Showing papers in "Mobile Dna in 2016"

Roles for retrotransposon insertions in human disease

Abstract: Over evolutionary time, the dynamic nature of a genome is driven, in part, by the activity of transposable elements (TE) such as retrotransposons. On a shorter time scale it has been established that new TE insertions can result in single-gene disease in an individual. In humans, the non-LTR retrotransposon Long INterspersed Element-1 (LINE-1 or L1) is the only active autonomous TE. In addition to mobilizing its own RNA to new genomic locations via a “copy-and-paste” mechanism, LINE-1 is able to retrotranspose other RNAs including Alu, SVA, and occasionally cellular RNAs. To date in humans, 124 LINE-1-mediated insertions which result in genetic diseases have been reported. Disease causing LINE-1 insertions have provided a wealth of insight and the foundation for valuable tools to study these genomic parasites. In this review, we provide an overview of LINE-1 biology followed by highlights from new reports of LINE-1-mediated genetic disease in humans.

Restricting retrotransposons: a review

Abstract: Retrotransposons have generated about 40 % of the human genome. This review examines the strategies the cell has evolved to coexist with these genomic “parasites”, focussing on the non-long terminal repeat retrotransposons of humans and mice. Some of the restriction factors for retrotransposition, including the APOBECs, MOV10, RNASEL, SAMHD1, TREX1, and ZAP, also limit replication of retroviruses, including HIV, and are part of the intrinsic immune system of the cell. Many of these proteins act in the cytoplasm to degrade retroelement RNA or inhibit its translation. Some factors act in the nucleus and involve DNA repair enzymes or epigenetic processes of DNA methylation and histone modification. RISC and piRNA pathway proteins protect the germline. Retrotransposon control is relaxed in some cell types, such as neurons in the brain, stem cells, and in certain types of disease and cancer, with implications for human health and disease. This review also considers potential pitfalls in interpreting retrotransposon-related data, as well as issues to consider for future research.

Endogenous retroviral promoter exaptation in human cancer

Abstract: Cancer arises from a series of genetic and epigenetic changes, which result in abnormal expression or mutational activation of oncogenes, as well as suppression/inactivation of tumor suppressor genes. Aberrant expression of coding genes or long non-coding RNAs (lncRNAs) with oncogenic properties can be caused by translocations, gene amplifications, point mutations or other less characterized mechanisms. One such mechanism is the inappropriate usage of normally dormant, tissue-restricted or cryptic enhancers or promoters that serve to drive oncogenic gene expression. Dispersed across the human genome, endogenous retroviruses (ERVs) provide an enormous reservoir of autonomous gene regulatory modules, some of which have been co-opted by the host during evolution to play important roles in normal regulation of genes and gene networks. This review focuses on the “dark side” of such ERV regulatory capacity. Specifically, we discuss a growing number of examples of normally dormant or epigenetically repressed ERVs that have been harnessed to drive oncogenes in human cancer, a process we term onco-exaptation, and we propose potential mechanisms that may underlie this phenomenon.

The contribution of transposable elements to size variations between four teleost genomes

Abstract: Teleosts are unique among vertebrates, with a wide range of haploid genome sizes in very close lineages, varying from less than 400 mega base pairs (Mb) for pufferfish to over 3000 Mb for salmon. The cause of the difference in genome size remains largely unexplained. In this study, we reveal that the differential success of transposable elements (TEs) correlates with the variation of genome size across four representative teleost species (zebrafish, medaka, stickleback, and tetraodon). The larger genomes represent a higher diversity within each clade (superfamily) and family and a greater abundance of TEs compared with the smaller genomes; zebrafish, representing the largest genome, shows the highest diversity and abundance of TEs in its genome, followed by medaka and stickleback; while the tetraodon, representing the most compact genome, displays the lowest diversity and density of TEs in its genome. Both of Class I (retrotransposons) and Class II TEs (DNA transposons) contribute to the difference of TE accumulation of teleost genomes, however, Class II TEs are the major component of the larger teleost genomes analyzed and the most important contributors to genome size variation across teleost lineages. The hAT and Tc1/Mariner superfamilies are the major DNA transposons of all four investigated teleosts. Divergence distribution revealed contrasting proliferation dynamics both between clades of retrotransposons and between species. The TEs within the larger genomes of the zebrafish and medaka represent relatively stronger activity with an extended time period during the evolution history, in contrast with the very young activity in the smaller stickleback genome, or the very low level of activity in the tetraodon genome. Overall, our data shows that teleosts represent contrasting profiles of mobilomes with a differential density, diversity and activity of TEs. The differences in TE accumulation, dominated by DNA transposons, explain the main size variations of genomes across the investigated teleost species, and the species differences in both diversity and activity of TEs contributed to the variations of TE accumulations across the four teleost species. TEs play major roles in teleost genome evolution.

Evidence for L1-associated DNA rearrangements and negligible L1 retrotransposition in glioblastoma multiforme

Abstract: LINE-1 (L1) retrotransposons are a notable endogenous source of mutagenesis in mammals. Notably, cancer cells can support unusual L1 retrotransposition and L1-associated sequence rearrangement mechanisms following DNA damage. Recent reports suggest that L1 is mobile in epithelial tumours and neural cells but, paradoxically, not in brain cancers. Here, using retrotransposon capture sequencing (RC-seq), we surveyed L1 mutations in 14 tumours classified as glioblastoma multiforme (GBM) or as a lower grade glioma. In four GBM tumours, we characterised one probable endonuclease-independent L1 insertion, two L1-associated rearrangements and one likely Alu-Alu recombination event adjacent to an L1. These mutations included PCR validated intronic events in MeCP2 and EGFR. Despite sequencing L1 integration sites at up to 250× depth by RC-seq, we found no tumour-specific, endonuclease-dependent L1 insertions. Whole genome sequencing analysis of the tumours carrying the MeCP2 and EGFR L1 mutations also revealed no endonuclease-dependent L1 insertions. In a complementary in vitro assay, wild-type and endonuclease mutant L1 reporter constructs each mobilised very inefficiently in four cultured GBM cell lines. These experiments altogether highlight the consistent absence of canonical L1 retrotransposition in GBM tumours and cultured cell lines, as well as atypical L1-associated sequence rearrangements following DNA damage in vivo.

Pinpointing the vesper bat transposon revolution using the Miniopterus natalensis genome

Abstract: Around 40 million years ago DNA transposons began accumulating in an ancestor of bats in the family Vespertilionidae. Since that time, Class II transposons have been continuously reinvading and accumulating in vespertilionid genomes at a rate that is unprecedented in mammals. Miniopterus (Miniopteridae), a genus of long-fingered bats that was recently elevated from Vespertilionidae, is the sister taxon to the vespertilionids and is often used as an outgroup when studying transposable elements in vesper bats. Previous wet-lab techniques failed to identify Helitrons, TcMariners, or hAT transposons in Miniopterus. Limitations of those methods and ambiguous results regarding the distribution of piggyBac transposons left some questions as to the distribution of Class II elements in this group. The recent release of the Miniopterus natalensis genome allows for transposable element discovery with a higher degree of precision. Here we analyze the transposable element content of M. natalensis to pinpoint with greater accuracy the taxonomic distribution of Class II transposable elements in bats. These efforts demonstrate that, compared to the vespertilionids, Class II TEs are highly mutated and comprise only a small portion of the M. natalensis genome. Despite the limited Class II content, M. natalensis possesses a limited number of lineage-specific, low copy number piggyBacs and shares several TcMariner families with vespertilionid bats. Multiple efforts to identify Helitrons, one of the major TE components of vesper bat genomes, using de novo repeat identification and structural based searches failed. These observations combined with previous results inform our understanding of the events leading to the unique Class II element acquisition that characterizes vespertilionids. While it appears that a small number of TcMariner and piggyBac elements were deposited in the ancestral Miniopterus + vespertilionid genome, these elements are not present in M. natalensis genome at high copy number. Instead, this work indicates that the vesper bats alone experienced the expansion of TEs ranging from Helitrons to piggyBacs to hATs.

Somatic retrotransposition is infrequent in glioblastomas

Abstract: Gliomas are the most common primary brain tumors in adults. We sought to understand the roles of endogenous transposable elements in these malignancies by identifying evidence of somatic retrotransposition in glioblastomas (GBM). We performed transposon insertion profiling of the active subfamily of Long INterspersed Element-1 (LINE-1) elements by deep sequencing (TIPseq) on genomic DNA of low passage oncosphere cell lines derived from 7 primary GBM biopsies, 3 secondary GBM tissue samples, and matched normal intravenous blood samples from the same individuals. We found and PCR validated one somatically acquired tumor-specific insertion in a case of secondary GBM. No LINE-1 insertions present in primary GBM oncosphere cultures were missing from corresponding blood samples. However, several copies of the element (11) were found in genomic DNA from blood and not in the oncosphere cultures. SNP 6.0 microarray analysis revealed deletions or loss of heterozygosity in the tumor genomes over the intervals corresponding to these LINE-1 insertions. These findings indicate that LINE-1 retrotransposon can act as an infrequent insertional mutagen in secondary GBM, but that retrotransposition is uncommon in these central nervous system tumors as compared to other neoplasias.

Active recombinant Tol2 transposase for gene transfer and gene discovery applications

Abstract: The revolutionary concept of “jumping genes” was conceived by McClintock in the late 1940s while studying the Activator/Dissociation (Ac/Ds) system in maize. Transposable elements (TEs) represent the most abundant component of many eukaryotic genomes. Mobile elements are a driving force of eukaryotic genome evolution. McClintock’s Ac, the autonomous element of the Ac/Ds system, together with hobo from Drosophila and Tam3 from snapdragon define an ancient and diverse DNA transposon superfamily named hAT. Other members of the hAT superfamily include the insect element Hermes and Tol2 from medaka. In recent years, genetic tools derived from the ‘cut’ and ‘paste’ Tol2 DNA transposon have been widely used for genomic manipulation in zebrafish, mammals and in cells in vitro. We report the purification of a functional recombinant Tol2 protein from E.coli. We demonstrate here that following microinjection using a zebrafish embryo test system, purified Tol2 transposase protein readily catalyzes gene transfer in both somatic and germline tissues in vivo. We show that purified Tol2 transposase can promote both in vitro cutting and pasting in a defined system lacking other cellular factors. Notably, our analysis of Tol2 transposition in vitro reveals that the target site preference observed for Tol2 in complex host genomes is maintained using a simpler target plasmid test system, indicating that the primary sequence might encode intrinsic cues for transposon integration. This active Tol2 protein is an important new tool for diverse applications including gene discovery and molecular medicine, as well as for the biochemical analysis of transposition and regulation of hAT transposon/genome interactions. The measurable but comparatively modest insertion site selection bias noted for Tol2 is largely determined by the primary sequence encoded in the target sequence as assessed through studying Tol2 protein-mediated transposition in a cell-free system.

Isolation and characterization of putative functional long terminal repeat retrotransposons in the Pyrus genome

Abstract: Long terminal repeat (LTR)-retrotransposons constitute 42.4 % of the genome of the ‘Suli’ pear (Pyrus pyrifolia white pear group), implying that retrotransposons have played important roles in Pyrus evolution. Therefore, further analysis of retrotransposons will enhance our understanding of the evolutionary history of Pyrus. We identified 1836 LTR-retrotransposons in the ‘Suli’ pear genome, of which 440 LTR-retrotransposons were predicted to contain at least two of three gene models (gag, integrase and reverse transcriptase). Because these were most likely to be functional transposons, we focused our analyses on this set of 440. Most of the LTR-retrotransposons were estimated to have inserted into the genome less than 2.5 million years ago. Sequence analysis showed that the reverse transcriptase component of the identified LTR-retrotransposons was highly heterogeneous. Analyses of transcripts assembled from RNA-Seq databases of two cultivars of Pyrus species showed that LTR-retrotransposons were expressed in the buds and fruit of Pyrus. A total of 734 coding sequences in the ‘Suli’ genome were disrupted by the identified LTR-retrotransposons. Five high-copy-number LTR-retrotransposon families were identified in Pyrus. These families were rarely found in the genomes of Malus and Prunus, but were distributed extensively in Pyrus and abundance varied between species. We identified potentially functional, full-length LTR-retrotransposons with three gene models in the ‘Suli’ genome. The analysis of RNA-seq data demonstrated that these retrotransposons are expressed in the organs of pears. The differential copy number of LTR-retrotransposon families between Pyrus species suggests that the transposition of retrotransposons is an important evolutionary force driving the genetic divergence of species within the genus.

Genomic analysis of mouse VL30 retrotransposons.

Abstract: Retrotransposons are mobile elements that have a high impact on shaping the mammalian genomes. Since the availability of whole genomes, genomic analyses have provided novel insights into retrotransposon biology. However, many retrotransposon families and their possible genomic impact have not yet been analysed. Here, we analysed the structural features, the genomic distribution and the evolutionary history of mouse VL30 LTR-retrotransposons. In total, we identified 372 VL30 sequences categorized as 86 full-length and 49 truncated copies as well as 237 solo LTRs, with non-random chromosomal distribution. Full-length VL30s were highly conserved elements with intact retroviral replication signals, but with no protein-coding capacity. Analysis of LTRs revealed a high number of common transcription factor binding sites, possibly explaining the known inducible and tissue-specific expression of individual elements. The overwhelming majority of full-length and truncated elements (82/86 and 40/49, respectively) contained one or two specific motifs required for binding of the VL30 RNA to the poly-pyrimidine tract-binding protein-associated splicing factor (PSF). Phylogenetic analysis revealed three VL30 groups with the oldest emerging ~17.5 Myrs ago, while the other two were characterized mostly by new genomic integrations. Most VL30 sequences were found integrated either near, adjacent or inside transcription start sites, or into introns or at the 3′ end of genes. In addition, a significant number of VL30s were found near Krueppel-associated box (KRAB) genes functioning as potent transcriptional repressors. Collectively, our study provides data on VL30s related to their: (a) number and structural features involved in their transcription that play a role in steroidogenesis and oncogenesis; (b) evolutionary history and potential for retrotransposition; and (c) unique genomic distribution and impact on gene expression.

The intron-enriched HERV-K(HML-10) family suppresses apoptosis, an indicator of malignant transformation

Abstract: Human endogenous retroviruses (HERVs) constitute 8% of the human genome and contribute substantially to the transcriptome. HERVs have been shown to generate RNAs that modulate host gene expression. However, experimental evidence for an impact of these regulatory transcripts on the cellular phenotype has been lacking. We characterized the previously little described HERV-K(HML-10) endogenous retrovirus family on a genome-wide scale. HML-10 invaded the ancestral genome of Old World monkeys about 35 Million years ago and is enriched within introns of human genes when compared to other HERV families. We show that long terminal repeats (LTRs) of HML-10 exhibit variable promoter activity in human cancer cell lines. One identified HML-10 LTR-primed RNA was in opposite orientation to the pro-apoptotic Death-associated protein 3 (DAP3). In HeLa cells, experimental inactivation of HML-10 LTR-primed transcripts induced DAP3 expression levels, which led to apoptosis. Its enrichment within introns suggests that HML-10 may have been evolutionary co-opted for gene regulation more than other HERV families. We demonstrated such a regulatory activity for an HML-10 RNA that suppressed DAP3-mediated apoptosis in HeLa cells. Since HML-10 RNA appears to be upregulated in various tumor cell lines and primary tumor samples, it may contribute to evasion of apoptosis in malignant cells. However, the overall weak expression of HML-10 transcripts described here raises the question whether our result described for HeLa represent a rare event in cancer. A possible function in other cells or tissues requires further investigation.

Convergent evolution of tRNA gene targeting preferences in compact genomes.

Abstract: In gene-dense genomes, mobile elements are confronted with highly selective pressure to amplify without causing excessive damage to the host. The targeting of tRNA genes as potentially safe integration sites has been developed by retrotransposons in various organisms such as the social amoeba Dictyostelium discoideum and the yeast Saccharomyces cerevisiae. In D. discoideum, tRNA gene-targeting retrotransposons have expanded to approximately 3 % of the genome. Recently obtained genome sequences of species representing the evolutionary history of social amoebae enabled us to determine whether the targeting of tRNA genes is a generally successful strategy for mobile elements to colonize compact genomes. During the evolution of dictyostelids, different retrotransposon types independently developed the targeting of tRNA genes at least six times. DGLT-A elements are long terminal repeat (LTR) retrotransposons that display integration preferences ~15 bp upstream of tRNA gene-coding regions reminiscent of the yeast Ty3 element. Skipper elements are chromoviruses that have developed two subgroups: one has canonical chromo domains that may favor integration in centromeric regions, whereas the other has diverged chromo domains and is found ~100 bp downstream of tRNA genes. The integration of D. discoideum non-LTR retrotransposons ~50 bp upstream (TRE5 elements) and ~100 bp downstream (TRE3 elements) of tRNA genes, respectively, likely emerged at the root of dictyostelid evolution. We identified two novel non-LTR retrotransposons unrelated to TREs: one with a TRE5-like integration behavior and the other with preference ~4 bp upstream of tRNA genes. Dictyostelid retrotransposons demonstrate convergent evolution of tRNA gene targeting as a probable means to colonize the compact genomes of their hosts without being excessively mutagenic. However, high copy numbers of tRNA gene-associated retrotransposons, such as those observed in D. discoideum, are an exception, suggesting that the targeting of tRNA genes does not necessarily favor the amplification of position-specific integrating elements to high copy numbers under the repressive conditions that prevail in most host cells.

Phylogenomic analysis reveals genome-wide purifying selection on TBE transposons in the ciliate Oxytricha.

Abstract: Transposable elements are a major player contributing to genetic variation and shaping genome evolution. Multiple independent transposon domestication events have occurred in ciliates, recruiting transposases to key roles in cellular processes. In the ciliate Oxytricha trifallax, the telomere-bearing elements (TBE), a Tc1/mariner transposon, occupy a significant portion of the germline genome and are involved in programmed genome rearrangements that produce a transcriptionally active somatic nucleus from a copy of the germline nucleus during development. Here we provide a thorough characterization of the distribution and sequences of TBE transposons in the Oxytricha germline genome. We annotate more than 10,000 complete and 24,000 partial TBE sequences. TBEs cluster into four major families and display a preference for either insertion into DNA segments that are retained in the somatic genome or their maintenance at such sites. The three TBE-encoded genes in all four families display dN/dS ratios much lower than 1, suggesting genome-wide purifying selection. We also identify TBE homologs in other ciliate species for phylogenomic analysis. This paper provides genome-wide characterization of a major class of ciliate transposons. Phylogenomic analysis reveals selective constraints on transposon-encoded genes, shedding light on the evolution and domesticated functions of these transposons.

The endonuclease domain of the LINE-1 ORF2 protein can tolerate multiple mutations

Abstract: Approximately 17 % of the human genome is comprised of the Long INterspersed Element-1 (LINE-1 or L1) retrotransposon, the only currently active autonomous family of retroelements. Though L1 elements have helped to shape mammalian genome evolution over millions of years, L1 activity can also be mutagenic and result in human disease. L1 expression has the potential to contribute to genomic instability via retrotransposition and DNA double-strand breaks (DSBs). Additionally, L1 is responsible for structural genomic variations induced by other transposable elements such as Alu and SVA, which rely on the L1 ORF2 protein for their propagation. Most of the genomic damage associated with L1 activity originates with the endonuclease domain of the ORF2 protein, which nicks the DNA in preparation for target-primed reverse transcription. Bioinformatic analysis of full-length L1 loci residing in the human genome identified numerous mutations in the amino acid sequence of the ORF2 endonuclease domain. Some of these mutations were found in residues which were predicted to be phosphorylation sites for cellular kinases. We mutated several of these putative phosphorylation sites in the ORF2 endonuclease domain and investigated the effect of these mutations on the function of the full-length ORF2 protein and the endonuclease domain (ENp) alone. Most of the single and multiple point mutations that were tested did not significantly impact expression of the full-length ORF2p, or alter its ability to drive Alu retrotransposition. Similarly, most of those same mutations did not significantly alter expression of ENp, or impair its ability to induce DNA damage and cause toxicity. Overall, our data demonstrate that the full-length ORF2p or the ENp alone can tolerate several specific single and multiple point mutations in the endonuclease domain without significant impairment of their ability to support Alu mobilization or induce DNA damage, respectively.

Functional opsin retrogene in nocturnal moth

Abstract: Retrotransposed genes are different to other types of genes as they originate from a processed mRNA and are then inserted back into the genome. For a long time, the contribution of this mechanism to the origin of new genes, and hence to the evolutionary process, has been questioned as retrogenes usually lose their regulatory sequences upon insertion and generally decay into pseudogenes. In recent years, there is growing evidence, notably in mammals, that retrotransposition is an important process driving the origin of new genes, but the evidence in insects remains largely restricted to a few model species. By sequencing the messenger RNA of three developmental stages (first and fifth instar larvae and adults) of the pest Helicoverpa armigera, we identified a second, intronless, long-wavelength sensitive opsin (that we called LWS2). We then amplified the partial CDS of LWS2 retrogenes from another six noctuid moths, and investigate the phylogenetic distribution of LWS2 in 15 complete Lepidoptera and 1 Trichoptera genomes. Our results suggests that LWS2 evolved within the noctuid. Furthermore, we found that all the LWS2 opsins have an intact ORF, and have an ω-value (ω = 0.08202) relatively higher compared to their paralog LWS1 (ω = 0.02536), suggesting that LWS2 opsins were under relaxed purifying selection. Finally, the LWS2 shows temporal compartmentalization of expression. LWS2 in H. armigera in adult is expressed at a significantly lower level compared to all other opsins in adults; while in the in 1st instar stage larvae, it is expressed at a significantly higher level compared to other opsins. Together the results of our evolutionary sequence analyses and gene expression data suggest that LWS2 is a functional gene, however, the relatively low level of expression in adults suggests that LWS2 is most likely not involved in mediating the visual process.

Non-canonical Helitrons in Fusarium oxysporum

Abstract: Helitrons are eukaryotic rolling circle transposable elements that can have a large impact on host genomes due to their copy-number and their ability to capture and copy genes and regulatory elements. They occur widely in plants and animals, and have thus far been relatively little investigated in fungi. Here, we comprehensively survey Helitrons in several completely sequenced genomes representing the F. oxysporum species complex (FOSC). We thoroughly characterize 5 different Helitron subgroups and determine their impact on genome evolution and assembly in this species complex. FOSC Helitrons resemble members of the Helitron2 variant that includes Helentrons and DINEs. The fact that some Helitrons appeared to be still active in FOSC provided the opportunity to determine whether Helitrons occur as a circular intermediate in FOSC. We present experimental evidence suggesting that at least one Helitron subgroup occurs with joined ends, suggesting a circular intermediate. We extend our analyses to other Pezizomycotina and find that most fungal Helitrons we identified group phylogenetically with Helitron2 and probably have similar characteristics. FOSC genomes harbour non-canonical Helitrons that are characterized by asymmetric terminal inverted repeats, show hallmarks of recent activity and likely transpose via a circular intermediate. Bioinformatic analyses indicate that they are representative of a large reservoir of fungal Helitrons that thus far has not been characterized.

Genome ARTIST: a robust, high-accuracy aligner tool for mapping transposon insertions and self-insertions

Abstract: A critical topic of insertional mutagenesis experiments performed on model organisms is mapping the hits of artificial transposons (ATs) at nucleotide level accuracy. Mapping errors may occur when sequencing artifacts or mutations as single nucleotide polymorphisms (SNPs) and small indels are present very close to the junction between a genomic sequence and a transposon inverted repeat (TIR). Another particular item of insertional mutagenesis is mapping of the transposon self-insertions and, to our best knowledge, there is no publicly available mapping tool designed to analyze such molecular events. We developed Genome ARTIST, a pairwise gapped aligner tool which works out both issues by means of an original, robust mapping strategy. Genome ARTIST is not designed to use next-generation sequencing (NGS) data but to analyze ATs insertions obtained in small to medium-scale mutagenesis experiments. Genome ARTIST employs a heuristic approach to find DNA sequence similarities and harnesses a multi-step implementation of a Smith-Waterman adapted algorithm to compute the mapping alignments. The experience is enhanced by easily customizable parameters and a user-friendly interface that describes the genomic landscape surrounding the insertion. Genome ARTIST is functional with many genomes of bacteria and eukaryotes available in Ensembl and GenBank repositories. Our tool specifically harnesses the sequence annotation data provided by FlyBase for Drosophila melanogaster (the fruit fly), which enables mapping of insertions relative to various genomic features such as natural transposons. Genome ARTIST was tested against other alignment tools using relevant query sequences derived from the D. melanogaster and Mus musculus (mouse) genomes. Real and simulated query sequences were also comparatively inquired, revealing that Genome ARTIST is a very robust solution for mapping transposon insertions. Genome ARTIST is a stand-alone user-friendly application, designed for high-accuracy mapping of transposon insertions and self-insertions. The tool is also useful for routine aligning assessments like detection of SNPs or checking the specificity of primers and probes. Genome ARTIST is an open source software and is available for download at www.genomeartist.ro and at GitHub ( https://github.com/genomeartist/genomeartist ).

Identification of polymorphic SVA retrotransposons using a mobile element scanning method for SVA (ME-Scan-SVA).

Abstract: Mobile element insertions are a major source of human genomic variation. SVA (SINE-R/VNTR/Alu) is the youngest retrotransposon family in the human genome and a number of diseases are known to be caused by SVA insertions. However, inter-individual genomic variations generated by SVA insertions and their impacts have not been studied extensively due to the difficulty in identifying polymorphic SVA insertions. To systematically identify SVA insertions at the population level and assess their genomic impact, we developed a mobile element scanning (ME-Scan) protocol we called ME-Scan-SVA. Using a nested SVA-specific PCR enrichment method, ME-Scan-SVA selectively amplify the 5′ end of SVA elements and their flanking genomic regions. To demonstrate the utility of the protocol, we constructed and sequenced a ME-Scan-SVA library of 21 individuals and analyzed the data using a new analysis pipeline designed for the protocol. Overall, the method achieved high SVA-specificity and over >90 % of the sequenced reads are from SVA insertions. The method also had high sensitivity (>90 %) for fixed SVA insertions that contain the SVA-specific primer-binding sites in the reference genome. Using candidate locus selection criteria that are expected to have a 90 % sensitivity, we identified 151 and 29 novel polymorphic SVA candidates under relaxed and stringent cutoffs, respectively (average 12 and 2 per individual). For six polymorphic SVAs that we were able to validate by PCR, the average individual genotype accuracy is 92 %, demonstrating a high accuracy of the computational genotype calling pipeline. The new approach allows identifying novel SVA insertions using high-throughput sequencing. It is cost-effective and can be applied in large-scale population study. It also can be applied for detecting potential active SVA elements, and somatic SVA retrotransposition events in different tissues or developmental stages.

A map of mobile DNA insertions in the NCI-60 human cancer cell panel.

Abstract: The National Cancer Institute-60 (NCI-60) cell lines are among the most widely used models of human cancer They provide a platform to integrate DNA sequence information, epigenetic data, RNA and protein expression, and pharmacologic susceptibilities in studies of cancer cell biology Genome-wide studies of the complete panel have included exome sequencing, karyotyping, and copy number analyses but have not targeted repetitive sequences Interspersed repeats derived from mobile DNAs are a significant source of heritable genetic variation, and insertions of active elements can occur somatically in malignancy We used Transposon Insertion Profiling by microarray (TIP-chip) to map Long INterspersed Element-1 (LINE-1, L1) and Alu Short INterspersed Element (SINE) insertions in cancer genes in NCI-60 cells We focused this discovery effort on annotated Cancer Gene Index loci We catalogued a total of 749 and 2,100 loci corresponding to candidate LINE-1 and Alu insertion sites, respectively As expected, these numbers encompass previously known insertions, polymorphisms shared in unrelated tumor cell lines, as well as unique, potentially tumor-specific insertions We also conducted association analyses relating individual insertions to a variety of cellular phenotypes These data provide a resource for investigators with interests in specific cancer gene loci or mobile element insertion effects more broadly Our data underscore that significant genetic variation in cancer genomes is owed to LINE-1 and Alu retrotransposons Our findings also indicate that as large numbers of cancer genomes become available, it will be possible to associate individual transposable element insertion variants with molecular and phenotypic features of these malignancies

Deciphering fact from artifact when using reporter assays to investigate the roles of host factors on L1 retrotransposition

Abstract: The Long INterspersed Element-1 (L1, LINE-1) is the only autonomous mobile DNA element in humans and has generated as much as half of the genome. Due to increasing clinical interest in the roles of L1 in cancer, embryogenesis and neuronal development, it has become a priority to understand L1-host interactions and identify host factors required for its activity. Apropos to this, we recently reported that L1 retrotransposition in HeLa cells requires phosphorylation of the L1 protein ORF1p at motifs targeted by host cell proline-directed protein kinases (PDPKs), which include the family of mitogen-activated protein kinases (MAPKs). Using two engineered L1 reporter assays, we continued our investigation into the roles of MAPKs in L1 activity. We found that the MAPK p38δ phosphorylated ORF1p on three of its four PDPK motifs required for L1 activity. In addition, we found that a constitutively active p38δ mutant appeared to promote L1 retrotransposition in HeLa cells. However, despite the consistency of these findings with our earlier work, we identified some technical concerns regarding the experimental methodology. Specifically, we found that exogenous expression of p38δ appeared to affect at least one heterologous promoter in an engineered L1 reporter, as well as generate opposing effects on two different reporters. We also show that two commercially available non-targeting control (NTC) siRNAs elicit drastically different effects on the apparent retrotransposition reported by both L1 assays, which raises concerns about the use of NTCs as normalizing controls. Engineered L1 reporter assays have been invaluable for determining the functions and critical residues of L1 open reading frames, as well as elucidating many aspects of L1 replication. However, our results suggest that caution is required when interpreting data obtained from L1 reporters used in conjunction with exogenous gene expression or siRNA.

TE studies in Japan: the fourth Japanese meeting on host-transposon interactions.

Abstract: The fourth Japanese meeting entitled “Biological Function and Evolution through Interactions between Hosts and Transposable Elements (TEs)” was held on August 20–21, 2018 at the National Institute of Genetics (NIG), Mishima, Japan. The meeting was supported by NIG, and its objective was to bring together researchers who study the diverse roles of TEs in genome evolution, as well as host defense systems against TE mobility, such as chromatin modifications, small RNAs, and others. Here, we present the highlights of the talks given by 14 invited speakers. Organizers: Kenji Ichiyanagi (chief), Kuniaki Saito, and Tetsuji Kakutani.

Virus-like attachment sites as structural landmarks of plants retrotransposons

Abstract: The genomic data available nowadays has enabled the study of repetitive sequences and their relationship to viruses. Among them, long terminal repeat retrotransposons (LTR-RTs) are the largest component of most plant genomes, the Gypsy and Copia superfamilies being the most common. Recently it has been found that Del lineage, an LTR-RT of Gypsy superfamily, has putative virus-like attachment (vl-att) sites. This signature, originally described for retroviruses, is recognized by retroviral integrase conferring specificity to the integration process. Here we retrieved 26,092 putative complete LTR-RTs from 10 lineages found in 10 fully sequenced angiosperm genomes and found putative vl-att sites that are a conserved structural landmark across these genomes. Furthermore, we reveal that each plant genome has a distinguishable LTR-RT lineage amplification pattern that could be related to the vl-att sites diversity. We used these patterns to generate a specific quick-response (QR) code for each genome that could be used as a barcode of identification of plants in the future. The universal distribution of vl-att sites represents a new structural feature common to plant LTR-RTs and retroviruses. This is an important finding that expands the information about the structural similarity between LTR-RT and retroviruses. We speculate that the sequence diversity of vl-att sites could be important for the life cycle of retrotransposons, as it was shown for retroviruses. All the structural vl-att site signatures are strong candidates for further functional studies. Moreover, this is the first identification of specific LTR-RT content and their amplification patterns in a large dataset of LTR-RT lineages and angiosperm genomes. These distribution patterns could be used in the future with biotechnological identification purposes.

Alu SINE analyses of 3,000-year-old human skeletal remains: a pilot study

Abstract: As Short Interspersed Elements (SINEs), human-specific Alu elements can be used for population genetic studies. Very recent inserts are polymorphic within and between human populations. In a sample of 30 elements originating from three different Alu subfamilies, we investigated whether they are preserved in prehistorical skeletal human remains from the Bronze Age Lichtenstein cave in Lower Saxony, Germany. In the present study, we examined a prehistoric triad of father, mother and daughter. For 26 of the 30 Alu loci investigated, definite results were obtained. We were able to demonstrate that presence/absence analyses of Alu elements can be conducted on individuals who lived 3,000 years ago. The preservation of the ancient DNA (aDNA) is good enough in two out of three ancient individuals to routinely allow the amplification of 500 bp fragments. The third individual revealed less well-preserved DNA, which results in allelic dropout or complete amplification failures. We here present an alternative molecular approach to deal with these degradation phenomena by using internal Alu subfamily specific primers producing short fragments of approximately 150 bp. Our data clearly show the possibility of presence/absence analyses of Alu elements in individuals from the Lichtenstein cave. Thus, we demonstrate that our method is reliably applicable for aDNA samples with good or moderate DNA preservation. This method will be very useful for further investigations with more Alu loci and larger datasets. Human population genetic studies and other large-scale investigations would provide insight into Alu SINE-based microevolutionary processes in humans during the last few thousand years and help us comprehend the evolutionary dynamics of our genome.

International Congress on Transposable elements (ICTE 2016) in Saint Malo: mobile elements under the sun of Brittany.

Abstract: The third international conference on Transposable Elements (ICTE) was held 16–19 April 2016 in Saint Malo, France. Organized by the French Transposition Community (Research group of the CNRS: “Mobile genetic elements: from mechanism to populations, an integrative approach”) and the French Society of Genetics, the conference’s goal was to bring together researchers who study transposition in diverse organisms, using multiple experimental approaches. The meeting gathered 180 participants from all around the world. Most of them contributed through poster presentations, invited talks and short talks selected from poster abstracts. The talks were organized into six scientific sessions: “Taming mobile DNA: self and non-self recognition”; “Trans-generational inheritance”; “Mobile DNA genome structure and organization, from molecular mechanisms to applications”; “Remembrance of (retro)transposon past: mobile DNA in genome evolution”; and finally “The yin and the yang of mobile DNA in human health”.

Distribution of the DNA transposon family, Pokey in the Daphnia pulex species complex

Abstract: The Pokey family of DNA transposons consists of two putatively autonomous groups, PokeyA and PokeyB, and two groups of Miniature Inverted-repeat Transposable Elements (MITEs), mPok1 and mPok2. This TE family is unusual as it inserts into a specific site in ribosomal (r)DNA, as well as other locations in Daphnia genomes. The goals of this study were to determine the distribution of the Pokey family in lineages of the Daphnia pulex species complex, and to test the hypothesis that unusally high PokeyA number in some isolates of Daphnia pulicaria is the result of recent transposition. To do this, we estimated the haploid number of Pokey, mPok, and rRNA genes in 45 isolates from five Daphnia lineages using quantitative PCR. We also cloned and sequenced partial copies of PokeyA from four isolates of D. pulicaria. Haploid PokeyA and PokeyB number is generally less than 20 and tends to be higher outside rDNA in four lineages. Conversely, the number of both groups is much higher outside rDNA (~120) in D. arenata, and PokeyB is also somewhat higher inside rDNA. mPok1 was only detected in D. arenata. mPok2 occurs both outside (~30) and inside rDNA (~6) in D. arenata, but was rare (≤2) outside rDNA in the other four lineages. There is no correlation between Pokey and rRNA gene number (mean = 240 across lineages) in any lineage. Variation among cloned partial PokeyA sequences is significantly higher in isolates with high number compared to isolates with an average number. The high Pokey number outside rDNA in D. arenata and inside rDNA in some D. pulicaria isolates is consistent with a recent increase in transposition rate. The D. pulicaria increase may have been triggered by insertion of PokeyA into a region of transcriptionally active rDNA. The expansion in D. arenata (thought to be of hybrid origin) may be a consequence of release from epigenetic repression following hybridization. Previous work found D. obtusa to be very different from the D. pulex complex; mean PokeyA is higher in rDNA (~75), rDNA array size is nearly twice as large (415), and the two are positively correlated. The predominance of Pokey in only one location could be explained by purifying selection against ectopic recombination between elements inside and outside rDNA.