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

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

<p>Abstract</p> <p>Background</p> <p>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.</p> <p>Results</p> <p>We performed sensitive comparative analysis of the genomes of a large set of Ty1/<it>copia </it>retrotransposons. We discovered that Sireviruses are unique among <it>Pseudoviridae </it>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.</p> <p>Conclusions</p> <p>Our comparative analysis of retrotransposon genomes using advanced <it>in silico </it>methods highlighted the unique genome organization of Sireviruses. Their structure may dictate a life cycle with different regulation and transmission strategy compared to other <it>Pseudoviridae</it>, which may contribute towards their pattern of distribution within and across plants.</p

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

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

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

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

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 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 longread 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 CENH3 occupancy and are densely DNA methylated, with satellite variants private to each chromosome. CENH3 preferentially occupies satellites that show least divergence and occur in higherorder repeats. The centromeres are invaded by ATHILA retrotransposons, which disrupt genetic and epigenetic organization. Centromeric crossover recombination is suppressed, yet low levels of meiotic DSBs occur that are regulated by DNA methylation. We propose that Arabidopsis centromeres are evolving via cycles of satellite homogenization and retrotransposon-driven diversification.BBSRC grants BB/S006842/1, BB/S020012/1 and BB/V003984/1

Is There an Upper Limit to Genome Size?

O.H. was supported by the Marie Skłodowska Curie Action Individual Fellowship program (CAPITULA, grant agreement 657918