Shaping eukaryotic epigenetic systems by horizontal gene transfer.

DNA methylation constitutes one of the pillars of epigenetics, relying on covalent bonds for addition and/or removal of chemically distinct marks within the major groove of the double helix. DNA methyltransferases, enzymes which introduce methyl marks, initially evolved in prokaryotes as components of restriction-modification systems protecting host genomes from bacteriophages and other invading foreign DNA. In early eukaryotic evolution, DNA methyltransferases were horizontally transferred from bacteria into eukaryotes several times and independently co-opted into epigenetic regulatory systems, primarily via establishing connections with the chromatin environment. While C5-methylcytosine is the cornerstone of plant and animal epigenetics and has been investigated in much detail, the epigenetic role of other methylated bases is less clear. The recent addition of N4-methylcytosine of bacterial origin as a metazoan DNA modification highlights the prerequisites for foreign gene co-option into the host regulatory networks, and challenges the existing paradigms concerning the origin and evolution of eukaryotic regulatory systems.

To Be Mobile or Not: The Variety of Reverse Transcriptases and Their Recruitment by Host Genomes.

Reverse transcriptases (RT), or RNA-dependent DNA polymerases, are unorthodox enzymes that originally added a new angle to the conventional view of the unidirectional flow of genetic information in the cell from DNA to RNA to protein. First discovered in vertebrate retroviruses, RTs were since re-discovered in most eukaryotes, bacteria, and archaea, spanning essentially all domains of life. For retroviruses, RTs provide the ability to copy the RNA genome into DNA for subsequent incorporation into the host genome, which is essential for their replication and survival. In cellular organisms, most RT sequences originate from retrotransposons, the type of self-replicating genetic elements that rely on reverse transcription to copy and paste their sequences into new genomic locations. Some retroelements, however, can undergo domestication, eventually becoming a valuable addition to the overall repertoire of cellular enzymes. They can be beneficial yet accessory, like the diversity-generating elements, or even essential, like the telomerase reverse transcriptases. Nowadays, ever-increasing numbers of domesticated RT-carrying genetic elements are being discovered. It may be argued that domesticated RTs and reverse transcription in general is more widespread in cellular organisms than previously thought, and that many important cellular functions, such as chromosome end maintenance, may evolve from an originally selfish process of converting RNA into DNA.

An Ancient Clade of Penelope-Like Retroelements with Permuted Domains Is Present in the Green Lineage and Protists, and Dominates Many Invertebrate Genomes.

Penelope-like elements (PLEs) are an enigmatic clade of retrotransposons whose reverse transcriptases (RTs) share a most recent common ancestor with telomerase RTs. The single ORF of canonical endonuclease (EN)+ PLEs encodes RT and a C-terminal GIY-YIG EN that enables intrachromosomal integration, whereas EN- PLEs lack EN and are generally restricted to chromosome termini. EN+ PLEs have only been found in animals, except for one case of horizontal transfer to conifers, whereas EN- PLEs occur in several kingdoms. Here, we report a new, deep-branching PLE clade with a permuted domain order, whereby an N-terminal GIY-YIG EN is linked to a C-terminal RT by a short domain with a characteristic CxC motif. These N-terminal EN+ PLEs share a structural organization, including pseudo-LTRs and complex tandem/inverted insertions, with canonical EN+ PLEs from Penelope/Poseidon, Neptune, and Nematis clades, and show insertion bias for microsatellites, but lack canonical hammerhead ribozyme motifs. However, their phylogenetic distribution is much broader. The Naiads, found in numerous invertebrate phyla, can reach tens of thousands of copies per genome. In spiders and clams, Naiads independently evolved to encode selenoproteins containing multiple selenocysteines. Chlamys, which lack the CCHH motif universal to PLE ENs, occur in green algae, spike mosses (targeting ribosomal DNA), and slime molds. Unlike canonical PLEs, RTs of N-terminal EN+ PLEs contain the insertion-in-fingers domain (IFD), strengthening the link between PLEs and telomerases. Additionally, we describe Hydra, a novel metazoan C-terminal EN+ clade. Overall, we conclude that PLE diversity, taxonomic distribution, and abundance are comparable with non-LTR and LTR-retrotransposons.

Neutral Theory, Transposable Elements, and Eukaryotic Genome Evolution.

Among the multitude of papers published yearly in scientific journals, precious few publications may be worth looking back in half a century to appreciate the significance of the discoveries that would later become common knowledge and get a chance to shape a field or several adjacent fields. Here, Kimura's fundamental concept of neutral mutation-random drift, which was published 50 years ago, is re-examined in light of its pervasive influence on comparative genomics and, more specifically, on the contribution of transposable elements to eukaryotic genome evolution.

Giant Reverse Transcriptase-Encoding Transposable Elements at Telomeres.

Transposable elements are omnipresent in eukaryotic genomes and have a profound impact on chromosome structure, function and evolution. Their structural and functional diversity is thought to be reasonably well-understood, especially in retroelements, which transpose via an RNA intermediate copied into cDNA by the element-encoded reverse transcriptase, and are characterized by a compact structure. Here, we report a novel type of expandable eukaryotic retroelements, which we call Terminons. These elements can attach to G-rich telomeric repeat overhangs at the chromosome ends, in a process apparently facilitated by complementary C-rich repeats at the 3'-end of the RNA template immediately adjacent to a hammerhead ribozyme motif. Terminon units, which can exceed 40 kb in length, display an unusually complex and diverse structure, and can form very long chains, with host genes often captured between units. As the principal polymerizing component, Terminons contain Athena reverse transcriptases previously described in bdelloid rotifers and belonging to the enigmatic group of Penelope-like elements, but can additionally accumulate multiple cooriented ORFs, including DEDDy 3'-exonucleases, GDSL esterases/lipases, GIY-YIG-like endonucleases, rolling-circle replication initiator (Rep) proteins, and putatively structural ORFs with coiled-coil motifs and transmembrane domains. The extraordinary length and complexity of Terminons and the high degree of interfamily variability in their ORF content challenge the current views on the structural organization of eukaryotic retroelements, and highlight their possible connections with the viral world and the implications for the elevated frequency of gene transfer.

Biochemical properties of bacterial reverse transcriptase-related (rvt) gene products: multimerization, protein priming, and nucleotide preference.

Cellular reverse transcriptase-related (rvt) genes represent a novel class of reverse transcriptases (RTs), which are only distantly related to RTs of retrotransposons and retroviruses, but, similarly to telomerase RTs, are immobilized in the genome as single-copy genes. They have been preserved by natural selection throughout the evolutionary history of large taxonomic groups, including most fungi, a few plants and invertebrates, and even certain bacteria, being the only RTs present across different domains of life. Bacterial rvt genes are exceptionally rare but phylogenetically related, consistent with common origin of bacterial rvt genes rather than eukaryote-to-bacteria transfer. To investigate biochemical properties of bacterial RVTs, we conducted in vitro studies of recombinant HaRVT protein from the filamentous gliding bacterium Herpetosiphon aurantiacus (Chloroflexi). Although HaRVT does not utilize externally added standard primer-template combinations, in the presence of divalent manganese, it can polymerize very short products, using dNTPs rather than NTPs, with a strong preference for dCTP incorporation. Furthermore, we investigated the highly conserved N- and C-terminal domains, which distinguish RVT proteins from other RTs. We show that the N-terminal coiled-coil motif, which is present in nearly all RVTs, is responsible for the ability of HaRVT to multimerize in solution, forming up to octamers. The C-terminal domain may be capable of protein priming, which is abolished by site-directed mutagenesis of the catalytic aspartate and greatly reduced in the absence of the conserved tyrosine residues near the C-terminus. The unusual biochemical properties displayed by RVT in vitro will provide the basis for understanding its biological function in vivo.

Mobile genetic elements: in silico, in vitro, in vivo.

Mobile genetic elements (MGEs), also called transposable elements (TEs), represent universal components of most genomes and are intimately involved in nearly all aspects of genome organization, function and evolution. However, there is currently a gap between the fast pace of TE discovery in silico, driven by the exponential growth of comparative genomic studies, and a limited number of experimental models amenable to more traditional in vitro and in vivo studies of structural, mechanistic and regulatory properties of diverse MGEs. Experimental and computational scientists came together to bridge this gap at a recent conference, 'Mobile Genetic Elements: in silico, in vitro, in vivo', held at the Marine Biological Laboratory (MBL) in Woods Hole, MA, USA.

A widespread class of reverse transcriptase-related cellular genes.

Reverse transcriptases (RTs) polymerize DNA on RNA templates. They fall into several structurally related but distinct classes and form an assemblage of RT-like enzymes that, in addition to RTs, also includes certain viral RNA-dependent RNA polymerases (RdRP) synthesizing RNA on RNA templates. It is generally believed that most RT-like enzymes originate from retrotransposons or viruses and have no specific function in the host cell, with telomerases being the only notable exception. Here we report on the discovery and properties of a unique class of RT-related cellular genes collectively named rvt. We present evidence that rvts are not components of retrotransposons or viruses, but single-copy genes with a characteristic domain structure that may contain introns in evolutionarily conserved positions, occur in syntenic regions, and evolve under purifying selection. These genes can be found in all major taxonomic groups including protists, fungi, animals, plants, and even bacteria, although they exhibit patchy phylogenetic distribution in each kingdom. We also show that the RVT protein purified from one of its natural hosts, Neurospora crassa, exists in a multimeric form and has the ability to polymerize NTPs as well as dNTPs in vitro, with a strong preference for NTPs, using Mn(2+) as a cofactor. The existence of a previously unknown class of single-copy RT-related genes calls for reevaluation of the current views on evolution and functional roles of RNA-dependent polymerases in living cells.

Internal initiation of reverse transcription in a Penelope-like retrotransposon.

Eukaryotic retroelements are generally divided into two classes: long terminal repeat (LTR) retrotransposons and non-LTR retrotransposons. A third class of eukaryotic retroelement, the Penelope-like elements (PLEs), has been well-characterized bioinformatically, but relatively little is known about the transposition mechanism of these elements. PLEs share some features with the R2 retrotransposon from Bombyx mori, which uses a target-primed reverse transcription (TPRT) mechanism, but their distinct phylogeny suggests PLEs may utilize a novel mechanism of mobilization. Using protein purified from E. coli, we report unique in vitro properties of a PLE from the green anole (Anolis carolinensis), revealing mechanistic aspects not shared by other retrotransposons. We found that reverse transcription is initiated at two adjacent sites within the transposon RNA that is not homologous to the cleaved DNA, a feature that is reflected in the genomic "tail" signature shared between and unique to PLEs. Our results for the first active PLE in vitro provide a starting point for understanding PLE mobilization and biology.

Retroelements containing introns in diverse invertebrate taxa.

We report that two structurally similar transposable elements containing reverse transcriptase (RT), Penelope in Drosophila virilis and Athena in bdelloid rotifers, have proliferated as copies containing introns. The ability of Penelope-like elements (PLEs) to retain introns, their separate phylogenetic placement and their peculiar structural features make them a novel class of eukaryotic retroelements.

An Overview of Best Practices for Transposable Element Identification, Classification, and Annotation in Eukaryotic Genomes.

Transposable elements (TEs) exert an increasingly diverse spectrum of influences on eukaryotic genome structure, function, and evolution. A deluge of genomic, transcriptomic, and proteomic data provides the foundation for turning essentially any non-model eukaryotic species into an emerging model to study any and all aspects of organismal biology, ultimately shaping future directions for biomedical, environmental, and biodiversity research. However, identification and annotation of the mobile genome component still lags behind the standards accepted for host gene annotation. To achieve the objective of providing every genome project with a comprehensive description of its mobilome component in addition to the standard genic and transcriptomic datasets, each step of TE identification, classification, and annotation should be focused on improving TE boundary designation, reducing identification error rates, and providing accurate information on the type and integrity of TE insertions. Here, we offer practical advice for generating TE models in de novo assemblies for non-model organisms, provide step-by-step instructions to guide inexperienced TE annotators through some of the commonly utilized TE analysis pipelines, and entertain suggestions for tool improvement which could be implemented by interested developers.

Meeting report: transposable elements at the crossroads of evolution, health and disease 2023.

The conference "Transposable Elements at the Crossroads of Evolution, Health and Disease" was hosted by Keystone Symposia in Whistler, British Columbia, Canada, on September 3-6, 2023, and was organized by Kathleen Burns, Harmit Malik and Irina Arkhipova. The central theme of the meeting was the incredible diversity of ways in which transposable elements (TEs) interact with the host, from disrupting the existing genes and pathways to creating novel gene products and expression patterns, enhancing the repertoire of host functions, and ultimately driving host evolution. The meeting was organized into six plenary sessions and two afternoon workshops with a total of 50 invited and contributed talks, two poster sessions, and a career roundtable. The topics ranged from TE roles in normal and pathological processes to restricting and harnessing TE activity based on mechanistic insights gained from genetic, structural, and biochemical studies.

Bacterial N4-methylcytosine as an epigenetic mark in eukaryotic DNA.

DNA modifications are used to regulate gene expression and defend against invading genetic elements. In eukaryotes, modifications predominantly involve C5-methylcytosine (5mC) and occasionally N6-methyladenine (6mA), while bacteria frequently use N4-methylcytosine (4mC) in addition to 5mC and 6mA. Here we report that 4mC can serve as an epigenetic mark in eukaryotes. Bdelloid rotifers, tiny freshwater invertebrates with transposon-poor genomes rich in foreign genes, lack canonical eukaryotic C5-methyltransferases for 5mC addition, but encode an amino-methyltransferase, N4CMT, captured from bacteria >60 Mya. N4CMT deposits 4mC at active transposons and certain tandem repeats, and fusion to a chromodomain shapes its "histone-read-DNA-write" architecture recognizing silent chromatin marks. Furthermore, amplification of SETDB1 H3K9me3 histone methyltransferases yields variants preferentially binding 4mC-DNA, suggesting "DNA-read-histone-write" partnership to maintain chromatin-based silencing. Our results show how non-native DNA methyl groups can reshape epigenetic systems to silence transposons and demonstrate the potential of horizontal gene transfer to drive regulatory innovation in eukaryotes.

A Survey of Transposon Landscapes in the Putative Ancient Asexual Ostracod Darwinula stevensoni.

How asexual reproduction shapes transposable element (TE) content and diversity in eukaryotic genomes remains debated. We performed an initial survey of TE load and diversity in the putative ancient asexual ostracod Darwinula stevensoni. We examined long contiguous stretches of DNA in clones from a genomic fosmid library, totaling about 2.5 Mb, and supplemented these data with results on TE abundance and diversity from an Illumina draft genome. In contrast to other TE studies in putatively ancient asexuals, which revealed relatively low TE content, we found that at least 19% of the fosmid dataset and 26% of the genome assembly corresponded to known transposons. We observed a high diversity of transposon families, including LINE, gypsy, PLE, mariner/Tc, hAT, CMC, Sola2, Ginger, Merlin, Harbinger, MITEs and helitrons, with the prevalence of DNA transposons. The predominantly low levels of sequence diversity indicate that many TEs are or have recently been active. In the fosmid data, no correlation was found between telomeric repeats and non-LTR retrotransposons, which are present near telomeres in other taxa. Most TEs in the fosmid data were located outside of introns and almost none were found in exons. We also report an N-terminal Myb/SANT-like DNA-binding domain in site-specific R4/Dong non-LTR retrotransposons. Although initial results on transposable loads need to be verified with high quality draft genomes, this study provides important first insights into TE dynamics in putative ancient asexual ostracods.

Evolutionary dynamics of transposable elements in bdelloid rotifers.

Transposable elements (TEs) are selfish genomic parasites whose ability to spread autonomously is facilitated by sexual reproduction in their hosts. If hosts become obligately asexual, TE frequencies and dynamics are predicted to change dramatically, but the long-term outcome is unclear. Here, we test current theory using whole-genome sequence data from eight species of bdelloid rotifers, a class of invertebrates in which males are thus far unknown. Contrary to expectations, we find a variety of active TEs in bdelloid genomes, at an overall frequency within the range seen in sexual species. We find no evidence that TEs are spread by cryptic recombination or restrained by unusual DNA repair mechanisms. Instead, we find that that TE content evolves relatively slowly in bdelloids and that gene families involved in RNAi-mediated TE suppression have undergone significant expansion, which might mitigate the deleterious effects of active TEs and compensate for the consequences of long-term asexuality.

A single-copy IS5-like transposon in the genome of a bdelloid rotifer.

In the course of sequencing telomeric chromosomal regions of the bdelloid rotifer Adineta vaga, we encountered an unusual DNA transposon. Unlike other bdelloid and, more generally, eukaryotic transposable elements (TEs), it exhibits similarity to prokaryotic insertion sequences (ISs). Phylogenetic analysis indicates that this transposon, named IS5_Av, is related to the ISL2 group of the IS5 family of bacterial IS elements. Despite the apparent intactness of the single open reading frame coding for a DDE transposase and the perfect identity of its 213-bp terminal inverted repeats (TIRs), the element is present in only one copy per diploid genome. It does not exhibit any detectable levels of transcription, so that its transposase gene appears to be silent in the bdelloid host. Although horizontal transfers of TEs between kingdoms are not known to happen in nature, it appears likely that IS5_Av underwent integration into the A. vaga genome relatively recently, but was not successful in adapting to the new host and failed to increase in copy number. Alternatively, it might be the only known member of a novel eukaryotic DNA TE superfamily which is so rare that its other members, if any, have not yet been identified in eukaryotic genomes sequenced to date.

Genome structure of bdelloid rotifers: shaped by asexuality or desiccation?

Bdelloid rotifers are microscopic invertebrate animals best known for their ancient asexuality and the ability to survive desiccation at any life stage. Both factors are expected to have a profound influence on their genome structure. Recent molecular studies demonstrated that, although the gene-rich regions of bdelloid genomes are organized as colinear pairs of closely related sequences and depleted in repetitive DNA, subtelomeric regions harbor diverse transposable elements and horizontally acquired genes of foreign origin. Although asexuality is expected to result in depletion of deleterious transposons, only desiccation appears to have the power to produce all the uncovered genomic peculiarities. Repair of desiccation-induced DNA damage would require the presence of a homologous template, maintaining colinear pairs in gene-rich regions and selecting against insertion of repetitive DNA that might cause chromosomal rearrangements. Desiccation may also induce a transient state of competence in recovering animals, allowing them to acquire environmental DNA. Even if bdelloids engage in rare or obscure forms of sexual reproduction, all these features could still be present. The relative contribution of asexuality and desiccation to genome organization may be clarified by analyzing whole-genome sequences and comparing foreign gene and transposon content in species which lost the ability to survive desiccation.

Metagenome Proteins and Database Contamination.

Continued influx of metagenome-derived proteins with misannotated taxonomy into conventional databases, including RefSeq, threatens to eliminate the value of taxonomy identifiers. To prevent this, urgent efforts should be undertaken by submitters of metagenomic data sets as well as by database managers.

Adaptation of gene loci to heterochromatin in the course of Drosophila evolution is associated with insulator proteins.

Pericentromeric heterochromatin is generally composed of repetitive DNA forming a transcriptionally repressive environment. Dozens of genes were embedded into pericentromeric heterochromatin during evolution of Drosophilidae lineage while retaining activity. However, factors that contribute to insusceptibility of gene loci to transcriptional silencing remain unknown. Here, we find that the promoter region of genes that can be embedded in both euchromatin and heterochromatin exhibits a conserved structure throughout the Drosophila phylogeny and carries motifs for binding of certain chromatin remodeling factors, including insulator proteins. Using ChIP-seq data, we demonstrate that evolutionary gene relocation between euchromatin and pericentric heterochromatin occurred with preservation of sites of insulation of BEAF-32 in evolutionarily distant species, i.e. D. melanogaster and D. virilis. Moreover, promoters of virtually all protein-coding genes located in heterochromatin in D. melanogaster are enriched with insulator proteins BEAF-32, GAF and dCTCF. Applying RNA-seq of a BEAF-32 mutant, we show that the impairment of BEAF-32 function has a complex effect on gene expression in D. melanogaster, affecting even those genes that lack BEAF-32 association in their promoters. We propose that conserved intrinsic properties of genes, such as sites of insulation near the promoter regions, may contribute to adaptation of genes to the heterochromatic environment and, hence, facilitate the evolutionary relocation of genes loci between euchromatin and heterochromatin.

Genomic signatures of recombination in a natural population of the bdelloid rotifer Adineta vaga.

Sexual reproduction is almost ubiquitous among extant eukaryotes. As most asexual lineages are short-lived, abandoning sex is commonly regarded as an evolutionary dead end. Still, putative anciently asexual lineages challenge this view. One of the most striking examples are bdelloid rotifers, microscopic freshwater invertebrates believed to have completely abandoned sexual reproduction tens of Myr ago. Here, we compare whole genomes of 11 wild-caught individuals of the bdelloid rotifer Adineta vaga and present evidence that some patterns in its genetic variation are incompatible with strict clonality and lack of genetic exchange. These patterns include genotype proportions close to Hardy-Weinberg expectations within loci, lack of linkage disequilibrium between distant loci, incongruent haplotype phylogenies across the genome, and evidence for hybridization between divergent lineages. Analysis of triallelic sites independently corroborates these findings. Our results provide evidence for interindividual genetic exchange and recombination in A. vaga, a species previously thought to be anciently asexual.