Open Access
Transposon-associated epigenetic silencing during Pleurotus ostreatus life cycle
(Oxford University Press, 2018) Borgognone, Alessandra; Castanera Andrés, Raúl; Morselli, Marco; López Varas, Leticia; Rubbi, Liudmilla; Pisabarro de Lucas, Gerardo; Pellegrini, Matteo; Ramírez Nasto, Lucía; Producción Agraria; Nekazaritza Ekoizpena; Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa
Transposable elements constitute an important fraction of eukaryotic genomes. Given their mutagenic potential, host-genomes have evolved epigenetic defense mechanisms to limit their expansion. In fungi, epigenetic modifications have been widely studied in ascomycetes, although we lack a global picture of the epigenetic landscape in basidiomycetes. In this study, we analysed the genome-wide epigenetic and transcriptional patterns of the white-rot basidiomycete Pleurotus ostreatus throughout its life cycle. Our results performed by using high-throughput sequencing analyses revealed that strain-specific DNA methylation profiles are primarily involved in the repression of transposon activity and suggest that 21 nt small RNAs play a key role in transposon silencing. Furthermore, we provide evidence that transposon-associated DNA methylation, but not sRNA production, is directly involved in the silencing of genes surrounded by transposons. Remarkably, we found that nucleus-specific methylation levels varied in dikaryotic strains sharing identical genetic complement but different subculture conditions. Finally, we identified key genes activated in the fruiting process through the comparative analysis of transcriptomes. This study provides an integrated picture of epigenetic defense mechanisms leading to the transcriptional silencing of transposons and surrounding genes in basidiomycetes. Moreover, our findings suggest that transcriptional but not methylation reprogramming triggers fruitbody development in P. ostreatus.
Open 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.
Open Access
Transposable elements versus the fungal genome: impact on whole-genome architecture and transcriptional profiles
(Public Library of Science, 2016) Castanera Andrés, Raúl; López Varas, Leticia; Borgognone, Alessandra; LaButti, Kurt; Lapidus, Alla; Schmutz, Jeremy; Grimwood, Jane; Pérez Garrido, María Gumersinda; Pisabarro de Lucas, Gerardo; Grigoriev, Igor V.; Stajich, Jason E.; Ramírez Nasto, Lucía; Producción Agraria; Nekazaritza Ekoizpena
Transposable elements (TEs) are exceptional contributors to eukaryotic genome diversity. Their ubiquitous presence impacts the genomes of nearly all species and mediates genome evolution by causing mutations and chromosomal rearrangements and by modulating gene expression. We performed an exhaustive analysis of the TE content in 18 fungal genomes, including strains of the same species and species of the same genera. Our results depicted a scenario of exceptional variability, with species having 0.02 to 29.8% of their genome consisting of transposable elements. A detailed analysis performed on two strains of Pleurotus ostreatus uncovered a genome that is populated mainly by Class I elements, especially LTR-retrotransposons amplified in recent bursts from 0 to 2 million years (My) ago. The preferential accumulation of TEs in clusters led to the presence of genomic regions that lacked intra- and inter-specific conservation. In addition, we investigated the effect of TE insertions on the expression of their nearby upstream and downstream genes. Our results showed that an important number of genes under TE influence are significantly repressed, with stronger repression when genes are localized within transposon clusters. Our transcriptional analysis performed in four additional fungal models revealed that this TE-mediated silencing was present only in species with active cytosine methylation machinery. We hypothesize that this phenomenon is related to epigenetic defense mechanisms that are aimed to suppress TE expression and control their proliferation.
Open Access
Somatic transposition and meiotically driven elimination of an active helitron family in Pleurotus ostreatus
(Oxford University Press, 2017) Borgognone, Alessandra; Castanera Andrés, Raúl; Muguerza Domínguez, Elaia; Pisabarro de Lucas, Gerardo; Ramírez Nasto, Lucía; Producción Agraria; Nekazaritza Ekoizpena; Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa
Helitrons constitute a superfamily of DNA transposons that were discovered in silico and are widespread in most eukaryotic genomes. They are postulated to mobilize through a “rollingcircle” mechanism, but the experimental evidence of their transposition has been described only recently. Here, we present the inheritance patterns of HELPO1 and HELPO2 helitron families in meiotically derived progeny of the basidiomycete Pleurotus ostreatus. We found distorted segregation patterns of HELPO2 helitrons that led to a strong under-representation of these elements in the progeny. Further investigation of HELPO2 flanking sites showed that gene conversion may contribute to the elimination of such repetitive elements in meiosis, favouring the presence of HELPO2 vacant loci. In addition, the analysis of HELPO2 content in a reconstructed pedigree of subclones maintained under different culture conditions revealed an event of helitron somatic transposition. Additional analyses of genome and transcriptome data indicated that P. ostreatus carries active RNAi machinery that could be involved in the control of transposable element proliferation. Our results provide the first evidence of helitron mobilization in the fungal kingdom and highlight the interaction between genome defence mechanisms and invasive DNA.

Borgognone, Alessandra

Email Address

person.page.identifierURI

Birth Date

Job Title

Last Name

First Name

person.page.departamento

person.page.instituteName

ORCID

person.page.observainves

person.page.upna

Name

Filters

Author

Subject

Date

Has files

Item Type

Type

Settings

Sort By

Results per page

Search Results