Tesis doctorales DPA - NES Doktoretza tesiak

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Browsing Tesis doctorales DPA - NES Doktoretza tesiak by Author "Castanera Andrés, Raúl"

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    PublicationOpen Access
    Characterization of transposon activity and genome-wide epigenetic regulation throughout the life cycle of Pleurotus ostreatus
    (2017) Borgognone, Alessandra; Ramírez Nasto, Lucía; Castanera Andrés, Raúl; Producción Agraria; Nekazaritza Ekoizpena; Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa
    Most prokaryotic and eukaryotic life forms have to deal with the presence of repetitive DNA sequences called transposable elements (TEs), whose ability to mobilize through the genome and insert at random position has an impact on genome stability and functionality. For a long time, TEs were described as ‘selfish’ DNA fragments, owing to their proliferation within the host genome without conferring any benefit or inducing detrimental effects when inserted in gene coding regions. Over time, however, this view was changed by the discovery of the contribution of transposons in genome integrity and evolution. Recent genome-wide characterizations have focused on the distribution of these mobile elements and the effects of active transposon copies within closely related genomes. Given their mutagenic potential, host genomes have evolved endogenous mechanisms to limit the mobilization and repress the transcriptional activity of transposons. In higher organisms, repeat sequences are transcriptionally silenced through epigenetic modifications, which modulate gene expression without producing permanent modifications along the nucleotide sequence. The integrated and dynamic nature of epigenetic pathways, including DNA methylation and RNA-silencing systems, can be regulated at different levels, leading to the targeted silencing of specific genomic regions. Thus, the revolutionary advent of high-throughput sequencing led to an unprecedented opportunity to generate genome-wide epigenetic profiles and extend the understanding of the contribution of TEs in genome evolution. The filamentous fungi, in particular, Neurospora crassa and other ascomycetes, have provided fundamental advances in many of the aforementioned areas. These organisms possess complex epigenetic pathways that are also conserved in other higher eukaryotes to efficiently shut down transposon activity. Despite their importance, the occurrence of epigenetic events as well as the impact of transposon activity in basidiomycetes have been poorly analyzed so far. Recent studies in Pleurotus ostreatus uncovered that the genome of this basidiomycete model is populated by a diverse set of TE families, providing a detailed picture of the distribution and importance of helitrons, which are a group of DNA transposons that mobilize through a rolling-circle mechanism. Therefore, the principal aim of this work is to investigate the inheritance of helitron transposons and profile the epigenetic and transcriptomic landscape of P. ostreatus at different growing stages. Both objectives have been performed through the use of molecular techniques integrated with subsequent Sanger or Next-Generation sequencing data analysis. This PhD dissertation is comprised of four main chapters. Chapter I describes the current state-of-the-art of genome sequencing, transposon identification and epigenetic mechanisms (DNA methylation and RNA silencing pathways); it also provides an introductory overview on the fungal kingdom and the model system P. ostreatus. Chapter II presents the inheritance patterns of HELPO1 and HELPO2 helitron families in the meiotically-derived progeny of P. ostreatus. Our results report the distorted segregation patterns of HELPO2 helitrons that lead to a strong under-representation of these elements in the progeny. Further analyses of the HELPO2 flanking sites showed that the meiotic process of gene conversion may contribute to the elimination of such repetitive elements, favoring the presence of HELPO2 vacant loci. Moreover, the analysis of HELPO2 content in a reconstructed pedigree of subclones maintained under different culture conditions revealed an event of helitron somatic transposition. Additional investigations of genome and transcriptome data indicated that P. ostreatus carries active RNAi machinery that could be involved in the control of transposable element proliferation. These findings provide the first evidence of helitron mobilization in the fungal kingdom and highlight the interaction between genome defense mechanisms and invasive DNA using P. ostreatus as a model. Chapter III describes the occurrence of epigenetic defense strategies in this fungal model. The analyses report a picture of genome-wide epigenetic (DNA methylation and small RNAs) and transcriptional (mRNA) patterns in P. ostreatus throughout its life cycle. High-throughput sequencing analyses (BS-seq, sRNA-seq and mRNA-seq) performed in monokaryon and dikaryon samples revealed epigenetic differences among strains but not within developmental stages. Our results uncovered strain-specific DNA methylation profiles (~ 2 to 7 % global methylation levels) and 21-22nt small RNA production primarily involved in the repression of transposon activity. Furthermore, our findings provide evidence that TE-associated DNA methylation—but not small RNA production—is directly involved in the silencing of genes surrounded by transposons. Finally, these findings also report key genes that are activated in the fruiting process through a comparative analysis of transcriptomes. A general discussion on findings presented in this thesis is given in Chapter IV. This last part reports a critical outlook on the epigenetic and transcriptional state of the two helitron families of the P. ostreatus strains that are differentially subcultured during several years, as well as nucleus-specific methylation variations in dikaryotic strains that share an identical genetic complement but different subculture conditions.
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    PublicationOpen Access
    Transposable elements in basidiomycete fungi: dynamics and impact on genome architecture and transcriptional profiles
    (2017) Castanera Andrés, Raúl; Ramírez Nasto, Lucía; Producción Agraria; Nekazaritza Ekoizpena
    Transposable elements (TE), also known as mobile elements or transposons, are enigmatic genetic units that have played important roles in the evolution of eukaryotes. Their impact on genome architecture and phenotypic traits has been widely studied in plants and animals, ever since their discovery in maize during the 1950s by Barbara McClintock. Their ability to move from one locus to another makes them natural tools for generating diversity, as this characteristic leads to genomic alterations with deleterious, neutral, or beneficial effects on hosts. Thus, their survival in the genome depends on the equilibrium between their own benefit and their host’s “permissibility.” Plant and animal TEs have received much attention, yet very little is known about their occurrence and impact on the fungal kingdom. In fact, the first fungal TE was described in Neurospora crassa in 1989, about 40 years later than the discovery of the first TE in plants. Today, revolutionary advances in genome sequencing have opened the possibility of studying non-model species at a whole-genome level. The number of fungal-sequenced genomes increases daily at an unprecedented rate, and most efforts are being concentrated on basidiomycetes, a group of fungi of great interest due to their role in natural ecosystems and their use in multiple industrial applications. In this sense, the amount of genomic information released offers a unique opportunity to start deciphering the effect that mobile, repetitive elements have on fungal genomes. At the time of the start of this PhD thesis (January 2013), very little information regarding basidiomycete TEs existed, as most research was focused on the functional characterization of protein-coding genes. In light of these precedents, the main topics covered in this work are the distribution, characteristics, and impact of transposons in fungal genomes, with an emphasis on basidiomycetes. Using Pleurotus ostreatus as a working model, bioinformatics pipelines have been developed to dig into the extensive genomic data to obtain high quality TE annotations. This approach has allowed for the quantification and characterization of the transposon load of many fungal species and for the testing of hypotheses about the effect that TE insertions produce at the genomic and transcriptomic level.