Long Terminal Repeat Retroelements Research Articles

Roles of endogenous retroviral elements in the establishment and maintenance of imprinted gene expression.

DNA methylation (DNAme) has long been recognized as a host defense mechanism, both in the restriction modification systems of prokaryotes as well as in the transcriptional silencing of repetitive elements in mammals. When DNAme was shown to be implicated as a key epigenetic mechanism in the regulation of imprinted genes in mammals, a parallel with host defense mechanisms was drawn, suggesting perhaps a common evolutionary origin. Here we review recent work related to this hypothesis on two different aspects of the developmental imprinting cycle in mammals that has revealed unexpected roles for long terminal repeat (LTR) retroelements in imprinting, both canonical and noncanonical. These two different forms of genomic imprinting depend on different epigenetic marks inherited from the mature gametes, DNAme and histone H3 lysine 27 trimethylation (H3K27me3), respectively. DNAme establishment in the maternal germline is guided by transcription during oocyte growth. Specific families of LTRs, evading silencing mechanisms, have been implicated in this process for specific imprinted genes. In noncanonical imprinting, maternally inherited histone marks play transient roles in transcriptional silencing during preimplantation development. These marks are ultimately translated into DNAme, notably over LTR elements, for the maintenance of silencing of the maternal alleles in the extraembryonic trophoblast lineage. Therefore, LTR retroelements play important roles in both establishment and maintenance of different epigenetic pathways leading to imprinted expression during development. Because such elements are mobile and highly polymorphic among different species, they can be coopted for the evolution of new species-specific imprinted genes.

Marsupial satellite DNA as faithful reflections of long-terminal repeat retroelement structure.

Long terminal repeat (LTR) retroelements, including endogenous retroviruses, are one of the origins of satellite DNAs. However, the vast majority of satellite DNAs originating from LTR retroelements consists of parts of the element. In addition, they frequently contain sequences unrelated to that element. Here we report a novel marsupial satellite DNA (named walbRep) that contains, and consists solely of, the entire sequence of an LTR retroelement (the walb element). As is common with LTR retroelements, walb copies exhibit length variation. We focused on the abundance of copies of a specific length (2.7kb) in the genome of the red-necked wallaby. Cloning and analyses of long genomic DNA fragments revealed a satellite DNA in which the LTR sequence (0.4kb) and the sequence of the internal region of a nonautonomous walb copy (2.3kb) were repeated alternately. The junctions between these two components exhibited the same end-to-end arrangements as those in the walb element. This satellite organization could be accounted for by a simple formation model that includes slippage during chromosome pairing followed by homologous recombination but does not invoke any other types of rearrangements. We discuss the possible reasons why satellite DNAs having such structures are rarely found in mammals.

Genome-Wide Analysis of Transposable Elements and Satellite DNAs in Spinacia Species to Shed Light on Their Roles in Sex Chromosome Evolution.

Sex chromosome evolution has mostly been studied in species with heteromorphic sex chromosomes. The Spinacia genus serves as an ideal model for investigating evolutionary mechanisms underlying the transition from homomorphic to heteromorphic sex chromosomes. Among evolutionary factors, repetitive sequences play multiple roles in sex chromosome evolution while their forces have not been fully explored in Spinacia species. Here, we identified major repetitive sequence classes in male and female genomes of Spinacia species and their ancestral relative sugar beet to elucidate the evolutionary processes of sex chromosome evolution using next-generation sequencing (NGS) data. Comparative analysis revealed that the repeat elements of Spinacia species are considerably higher than of sugar beet, especially the Ty3/Gypsy and Ty1/Copia retrotransposons. The long terminal repeat retroelements (LTR) Angela, Athila, and Ogre may be accounted for the higher proportion of repeats in the spinach genome. Comparison of the repeats proportion between female and male genomes of three Spinacia species indicated the different representation in Spinacia tetrandra samples but not in the S. oleracea or S. turkestanica samples. From these results, we speculated that emergence of repetitive DNA sequences may correlate the formation of sex chromosome and the transition from homomorphic sex chromosomes to heteromorphic sex chromosomes as heteromorphic sex chromosomes exclusively existed in Spinacia tetrandra. Three novel sugar beet-specific satellites were identified and confirmed by fluorescence in situ hybridization (FISH); six out of eight new spinach-specific satellites were mapped to the short arm of sex chromosomes. A total of 141 copies of SolSat01-171-s were found in the sex determination region (SDR). Thus, the accumulation of satellite DNA on the short arm of chromosome 1 may be involved in the sex chromosome evolution in Spinacia species. Our study provides a fundamental resource for understanding repeat sequences in Spinacia species and their roles in sex chromosome evolution.

RepeatModeler2 for automated genomic discovery of transposable element families

The accelerating pace of genome sequencing throughout the tree of life is driving the need for improved unsupervised annotation of genome components such as transposable elements (TEs). Because the types and sequences of TEs are highly variable across species, automated TE discovery and annotation are challenging and time-consuming tasks. A critical first step is the de novo identification and accurate compilation of sequence models representing all of the unique TE families dispersed in the genome. Here we introduce RepeatModeler2, a pipeline that greatly facilitates this process. This program brings substantial improvements over the original version of RepeatModeler, one of the most widely used tools for TE discovery. In particular, this version incorporates a module for structural discovery of complete long terminal repeat (LTR) retroelements, which are widespread in eukaryotic genomes but recalcitrant to automated identification because of their size and sequence complexity. We benchmarked RepeatModeler2 on three model species with diverse TE landscapes and high-quality, manually curated TE libraries: Drosophila melanogaster (fruit fly), Danio rerio (zebrafish), and Oryza sativa (rice). In these three species, RepeatModeler2 identified approximately 3 times more consensus sequences matching with >95% sequence identity and sequence coverage to the manually curated sequences than the original RepeatModeler. As expected, the greatest improvement is for LTR retroelements. Thus, RepeatModeler2 represents a valuable addition to the genome annotation toolkit that will enhance the identification and study of TEs in eukaryotic genome sequences. RepeatModeler2 is available as source code or a containerized package under an open license (https://github.com/Dfam-consortium/RepeatModeler, http://www.repeatmasker.org/RepeatModeler/).

LTR retroelement expansion of the human cancer transcriptome and immunopeptidome revealed by de novo transcript assembly.

Dysregulated endogenous retroelements (EREs) are increasingly implicated in the initiation, progression, and immune surveillance of human cancer. However, incomplete knowledge of ERE activity limits mechanistic studies. By using pan-cancer de novo transcript assembly, we uncover the extent and complexity of ERE transcription. The current assembly doubled the number of previously annotated transcripts overlapping with long-terminal repeat (LTR) elements, several thousand of which were expressed specifically in one or a few related cancer types. Exemplified in melanoma, LTR-overlapping transcripts were highly predictable, disease prognostic, and closely linked with molecularly defined subtypes. They further showed the potential to affect disease-relevant genes, as well as produce novel cancer-specific antigenic peptides. This extended view of LTR elements provides the framework for functional validation of affected genes and targets for cancer immunotherapy.

Genome-Wide Analysis Elucidates the Role of CONSTANS-like Genes in Stress Responses of Cotton.

The CONSTANS (CO)-like gene family has been well studied for its role in the regulation of plant flowering time. However, their role remains poorly understood in cotton. To better understand the possible roles of CO-like in cotton, we performed a comprehensive genome-wide analysis of CO-like genes in cotton. Phylogenetic tree analysis showed that CO-like genes naturally clustered into three groups. Segmental duplication and whole genome duplication (WGD), which occurred before polyploidy, were important contributors to its expansion within the At (“t” indicates tetraploid) and Dt subgenomes, particularly in Group III. Long-terminal repeat retroelements were identified as the main transposable elements accompanying 18 genes. The genotype of GhCOL12_Dt displayed low diversity; it was a candidate involved in domestication. Selection pressure analyses indicated that relaxed purifying selection might have provided the main impetus during the evolution of CO-like genes in upland cotton. In addition, the high expression in the torus and calycle indicated that CO-like genes might affect flowering. The genes from Group II, and those from Group III involved in segmental duplication or WGD, might play important roles in response to drought and salt stress. Overall, this comprehensive genome-wide study of the CO-like gene family would facilitate further detailed studies in cotton.

Genome-Wide Study of YABBY Genes in Upland Cotton and Their Expression Patterns under Different Stresses.

Members of the YABBY gene family, a small plant-specific family of genes, have been proposed to function in specifying abaxial cell fate. Although to date little has been learned about cotton YABBY genes, completion of the cotton genome enables a comprehensive genome-wide analysis of YABBY genes in cotton. Here, a total of 12, 12, and 23 YABBY genes were identified in Gossypium arboreum (2n = 26, A2), G. raimondii (2n = 26, D5), and G. hirsutum (2n = 4x = 52, [AD]t), respectively. Sequence analysis showed that the N-terminal zinc-finger and C-terminal YABBY domains in YABBY proteins are highly conserved among cotton, Arabidopsis, and rice. Eighty-five genes from eight sequenced species naturally clustered into five groups, and the YAB2-like group could be divided into three sub-groups, indicating that YABBYs are highly conserved among the examined species. Orthologs from the At and Dt sub-genomes (where “t” indicates tetraploid) showed good collinearity, indicating that YABBY loci are highly conserved between these two sub-genomes. Whole-genome duplication was the primary cause of upland cotton YABBY gene expansion, segmental duplication played important roles in YABBY gene expansion within the At and Dt sub-genomes, and the YAB5-like group was mainly generated by segmental duplication. The long-terminal repeat retroelements Copia and Gypsy were identified as major transposable elements accompanying the appearance of duplicated YABBY genes, suggesting that transposable element expansion might be involved in gene duplication. Selection pressure analyses using PAML revealed that relaxed purifying selection might be the main impetus during evolution of YABBY genes in the examined species. Furthermore, exon/intron pattern and motif analyses indicated that genes within the same group were significantly conserved between Arabidopsis and cotton. In addition, the expression patterns in different tissues suggest that YABBY proteins may play roles in ovule development because YABBYs are highly expressed in ovules. The expression pattern of YABBY genes showed that approximately half of the YABBYs were down-regulated under different stress treatments. Collectively, our results represent a comprehensive genome-wide study of the YABBY gene family, which should be helpful in further detailed studies on the gene function and evolution of YABBY genes in cotton.

Host factors that promote retrotransposon integration are similar in distantly related eukaryotes.

Retroviruses and Long Terminal Repeat (LTR)-retrotransposons have distinct patterns of integration sites. The oncogenic potential of retrovirus-based vectors used in gene therapy is dependent on the selection of integration sites associated with promoters. The LTR-retrotransposon Tf1 of Schizosaccharomyces pombe is studied as a model for oncogenic retroviruses because it integrates into the promoters of stress response genes. Although integrases (INs) encoded by retroviruses and LTR-retrotransposons are responsible for catalyzing the insertion of cDNA into the host genome, it is thought that distinct host factors are required for the efficiency and specificity of integration. We tested this hypothesis with a genome-wide screen of host factors that promote Tf1 integration. By combining an assay for transposition with a genetic assay that measures cDNA recombination we could identify factors that contribute differentially to integration. We utilized this assay to test a collection of 3,004 S. pombe strains with single gene deletions. Using these screens and immunoblot measures of Tf1 proteins, we identified a total of 61 genes that promote integration. The candidate integration factors participate in a range of processes including nuclear transport, transcription, mRNA processing, vesicle transport, chromatin structure and DNA repair. Two candidates, Rhp18 and the NineTeen complex were tested in two-hybrid assays and were found to interact with Tf1 IN. Surprisingly, a number of pathways we identified were found previously to promote integration of the LTR-retrotransposons Ty1 and Ty3 in Saccharomyces cerevisiae, indicating the contribution of host factors to integration are common in distantly related organisms. The DNA repair factors are of particular interest because they may identify the pathways that repair the single stranded gaps flanking the sites of strand transfer following integration of LTR retroelements.

Transcriptionally active LTR retroelement-related sequences and their relationship with small RNA in moso bamboo (Phyllostachys edulis)

Many different cultivars or forms with diverse phenotypes of moso bamboo have been produced during its long cultivation history. The diverse phenotypes of moso bamboo are mainly reversible and unpredictable during cultivation, which lead to the hypothesis of their epigenetic origin. Earlier studies have shown that transposable elements might be involved in the different expression patterns of moso bamboo genes. LTR (long terminal repeat) retroelement populations are the main components of moso bamboo genomes. In the present study, a genome-wide analysis addressing their potential impact on host gene expression and the regulatory network was carried out. The results show that LTR retroelements are usually inserted far away from the gene regions. Transcriptional activity appears to be common in some moso bamboo retroelement-related sequences. The expression level and number of retroelement-related sequences tend to decrease with increasing distance from the closest genes, indicating that the interaction between retroelement-related sequences and near genes might play a role in the expression pattern of retroelement-related sequences. Retroelement-related sequences generate more than 30% of the siRNAs in moso bamboo. Both 21-nt siRNA and 24-nt siRNA mainly target within LTR regions of the bamboo LTR retroelements. Given the high copy number of LTR retroelements, the transcriptional activity of LTR retroelements, and the high number of siRNAs derived from the LTR retroelements, moso bamboo LTR retroelements might be involved in the transcriptional regulation of host genes, and this may be responsible for the diverse phenotypes of moso bamboo.

Molecular cytogenetic characterization of chromosome site-specific repetitive sequences in the Arctic lamprey (Lethenteron camtschaticum, Petromyzontidae).

All extant lamprey karyotypes are characterized by almost all dot-shaped microchromosomes. To understand the molecular basis of chromosome structure in lampreys, we performed chromosome C-banding and silver staining and chromosome mapping of the 18S–28S and 5S ribosomal RNA (rRNA) genes and telomeric TTAGGG repeats in the Arctic lamprey (Lethenteron camtschaticum). In addition, we cloned chromosome site-specific repetitive DNA sequences and characterized them by nucleotide sequencing, chromosome in situ hybridization, and filter hybridization. Three types of repetitive sequences were detected; a 200-bp AT-rich repetitive sequence, LCA-EcoRIa that co-localized with the 18S–28S rRNA gene clusters of 3 chromosomal pairs; a 364-bp AT-rich LCA-EcoRIb sequence that showed homology to the EcoRI sequence family from the sea lamprey (Petromyzon marinus), which contains short repeats as centromeric motifs; and a GC-rich 702-bp LCA-ApaI sequence that was distributed on nearly all chromosomes and showed significant homology with the integrase-coding region of a Ty3/Gypsy family long terminal repeat (LTR) retrotransposon. All three repetitive sequences are highly conserved within the Petromyzontidae or within Petromyzontidae and Mordaciidae. Molecular cytogenetic characterization of these site-specific repeats showed that they may be correlated with programed genome rearrangement (LCA-EcoRIa), centromere structure and function (LCA-EcoRIb), and site-specific amplification of LTR retroelements through homogenization between non-homologous chromosomes (LCA-ApaI).

Heterogeneous evolution of Ty3-gypsy retroelements among bamboo species.

Ty3-gypsy long-terminal repeat retroelements are ubiquitously found in many plant genomes. This study reports the occurrence of heterogeneous Ty3-gypsy retroelements in four representative bamboo species: Phyllostachys heterocycla (Carr.) Mitford cv. pubescens, P. heterocycla (Carr.) Mitford cv. heterocycla, Dendrocalamopsis oldhami, and Pleioblastus fortunei. Using degenerate oligonucleotide primers corresponding to the conserved domains of reverse transcriptase (rt) genes of Ty3-gypsy retroelements, 165 distinct sequences were amplified from genomic DNA. The length of the nucleotide sequences varied from 366 to 438 bp. The sequences demonstrated a high heterogeneity, with homology ranging from 52.2 to 99.8%. A phylogenetic tree was constructed, including Arabidopsis thaliana and Oryza sativa. Bamboo Ty3-gypsy sequences formed three distinct retroelement clusters (gypsy I-III). Further analysis indicated that there were not only nearly identical Ty3-gypsy retroelements found in distantly related species, but also highly diverse Ty3-gypsy retroelements observed in closely related species. The results of this study provide genetic and evolutionary information about the bamboo genome that could contribute to further studies of repetitive elements in bamboo as well as in other species.

The Ty1-copia LTR retroelement family PARTC is highly conserved in conifers over 200 MY of evolution

Long Terminal Repeat retroelements (LTR-RTs) are a major component of many plant genomes. Although well studied and described in angiosperms, their features and dynamics are poorly understood in gymnosperms. Representative complete copies of a Ty1-copia element isolate in Picea abies and named PARTC were identified in six other conifer species (Picea glauca, Pinus sylvestris, Pinus taeda, Abies sibirica, Taxus baccata and Juniperus communis) covering more than 200 million years of evolution. Here we characterized the structure of this element, assessed its abundance across conifers, studied the modes and timing of its amplification, and evaluated the degree of conservation of its extant copies at nucleotide level over distant species. We demonstrated that the element is ancient, abundant, widespread and its paralogous copies are present in the genera Picea, Pinus and Abies as an LTR-RT family. The amplification leading to the extant copies of PARTC occurred over long evolutionary times spanning 10s of MY and mostly took place after the speciation of the conifers analyzed. The level of conservation of PARTC is striking and may be explained by low substitution rates and limited removal mechanisms for LTR-RTs. These PARTC features and dynamics are representative of a more general scenario for LTR-RTs in gymnosperms quite different from that characterizing the vast majority of LTR-RT elements in angiosperms.

A computational study of the dynamics of LTR retrotransposons in the Populus trichocarpa genome

Retrotransposons are an ubiquitous component of plant genomes, especially abundant in species with large genomes. Populus trichocarpa has a relatively small genome, which was entirely sequenced; however, studies focused on poplar retrotransposons dynamics are rare. With the aim to study the retrotransposon component of the poplar genome, we have scanned the complete genome sequence searching full-length long-terminal repeat (LTR) retrotransposons, i.e., characterised by two long terminal repeats at the 5′ and 3′ ends. A computational approach based on detection of conserved structural features, on building multiple alignments, and on similarity searches was used to identify 1,479 putative full-length LTR retrotransposons. Ty1-copia elements were more numerous than Ty3-gypsy. However, many LTR retroelements were not assigned to any superfamily because lacking of diagnostic features and non-autonomous. LTR retrotransposon remnants were by far more numerous than full-length elements, indicating that during the evolution of poplar, large amplification of these elements was followed by DNA loss. Within superfamilies, Ty3-gypsy families are made of more members than Ty1-copia ones. Retrotransposition occurred with increasing frequency following the separation of Populus sections, with different waves of retrotransposition activity between Ty3-gypsy and Ty1-copia elements. Recently inserted elements appear more frequently expressed than older ones. Finally, different levels of activity of retrotransposons were observed according to their position and their density in the linkage groups. On the whole, the results support the view of retrotransposons as a community of different organisms in the genome, whose activity (both retrotransposition and DNA loss) has heavily impacted and probably continues to impact poplar genome structure and size.

The Reverse Transcriptase of the Tf1 Retrotransposon Has a Specific Novel Activity for Generating the RNA Self-Primer That Is Functional in cDNA Synthesis

The Tf1 retrotransposon of Schizosaccharomyces pombe represents a group of eukaryotic long terminal repeat (LTR) retroelements that, based on their sequences, were predicted to use an RNA self-primer for initiating reverse transcription while synthesizing the negative-sense DNA strand. This feature is substantially different from the one typical to retroviruses and other LTR retrotransposons that all exhibit a tRNA-dependent priming mechanism. Genetic studies have suggested that the self-primer of Tf1 can be generated by a cleavage between the 11th and 12th bases of the Tf1 RNA transcript. The in vitro data presented here show that recombinant Tf1 reverse transcriptase indeed introduces a nick at the end of a duplexed region at the 5' end of Tf1 genomic RNA, substantiating the prediction that this enzyme is responsible for generating this RNA self-primer. The 3' end of the primer, generated in this manner, can then be extended upon the addition of deoxynucleoside triphosphates by the DNA polymerase activity of the same enzyme, synthesizing the negative-sense DNA strand. This functional primer must have been generated by the RNase H activity of Tf1 reverse transcriptase, since a mutant enzyme lacking this activity has lost its ability to generate the self-primer. It was also found here that the reverse transcriptases of human immunodeficiency virus type 1 and of murine leukemia virus do not exhibit this specific cleavage activity. In all, it is likely that the observed unique mechanism of self-priming in Tf1 represents an early advantageous form of initiating reverse transcription in LTR retroelements without involving cellular tRNAs.

Colinearity and its exceptions in orthologous adh regions of maize and sorghum.

Orthologous adh regions of the sorghum and maize genomes were sequenced and analyzed. Nine known or candidate genes, including adh1, were found in a 225-kilobase (kb) maize sequence. In a 78-kb space of sorghum, the nine homologues of the maize genes were identified in a colinear order, plus five additional genes. The major fraction of DNA in maize, occupying 166 kb (74%), is represented by 22 long terminal repeat (LTR) retrotransposons. About 6% of the sequence belongs to 33 miniature inverted-repeat transposable elements (MITEs), remnants of DNA transposons, 4 simple sequence repeats, and low-copy-number DNAs of unknown origin. In contrast, no LTR retroelements were detected in the orthologous sorghum region. The unconserved sorghum DNA is composed of 20 putative MITEs, transposon-like elements, 5 simple sequence repeats, and low-copy-number DNAs of unknown origin. No MITEs were discovered in the 166 kb of DNA occupied by the maize LTR retrotransposons. In both species, MITEs were found in the space between genes and inside introns, indicating specific insertion and/or retention for these elements. Two adjacent sorghum genes, including one gene missing in maize, had colinear homologues on Arabidopsis chromosome IV, suggesting two rearrangements in the sorghum and three in the maize genome in comparison to a four-gene region of Arabidopsis. Hence, multiple small rearrangements may be present even in largely colinear genomic regions. These studies revealed a much higher degree of diversity at a microstructural level than predicted by genetic mapping studies for closely related grass species, as well as for comparisons of monocots and dicots.