Diss. (sammanfattning) Stockholm : Univ., 2007. / Härtill 4 uppsatser.

Transposable elements (TEs) are mutagenic mobile DNA sequences whose excision and insertion are powerful drivers of evolution. Some TE families are known to target specific genome features, and studying their insertion preferences can provide information about both TE biology and the state of the genome at these locations. To investigate this, collecting large numbers of insertion sites for TEs in natural populations is required. Genome resequencing data can potentially provide a rich source of such insertion sites. The field of detecting these "non-reference" TE insertions is an active area of research, with many methods being released and no comprehensive review performed. To drive forward knowledge of TE biology and the field of non-reference TE detection, we created McClintock, an integrated pipeline of six TE detection methods. McClintock lowers the barriers against use of these methods by automating the creation of the diverse range of input files required whilst also setting up all methods to run simultaneously and standardising the output. To test McClintock and its component methods, it was run on both simulated and real Saccharomyces cerevisiae data. Tests on simulated data reveal the general properties of component methods' predictions as well as the limitations of simulated data for testing software systems. Overlap between results from the McClintock component methods show many insertions detected by only one method, highlighting the need to run multiple TE detection methods to fully understand a resequenced sample. Utilising the well characterised properties of S. cerevisiae TE insertion preferences, real yeast population resequencing data can act as a biological validation for the predictions of McClintock. All component methods recreated previously known biological properties of S. cerevisiae TE insertions in natural population data. To demonstrate the versatility of McClintock, we applied the system to Drosophila melanogaster resequencing data. 27 Schneider's cell lines were sequenced and analysed with McClintock. In addition to demonstrating the scalability of McClintock to larger genomes with more TE families, this exposed ongoing transposition in S2 cell lines. Likewise, the use of non-reference TE insertions as variable sites allowed us to recreate the relationships between S2 sub-lines, confirming that S1, S2, and S3 were most likely established separately. The results also suggest that there are several S2 sub-lines in use and that these sub-lines can differ from each other in TE content by hundreds of non-reference TE copies. Overall this thesis demonstrates that the McClintock pipeline can highlight problems in TE detection from genome data as well as revealing that much can still be learned from this data source.

Since their first discovery in the 19th century, microsporidian species have been found to be successful obligate intracellular parasites capable of infecting a wide variety of hosts including economically and ecologically important organisms as well as model organisms for biomedical research. Recently, cases of infection of the widely used animal model Danio rerio, commonly known as the zebrafish, by the newly described microsporidium Pseudoloma neurophilia have been reported in an increasing number of research facilities. Current knowledge of the biology of this parasite found in 75% of the Zebrafish Resource Center facilities is limited to microscopic analyses on its lifecycle as well as its physical, behavioral and psychic impact on its hosts. Despite the growing relevance of this parasite in biomedical research no current data is available on its genome. In this dissertation, I provide additional knowledge on the basic biology of P. neurophilia by acquiring and exploring the content and structure of the first genome draft of the zebrafish parasite. My findings reveal that the 5.25 Mb genome of P. neurophilia harbors an unusually high amount of transposable elements as well as numerous inserts found in coding regions typically conserved in microsporidia and other organisms. This peculiar obligate parasite demonstrates strong phylogenetic and genetic relationships with other fish-mosquito microsporidia. Similar to what is observed in closely related species, intra-genomic analyses of P. neurophilia’s genome suggest that it is diploid and possesses a large repertoire of over a thousand putative genes unique to this specie. Overall, my findings provide new insights into the basic biology of this parasite and represent a milestone in the understanding of P. neurophilia and D. rerio host-parasite interaction and ultimately in the development of treatments against this parasite that has been infecting the zebrafish research industry for the past decades.

Transposable elements (TEs) are mobile DNA sequences that are a source of mutations and can target specific sites in host genome. Understanding the molecular mechanisms of TE target site preferences is a fundamental challenge in functional and evolutionary genomics. Here we used accurately mapped TE insertions in the Drosophila melanogaster genome, from large-scale gene disruption and resequencing projects, to better understand TE insertion site mechanisms. First we test predictions of the palindromic target site model for DNA transposon insertion using artificially generated P-element insertions. We provide evidence that the P-element targets a 14 bp palindromic motif that can be identified at the primary sequence level that differs significantly from random base composition in the D. melanogaster genome. This sequence also predicts local spacing, hotspots and strand orientation of P-element insertions. Next, we combine artificial P-element insertions with data from genome- wide studies on sequence properties of promoter regions, in an attempt to decode the genomic factors associated with P-element promoter targeting. Our results indicate that the P-element insertions are affected by nucleosome positioning and the presence of chromatin marks made by the Polycomb and trithorax protein groups. We provide the first genome-wide study which shows that core promoter architecture and chromatin structure impact P-element target preferences shedding light on the nuclear processes that influence its pattern of TE insertions across the D. melanogaster genome. In an effort to understand the natural insertion preferences of a wide range of TEs, we then used genome resequencing data to identify insertions sites not present in the reference strain. We found that both Illumina and 454 sequencing platforms showed consistent results in terms of target site duplication (TSD) and target site motif (TSM) discovery. We found that TSMs typically extend the TSD and are palindromic for both DNA and LTR elements with a variable center that depends on the length of the TSD. Additionally, we found that TEs from the same subclass present similar TSDs and TSMs. Finally, by correlating results on P-element insertion sites from natural strains with gene disruption experiments, we show that there is an overlap in target site preferences between artificial and natural insertion events and that P-element targeting of promoter regions of genes is a natural characteristic of this element that is influenced by the same features has the artificially generated insertions. Together, the results presented in this thesis provide important new findings about the target preferences of TEs in one of the best-studied and most important model organisms, and provide a platform for understanding target site preferences of TEs in other species using genomic data.

Os elementos transponíveis (TEs) constituem grande parte do material genético de diversos eucariotos, alcançando entre 50-80% do genoma de gramíneas. Os projetos genoma proporcionaram um aumento das informações disponíveis sobre estes elementos, o que evidenciou sua importância e possibilitou o desenvolvimento de novas abordagens para seu estudo. O sistema Mutator (Mu) de milho é o mais ativo e mutagênico transposon de plantas. Além do elemento autônomo, MuDR, o sistema compreende ainda um conjunto de elementos bastante heterogêneo em sua seqüência e estrutura, chamados MuLEs, que podem conter até mesmo fragmentos de genes do hospedeiro. As seqüências de transposons mais abundantemente expressas no transcriptoma de cana-de-açúcar são relacionadas a MuDR e se agrupam em quatro clados (nomeados Classes I, II, II e IV), existentes antes da divergência entre Mono e Eudicotiledôneas. O trabalho apresentado aqui teve o objetivo de aprofundar o conhecimento sobre o sistema Mutator em cana-de-açúcar a partir da análise comparativa entre seqüências dessa planta e de arroz (cujo genoma está totalmente seqüenciado). Foi possível avaliar a abundância e diversidade do sistema Mu em gramíneas, ficando evidente uma amplificação de elementos clado-específica, tendo a Classe II sofrido uma explosão no número de cópias ao longo da evolução destas plantas. Análises estruturais revelaram que, enquanto as Classes I e II compreendem elementos com características de transposons, as Classes III e IV são, na verdade, transposases domesticadas. Foram completamente seqüenciados dois clones de BAC de cana-de-açúcar, um proveniente de cada parental do híbrido (Saccharum officinarum e Saccharum spontaneum), ambos contendo elementos da Classe III. Estes elementos foram caracterizados e a seqüência genômica de cana foi comparada com sua ortóloga em arroz, revelando um acúmulo de TEs nas regiões intergênicas. / Transposable elements (TEs) constitute great part of eukaryote genetic material, in grasses, they comprise between 50-80% of the genome. Genome projects have significantly increased the amount of information about these elements, revealing their importance and allowing the development of new approaches for their study. The Mutator system (Mu) of maize is the most active and mutagenic plant transposon. Beyond the autonomous element, MuDR, the system comprises a very heterogeneous, in sequence and structure, set of elements, called MuLEs, that can contain even host gene fragments. The most abundant transposon related sequences expressed in sugarcane transcriptoma are the MuDR-like. They group into four clades (called Classes I, II, III and IV) that exist prior to the Mono and Eudicot split. The aim of this work is to gain knowledge about the Mutator system in sugarcane through the comparative analysis against rice (whose genome is completely sequenced). The results described the abundance and diversity of the Mu system in grasses, evidencing a clado-specific amplification with a burst of Class II along the evolution of this plant group. Structural analyses showed that, while Classes I and II comprise elements with transposon characteristics, Classes III and IV are domesticated transposases. One BAC clone from each sugarcane parental genotype (Saccharum officinarum and Saccharum spontaneum) have been completely sequenced, both containing Class III elements. These elements have been characterized and the sugarcane genomic sequences were compared with their orthologues in rice. The comparative analyses showed an accumulation of TEs in the intergenic regions.

The components contributing to cancer progression, especially the transition from early to invasive are unknown. Consequently, the biological reasons are unclear as to why some patients diagnosed with atypia and ductal carcinoma in situ (DCIS) never progress into invasive breast cancer. The “one gene at a time” approach does not sufficiently predict progression. To elucidate the early stage progression to invasive ductal cancer, expression signature of transcripts and transposable elements in micropunched samples of formalin-fixed, paraffin embedded (FFPE) tissue was conducted. A bioinformatics pipeline to analyze poor quality, short reads (>36 nts) from RNA-Seq data was created to compare the most common tools for alignment and differential expression. Most samples from patients prepared for RNA-seq analysis are acquired through archived FFPE tissue collections, which have low RNA quality. The pipeline analytics revealed that STAR alignment software outperformed others. Furthermore, our comparison revealed both DESeq2 and edgeR, with the estimateDisp function applied, both perform well when analyzing greater than 12 replicates. Transcriptome analysis revealed progressive diversification into known oncogenic pathways, a few novel biochemical pathways, in addition to antiviral and interferon activation. Furthermore, the transposable element (TE) signature during breast cancer progression at early stages indicated long terminal repeat (LTRs) as the most abundantly differentially expressed TEs. LTRs belong to endogenous retroviruses (ERV), a subclass of TEs. The retroviral and innate immune response activity in DCIS, which indirectly corroborates the increase in ERV expression in this pre-malignant stage. Finally, to demonstrate the potential role of TEs in the transition from pre-malignant to malignant breast cancer we used pharmacological approaches to alter global TE expression and inhibit retrotransposition activity in control and breast cancer cell lines. It was expected that dysregulation of TEs be associated with increased invasiveness and growth. However, our results indicated that DNA methyltransferase inhibitor 5-Azacytidine (AZA) consistently retarded cell migration and growth. While unexpected, these findings corroborate recent studies that AZA may induce an interferon response in cancer via increased ERV expression. This body of work illustrates the importance of understanding bioinformatics methods used in RNA-seq analysis of common clinical samples. These studies suggest the potential for TEs as biomarkers for disease progression and novel therapeutic approach to investigate in additional model systems.

Every day more than half of the world consumes rice as a primary dietary resource. Thus, rice is one of the most important food crops in the world. Rice and its wild relatives are part of the genus Oryza. Studying the genome structure, function, and evolution of Oryza species in a comparative genomics framework is a useful approach to provide a wealth of knowledge that can significantly improve valuable agronomic traits. The Oryza genus includes 27 species, with 11 different genome types as identified by genetic and cytogenetic analyses. Six genome types, including that of domesticated rice - O. sativa and O. glaberrima, are diploid, and the remaining 5 are tetraploids. Three of the tetraploid species contain the CCDD genome types (O. grandiglumis, O. latifolia, and O. alta), which arose less than 2 million years ago. Polyploidization is one of the major contributors to evolutionary divergence and can thereby lead to adaptation to new environmental niches. An important first step in the characterization of the polyploid Oryza species is the generation of a high-quality reference genome sequence. Unfortunately, up until recently, the generation of such an important and fundamental resource from polyploid species has been challenging, primarily due to their genome complexity and repetitive sequence content. In this project, I assembled two high-quality genomes assemblies for O. grandiglumis and O. latifolia using PacBio long-read sequencing technology and an assembly pipeline that employed 3 genome assemblers (i.e., Canu/2.0, Mecat2, and Flye/2.5) and multiple rounds of sequence polishing with both Arrow and Pilon/1.23. After the primary assembly, sequence contigs were arranged into pseudomolecules, and homeologous chromosomes were assigned to their respective genome types (i.e., CC or DD). Finally, the assemblies were extensively edited manually to close as many gaps as possible. Both assemblies were then analyzed for transposable element and structural variant content between species and homoeologous chromosomes. This enabled us to study the evolutionary divergence of those two genomes, and to explore the possibility of neo-domesticating either species in future research for my PhD dissertation.

La maturation des ARN implique un grand nombre d’évènements post-transcriptionnels, parmi lesquels la polyadénylation qui constitue une étape clé. Chez Arabidopsis, la présence de l’hétérochromatine au niveau des introns de certains gènes peut influencer considérablement la polyadénylation de leur transcrits. INCREASED IN BONSAI METHYLATION2 (IBM2) est une protéinequi contrôle cette catégorie de gènes en reconnaissant l’hétérochromatine au niveau des introns via son domaine BOMO-ADJACENT HOMOLOGY (BAH). IBM2 se lie à l’ARNm par son motif RNA RECOGNOTION (RRM), afin d’assurer la transcription complète de ces gènes cibles en favorisant l’utilisation d’un site distal de polyadénylation. Par conséquent, en mutant IBM2, des plus transcrits courts sont synthétisés suite à une polyadénylation précoce au niveau de la régionhétérochromatique. Durant ma thèse, j’ai cherché à comprendre les mécanismes moléculaires sous-jacents de cette régulation tout en étudiant le rôle du complexe protéique IBM2. Nous avons identifié des protéines partenaires d’IBM2 déjà étudiées telle que ENHANCED DOWNY MILDEW2 (EDM2) et ASI-IMMUNOPRECIPITATED PROTEIN1 (AIPP1), ainsi qu’une nouvelle protéine interagissant physiquement avec IBM2 et d’autres protéines. La mutation du gène correspondant à cette protéine conduit à une réduction de l’expression globale des cibles d’IBM2testées, accompagnée d’un niveau réduit de transcrits longs fonctionnels. Moyennant un crible génétique des suppresseurs de la mutation ibm2, nous avons identifié plusieurs facteurs agissant en amont de la voie IBM2, notamment la protéine FLOWERING TIME CONTROL (FPA). FPA est une protéine capable de s’associer à l’ARN pour favoriser l’utilisation de sites proximaux de polyadénylation de plusieurs gènes cibles, avec parmi eux des gènes contrôlés par IBM2, ce qui suggère que la transcription complète de ces gènes dépend étroitement des actions antagonistes entre IBM2 et FPA. Nos résultats ont montré que le choix du site de polyadénylation de gènes contenant de l’hétérochromatine dépend de plusieurs protéines agissant en différents complexes ainsi que l’interconnexion avec d’autres voies. / RNA maturation implies numerous post-transcriptional modifications in whichpolyadenylation is a key step. In Arabidopsis, the heterochromatin found within introns(intronic-HC) can impact transcripts polyadenylation of host genes. INCREASED IN BONSAI METHYLATION2 (IBM2), an RNA-binding protein containing a bromo-adjacent homology (BAH) domain, interacts with intronic-HC to produce functional full-length transcripts by promoting distal polyadenylation. Loss of IBM2 function triggers short transcripts production due to premature polyadenylation from the heterochromatic region. During my thesis, I investigated the role of proteins that may belong to different sub-complexes in the regulation of intronic-HC containing genes. We identified IBM2 partners, including ENHANCED DOWNY MILDEW 2 (EDM2) and ASI-IMMUNOPRECIPITATED PROTEIN1 (AIPP1), and a novel partner that interacts directly with IBM2 and other proteins. Mutating the corresponding gene of the novel partner results in decreased expression of tested IBM2-targets such as IBM1 encoding an H3K9demethylase and the disease resistance gene RECOGNITION OF PERONOSPORA PARASITICA 7 (RPP7), accompanied with compromised use of their distal polyadenylation sites. By conducting a genetic screen of ibm2 mutation suppressors, we identified factors belonging to different pathways that act upstream of IBM2, among them the FLOWERING TIME CONTROL PROTEIN (FPA). FPA is an RNA-binding protein that promotes the use of proximal polyadenylation sitesof several genes such as IBM1. Our data bring evidence that antagonistic actions of FPA and IBM2 regulates polyadenylation sites choice at intronic-HC containing genes. These results provide new insights to understand the interplay between heterochromatin and RNA processing.

No description available.

This research focuses on non-Long Terminal Repeat (non-LTR) retrotransposons in the African malaria mosquito, Anopheles gambiae and other mosquito species. An unprecedented diversity of non-LTRs was discovered by genome analysis of the An. gambiae genome assembly. One hundred and four families were found by a reiterative and comprehensive search using the conserved reverse transcriptase domains of known non-LTRs from a number of organisms as the starting queries. These families range in copy number from a few to approximately 2000 and occupy at least 3% of the genome. An. gambiae non-LTRs represent 8 of the 15 previously defined clades, plus two novel clades, Loner and Outcast, raising the total number of known clades to 17. The first invertebrate L1 clade representatives were also found. All clades except one have families with sequence characteristics suggesting recent activity. Juan, a non-LTR of the Jockey clade originally discovered in the mosquito Culex pipiens quinquefasciatus (Mouches et al. 1991), has been implicated in horizontal transfer in three non-sibling species of the Aedes genus (Mouches, Bensaadi, and Salvado 1992). PCR was used to obtain sequences from 18 mosquito species of six genera. Phylogenetic analysis demonstrates predominant vertical inheritance of Juan elements among these species. There is strong evidence from sequence analysis supporting the recent activity of Juan in several divergent species. We hypothesize that the sustained activity (versus quick inactivation) of non-LTRs in mosquitoes may contribute to the diversity we observe in the An. gambiae genome today. Promoter and transcriptional analyses were performed for several families previously identified as potentially active elements based on sequence analysis. RT-PCR results indicate that transcripts are present in An. gambiae cell lines that contain sequences corresponding to 13 of 15 tested non-LTR families. The 5' UTRs of An. gambiae non-LTRs from the I, Jockey, and L1 clades support basal transcription in divergent mosquito cell lines from 3 species. The Jen-1 5'UTR did not support transcription in Ae. aegypti and had low activity in Ae. albopictus. In summary, this research shows that Non-LTRs have been highly successful genomic elements that have flourished in many divergent mosquito species. / Ph. D.