Transposable Elements: Epigenetic Silencing Mechanisms or Modulating Tools for Vertebrate Adaptations? Two Sides of the Same Coin.

Transposable elements constitute one of the main components of eukaryotic genomes. In vertebrates, they differ in content, typology, and family diversity and played a crucial role in the evolution of this taxon. However, due to their transposition ability, TEs can be responsible for genome instability, and thus silencing mechanisms were evolved to allow the coexistence between TEs and eukaryotic host-coding genes. Several papers are highlighting in TEs the presence of regulatory elements involved in regulating nearby genes in a tissue-specific fashion. This suggests that TEs are not sequences merely to silence; rather, they can be domesticated for the regulation of host-coding gene expression, permitting species adaptation and resilience as well as ensuring human health. This review presents the main silencing mechanisms acting in vertebrates and the importance of exploiting these mechanisms for TE control to rewire gene expression networks, challenging the general view of TEs as threatening elements.

Characterization of influenza A virus induced transposons reveals a subgroup of transposons likely possessing the regulatory role as eRNAs.

Although many studies have observed genome-wide host transposon expression alteration during viral infection, the mechanisms of induction and the impact on the host remain unclear. Utilizing recently published influenza A virus (IAV) time series data and ENCODE functional genomics data, we characterized virus induced host differentially expressed transposons (virus-induced-TE) by investigating genome-wide spatial and functional relevance between the virus-induced-TEs and epigenomic markers (e.g. histone modification and chromatin remodelers). We found that a significant fraction of virus-induced-TEs are derived from host enhancer regions, where CHD4 binding and/or H3K27ac occupancy is high or H3K9me3 occupancy is low. By overlapping virus-induced-TEs to human enhancer RNAs (eRNAs), we discovered that a proportion of virus-induced-TEs are either eRNAs or part of enhancer RNAs. Upon further analysis of the eRNA targeted genes, we found that the virus-induced-TE related eRNA targets are overrepresented in differentially expressed host genes of IAV infected samples. Our results suggest that changing chromatin accessibility from repressive to permissive in the transposon docked enhancer regions to regulate host downstream gene expression is potentially one of the virus and host cell interaction mechanisms, where transposons are likely important regulatory genomic elements. Our study provides a new insight into the mechanisms of virus-host interaction and may lead to novel strategies for prevention and therapeutics of IAV and other virus infectious diseases.

Transposons: Unexpected players in cancer.

Transposons are repetitive DNA sequences encompassing about half of the human genome. They play a vital role in genome stability maintenance and contribute to genomic diversity and evolution. Their activity is regulated by various mechanisms considering the deleterious effects of these mobile elements. Various genetic risk factors and environmental stress conditions affect the regulatory pathways causing alteration of transposon expression. Our knowledge of the biological role of transposons is limited especially in various types of cancers. Retrotransposons of different types (LTR-retrotransposons, LINEs and SINEs) regulate a plethora of genes that have a role in cell reprogramming, tumor suppression, cell cycle, apoptosis, cell adhesion and migration, and DNA repair. The regulatory mechanisms of transposons, their deregulation and different mechanisms underlying transposon-mediated carcinogenesis in humans focusing on the three most prevalent types, lung, breast and colorectal cancers, were reviewed. The modes of regulation employed include alternative splicing, deletion, insertion, duplication in genes and promoters resulting in upregulation, downregulation or silencing of genes.

Metagenomic discovery of CRISPR-associated transposons.

CRISPR-associated Tn7 transposons (CASTs) co-opt cas genes for RNA-guided transposition. CASTs are exceedingly rare in genomic databases; recent surveys have reported Tn7-like transposons that co-opt Type I-F, I-B, and V-K CRISPR effectors. Here, we expand the diversity of reported CAST systems via a bioinformatic search of metagenomic databases. We discover architectures for all known CASTs, including arrangements of the Cascade effectors, target homing modalities, and minimal V-K systems. We also describe families of CASTs that have co-opted the Type I-C and Type IV CRISPR-Cas systems. Our search for non-Tn7 CASTs identifies putative candidates that include a nuclease dead Cas12. These systems shed light on how CRISPR systems have coevolved with transposases and expand the programmable gene-editing toolkit.

Impact of transposable elements on the evolution of complex living systems and their epigenetic control.

Transposable elements (TEs) contribute to genomic innovations, as well as genome instability, across a wide variety of species. Popular designations such as 'selfish DNA' and 'junk DNA,' common in the 1980s, may be either inaccurate or misleading, while a more enlightened view of the TE-host relationship covers a range from parasitism to mutualism. Both plant and animal hosts have evolved epigenetic mechanisms to reduce the impact of TEs, both by directly silencing them and by reducing their ability to transpose in the genome. However, TEs have also been co-opted by both plant and animal genomes to perform a variety of physiological functions, ranging from TE-derived proteins acting directly in normal biological functions to innovations in transcription factor activity and also influencing gene expression. Their presence, in fact, can affect a range of features at genome, phenotype, and population levels. The impact TEs have had on evolution is multifaceted, and many aspects still remain unexplored. In this review, the epigenetic control of TEs is contextualized according to the evolution of complex living systems.

Overexpression of transposable elements is associated with immune evasion and poor outcome in colorectal cancer.

AIM: High immune cell infiltration of the tumour microenvironment is generally associated with a good prognosis in solid cancers. However, a subset of patients with colorectal cancer (CRC) tumours with high immune cell infiltration have a poor outcome. These tumours have a high level of T cell infiltration and are also characterised by increased expression of programmed death-ligand 1 (PD-L1). As these tumours comprise both microsatellite instability and microsatellite stable subtypes, the mechanism underlying this phenotype is unknown. METHODS: Using RNA-seq data from The Cancer Genome Atlas, we quantified transposable element (TE) expression and developed a TE expression score that is predictive of prognosis and immune infiltration independent of microsatellite instability status and tumour staging in CRC. RESULTS: Tumours with the highest TE expression score showed increased immune cell infiltration with upregulation of interferon (IFN) signalling pathways and downstream activation of IFN-simulated genes. As expected, cell lines treated with DNA methyltransferase inhibitor mimicked patient tumours with increased TE expression and IFN signalling. However, surprisingly, unlike high TE expressing CRC, there is little evidence for the activation of JAK-STAT signalling and PD-L1 expression in DNA methyltransferase inhibitor-treated cells. Single-cell RNA-seq analysis of CRC samples showed that PD-L1 expression is mainly confined to tumour-associated macrophages and T cells, suggesting that TE mediated IFN signalling is triggering expression of PD-L1 in immune cells rather than in tumour cells. CONCLUSIONS: Our study uncovers a novel mechanism of TE driven immune evasion and highlights TE expression as an important factor for patient prognosis in CRC.

More than causing (epi)genomic instability: emerging physiological implications of transposable element modulation.

Transposable elements (TEs) initially attracted attention because they comprise a major portion of the genomic sequences in plants and animals. TEs may jump around the genome and disrupt both coding genes as well as regulatory sequences to cause disease. Host cells have therefore evolved various epigenetic and functional RNA-mediated mechanisms to mitigate the disruption of genomic integrity by TEs. TE associated sequences therefore acquire the tendencies of attracting various epigenetic modifiers to induce epigenetic alterations that may spread to the neighboring genes. In addition to posting threats for (epi)genome integrity, emerging evidence suggested the physiological importance of endogenous TEs either as cis-acting control elements for controlling gene regulation or as TE-containing functional transcripts that modulate the transcriptome of the host cells. Recent advances in long-reads sequence analysis technologies, bioinformatics and genetic editing tools have enabled the profiling, precise annotation and functional characterization of TEs despite their challenging repetitive nature. The importance of specific TEs in preimplantation embryonic development, germ cell differentiation and meiosis, cell fate determination and in driving species specific differences in mammals will be discussed.

THAP9 Transposase Cleaves DNA via Conserved Acidic Residues in an RNaseH-Like Domain.

The catalytic domain of most 'cut and paste' DNA transposases have the canonical RNase-H fold, which is also shared by other polynucleotidyl transferases such as the retroviral integrases and the RAG1 subunit of V(D)J recombinase. The RNase-H fold is a mixture of beta sheets and alpha helices with three acidic residues (Asp, Asp, Glu/Asp-DDE/D) that are involved in the metal-mediated cleavage and subsequent integration of DNA. Human THAP9 (hTHAP9), homologous to the well-studied Drosophila P-element transposase (DmTNP), is an active DNA transposase that, although domesticated, still retains the catalytic activity to mobilize transposons. In this study we have modeled the structure of hTHAP9 using the recently available cryo-EM structure of DmTNP as a template to identify an RNase-H like fold along with important acidic residues in its catalytic domain. Site-directed mutagenesis of the predicted catalytic residues followed by screening for DNA excision and integration activity has led to the identification of candidate Ds and Es in the RNaseH fold that may be a part of the catalytic triad in hTHAP9. This study has helped widen our knowledge about the catalytic activity of a functionally uncharacterized transposon-derived gene in the human genome.

Maize decrease in DNA methylation 1 targets RNA-directed DNA methylation on active chromatin.

DNA methylation plays vital roles in repressing transposable element activity and regulating gene expression. The chromatin-remodeling factor Decrease in DNA methylation 1 (DDM1) is crucial for maintaining DNA methylation across diverse plant species, and is required for RNA-directed DNA methylation (RdDM) to maintain mCHH islands in maize (Zea mays). However, the mechanisms by which DDM1 is involved in RdDM are not well understood. In this work, we used chromatin immunoprecipitation coupled with high-throughput sequencing to ascertain the genome-wide occupancy of ZmDDM1 in the maize genome. The results revealed that ZmDDM1 recognized an 8-bp-long GC-rich degenerate DNA sequence motif, which is enriched in transcription start sites and other euchromatic regions. Meanwhile, 24-nucleotide siRNAs and CHH methylation were delineated at the edge of ZmDDM1-occupied sites. ZmDDM1 co-purified with Argonaute 4 (ZmAGO4) proteins, providing further evidence that ZmDDM1 is a component of RdDM complexes in planta. Consistent with this, the vast majority of ZmDDM1-targeted regions co-localized with ZmAGO4-bound genomic sites. Overall, our results suggest a model that ZmDDM1 may be recruited to euchromatic regions via recognition of a GC-rich motif, thereby remodeling chromatin to provide access for RdDM activities in maize.

Dual modes of CRISPR-associated transposon homing.

Tn7-like transposons have co-opted CRISPR systems, including class 1 type I-F, I-B, and class 2 type V-K. Intriguingly, although these CRISPR-associated transposases (CASTs) undergo robust CRISPR RNA (crRNA)-guided transposition, they are almost never found in sites targeted by the crRNAs encoded by the cognate CRISPR array. To understand this paradox, we investigated CAST V-K and I-B systems and found two distinct modes of transposition: (1) crRNA-guided transposition and (2) CRISPR array-independent homing. We show distinct CAST systems utilize different molecular mechanisms to target their homing site. Type V-K CAST systems use a short, delocalized crRNA for RNA-guided homing, whereas type I-B CAST systems, which contain two distinct target selector proteins, use TniQ for RNA-guided DNA transposition and TnsD for homing to an attachment site. These observations illuminate a key step in the life cycle of CAST systems and highlight the diversity of molecular mechanisms mediating transposon homing.

Essential Gene Analysis in Acinetobacter baumannii by High-Density Transposon Mutagenesis and CRISPR Interference.

Acinetobacter baumannii is a poorly understood bacterium capable of life-threatening infections in hospitals. Few antibiotics remain effective against this highly resistant pathogen. Development of rationally designed antimicrobials that can target A. baumannii requires improved knowledge of the proteins that carry out essential processes allowing growth of the organism. Unfortunately, studying essential genes has been challenging using traditional techniques, which usually require time-consuming recombination-based genetic manipulations. Here, we performed saturating mutagenesis with dual transposon systems to identify essential genes in A. baumannii, and we developed a CRISPR interference (CRISPRi) system for facile analysis of these genes. We show that the CRISPRi system enables efficient transcriptional silencing in A. baumannii. Using these tools, we confirmed the essentiality of the novel cell division protein AdvA and discovered a previously uncharacterized AraC family transcription factor (ACX60_RS03245) that is necessary for growth. In addition, we show that capsule biosynthesis is a conditionally essential process, with mutations in late-acting steps causing toxicity in strain ATCC 17978 that can be bypassed by blocking early-acting steps or activating the BfmRS stress response. These results open new avenues for analysis of essential pathways in A. baumannii. IMPORTANCE New approaches are urgently needed to control A. baumannii, one of the most drug-resistant pathogens known. To facilitate the development of novel targets that allow inhibition of the pathogen, we performed a large-scale identification of genes whose products the bacterium needs for growth. We also developed a CRISPR-based gene knockdown tool that operates efficiently in A. baumannii, allowing rapid analysis of these essential genes. We used these methods to define multiple processes vital to the bacterium, including a previously uncharacterized gene regulatory factor and export of a protective polymeric capsule. These tools will enhance our ability to investigate processes critical for the essential biology of this challenging hospital-acquired pathogen.

A transposon surveillance mechanism that safeguards plant male fertility during stress.

Although plants are able to withstand a range of environmental conditions, spikes in ambient temperature can impact plant fertility causing reductions in seed yield and notable economic losses1,2. Therefore, understanding the precise molecular mechanisms that underpin plant fertility under environmental constraints is critical to safeguarding future food production3. Here, we identified two Argonaute-like proteins whose activities are required to sustain male fertility in maize plants under high temperatures. We found that MALE-ASSOCIATED ARGONAUTE-1 and -2 associate with temperature-induced phased secondary small RNAs in pre-meiotic anthers and are essential to controlling the activity of retrotransposons in male meiocyte initials. Biochemical and structural analyses revealed how male-associated Argonaute activity and its interaction with retrotransposon RNA targets is modulated through the dynamic phosphorylation of a set of highly conserved, surface-located serine residues. Our results demonstrate that an Argonaute-dependent, RNA-guided surveillance mechanism is critical in plants to sustain male fertility under environmentally constrained conditions, by controlling the mutagenic activity of transposons in male germ cells.

Whole-genome characterization of chronological age-associated changes in methylome and circular RNAs in moso bamboo (Phyllostachys edulis) from vegetative to floral growth.

In mammals, DNA methylation is associated with aging. However, age-related DNA methylation changes during phase transitions largely remain unstudied in plants. Moso bamboo (Phyllostachys edulis) requires a very long time to transition from the vegetative to the floral phase. To comprehensively investigate the association of DNA methylation with aging, we present here single-base-resolution DNA methylation profiles using both high-throughput bisulfite sequencing and single-molecule nanopore-based DNA sequencing, covering the long period of vegetative growth and transition to flowering in moso bamboo. We discovered that CHH methylation gradually accumulates from vegetative to reproductive growth in a time-dependent fashion. Differentially methylated regions, correlating with chronological aging, occurred preferentially at both transcription start sites and transcription termination sites. Genes with CG methylation changes showed an enrichment of Gene Ontology (GO) categories in 'vegetative to reproductive phase transition of meristem'. Combining methylation data with mRNA sequencing revealed that DNA methylation in promoters, introns and exons may have different roles in regulating gene expression. Finally, circular RNA (circRNA) sequencing revealed that the flanking introns of circRNAs are hypermethylated and enriched in long terminal repeat (LTR) retrotransposons. Together, the observations in this study provide insights into the dynamic DNA methylation and circRNA landscapes, correlating with chronological age, which paves the way to study further the impact of epigenetic factors on flowering in moso bamboo.

Transposable element persistence via potential genome-level ecosystem engineering.

BACKGROUND: The nuclear genomes of eukaryotes vary enormously in size, with much of this variability attributable to differential accumulation of transposable elements (TEs). To date, the precise evolutionary and ecological conditions influencing TE accumulation remain poorly understood. Most previous attempts to identify these conditions have focused on evolutionary processes occurring at the host organism level, whereas we explore a TE ecology explanation. RESULTS: As an alternative (or additional) hypothesis, we propose that ecological mechanisms occurring within the host cell may contribute to patterns of TE accumulation. To test this idea, we conducted a series of experiments using a simulated asexual TE/host system. Each experiment tracked the accumulation rate for a given type of TE within a particular host genome. TEs in this system had a net deleterious effect on host fitness, which did not change over the course of experiments. As one might expect, in the majority of experiments TEs were either purged from the genome or drove the host population to extinction. However, in an intriguing handful of cases, TEs co-existed with hosts and accumulated to very large numbers. This tended to occur when TEs achieved a stable density relative to non-TE sequences in the genome (as opposed to reaching any particular absolute number). In our model, the only way to maintain a stable density was for TEs to generate new, inactive copies at a rate that balanced with the production of active (replicating) copies. CONCLUSIONS: From a TE ecology perspective, we suggest this could be interpreted as a case of ecosystem engineering within the genome, where TEs persist by creating their own "habitat".

Heat stress-induced transposon activation correlates with 3D chromatin organization rearrangement in Arabidopsis.

In higher eukaryotes, heterochromatin is mainly composed of transposable elements (TEs) silenced by epigenetic mechanisms. But, the silencing of certain heterochromatin-associated TEs is disrupted by heat stress. By comparing genome-wide high-resolution chromatin packing patterns under normal or heat conditions obtained through Hi-C analysis, we show here that heat stress causes global rearrangement of the 3D genome in Arabidopsis thaliana. Contacts between pericentromeric regions and distal chromosome arms, as well as proximal intra-chromosomal interactions along the chromosomes, are enhanced. However, interactions within pericentromeres and those between distal intra-chromosomal regions are decreased. Many inter-chromosomal interactions, including those within the KNOT, are also reduced. Furthermore, heat activation of TEs exhibits a high correlation with the reduction of chromosomal interactions involving pericentromeres, the KNOT, the knob, and the upstream and downstream flanking regions of the activated TEs. Together, our results provide insights into the relationship between TE activation and 3D genome reorganization.

The genetics of evolutionary radiations.

With the realization that much of the biological diversity on Earth has been generated by discrete evolutionary radiations, there has been a rapid increase in research into the biotic (key innovations) and abiotic (key environments) circumstances in which such radiations took place. Here we focus on the potential importance of population genetic structure and trait genetic architecture in explaining radiations. We propose a verbal model describing the stages of an evolutionary radiation: first invading a suitable adaptive zone and expanding both spatially and ecologically through this zone; secondly, diverging genetically into numerous distinct populations; and, finally, speciating. There are numerous examples of the first stage; the difficulty, however, is explaining how genetic diversification can take place from the establishment of a, presumably, genetically depauperate population in a new adaptive zone. We explore the potential roles of epigenetics and transposable elements (TEs), of neutral process such as genetic drift in combination with trait genetic architecture, of gene flow limitation through isolation by distance (IBD), isolation by ecology and isolation by colonization, the possible role of intra-specific competition, and that of admixture and hybridization in increasing the genetic diversity of the founding populations. We show that many of the predictions of this model are corroborated. Most radiations occur in complex adaptive zones, which facilitate the establishment of many small populations exposed to genetic drift and divergent selection. We also show that many radiations (especially those resulting from long-distance dispersal) were established by polyploid lineages, and that many radiating lineages have small genome sizes. However, there are several other predictions which are not (yet) possible to test: that epigenetics has played a role in radiations, that radiations occur more frequently in clades with small gene flow distances, or that the ancestors of radiations had large fundamental niches. At least some of these may be testable in the future as more genome and epigenome data become available. The implication of this model is that many radiations may be hard polytomies because the genetic divergence leading to speciation happens within a very short time, and that the divergence history may be further obscured by hybridization. Furthermore, it suggests that only lineages with the appropriate genetic architecture will be able to radiate, and that such a radiation will happen in a meta-population environment. Understanding the genetic architecture of a lineage may be an essential part of accounting for why some lineages radiate, and some do not.

Silencing of Euchromatic Transposable Elements as a Consequence of Nuclear Lamina Dysfunction.

Transposable elements (TEs) are mobile genomic sequences that are normally repressed to avoid proliferation and genome instability. Gene silencing mechanisms repress TEs by RNA degradation or heterochromatin formation. Heterochromatin maintenance is therefore important to keep TEs silent. Loss of heterochromatic domains has been linked to lamin mutations, which have also been associated with derepression of TEs. In fact, lamins are structural components of the nuclear lamina (NL), which is considered a pivotal structure in the maintenance of heterochromatin domains at the nuclear periphery in a silent state. Here, we show that a lethal phenotype associated with Lamin loss-of-function mutations is influenced by Drosophilagypsy retrotransposons located in euchromatic regions, suggesting that NL dysfunction has also effects on active TEs located in euchromatic loci. In fact, expression analysis of different long terminal repeat (LTR) retrotransposons and of one non-LTR retrotransposon located near active genes shows that Lamin inactivation determines the silencing of euchromatic TEs. Furthermore, we show that the silencing effect on euchromatic TEs spreads to the neighboring genomic regions, with a repressive effect on nearby genes. We propose that NL dysfunction may have opposed regulatory effects on TEs that depend on their localization in active or repressed regions of the genome.

Chromatin Organization in Early Land Plants Reveals an Ancestral Association between H3K27me3, Transposons, and Constitutive Heterochromatin.

Genome packaging by nucleosomes is a hallmark of eukaryotes. Histones and the pathways that deposit, remove, and read histone modifications are deeply conserved. Yet, we lack information regarding chromatin landscapes in extant representatives of ancestors of the main groups of eukaryotes, and our knowledge of the evolution of chromatin-related processes is limited. We used the bryophyte Marchantia polymorpha, which diverged from vascular plants circa 400 mya, to obtain a whole chromosome genome assembly and explore the chromatin landscape and three-dimensional genome organization in an early diverging land plant lineage. Based on genomic profiles of ten chromatin marks, we conclude that the relationship between active marks and gene expression is conserved across land plants. In contrast, we observed distinctive features of transposons and other repetitive sequences in Marchantia compared with flowering plants. Silenced transposons and repeats did not accumulate around centromeres. Although a large fraction of constitutive heterochromatin was marked by H3K9 methylation as in flowering plants, a significant proportion of transposons were marked by H3K27me3, which is otherwise dedicated to the transcriptional repression of protein-coding genes in flowering plants. Chromatin compartmentalization analyses of Hi-C data revealed that repressed B compartments were densely decorated with H3K27me3 but not H3K9 or DNA methylation as reported in flowering plants. We conclude that, in early plants, H3K27me3 played an essential role in heterochromatin function, suggesting an ancestral role of this mark in transposon silencing.

Our Conflict with Transposable Elements and Its Implications for Human Disease.

Our genome is a historic record of successive invasions of mobile genetic elements. Like other eukaryotes, we have evolved mechanisms to limit their propagation and minimize the functional impact of new insertions. Although these mechanisms are vitally important, they are imperfect, and a handful of retroelement families remain active in modern humans. This review introduces the intrinsic functions of transposons, the tactics employed in their restraint, and the relevance of this conflict to human pathology. The most straightforward examples of disease-causing transposable elements are germline insertions that disrupt a gene and result in a monogenic disease allele. More enigmatic are the abnormal patterns of transposable element expression in disease states. Changes in transposon regulation and cellular responses to their expression have implicated these sequences in diseases as diverse as cancer, autoimmunity, and neurodegeneration. Distinguishing their epiphenomenal from their pathogenic effects may provide wholly new perspectives on our understanding of disease.