioration des espèces cultivées. / Annual teosintes, the closest wild relatives of maize, are ideal systems to study local adaptation because their distribution spans a wide range of environmental conditions. Zea mays ssp. parviglumis is distributed in warm and mesic conditions below 1800 m, while Zea mays ssp. mexicana thrives in dry and cool conditions at higher altitudes. We combined reverse ecology and association mapping to mine the determinants of local adaptation in annual teosintes. Based on high throughput sequencing (HTS) data from six populations encompassing lowland and highland populations growing along two elevation gradients, a previous study has identified candidate regions displaying signals of selection. Within those regions a subset of 171 candidate single nucleotide polymorphisms (SNPs) was selected to test their association to phenotypic variation at 18 traits. Our association panel encompassed 1663 plants from seeds collected from eleven populations sampled along the elevation gradients. We benefit from phenotypic characterization of all the plants in two common gardens located at mid-altitude for two years. In addition, we controlled for neutral structure of the association panel using 18 microsatellite markers. Phenotypic variation revealed the components of an altitudinal “syndrome” constituted of ten traits evolving under spatially-varying selection. Plants flowered earlier, produced less tillers, displayed lower stomata density and carried larger, longer and heavier grains with increasing elevation of population collection site. This syndrome evolved in spite of detectable gene flow among populations. The percentage of candidate SNPs associated with traits largely depended on whether we corrected for five genetic groups (71.7%) or eleven populations (11.5%), thereby indicating a complex stratification in our association panel. We analyzed correlations between environmental variables and allele frequencies of candidate SNPs on a larger set of 28 populations. We found enrichment for SNPs displaying phenotypic associations and environmental correlations in three Mb-scale chromosomal inversions, confirming the role of these inversions in local adaptation. To further explore the contribution of structural variation to adaptive evolution, we focused on transposable element (TE) content of the HTS populations. TEs constitute ~85% of the maize genome and contribute to its functional variability via gene inactivation and modulation of gene expression. We performed the first population-level description of TEs in teosintes for two categories of insertions, those present and those absent from the maize reference genome. We next searched for TE polymorphisms with contrasted allele frequencies between lowland and highland populations. We pinpointed a subset of adaptive candidate insertions. Finally, we genotyped in our association panel TE insertions known to have contributed to maize phenotypic evolution. In contrast to what was found in maize, some of these insertions displayed no measurable phenotypic effects in teosintes, suggesting that their effect depends on the genetic background. Altogether our study brings new insights into plant altitudinal adaptation. It opens discussions on the challenges raised by the use (1) of population genomic tools to discover adaptive variation, (2) of natural populations in association mapping, and (1) of wild genetic resources in crop breeding.