Miniature Inverted-repeat Transposable Elements (MITEs) are a type of transposable element (TE) that lacks coding capacity. It has been established that in rice that certain Stowaway MITEs are mobilized by transposases from the Tc1/Mariner superfamily of TEs. To retrieve all Tc1/Mariner TEs from the genome, bioinformatic approaches were performed. A total of 295 Tc1/Mariner TEs that encoded a full or partial transposase were recorded which 100 were newly described. Sequence alignment, and identification of the catalytic motif placed these transposases into eight groups. A functional link was established by comparing the terminal sequences of the Stowaway-like MITEs to the termini of the terminal sequences of Tc1/Mariner TEs. A yeast excision assay was used to experimentally test these functional links. Majority of the Stowaway-like MITE and transposase combinations tested did not indicate a functional link. However, a possible functional link was observed between the AATp3-13 transposase and AAStow-5 Stowaway-like MITEs.

Miniature Inverted-repeat Transposable Elements (MITEs) are a type of transposable element (TE) that lacks coding capacity. It has been established that in rice that certain Stowaway MITEs are mobilized by transposases from the Tc1/Mariner superfamily of TEs. To retrieve all Tc1/Mariner TEs from the genome, bioinformatic approaches were performed. A total of 295 Tc1/Mariner TEs that encoded a full or partial transposase were recorded which 100 were newly described. Sequence alignment, and identification of the catalytic motif placed these transposases into eight groups. A functional link was established by comparing the terminal sequences of the Stowaway-like MITEs to the termini of the terminal sequences of Tc1/Mariner TEs. A yeast excision assay was used to experimentally test these functional links. Majority of the Stowaway-like MITE and transposase combinations tested did not indicate a functional link. However, a possible functional link was observed between the AATp3-13 transposase and AAStow-5 Stowaway-like MITEs.

Nearly one half of the human genome is composed of transposable elements (TEs). Once dismissed as 'selfish' or 'junk' DNA, TEs have also been implicated in a numerous functions that serve the needs of their host genome. I have evaluated the role of TEs in mediating the epigenetic mechanisms that serve to regulate human gene expression. These findings can be broadly divided into two major mechanisms by which TEs affect human gene expression; by modulating nucleosome binding in the promoter regions and by recruiting epigenetic histone modifications that enable them to serve as promoters and enhancers. Thus. the studies encompassed in this thesis elucidate the contributions of TEs in epigenetically regulating human gene expression on a global as well as local scale.

Thesis (Ph. D.)--University of Washington, 2001. / Vita. Includes bibliographical references (leaves 112-133).

Transposable elements (TEs) have had immense impact on the structure, function and evolution of eukaryotic genomes. The work in this thesis identified Kat1, a novel domesticated DNA transposase of the hAT family in the yeast Kluyveromyces lactis. Kat1 triggers a genome rearrangement that results in a switch of mating type from MATa to MATα. Furthermore, Kat1 acts on sequences that presumably are ancient remnants of a long-lost transposable element. Therefore, Kat1 provides a remarkable example of the intricate relationship between transposable elements and their hosts. We showed that Kat1 generates two DNA double strand breaks (DSBs) in MATa and that the DDE motif and several other conserved amino acid residues are important for Kat1 cleavage activity. DNA hairpins were formed on one end of the DSBs whereas the DNA between the DSBs was joined into a circle. Kat1 was transcriptionally activated by nutrient limitation through the transcription factor Mts1 and negatively regulated by translational frameshifting. In conclusion, Kat1 is a highly regulated domesticated transposase that induces sexual differentiation.  In another study, we developed an assay to measure switching rates in K. lactis and found that the switching rate was ~6x10-4 events/generation. In a genetic screen for mutations that increased mating-type switching, we found mutations in the RAS1 gene. The small GTPase Ras1 regulates cellular cyclic AMP levels and we demonstrated that Mts1 transcription is regulated by the RAS/cAMP pathway and the transcription factor Msn2. Since Ras activity is regulated by nutrient availability, these data likely explains why nutrient limitation induces mating-type switching.

No description available.

Thesis (Ph. D.)--University of Washington, 2006. / Vita. Includes bibliographical references (leaves 69-76).

La spermatogenèse est le processus par lequel les cellules germinales se différencient pour former les spermatozoides. Elle se déroule à l’intérieur des tubes séminifères. Pendant la période embryonnaire, les précurseurs des cellules germinales adultes constituent un pool de cellules appelées cellules germinales primordiales (Primordial Germ Cells, PGCs), qui vont migrer pour aller coloniser les gonades (Durcova-Hills and Capel, 2008; Surani et al., 2008). Au cours de leur migration, les PGCs vont subir une reprogrammation épigénétique de l’ensemble de leur génome, qui leur sera nécessaire pour passer de l’état somatique à l’état de totipotence (Ohinata et al., 2005). Durant cette reprogrammation, l’ADN est massivement démethylé, entrainant l’activation de plusieurs gènes qui sont importants pour le développement des PGCs, mais également l’activation des éléments transposables (ETs) (Hajkova et al., 2008; Sasaki and Matsui, 2008; Surani and Hajkova, 2010). Ces derniers constituent environ 50% du génome des mammifères. Ils sont subdivisés en deux classes et sont connus par leur capacité à être mobilisés dans le génome (Zamudio and Bourc'his, 2010). Cette mobilisation se fait au hasard et constitue ainsi un risque considérable de mutations, qui peuvent provoquer des tumeurs, des pathologies de développement et une infertilité (Zamudio and Bourc'his, 2010). Pour cela, leur expression doit être contrôlée pour maintenir l’intégrité du génome de la lignée germinale. Pour toutes ces raisons, les PGCs ainsi que les cellules germinales en cours de méiose ont développé des stratégies de défenses pour contrôler la mobilisation et l’expression des ETs basées entre autre sur la voie des piwi-interacting RNA (piRNA) (Chuma and Pillai, 2009; Pillai and Chuma, 2012b). Dans le laboratoire du Pr. Stéphane Viville, mes travaux de thèse se sont concentrés sur l’étude d’un gène nommé Tex19 pour Testis Expressed gene chez la souris. Nous avons démontré que ce gène est spécifique des mammifères et est dupliqué chez le rat et la souris en deux paralogues nommés Tex19.1 et Tex19.2. Deux domaines hautement conservés ont été identifiés par alignement multiple des protéines TEX19 et nommés MCP et VPTEL. Ces domaines ne présentent aucune homologie avec des domaines déjà caractérisés, prévenant ainsi toute prédiction de leurs fonctions (Kuntz et al., 2008). L’étude du profil d’expression de Tex19.1 et Tex19.2 a montré que ces deux gènes sont exprimés dans l’ectoderme et les PGCs. Ils sont aussi co-exprimés dans le testicule de l’âge embryonnaire à l’âge adulte. Néanmoins, seul Tex19.1 est exprimé dans les ovaires et le précurseur du placenta appelé cône ectoplacentaire (Celebi et al., 2012). Le knockout (KO) de Tex19.1 provoque une infertilité masculine chez la souris avec un arrêt de la spermatogenèse au stade pachytène, accompagnée d’une surexpression d’un rétrotransposon, MMERVK10C (Ollinger et al., 2008). Récemment, il a été démontré que Tex19.1 joue aussi un rôle dans le développement du placenta (Reichmann et al., 2013). Au cours de mes trois années de thèse, nous avons approfondie l’étude du KO de Tex19.1dans le testicule, les cellules embryonnaires souches (Embryonic Stem Cells, ESCs) et le placenta (Tarabay et al., 2013). Nous avons également étudié le phénotype observé suite au double KO de Tex19.1 et Tex19.2. [...] / We recently characterized two new mammalian specific genes, Tex19.1 and its paralog Tex19.2. Both genes are expressed in pachytene spermatocytes in adult testes. In addition, Tex19.1 is expressed in pluripotent cells (ES, EG, iPS and PGC cells), the inner cell mass of the blastocysts and the placenta. In order to decipher Tex19 functions, we generate three types of knockout (KO): i) KO of Tex19.1 ii) KO of Tex19.2 iii) double KO (DKO) of both genes. All Tex19.1-/- KO animals are growth-retarded and half of them die just after birth. This phenotype is probably linked to placenta defects. Surviving adults Tex19.1-/- KO males display a variable spermatogenesis phenotype, associated with an up-regulation of one endogenous retrovirus, MMERVK10C. Tex19.2 KO mice exhibit a subtle phenotype. Few seminiferous epitheliums are degenerated while the rest appear normal. DKO show a fully penetrant phenotype similar to the most severe Tex19.1-/- phenotype. DKO males exhibit small testes. Despite the presence of spermatogonia and spermatocytes, spermatogenesis is blocked at the pachytene stage. By RNA deep-sequencing on 10 days old DKO and WT testes, prior to histological phenotype, 114 genes are significantly up-regulated and 320 genes significantly down-regulated in the DKO compared to the WT. Gene ontology analyses show that among of these genes, two essential pathways are altered: meiosis and the piRNA pathway. Consistent with that, GST-pulldown and immunoprecipitation experiments demonstrate that MIWI, MILI, MAEL and MVH are partners of TEX19. Considering PIWI proteins function in the silencing of transposable elements through the piRNA pathway, we checked if TEX19 paralogs bind piRNA. By immunoprecipitation using WT and KO testes, we show that both TEX19.1 and TEX19.2 bind small RNA of 30 nucleotides through their VPTEL domain. This study highlights the pivot role of Tex19 paralogs in three essential functions of mammalian life cycle, i.e. pluripotency, placenta-supported in utero growth and fertility. The functional similarities of both paralogs, through the expression control of one endogenous retrovirus and the binding of piRNAs, lead us to propose that Tex19 paralogs are new members of the piRNA pathway.

Elementos transponíveis (TEs) são sequências de DNA que possuem a capacidade de transposição no genoma hospedeiro. O principal objetivo deste trabalho reside na investigação em torno de possíveis contribuições de TEs nos genomas das algas C. reinhardtii e V. carteri, mais especificamente, na arquitetura dos genes ortólogos nestas espécies. Neste contexto, análises em sílico em larga escala foram realizadas, buscando-se identificar associações entre TEs e os genes ortólogos. Os resultados indicaram que os genes em C. reinhardtii tendem a acumular mais cópias de TEs em relação aos seus ortólogos em V. carteri. C. reinhardtii apresentou maior densidade de cópias de TEs para as regiões flanqueadora 5´ , flanqueadora 3´ e intrônica quando comparada a V. carteri; o inverso foi verificado quando analisada a densidade de TEs nas regiões codificantes. Análises para apurar a distribuição dos elementos em regiões intergênicas e intragênicas foram estabelecidas, nas quais a frequência observada dos elementos foi comparada à frequência esperada segundo a distribuição randômica de TEs no genoma, simulada computacionalmente. Foram constatadas regiões em que a presença dos elementos encontra-se significativamente abaixo do esperado, a exemplo de intervalos adjacentes ao início e ao término dos genes, o que provavelmente reflete a seleção negativa de eventos de integração nestas delimitações, em virtude dos efeitos deletérios associados à disrupção de estruturas de regulação da expressão gênica. De forma geral, nas regiões flanqueadoras 5´ e 3´, foi identificada a tendência de elevação da frequência padronizada de TEs à medida que a classe de distância avaliada se distancia do início e do término do gene, respectivamente. A baixa representatividade dos elementos também foi constatada em regiões intragênicas. O estudo da distribuição de TEs nos íntrons dos genes ortólogos indicou a preservação destas regiões quanto à fixação de TEs, sendo a representatividade abaixo do esperado mais evidente em intervalos adjacentes ao éxon, o que minimiza a chance de ruptura no padrão de splicing dos genes. Em sequências codificantes, a escassez de TEs - esperada devido ao provável efeito deletério destes eventos para a função do gene - foi constatada nos ortólogos das duas espécies. No entanto, inovações decorrentes da integração dos elementos em regiões codificantes podem resultar em efeitos evolutivos positivos, embora estes eventos sejam raros. Nas espécies analisadas foram identificados dois casos, de especial interesse, em que um domínio da sequência peptídica encontra-se localizado em região derivada de TE: o primeiro refere-se ao gene Cre06.g262800, em C. reinhardtii, no qual foi identificado o domínio PHD-finger associadao ao elemento Gypsy-5-LTR_CR; o segundo remete ao gene Vocar20001092m.g, em V. carteri, no qual o domínio zinc knuckle foi reconhecido em região derivada do elemento Gypsy3-LTR_VC. Estes genes constituem exemplos da contribuição de TEs na evolução de sequências codificadoras nas espécies C. reinhardtii e V. carteri, corroborando a hipótese de que os TEs podem contribuir na evolução da arquitetura dos genes, apesar do efeito disruptivo inerente à integração dos mesmos em regiões gênicas. / Transposable elements (TEs) are DNA sequences able to transpose in the host genome. The aim of this study resides in the investigation of TEs contributions in the algae C. reinhardtii and V. carteri genomes, more specifically in the architecture of orthologous genes in these species. In this context, large scale in silico analysis were performed to identify associations between TEs and orthologous genes. The results indicated that genes in the C. reinhardtii specie tend to accumulate more TEs copies than orthologous genes in V. carteri. C. reinhardtii showed higher density of TEs copies in the 5´ flanking, 3´ flanking and intronic regions when compared to V. carteri; the opposite was observed in coding regions. Investigation of the elements distribution in the intergenic and intragenic regions was performed, in which the observed TE frequency was compared to expected TE frequency from the simulated random distribution of the elements in the genome. It was verified regions where TE frequency was significantly lower than expected, as in gene boundaries adjacencies, probably reflecting a negative selection of the TE integration events in these delimitations due to deleterious effects associated with disruption of gene regulatory structures. In general terms, it was observed an increasing standardized frequency in the 5´ and 3´ flanking regions as the distance from gene start and gene end, respectively, increases. TEs underrepresentation was also verified in the intragenic regions. The study of TEs distribution in the introns of orthologous genes revealed the preservation of these structures in relation to TEs fixation, with a stronger underrepresentation near exon, which minimizes the chance of gene splicing pattern disruption. In the coding sequences, the TEs scarcity - expected due the likely deleterious effects to gene function - was verified in the orthologous of both species. However, in rare instances, innovations mediated by TEs integration in the coding regions can lead to positive evolutionary effects. In the species analyzed two instances of particular interest were observed, in which the domain of peptide sequence is located in the region derived from TE. The first one refers to the Cre06.g262800 gene, in the C. reinhardtii specie, which has a PHD-finger domain associated with Gypsy-5-LTR_CR element. The second one refers to the Vocar20001092m.g gene, in V. carteri, in which the zinc knuckle was recognized in region derived from Gypsy3-LTR_VC element. These genes are examples of TEs contributions in the evolution of coding sequences in the C. reinhardtii and V. carteri species, corroborating the hypothesis that TEs can contribute to the evolution of gene architecture, despite the inherent disruptive effect in their integration in the gene regions.

SETMAR est un gène chimérique constitué d’un domaine SET (codant des fonctions d’histone méthylase) et du domaine MAR (ayant conservé certaines fonctions de la transposase HsMAR1). Des études ont montré que les deux domaines sont biologiquement actifs et sont impliqués dans la stabilité et/ou dans la régulation de l’expression du génome humain. La littérature suggère que l’expression de SETMAR est plus forte dans les cellules cancéreuses que dans les cellules saines. Notre hypothèse de travail est que la protéine SETMAR est surexprimée en conditions pathologiques, permettant aux cellules de franchir les points de contrôle du cycle cellulaire, contribuant ainsi à augmenter l’instabilité génétique. Notre objectif est d’étudier la régulation de l’expression de SETMAR et son implication dans l’oncogenèse, gliale en particulier. / SETMAR is a chimeric gene consisting of a SET domain (encoding methylase histone functions) and a MAR domain (having retained some of the of the HsMAR1 transposase functions). Studies have shown that the two domains are biologically active and are involved in the stability and / or in the regulation of the human genome expression. The literature suggests that SETMAR expression is higher in cancer cells than in normal cells. Our working hypothesis is that SETMAR protein is overexpressed in pathological conditions, allowing cells to overcome the cellular cycle checkpoints, helping to increase the genetic instability. Our goal is to study the regulation of the SETMAR expression and its involvement in oncogenesis, glial in particular.