Recent technological developments-in genomics, bioinformatics and high-throughput experimental techniques-are providing opportunities to study ongoing human transposable element (TE) activity at an unprecedented level of detail. It is now possible to characterize genome-wide collections of TE insertion sites for multiple human individuals, within and between populations, and for a variety of tissue types. Comparison of TE insertion site profiles between individuals captures the germline activity of TEs and reveals insertion site variants that segregate as polymorphisms among human populations, whereas comparison among tissue types ascertains somatic TE activity that generates cellular heterogeneity. In this review, we provide an overview of these new technologies and explore their implications for population and clinical genetic studies of human TEs. We cover both recent published results on human TE insertion activity as well as the prospects for future TE studies related to human evolution and health.
ABSTRACT Transposable elements (TEs) are mobile genetic elements that can mobilize within host genomes. As TEs comprise more than 40% of the human genome and are linked to numerous diseases, understanding their mechanisms of mobilization and regulation is important. Drosophila melanogaster is an ideal model organism for the study of eukaryotic TEs as its genome contains a diverse array of active TEs. TEs universally impact host genome size via transposition and deletion events, but may also adopt unique functional roles in host organisms. There are 2 main classes of TEs: DNA transposons and retrotransposons. These classes are further divided into subgroups of TEs with unique structural and functional characteristics, demonstrating the significant variability among these elements. Despite this variability, D. melanogaster and other eukaryotic organisms utilize conserved mechanisms to regulate TEs. This review focuses on the transposition mechanisms and regulatory pathways of TEs, and their functional roles in D. melanogaster.
A large proportion of the genome of most eukaryotic organisms consists of highly repetitive mobile genetic elements. The sum of these elements is called the "mobilome," which in eukaryotes is made up mostly of transposons. Transposable elements contribute to disease, evolution, and normal physiology by mediating genetic rearrangement, and through the "domestication" of transposon proteins for cellular functions. Although 'omics studies of mobilome genomes and transcriptomes are common, technical challenges have hampered high-throughput global proteomics analyses of transposons. In a recent paper, we overcame these technical hurdles using a technique called "proteomics informed by transcriptomics" (PIT), and thus published the first unbiased global mobilome-derived proteome for any organism (using cell lines derived from the mosquito Aedes aegypti). In this commentary, we describe our methods in more detail, and summarise our major findings. We also use new genome sequencing data to show that, in many cases, the specific genomic element expressing a given protein can be identified using PIT. This proteomic technique therefore represents an important technological advance that will open new avenues of research into the role that proteins derived from transposons and other repetitive and sequence diverse genetic elements, such as endogenous retroviruses, play in health and disease.
It is becoming increasingly recognized that Long Interspersed Nuclear Element, 1 (LINE-1), the most ubiquitous repetitive element in the mammalian genomes, plays an important role in the pathogenesis of disease and in the response to exposure to environmental stressors. Ionizing radiation is a known genotoxic stressor, but it is capable of targeting the cellular epigenome as well. Radiation-induced alterations in LINE-1 DNA methylation are the most frequently observed epigenetic effects of exposure. The extent of this aberrant DNA methylation, however, strongly depends on a number of factors, including the type and dose of radiation. Two other factors are being discussed in this commentary - the evolutionary age and type of the LINE-1 promoter, as well as the type of irradiated cell. This knowledge will further aid in elucidating the mechanisms of response to ionizing radiation exposure, as well in understanding the pathogenesis of the negative health effects associated with exposure.
The functional relationship between tRNAs and retrotransposons have been known for more than 35 years. tRNAs are used as primer molecules to guide the reverse transcription of retrotransposons. Recently, tRNAs have also emerge as important players in the postranscriptional regulation of retrotransposons by means of tRNA-derived small RNAs. This surprisingly new layer of regulation indicates that tRNAs are used both in the promotion and the suppression of the reverse transcription of retrotransposons indicating their primary role in the life cycle of LTR retrotransposons. This adds another level of translational control to tRNAs. Here we review the different known levels of interactions of tRNAs and retrotransposons and highlight the unknown parts of this interaction.
Horizontal gene transfer plays a significant role in spreading antimicrobial resistance and virulence genes throughout the genus Staphylococcus, which includes species of clinical relevance to humans and animals. While phages and plasmids are the most well-studied agents of horizontal gene transfer in staphylococci, the contribution of integrative conjugative elements (ICEs) has been mostly overlooked. Experimental work demonstrating the activity of ICEs in staphylococci remained frozen for years after initial work in the 1980s that showed Tn916 was capable of transfer from Enterococcus to Staphylococcus. However, recent work has begun to thaw this field. To date, 2 families of ICEs have been identified among staphylococci - Tn916 that includes the Tn5801 subfamily, and ICE6013 that includes at least 7 subfamilies. Both Tn5801 and ICE6013 commonly occur in clinical strains of S. aureus. Tn5801 is the most studied of the Tn916 family elements in staphylococci and encodes tetracycline resistance and a protein that, when expressed in Escherichia coli, inhibits restriction barriers to incoming DNA. ICE6013 is among the shortest known ICEs, but it still includes many uncharacterized open reading frames. This element uses an IS30-like transposase as its recombinase, providing some versatility in integration sites. ICE6013 also conjugatively transfers among receptive S. aureus strains at relatively higher frequency than Tn5801. Continued study of these mobile genetic elements may reveal the full extent to which ICEs impact horizontal gene transfer and the evolution of staphylococci.
Mobile genetic elements are near ubiquitous DNA segments that revealed a surprising variety of strategies for their propagation among prokaryotes and between eukaryotes. In bacteria, conjugative elements were shown to be key drivers of evolution and adaptation by efficiently disseminating genes involved in pathogenicity, symbiosis, metabolic pathways, and antibiotic resistance. Conjugative plasmids of the incompatibility groups A and C (A/C) are important vehicles for the dissemination of antibiotic resistance and the consequent global emergence and spread of multi-resistant pathogenic bacteria. Beyond their own mobility, A/C plasmids were also shown to drive the mobility of unrelated non-autonomous mobilizable genomic islands, which may also confer further advantageous traits. In this commentary, we summarize the current knowledge on different classes of A/C-dependent mobilizable genomic islands and we discuss other DNA hitchhikers and their implication in bacterial evolution. Furthermore, we glimpse at the complex genetic network linking autonomous and non-autonomous mobile genetic elements, and at the associated flow of genetic information between bacteria.
The somatic mobilization of transposable elements is more common than previously thought. In this review we discuss how the intensity and the biologic consequences of somatic mobilization are dependent on the transposable elements landscapes of each genome, and on the "momentum" of each particular TE with respect to the mechanisms that control its transposition and the possibility to escape this control. Additionally, the biologic consequences of somatic mobilization vary among organisms that show an early separation between the germline and somatic cells and those organisms that do not exhibit this separation or that reproduce asexually. In the former, somatic transposition can be involved in phenotypic plasticity, detrimental conditions such as disease, or processes such as aging. For the organisms without separation between the germ and soma, somatic mobilization can be a source of genetic variability.
Transposon-insertion sequencing methods are finding their way into the molecular toolbox of many fields of microbiology. These methods can identify the genomic locations and density of transposon insertions in saturated transposon mutant libraries and can be used to make inferences on gene function. For example, where no insertions or very few insertions are identified within a gene in a mutant library grown under permissive conditions, the gene may be essential. Furthermore, where mutations are enriched or lost in a gene after passaging the library through a selective process, the gene is likely to be involved in phenotypes linked to the process. Typically, a fitness based selection such as a stress condition is used in these experiments and the processed sequencing data are used to identify genes required for fitness under the selection. Our research team recently expanded the utility of the transposon directed insertion sequencing (TraDIS) method by applying a physical separation of a transposon mutant library mediated by fluorescence activated cell sorting, rather than a fitness-based selection. This approach, which we have named "TraDISort" is significant because it allows the study of phenotypes that are not linked to cell survival. The TraDISort approach has a broad range of future applications, in drug development, metabolic engineering and in studies of basic bacterial cell physiology.
The Long Interspersed Element 1 (LINE1 or L1) ORF2 protein (ORF2p) can cause DNA damage through the activity of its endonuclease domain (EN). The DNA double-strand breaks (DSB) introduced by the ORF2p EN have the potential to be mutagenic. Previously, our lab has shown that ORF2p fragments containing the EN domain could be expressed in mammalian cells and have variable cytotoxicity. Inclusion of the ORF2p sequence C-terminal to the EN domain in these fragments both reduced the cytotoxicity of these fragments and increased their presence in the nucleus as detected by Western blot analysis. Here, we identify the amino acids (aa 270-274) in the newly-identified ORF2p Cryptic region (Cry) that may be important to the subcellular localization and cytotoxic potential of these EN-containing ORF2p fragments.
BACKGROUND:Retrotransposition of long interspersed nuclear element-1 (L1-RTP) is proposed to contribute to central nervous system (CNS) plasticity by inducing mosaicism of neuronal cells. Clinical studies have identified increased L1 copy numbers in the brains of patients with psychiatric disorders. These observations implicate that L1-RTP is important for neurogenesis and that its deregulation represents a risk factor for mental disorders. However, no supportive evidence is available for understanding the importance of L1-RTP in CNS function.FINDINGS:To explore the physiological role of L1-RTP in CNS, we examined the L1 copy number during maturation. Interestingly, the L1 copy number increased after birth in the mouse hippocampus, but not the frontal lobe, with maximal copy numbers found in 8-week-old mice. This age-dependent L1 increase was abolished by administration of a reverse-transcriptase inhibitor, stavudine (d4T), which showed no toxic effects. Notably, the age-dependent L1 increase was attenuated by post-weaning social isolation (SI) stress, a well-known intervention for inducing psychiatric disorders in mice, or deletion of the NR2A gene that encodes a subunit of the glutamate receptor. Moreover, the negative effects of SI stress on L1-RTP were partially restored by environmental enrichment with voluntary running, but not by fluoxetine, a commonly used anti-psychiatric drug. Finally, behavioral experiments revealed that learning memory was defective in d4T-treated mice, which was similarly observed in mice raised under SI stress.CONCLUSION:We detected the modulation of L1-RTP in the hippocampus during maturation of the CNS. In a recent study, we demonstrated that stimulants such as methamphetamine and cocaine were active in the induction of L1-RTP in neuronal cells, and previous studies have shown that NR2A-deficient mice are susceptible to mental abnormality. Herein, our data support the notion that the age-dependent modulation of L1-RTP is involved in genome differentiation in the hippocampus, and that modulation defects are linked to the development of psychiatric disorders.
The horizontal gene transfer facilitated by mobile genetic elements impacts almost all areas of bacterial evolution, including the accretion and dissemination of antimicrobial-resistance genes in the human and animal pathogen Staphylococcus aureus. Genome surveys of staphylococcal plasmids have revealed an unexpected paucity of conjugation and mobilization loci, perhaps suggesting that conjugation plays only a minor role in the evolution of this genus. In this letter we present the DNA sequences of historically documented staphylococcal conjugative plasmids and highlight that at least 3 distinct and widely distributed families of conjugative plasmids currently contribute to the dissemination of antimicrobial resistance in Staphylococcus. We also review the recently documented "relaxase-in trans" mechanism of conjugative mobilization facilitated by conjugative plasmids pWBG749 and pSK41, and discuss how this may facilitate the horizontal transmission of around 90% of plasmids that were previously considered non-mobilizable. Finally, we enumerate unique sequenced S. aureus plasmids with a potential mechanism of mobilization and predict that at least 80% of all non-conjugative S. aureus plasmids are mobilizable by at least one mechanism. We suggest that a greater research focus on the molecular biology of conjugation is essential if we are to recognize gene-transfer mechanisms from our increasingly in silico analyses.
The long terminal repeat (LTR) and non-LTR retrotransposons comprise approximately half of the human genome, and we are only beginning to understand their influence on genome function and evolution. The LTR retrotransposon Ty1 is the most abundant mobile genetic element in the S. cerevisiae reference genome. Ty1 replicates via an RNA intermediate and shares several important structural and functional characteristics with retroviruses. However, unlike retroviruses Ty1 retrotransposition is not infectious. Retrotransposons integrations can cause mutations and genome instability. Despite the fact that S. cerevisiae lacks eukaryotic defense mechanisms such as RNAi, they maintain a relatively low copy number of the Ty1 retrotransposon in their genomes. A novel restriction factor derived from the C-terminal half of Gag (p22/p18) and encoded by internally initiated transcript inhibits retrotransposition in a dose-dependent manner. Therefore, Ty1 evolved a specific GAG organization and expression strategy to produce products both essential and antagonistic for retrotransposon movement. In this commentary we discuss our recent research aimed at defining steps of Ty1 replication influenced by p22/p18 with particular emphasis on the nucleic acid chaperone functions carried out by Gag and the restriction factor.
A conjugation assay was used to determine the effects of phenotypic resistance to one to up to 5 antibiotics, sampling site of origin, presence or absence of class 1 and/or class 2 integrase (intI) genes (intI1 and intI2), and the number of sulfamethoxazole resistance (sul) and trimethoprim resistance (dfr) genes on the transfer frequencies of plasmids from environmental, antibiotic-resistant Escherichia coli. Of 51 sulfamethoxazole and trimethoprim-resistant E. coli isolates conferring at least one mob gene (mobP51, mobF11, mobF12, mobQ11, mobQ12 , or mobQu ), 38 produced transconjugants with an overall mean frequency of 1.60 × 10-3 transconjugants/ donors (T/D) or 5.89 × 10-3 transconjugants/recipients (T/R). The presence or absence of intI1 and intI2 and the presence or absence of different targeted dfr genes (dfrA1, dfrA8, dfrA12, dfrA14, dfrA17, and/or dfrB3) were not statistically related to plasmid transfer frequencies as determined by ANOVA (P ≥ 0.05). However, E. coli isolates recovered 2 km downstream of wastewater treatment plant effluent input, and those possessing resistance to 3 antibiotics had significantly greater plasmid transfer frequency than their counterparts when calculated as T/D (ANOVA followed by Fisher's least significant difference means comparison, P < 0.05). Greater plasmid transfer frequency calculated as T/D was also measured for E. coli possessing 3 compared to a single sul gene. The in-vitro frequency suggests that horizontal gene transfer of conjugative mediated-antibiotic (sul) resistance genes may be significant among resistant, stream bacteria.
Researchers contest the importance of gene flow in bacterial core genomes, as traditionalists view microbes as predominantly clonal, asexually reproducing organisms. Contrary to the traditional perspective, Escherichia coli core genes vary greatly in their levels of synonymous genetic diversity. This observation indicates that the relative importance of evolutionary forces such as mutation, selection, and recombination varies from gene to gene. In this paper, I highlight why the synonymous diversity observation is broadly relevant to researchers interested in the evolutionary dynamics of microbial populations and communities. I explain how a model of evolution called the coalescent relates neutral diversity (i.e. mutations with negligible fitness effects) to mutation rates, evolutionary time, and a parameter called effective population size. I then describe the possible ways in which mutation, selection, and recombination can explain observed patterns of synonymous diversity in E. coli. Finally, I describe a model for E. coli genome evolution in which different loci are subject to varying levels of gene flow among co-occurring microbes and viruses in the environment. Researchers can falsify the gene flow hypothesis by sequencing genes and strains isolated from stable microbiomes or by carrying out evolution experiments that trace gene genealogies in real-time.
Transposable elements, the class of mobile DNA sequences that change their copies or positions within the genome have an ever increasing role in shaping the genetic and evolutionary landscape. Approximately half of the mammalian genome is composed of repetitive elements, including LINE-1 (L1) elements. Because of their ability to "copy and paste" into other regions of the genome, their activation represent an opportunity as well as a threat, as L1-induced mutations results in genomic instability and plasticity. On one hand L1 retrotransposition and integration fosters genomic diversity and on the other, de-repressed L1 functions as a driver of diseases such as cancer. The regulation of L1 is an area of intense research and novel epigenetic mechanisms have recently been discovered to now include DNA methylation, histone modifications, and miR-induced L1 silencing. During development, reprogramming and in transformed cells, specific classes of repetitive elements are upregulated, presumably due to the loss of epigenetic regulation in this process, increasing the risk of L1-induced mutations. Here we discuss how miR regulation of L1 activation fits into the complex picture of L1 repression in somatic cells and touch on some of the possible implications.
Transposable element (TE) science has been significantly influenced by the pioneering ideas of David Finnegan near the end of the last century, as well as by the classification systems that were subsequently developed. Today, whole genome TE annotation is mostly done using tools that were developed to aid gene annotation rather than to specifically study TEs. We argue that further progress in the TE field is impeded both by current TE classification schemes and by a failure to recognize that TE biology is fundamentally different from that of multicellular organisms. Novel genome wide TE annotation methods are helping to redefine our understanding of TE sequence origins and evolution. We briefly discuss some of these new methods as well as ideas for possible alternative classification schemes. Our hope is to encourage the formation of a society to organize a larger debate on these questions and to promote the adoption of standards for annotation and an improved TE classification.
Genes of the major histocompatibility complex (MHC; also called HLA in human) are polymorphic elements in the genomes of sharks to humans. Class-I and class-II MHC loci appear responsible for much of the genetic linkage to myriad disease states via the capacity to bind short (~8-15 a.a.) peptides of a given pathogen's proteome, or in some cases, the altered proteomes of cancerous cells, and even (in autoimmunity) certain nominal 'self' peptides (Janeway, 2004).(1) Unfortunately, little is known about how the canonical structure of the MHC-I/-II peptide-presenting gene evolved, particularly since beyond ~500 Mya (sharks) no paralogs exist.(2,3) We previously reported that HLA-A isotype alleles with the α1-helix, R65 motif, are wide-spread in phylogeny, but that the α 2-helix, H151R motif, has apparently segregated out of most species. Surprisingly, an uncharacterized orf in T. syrichta (Loc-103275158) encoded R151, but within a truncated A-23 like gene containing 5'- and 3'- footprints of the transposon (TE), tigger-1; the extant tarsier A-23 allele is totally missing exon-3 and part-of exon-4; together, suggesting TE-mediated inactivation of an intact/ancestral A-23 allele (Murray, 2015a).(4) The unique Loc-103275158 orf encodes a putative 15-exon transcript with no apparent paralogs throughout phylogeny. However, an HLA-A11 like gene in M. leucophaeus with a shortened C-terminal domain, and an HLA-A like orf in C. atys with two linked α1/α2/α3 domains, both contain a second transmembrane segment, which is conserved in Loc-103275158. Thus, we could model the putative protein with its Nef-like tail domain docked to its MHC-I like α3 domain (i.e., on the same side of a membrane). This modeled tertiary structure is strikingly similar to the solved structure of the Nef:MHC-I CD:AP1mu transporter (Jia, 2012).(5) Nef:AP1mu binds the CD of MHC-I in trafficking MHC-I away from the trans-golgi and into the endocytic pathway in HIV-1 infected cells. The CD loop of the Loc-103275158 provisional protein conserved the nominal MHC-I CD tyrosine phosphorylation site, and it has an N-terminal SH3 domain that we docked in one conformation to its internal Nef-like domain. Here, we suggest that phosphorylation of the protein's CD-loop signals an exchange between the internal Nef-like domain and a lentiviral-Nef for binding the N-terminal SH3 domain - freeing the Nef-like domain to bind MHC-I CD. Since the 5'-tigger sequence encodes part of the pseudo α1/α2 MHC-I domain, and the 3'-tigger part of the Nef-like domain, we speculate that transposition proceeded phylogenetically disparate horizontal transfers, involving adjacent 5'- and 3'- parasitic footprints, which we also found in the Loc-103275158 orf.
Retrotransposons play significant role in genome remodelling of T. brucei and about 5% of its genome consists of retrotransposons including INGI/RIME elements. INGI is one of the dispersed repetitive elements in T. brucei genome which is found distributed throughout all the chromosomes. SLACS (Spliced Leader Associated Conserved Sequence) however, is more conserved in its nature and lacks the typical poly-distributional pattern of LINE like transposons. We have found total 589 copies of these TEs with only 17.06 % (104 copies out of 589) copies with both ends intact thus showing a majority of truncated copies. Complete SLACS CRE were found only on chromosome 9, whereas, complete INGI/RIME were more ubiquitously distributed. The 50 - 70 bp upstream flanking sequence of these elements shows suitable nucleotide biophysical properties to favor transposition.
