Cycles of satellite and transposon evolution in Arabidopsis centromeres.

Centromeres are critical for cell division, loading CENH3 or CENPA histone variant nucleosomes, directing kinetochore formation and allowing chromosome segregation1,2. Despite their conserved function, centromere size and structure are diverse across species. To understand this centromere paradox3,4, it is necessary to know how centromeric diversity is generated and whether it reflects ancient trans-species variation or, instead, rapid post-speciation divergence. To address these questions, we assembled 346 centromeres from 66 Arabidopsis thaliana and 2 Arabidopsis lyrata accessions, which exhibited a remarkable degree of intra- and inter-species diversity. A. thaliana centromere repeat arrays are embedded in linkage blocks, despite ongoing internal satellite turnover, consistent with roles for unidirectional gene conversion or unequal crossover between sister chromatids in sequence diversification. Additionally, centrophilic ATHILA transposons have recently invaded the satellite arrays. To counter ATHILA invasion, chromosome-specific bursts of satellite homogenization generate higher-order repeats and purge transposons, in line with cycles of repeat evolution. Centromeric sequence changes are even more extreme in comparison between A. thaliana and A. lyrata. Together, our findings identify rapid cycles of transposon invasion and purging through satellite homogenization, which drive centromere evolution and ultimately contribute to speciation.

miRNA-like secondary structures in maize (Zea mays) genes and transposable elements correlate with small RNAs, methylation, and expression.

RNA molecules carry information in their primary sequence and also their secondary structure. Secondary structure can confer important functional information, but it is also a signal for an RNAi-like host epigenetic response mediated by small RNAs (smRNAs). In this study, we used two bioinformatic methods to predict local secondary structures across features of the maize genome, focusing on small regions that had similar folding properties to pre-miRNA loci. We found miRNA-like secondary structures to be common in genes and most, but not all, superfamilies of RNA and DNA transposable elements (TEs). The miRNA-like regions map to a higher diversity of smRNAs than regions without miRNA-like structure, explaining up to 27% of variation in smRNA mapping for some TE superfamilies. This mapping bias is more pronounced among putatively autonomous TEs relative to nonautonomous TEs. Genome-wide, miRNA-like regions are also associated with elevated methylation levels, particularly in the CHH context. Among genes, those with miRNA-like secondary structure are 1.5-fold more highly expressed, on average, than other genes. However, these genes are also more variably expressed across the 26 nested association mapping founder lines, and this variability positively correlates with the number of mapping smRNAs. We conclude that local miRNA-like structures are a nearly ubiquitous feature of expressed regions of the maize genome, that they correlate with higher smRNA mapping and methylation, and that they may represent a trade-off between functional requirements and the potentially negative consequences of smRNA production.

A role for palindromic structures in the cis-region of maize Sirevirus LTRs in transposable element evolution and host epigenetic response.

Transposable elements (TEs) proliferate within the genome of their host, which responds by silencing them epigenetically. Much is known about the mechanisms of silencing in plants, particularly the role of siRNAs in guiding DNA methylation. In contrast, little is known about siRNA targeting patterns along the length of TEs, yet this information may provide crucial insights into the dynamics between hosts and TEs. By focusing on 6456 carefully annotated, full-length Sirevirus LTR retrotransposons in maize, we show that their silencing associates with underlying characteristics of the TE sequence and also uncover three features of the host-TE interaction. First, siRNA mapping varies among families and among elements, but particularly along the length of elements. Within the cis-regulatory portion of the LTRs, a complex palindrome-rich region acts as a hotspot of both siRNA matching and sequence evolution. These patterns are consistent across leaf, tassel, and immature ear libraries, but particularly emphasized for floral tissues and 21- to 22-nt siRNAs. Second, this region has the ability to form hairpins, making it a potential template for the production of miRNA-like, hairpin-derived small RNAs. Third, Sireviruses are targeted by siRNAs as a decreasing function of their age, but the oldest elements remain highly targeted, partially by siRNAs that cross-map to the youngest elements. We show that the targeting of older Sireviruses reflects their conserved palindromes. Altogether, we hypothesize that the palindromes aid the silencing of active elements and influence transposition potential, siRNA targeting levels, and ultimately the fate of an element within the genome.

The turbulent life of Sirevirus retrotransposons and the evolution of the maize genome: more than ten thousand elements tell the story.

Sireviruses are one of the three genera of Copia long terminal repeat (LTR) retrotransposons, exclusive to and highly abundant in plants, and with a unique, among retrotransposons, genome structure. Yet, perhaps due to the few references to the Sirevirus origin of some families, compounded by the difficulty in correctly assigning retrotransposon families into genera, Sireviruses have hardly featured in recent research. As a result, analysis at this key level of classification and details of their colonization and impact on plant genomes are currently lacking. Recently, however, it became possible to accurately assign elements from diverse families to this genus in one step, based on highly conserved sequence motifs. Hence, Sirevirus dynamics in the relatively obese maize genome can now be comprehensively studied. Overall, we identified >10 600 intact and approximately 28 000 degenerate Sirevirus elements from a plethora of families, some brought into the genus for the first time. Sireviruses make up approximately 90% of the Copia population and it is the only genus that has successfully infiltrated the genome, possibly by experiencing intense amplification during the last 600 000 years, while being constantly recycled by host mechanisms. They accumulate in chromosome-distal gene-rich areas, where they insert in between gene islands, mainly in preferred zones within their own genomes. Sirevirus LTRs are heavily methylated, while there is evidence for a palindromic consensus target sequence. This work brings Sireviruses in the spotlight, elucidating their lifestyle and history, and suggesting their crucial role in the current genomic make-up of maize, and possibly other plant hosts.

MASiVEdb: the Sirevirus Plant Retrotransposon Database.

BACKGROUND: Sireviruses are an ancient genus of the Copia superfamily of LTR retrotransposons, and the only one that has exclusively proliferated within plant genomes. Based on experimental data and phylogenetic analyses, Sireviruses have successfully infiltrated many branches of the plant kingdom, extensively colonizing the genomes of grass species. Notably, it was recently shown that they have been a major force in the make-up and evolution of the maize genome, where they currently occupy ~21% of the nuclear content and ~90% of the Copia population. It is highly likely, therefore, that their life dynamics have been fundamental in the genome composition and organization of a plethora of plant hosts. To assist studies into their impact on plant genome evolution and also facilitate accurate identification and annotation of transposable elements in sequencing projects, we developed MASiVEdb (Mapping and Analysis of SireVirus Elements Database), a collective and systematic resource of Sireviruses in plants. DESCRIPTION: Taking advantage of the increasing availability of plant genomic sequences, and using an updated version of MASiVE, an algorithm specifically designed to identify Sireviruses based on their highly conserved genome structure, we populated MASiVEdb (http://bat.infspire.org/databases/masivedb/) with data on 16,243 intact Sireviruses (total length >158Mb) discovered in 11 fully-sequenced plant genomes. MASiVEdb is unlike any other transposable element database, providing a multitude of highly curated and detailed information on a specific genus across its hosts, such as complete set of coordinates, insertion age, and an analytical breakdown of the structure and gene complement of each element. All data are readily available through basic and advanced query interfaces, batch retrieval, and downloadable files. A purpose-built system is also offered for detecting and visualizing similarity between user sequences and Sireviruses, as well as for coding domain discovery and phylogenetic analysis. CONCLUSION: MASiVEdb is currently the most comprehensive directory of Sireviruses, and as such complements other efforts in cataloguing plant transposable elements and elucidating their role in host genome evolution. Such insights will gradually deepen, as we plan to further improve MASiVEdb by phylogenetically mapping Sireviruses into families, by including data on fragments and solo LTRs, and by incorporating elements from newly-released genomes.

Patterns of selection in the evolution of a transposable element.

Transposable elements are a major component of most eukaryotic genomes. Here, we present a new approach which allows us to study patterns of natural selection in the evolution of transposable elements over short time scales. The method uses the alignment of all elements with intact gag/pol genes of a transposable element family from a single genome. We predict that the ratio of nonsynonymous to synonymous variants in the alignment should decrease as a function of the frequency of the variants, because elements with nonsynonymous variants that reduce transposition will have fewer progeny. We apply our method to Sirevirus long-terminal repeat retrotransposons that are abundant in maize and other plant species and show that nonsynonymous to synonymous variants declines as variant frequency increases, indicating that negative selection is acting strongly on the Sirevirus genome. The asymptotic value of nonsynonymous to synonymous variants suggests that at least 85% of all nonsynonymous mutations in the transposable element reduce transposition. Crucially, these patterns in nonsynonymous to synonymous variants are only predicted to occur if the gene products from a particular transposable element insertion preferentially promote the transposition of the same insertion. Overall, by using large numbers of intact elements, this study sheds new light on the selective processes that act on transposable elements.

Gene capture by transposable elements leads to epigenetic conflict in maize.

Transposable elements (TEs) regularly capture fragments of genes. When the host silences these TEs, siRNAs homologous to the captured regions may also target the genes. This epigenetic crosstalk establishes an intragenomic conflict: silencing the TEs has the cost of silencing the genes. If genes are important, however, natural selection may maintain function by moderating the silencing response, which may also advantage the TEs. In this study, we examined this model by focusing on Helitrons, Pack-MULEs, and Sirevirus LTR retrotransposons in the maize genome. We documented 1263 TEs containing exon fragments from 1629 donor genes. Consistent with epigenetic conflict, donor genes mapped more siRNAs and were more methylated than genes with no evidence of capture. However, these patterns differed between syntelog versus translocated donor genes. Syntelogs appeared to maintain function, as measured by gene expression, consistent with moderation of silencing for functionally important genes. Epigenetic marks did not spread beyond their captured regions and 24nt crosstalk siRNAs were linked with CHH methylation. Translocated genes, in contrast, bore the signature of silencing. They were highly methylated and less expressed, but also overrepresented among donor genes and located away from chromosomal arms, which suggests a link between capture and gene movement. Splitting genes into potential functional categories based on evolutionary constraint supported the synteny-based findings. TE families captured genes in different ways, but the evidence for their advantage was generally less obvious; nevertheless, TEs with captured fragments were older, mapped fewer siRNAs, and were slightly less methylated than TEs without captured fragments. Collectively, our results argue that TE capture triggers an intragenomic conflict that may not affect the function of important genes but may lead to the pseudogenization of less-constrained genes.

The genetic and epigenetic landscape of the Arabidopsis centromeres.

Centromeres attach chromosomes to spindle microtubules during cell division and, despite this conserved role, show paradoxically rapid evolution and are typified by complex repeats. We used long-read sequencing to generate the Col-CEN Arabidopsis thaliana genome assembly that resolves all five centromeres. The centromeres consist of megabase-scale tandemly repeated satellite arrays, which support CENTROMERE SPECIFIC HISTONE H3 (CENH3) occupancy and are densely DNA methylated, with satellite variants private to each chromosome. CENH3 preferentially occupies satellites that show the least amount of divergence and occur in higher-order repeats. The centromeres are invaded by ATHILA retrotransposons, which disrupt genetic and epigenetic organization. Centromeric crossover recombination is suppressed, yet low levels of meiotic DNA double-strand breaks occur that are regulated by DNA methylation. We propose that Arabidopsis centromeres are evolving through cycles of satellite homogenization and retrotransposon-driven diversification.

Highly conserved motifs in non-coding regions of Sirevirus retrotransposons: the key for their pattern of distribution within and across plants?

BACKGROUND: Retrotransposons are key players in the evolution of eukaryotic genomes. Moreover, it is now known that some retrotransposon classes, like the abundant and plant-specific Sireviruses, have intriguingly distinctive host preferences. Yet, it is largely unknown if this bias is supported by different genome structures. RESULTS: We performed sensitive comparative analysis of the genomes of a large set of Ty1/copia retrotransposons. We discovered that Sireviruses are unique among Pseudoviridae in that they constitute an ancient genus characterized by vastly divergent members, which however contain highly conserved motifs in key non-coding regions: multiple polypurine tract (PPT) copies cluster upstream of the 3' long terminal repeat (3'LTR), of which the terminal PPT tethers to a distinctive attachment site and is flanked by a precisely positioned inverted repeat. Their LTRs possess a novel type of repeated motif (RM) defined by its exceptionally high copy number, symmetry and core CGG-CCG signature. These RM boxes form CpG islands and lie a short distance upstream of a conserved promoter region thus hinting towards regulatory functions. Intriguingly, in the envelope-containing Sireviruses additional boxes cluster at the 5' vicinity of the envelope. The 5'LTR/internal domain junction and a polyC-rich integrase signal are also highly conserved domains of the Sirevirus genome. CONCLUSIONS: Our comparative analysis of retrotransposon genomes using advanced in silico methods highlighted the unique genome organization of Sireviruses. Their structure may dictate a life cycle with different regulation and transmission strategy compared to other Pseudoviridae, which may contribute towards their pattern of distribution within and across plants.

Absence of close-facing retrotransposons: a comparison of molecular data and theory.

Retrotransposons occur in extremely large numbers in many eukaryotic genomes. However, little is known of the factors which affect the distribution of close proximity elements. In this work we investigate the frequency of close facing retrotransposons in a plant species with extremely high numbers of retrotransposons. Molecular observations are compared with predictions of a mathematical model that assumes a uniform probability of retrotransposon insertion into the genome. The mathematical model plays the role of a null hypothesis. We find that compared with the predictions of the model, there is a statistically significant deficit of identical copies of facing retroelements that are close to one another. This suggests that an efficient mechanism exists that removes or limits close facing retroelements.

Integrating transposable elements in the 3D genome.

Chromosome organisation is increasingly recognised as an essential component of genome regulation, cell fate and cell health. Within the realm of transposable elements (TEs) however, the spatial information of how genomes are folded is still only rarely integrated in experimental studies or accounted for in modelling. Whilst polymer physics is recognised as an important tool to understand the mechanisms of genome folding, in this commentary we discuss its potential applicability to aspects of TE biology. Based on recent works on the relationship between genome organisation and TE integration, we argue that existing polymer models may be extended to create a predictive framework for the study of TE integration patterns. We suggest that these models may offer orthogonal and generic insights into the integration profiles (or "topography") of TEs across organisms. In addition, we provide simple polymer physics arguments and preliminary molecular dynamics simulations of TEs inserting into heterogeneously flexible polymers. By considering this simple model, we show how polymer folding and local flexibility may generically affect TE integration patterns. The preliminary discussion reported in this commentary is aimed to lay the foundations for a large-scale analysis of TE integration dynamics and topography as a function of the three-dimensional host genome.

The genome-wide dynamics of purging during selfing in maize.

Self-fertilization (also known as selfing) is an important reproductive strategy in plants and a widely applied tool for plant genetics and plant breeding. Selfing can lead to inbreeding depression by uncovering recessive deleterious variants, unless these variants are purged by selection. Here we investigated the dynamics of purging in a set of eleven maize lines that were selfed for six generations. We show that heterozygous, putatively deleterious single nucleotide polymorphisms are preferentially lost from the genome during selfing. Deleterious single nucleotide polymorphisms were lost more rapidly in regions of high recombination, presumably because recombination increases the efficacy of selection by uncoupling linked variants. Overall, heterozygosity decreased more slowly than expected, by an estimated 35% to 40% per generation instead of the expected 50%, perhaps reflecting pervasive associative overdominance. Finally, three lines exhibited marked decreases in genome size due to the purging of transposable elements. Genome loss was more likely to occur for lineages that began with larger genomes with more transposable elements and chromosomal knobs. These three lines purged an average of 398 Mb from their genomes, an amount equivalent to three Arabidopsis thaliana genomes per lineage, in only a few generations.

Modeling Interactions between Transposable Elements and the Plant Epigenetic Response: A Surprising Reliance on Element Retention.

Transposable elements (TEs) compose the majority of angiosperm DNA. Plants counteract TE activity by silencing them epigenetically. One form of epigenetic silencing requires 21-22 nt small interfering RNAs that act to degrade TE mRNA and may also trigger DNA methylation. DNA methylation is reinforced by a second mechanism, the RNA-dependent DNA methylation (RdDM) pathway. RdDM relies on 24 nt small interfering RNAs and ultimately establishes TEs in a quiescent state. These host factors interact at a systems level, but there have been no system level analyses of their interactions. Here, we define a deterministic model that represents the propagation of active TEs, aspects of the host response and the accumulation of silenced TEs. We describe general properties of the model and also fit it to biological data in order to explore two questions. The first is why two overlapping pathways are maintained, given that both are likely energetically expensive. Under our model, RdDM silenced TEs effectively even when the initiation of silencing was weak. This relationship implies that only a small amount of RNAi is needed to initiate TE silencing, but reinforcement by RdDM is necessary to efficiently counter TE propagation. Second, we investigated the reliance of the host response on rates of TE deletion. The model predicted that low levels of deletion lead to few active TEs, suggesting that silencing is most efficient when methylated TEs are retained in the genome, thereby providing one explanation for the large size of plant genomes.

Considerations and complications of mapping small RNA high-throughput data to transposable elements.

BACKGROUND: High-throughput sequencing (HTS) has revolutionized the way in which epigenetic research is conducted. When coupled with fully-sequenced genomes, millions of small RNA (sRNA) reads are mapped to regions of interest and the results scrutinized for clues about epigenetic mechanisms. However, this approach requires careful consideration in regards to experimental design, especially when one investigates repetitive parts of genomes such as transposable elements (TEs), or when such genomes are large, as is often the case in plants. RESULTS: Here, in an attempt to shed light on complications of mapping sRNAs to TEs, we focus on the 2,300 Mb maize genome, 85% of which is derived from TEs, and scrutinize methodological strategies that are commonly employed in TE studies. These include choices for the reference dataset, the normalization of multiply mapping sRNAs, and the selection among sRNA metrics. We further examine how these choices influence the relationship between sRNAs and the critical feature of TE age, and contrast their effect on low copy genomic regions and other popular HTS data. CONCLUSIONS: Based on our analyses, we share a series of take-home messages that may help with the design, implementation, and interpretation of high-throughput TE epigenetic studies specifically, but our conclusions may also apply to any work that involves analysis of HTS data.

Mechanistic and evolutionary questions about epigenetic conflicts between transposable elements and their plant hosts.

Transposable elements (TEs) constitute the majority of plant genomes, but most are epigenetically inactivated by their host. Research over the last decade has elucidated many of the molecular components that are required for TE silencing. In contrast, the evolutionary dynamics between TEs and silencing pathways are less clear. Here, we discuss current information about these dynamics from both mechanistic and evolutionary perspectives. We highlight new evidence that palindromic sequences within TEs may act as signals for host recognition and that cis-regulatory regions of TEs may be sites of ongoing arms races with host defenses. We also discuss patterns of TE aging after they are silenced; while there is not yet a consensus, it appears that TEs are removed more rapidly near genes, such that older TE insertions tend to be farther from genes. We conclude by discussing the energetic costs for maintaining silencing pathways, which appear to be substantive. The maintenance of silencing pathways across many species suggests that epigenetic emergencies are frequent.

Sirevirus LTR retrotransposons: phylogenetic misconceptions in the plant world.

Sireviruses are an ancient and plant-specific LTR retrotransposon genus. They possess a unique genome structure that is characterized by a plethora of highly conserved sequence motifs in key domains of the non-coding genome, and often, by the presence of an envelope-like gene. Recently, their crucial role in the organization of the maize genome, where Sireviruses occupy approximately 21% of its nuclear content, was revealed, followed by an analysis of their distribution across the plant kingdom. It is now suggested that Sireviruses have been a major mediator of the evolution of many plant genomes. However, the name 'Sirevirus' has caused confusion in the scientific community in regards to their classification within the LTR retrotransposon order and their relationship with viruses - a situation that is not unique to Sireviruses, but also affects other LTR retrotransposon genera. Here, we clarify the phylogenetic position of Sireviruses as typical LTR retrotransposons of the Copia superfamily and explain that the confusion stems from the discrepancy in the categorization of LTR retrotransposons by the two main classification systems: the International Committee on the Taxonomy of Viruses (ICTV) system and the unified classification system for eukaryotic transposable elements. While the name 'Sirevirus' has been given by ICTV, we show that the transposable element system, which is more suitable for eukaryotic genome studies, lacks an appropriate taxonomic level for describing them. We urge for this inconsistency to be addressed. Finally, we provide data suggesting that of the three ICTV-proposed genera of the Pseudoviridae (that is, Copia) family, only Sireviruses form a monophyletic group, while the phylogenetic distinction between Pseudoviruses and Hemiviruses is unclear. We conclude that because of their ongoing important contribution to the classification of transposable elements, these schemes need to be frequently revisited and revised - as shown by the example of the Sirevirus LTR retrotransposon genus.