L1 Elements Research Articles

Characterizing inhibitors of the human mobile element L1 endonuclease

The Long interspersed element‐1 (L1) an active autonomous retroelement within the human genome. L1 replicates itself through a process known as retrotransposition. While L1 mutagenic L1 insertion events have been associated with diseases like breast and colon cancer, the full effect of L1 endonuclease activity on the stability of the genome is undetermined. We know that L1 endonuclease expression is associated with a DNA double strand break (DSB) and that the DNA DSB must occur during retrotransposition prior to the integration of novel L1 elements. However, not all of the L1‐associated DNA DSBs are repaired by insertion of a novel L1 element, and the breaks must be repaired faithfully or DNA mutations can occur. Thus, we hypothesize that an inhibitor of L1 endonuclease would be beneficial for estimating L1‐associated damage and to potentially minimize L1‐related genetic instability. We have tested novel small molecules for their ability to inhibit L1 endonuclease using in vitro endonuclease cleavage assays and cell based L1 retrotransposition assays. We have thus far identified a subset of small molecules that inhibit the L1 endonuclease. We are still in the process of confirming these inhibitors through additional testing and locating more inhibitors. Once the inhibitor is located we can begin to answer the question of whether DNA damage associated with the activity of the LINE1 endonuclease leads to mutation and disease.

LINE-1 induces hTERT and ensures telomere maintenance in tumour cell lines.

A hallmark of cancer cells is an activated telomere maintenance mechanism, which allows prolonged survival of the malignant cells. In more than 80% of tumours, telomeres are elongated by the enzyme telomerase, which adds de novo telomere repeats to the ends of chromosomes. Cancer cells are also characterized by expression of active LINE-1 elements (L1s, long interspersed nuclear elements-1). L1 elements are abundant retrotransposons in the eukaryotic genome that are primarily known for facilitating aberrant recombination. Using L1-knockdown (KD), we show for the first time that L1 is critical for telomere maintenance in telomerase-positive tumour cells. The reduced length of telomeres in the L1-KD-treated cells correlated with an increased rate of telomere dysfunction foci, a reduced expression of shelterin proteins and an increased rate of anaphase bridges. The decreased telomere length was associated with a decreased telomerase activity and decreased telomerase mRNA level; the latter was increased upon L1 overexpression. L1-KD also led to a decrease in mRNA and protein expression of cMyc and KLF-4, two main transcription factors of telomerase and altered mRNA levels of other stem-cell-associated proteins such as CD44 and hMyb, as well as a corresponding reduced growth of spheroids. The KD of KLF-4 or cMyc decreased the level of L1-ORF1 mRNA, suggesting a specific reciprocal regulation with L1. Thus, our findings contribute to the understanding of L1 as a pathogenicity factor in cancer cells. As L1 is only expressed in pathophysiological conditions, L1 now appears to be target in the rational treatment of telomerase-positive cancer.

Sequencing, identification and mapping of primed L1 elements (SIMPLE) reveals significant variation in full length L1 elements between individuals.

BackgroundThere are over a half a million copies of L1 retroelements in the human genome which are responsible for as much as 0.5% of new human genetic diseases. Most new L1 inserts arise from young source elements that are polymorphic in the human genome. Highly active polymorphic “hot” L1 source elements have been shown to be capable of extremely high levels of mobilization and result in numerous instances of disease. Additionally, hot polymorphic L1s have been described to be highly active within numerous cancer genomes. These hot L1s result in mutagenesis by insertion of new L1 copies elsewhere in the genome, but also have been shown to generate additional full length L1 insertions which are also hot and able to further retrotranspose. Through this mechanism, hot L1s may amplify within a tumor and result in a continued cycle of mutagenesis.Results and conclusionsWe have developed a method to detect full-length, polymorphic L1 elements using a targeted next generation sequencing approach, Sequencing Identification and Mapping of Primed L1 Elements (SIMPLE). SIMPLE has 94% sensitivity and detects nearly all full-length L1 elements in a genome. SIMPLE will allow researchers to identify hot mutagenic full-length L1s as potential drivers of genome instability. Using SIMPLE we find that the typical individual has approximately 100 non-reference, polymorphic L1 elements in their genome. These elements are at relatively low population frequencies relative to previously identified polymorphic L1 elements and demonstrate the tremendous diversity in potentially active L1 elements in the human population.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1374-y) contains supplementary material, which is available to authorized users.

Effect of Copper Sulfate on Expression of Endogenous L1 Retrotransposons in HepG2 Cells (Hepatocellular Carcinoma).

The long interspersed element-1 (LINE-1 or L1) constitutes approximately 17% of human genome. The expression of these elements is deregulated upon exposure to environmental exposures resulting to genomic instability and cancer promotion. The effect of copper as essential elements in regulation of L1 expression remained to be elucidated. Using non-cytotoxic concentrations of the copper, the expression of endogenous L1 was analyzed by qPCR after 6 days of copper pretreatment in human hepatocellular carcinoma cells (HepG2). The results indicated that the expression of active L1 elements are significantly downregulated at concentrations of 12.5, 25, and 50 μM (p < 0.005). Our data imply that low-level copper exposure may have a protective effect to suppress the induction of L1 activity and decrease incidence of cancer-associated L1 mutagenesis. If this achievement is confirmed by further studies, it can be applied in the long-term goals of cancer prevention.

Evolutionary histories of transposable elements in the genome of the largest living marsupial carnivore, the Tasmanian devil.

The largest living carnivorous marsupial, the Tasmanian devil (Sarcophilus harrisii), is the sole survivor of a lineage originating about 12 Ma. We set out to investigate the spectrum of transposable elements found in the Tasmanian devil genome, the first high-coverage genome of an Australian marsupial. Marsupial genomes have been shown to have the highest amount of transposable elements among vertebrates. We analyzed the horizontally transmitted DNA transposons OC1 and hAT-1_MEu in the Tasmanian devil genome. OC1 is present in all carnivorous marsupials, while having a very limited distribution among the remaining Australian marsupial orders. In contrast, hAT-1_MEu is present in all Australian marsupial orders, and has so far only been identified in a few placental mammals. We screened 158 introns for phylogenetically informative retrotransposons in the order Dasyuromorphia, and found that the youngest SINE (Short INterspersed Element), WSINE1, is no longer active in the subfamily Dasyuridae. The lack of detectable WSINE1 activity in this group may be due to a retrotransposon inactivation event approximately 30 Ma. We found that the Tasmanian devil genome contains a relatively low number of continuous full-length LINE-1 (Long INterspersed Element 1, L1) retrotransposons compared with the opossum genome. Furthermore, all L1 elements in the Tasmanian devil appeared to be nonfunctional. Hidden Markov Model approaches suggested that other potential sources of functional reverse transcriptase are absent from the genome. We discuss the issues associated with assembling long, highly similar L1 copies from short read Illumina data and describe how assembly artifacts can potentially lead to erroneous conclusions.

Transposable elements and genomics

Once described as selfish DNA, transposable elements (TEs) have been revisited due to a plethora of new genomic information. TEs are considered indispensable genomic treasures during eukaryotic species evolution and supply raw materials for evolution by creating novel variation and allelic diversity (Volff 2006; Oliver et al. 2013). TEs are found ubiquitously with high copies in nearly all eukaryotic species. TEs are often present in major genomic denizens in some species by occupying more than half of the genome, contributing to the C-value paradox in eukaryotes. In this issue, six articles are published describing several aspects of transposable elements. Four review articles and two research articles are presented in this special issue. TEs are classified into class I TEs and class II TEs based on transposition mechanisms. Class I TEs are retrotransposons that retrotranspose via RNA intermediate semi-conservative manner. Class II TEs transpose via DNA intermediate conservative manner. Rajput (2015) described class I retrotransposons as the intrinsic genomic evolutionist as ‘‘Retrotransposons: the intrinsic genomic evolutionist’’. He reviewed essential information on retrotransposons. Retrotransposons are major agents in genome evolution and diversity by inducing rearrangements and restructuring genomes. Retrotransposons affect many aspects of biological organization, from gene regulation to genome obesity, which are critical in shaping current genomes. He also discusses the similarity between retrotransposons and endo-retroviruses, bringing the retrotransposons into a closer relationship to understand phylogenetics. Bae et al. contributed ‘‘Biological changes of transposable elements by radiation’’ (Bae et al. 2015). Human endogenous retroviruses (HERV) are a group of LTR (long terminal repeat) retrotransposons in the human genome. Transcriptionally-active TEs can move around in the genome and cause genome instability. HERV insertion into critical genes may lead to the progression of some diseases, including cancer. These authors reviewed cases of human disease due to HERV insertion. Radiation therapy manifests diverse, complex, and multicellular responses, including unmasking epigenetic suppression that switches silenced TEs into active ones. In the review, the authors summarized the recent understanding on radiationinduced DNA double-strand breaks (DSBs) and subsequent non-homologous end joining (NHEJ) repair. They also presented their recent results on activation of L1 elements by radiation in cancer cells, in which the methylation level of L1 was increased by relatively low radiation doses. Based on these results, the authors proposed that radiation exposure may lead directly to mutation as well as to methylation changes in TEs, which are considered important factors in cancer radiation therapy. Lee et al. contributed ‘‘Composition and evolutionary importance of transposable elements in humans and primates’’ (Lee et al. 2015). Primates, including humans, are not immune to the evolutionary impact of TEs on their genomes. Approximately half of the sequences of primate genomes are TErelated sequences. In their review, comparisons of TE constitution among primate species are extensively explained, and TE dispersions in the genome were analyzed between human and primates. These authors proposed that TE-driven structural variations are a major force in primate evolution. Roy et al. examined TEs in different aspects of molecular markers in ‘‘Marker utility of transposable elements for plant genetics, breeding, and N.-S. Kim (&) Department of Molecular Bioscience, Institute of Bioscience and Biotechnology, Kangwon National University, Chuncheon 200-701, Korea e-mail: kimnamsu@kangwon.ac.kr; nsk6472@yahoo.ca

Identification of PLZF-Dmrs in Hematopoiesis Highlight a Novel Role of PLZF As a Guardian of Hematopoietic Genome Integrity

Background: The eucaryotic genome is organized in chromatin domains which affect its function. This organization is partly established by special architectural and transcriptional factors specific to the cellular context. In an hematopoietic context, PLZF (Promyelocytic Leukemia Zinc Finger protein), a member of the family of POK repressor proteins, is directly implicated in the regulation of epigenetic modifications by tethering DNA methyltransferases (DNMT) and histone deacetylases (HDAC) to specific genomic targets. We have previously shown that PLZF transcriptional activation is controlled by acetylation of specific lysine residues (K647/650/653) affecting its nuclear localization. PLZF is mainly expressed in CD34 positive cells and has been shown to be crucial in the maintenance of hematopoietic stem cells (HSC). However, the epigenetic role of PLZF in HSC protection and maintenance is not yet understood.Methodology: We created PLZF functional knock-in mouse models by introducing PLZF lysine mutants with altered epigenetic functions at the PLZF locus. We used purified bone marrow (BM) cells from 12 week old mice to assess their self-renewal capacity by methylcellulose-based medium serial replating assays to detect and quantify hematopoietic progenitors in colony-forming unit (CFU) experiments and long-term self-renewal. In parallel, Methyl DNA immunoprecipitation followed by deep-sequencing (MeDiP-seq) was performed in order to establish DNA methylation pattern and characterize PLZF induced DNA methylation regions (PLZF-DMR). Retrotransposon activity was determined by quantifying the expression of L1 retrotransposon mRNAs in mouse cells and by retrotransposon assay using a GFP-L1 reporter in human 293T cells. Validation and functions of PLZF genomic targets found in mouse cells were tested in KG1a CD34+ hematopoietic human cells by Chromatin immunoprecipitation (ChIP) and reporter gene assays using PLZF-DMR luciferase vectors containing L1 and also Alu and telomeric sequences targeted by PLZF.Results: We have created two PLZF functional mutants, PLZFON mutant constitutively binding DNA and PLZFOFF mutant unable to interact with DNA, and shown that PLZFOFF mice are infertile due to loss of germinal cells recapitulating the PLZF knock-out mouse model phenotype. In both PLZFOFF and PLZFON 12 week old mice the number of myeloid colonies decreased by 62% compared to PLZFWT and a total absence of self-renewal capacity at the 1st replating in PLZFOFF was noted. In BM cells, establishment of differential DNA methylation profiles by MEDIP-seq of PLZFOFF, PLZFON and PLZFWT mice allowed to identify more than 500 PLZF-DMRs. We found that primary PLZF genomic targets are repeat elements scattered throughout the genome (75% of all PLZF-DMRs). These repeat elements include retrotransposons (L1, 21.9%), Alu sequences (14.4%), retroviruses (22.1%) and sub-telomeric/telomeric regions (10%). We first investigated the impact of PLZF on L1 elements because of their involvment in genome instability, gene control and cancer through retrotransposition and methylation events: -a) L1 and PLZF interact through an 8bp conserved binding site; -b) PLZF binds to full length and truncated L1 DNA sequences, inducing DNA methylation and histone deacetylation propagation by recruiting of DNMT and HDAC proteins; L1 mRNA expression is increased PLZFOFF BM cells. Furthermore, in a retrotransposition assay using a GFP-L1 reporter in human cells we demonstrate that PLZF inhibits L1 retrotransposition and that PLZF-DMRs containing L1, Alu and telomeric sequences induce transcriptional repression by creating heterochromatin boundaries.Conclusions: These results, using unique mouse models of PLZF inactivation, with loss of HSC maintenance show that PLZF may be a guardian of genome integrity of long living hematopoietic cells by establishing DNA methylation patterning, associated with constitutive heterochromatin domains involved in retrotransposition silencing and centromere/telomere stability. DisclosuresNo relevant conflicts of interest to declare.

Guanine quadruplexes are formed by specific regions of human transposable elements

BackgroundTransposable elements form a significant proportion of eukaryotic genomes. Recently, Lexa et al. (Nucleic Acids Res 42:968-978, 2014) reported that plant long terminal repeat (LTR) retrotransposons often contain potential quadruplex sequences (PQSs) in their LTRs and experimentally confirmed their ability to adopt four-stranded DNA conformations.ResultsHere, we searched for PQSs in human retrotransposons and found that PQSs are specifically localized in the 3’-UTR of LINE-1 elements, in LTRs of HERV elements and are strongly accumulated in specific regions of SVA elements. Circular dichroism spectroscopy confirmed that most PQSs had adopted monomolecular or bimolecular guanine quadruplex structures. Evolutionarily young SVA elements contained more PQSs than older elements and their propensity to form quadruplex DNA was higher. Full-length L1 elements contained more PQSs than truncated elements; the highest proportion of PQSs was found inside transpositionally active L1 elements (PA2 and HS families).ConclusionsConservation of quadruplexes at specific positions of transposable elements implies their importance in their life cycle. The increasing quadruplex presence in evolutionarily young LINE-1 and SVA families makes these elements important contributors toward present genome-wide quadruplex distribution.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-1032) contains supplementary material, which is available to authorized users.

Mobster: accurate detection of mobile element insertions in next generation sequencing data.

Mobile elements are major drivers in changing genomic architecture and can cause disease. The detection of mobile elements is hindered due to the low mappability of their highly repetitive sequences. We have developed an algorithm, called Mobster, to detect non-reference mobile element insertions in next generation sequencing data from both whole genome and whole exome studies. Mobster uses discordant read pairs and clipped reads in combination with consensus sequences of known active mobile elements. Mobster has a low false discovery rate and high recall rate for both L1 and Alu elements. Mobster is available at http://sourceforge.net/projects/mobster.Electronic supplementary materialThe online version of this article (doi:10.1186/s13059-014-0488-x) contains supplementary material, which is available to authorized users.

P.1.a.016 Emotionally painful stress causes changes in L1 insertion pattern in the hippocampus in rats with different nervous system excitability

Stress can induce structural changes in the brain and contribute to a variety of chronic diseases from post-traumatic stress disorder to depression. The hippocampus is a highly plastic brain region particularly susceptible to the effects of environmental stress. A genomic stress response partly consists in changes in insertion activity of transposable elements. Retrotransposons account for ~45% of the mammalian genome. Mechanisms of action of stress and the formation of posttraumatic stress disorder (PTSD) in human are associated with retrotransposons (LINE1, L1) activity in various tissues. Endogenous retrotransposition of L1 elements has been proposed as one potential mechanism generating neuronal genome diversity. They are capable of inserting into new genomic locations, which can result in deleterious outcomes. The greatest interest is the study of these processes in the brain due to individual variability of neural processes to effective pharmacotherapy path finding. The purpose of this work was to investigate the effect of stress on the LINE1 insertion pattern in the hippocampus. We used the model of emotional stress for the PTSD-like state formation in two lines of rats with genetically determined differences in the level of excitability of the nervous system (low and high) to study of emotionally painful stress (3 types: short-term, long-term and massive stress) effect on LINE1 retrotransposon insertion polymorphism in the dentate gyrus of the hippocampus. L1 insertions in the promoter of gene GRIN1 [glutamate receptor ionotropic N-methyl-D-aspartate (NMDA) subtype, subunit 1] examined by two-step PCR (general and directed). Glutamate NMDA receptors play a key role in synaptic plasticity, synaptogenesis, excitotoxicity, memory acquisition and learning. These receptors mediate neuronal functions in glutamate neurotransmission. NMDAR-mediated neurotransmission in the hippocampus is implicated in cognitive and emotional disturbances during stressrelated disorders. NMDA receptor subunit 1 is a core molecule in the NMDA receptor complex. GRIN1 expression is associated with neurotransmission efficiency and state of the nervous system. We suppose that L1 retrotransposon insertions to promoter of GRIN1 gene will alter GRIN1 expression. It was shown that L1 insertion pattern depends on the level of excitability of the nervous system, intensity and duration of stress exposure. The most variable spectrum of L1 transposable elements activity (L1 insertion pattern) is detected under the influence of massive stress in the rats with low excitability of the nervous system. We assume that L1 retrotransposon insertions take part in the regulation of expression of candidate genes associated with the stress response and the formation of psychopathology. P.1.a.017 Genetic variants in kinesin family member 13A (KIF13A) affect susceptibility to schizophrenia in Korean population

SIRT6 represses LINE1 retrotransposons by ribosylating KAP1 but this repression fails with stress and age

L1 retrotransposons are an abundant class of transposable elements which pose a threat to genome stability and may play a role in age-related pathologies such as cancer. Recent evidence indicates that L1s become more active in somatic tissues during the course of aging; the mechanisms underlying this phenomenon remain unknown, however. Here we report that the longevity regulating protein, SIRT6, is a powerful repressor of L1 activity. Specifically, SIRT6 binds to the 5′UTR of L1 loci, where it mono-ADP ribosylates the nuclear corepressor protein, KAP1, and facilitates KAP1 interaction with the heterochromatin factor, HP1α, thereby contributing to the packaging of L1 elements into transcriptionally repressive heterochromatin. During the course of aging, and also in response to DNA damage, however, we find that SIRT6 is depleted from L1 loci, allowing for the activation of these previously silenced retroelements.

Tangram: a comprehensive toolbox for mobile element insertion detection.

BackgroundMobile elements (MEs) constitute greater than 50% of the human genome as a result of repeated insertion events during human genome evolution. Although most of these elements are now fixed in the population, some MEs, including ALU, L1, SVA and HERV-K elements, are still actively duplicating. Mobile element insertions (MEIs) have been associated with human genetic disorders, including Crohn’s disease, hemophilia, and various types of cancer, motivating the need for accurate MEI detection methods. To comprehensively identify and accurately characterize these variants in whole genome next-generation sequencing (NGS) data, a computationally efficient detection and genotyping method is required. Current computational tools are unable to call MEI polymorphisms with sufficiently high sensitivity and specificity, or call individual genotypes with sufficiently high accuracy.ResultsHere we report Tangram, a computationally efficient MEI detection program that integrates read-pair (RP) and split-read (SR) mapping signals to detect MEI events. By utilizing SR mapping in its primary detection module, a feature unique to this software, Tangram is able to pinpoint MEI breakpoints with single-nucleotide precision. To understand the role of MEI events in disease, it is essential to produce accurate individual genotypes in clinical samples. Tangram is able to determine sample genotypes with very high accuracy. Using simulations and experimental datasets, we demonstrate that Tangram has superior sensitivity, specificity, breakpoint resolution and genotyping accuracy, when compared to other, recently developed MEI detection methods.ConclusionsTangram serves as the primary MEI detection tool in the 1000 Genomes Project, and is implemented as a highly portable, memory-efficient, easy-to-use C++ computer program, built under an open-source development model.

Identification of L1ASP-derived chimeric transcripts in lung cancer

Long interspersed elements (LINEs or L1 s) are an abundant class of autonomous retrotransposons in the human genome. Full-length L1 elements have two transcriptional regulatory regions: an internal sense promoter and an antisense promoter (ASP). The ASP of L1 elements can generate chimeric RNAs via direct transcription of adjacent genomic sequences. Chimeric transcripts derived from the L1ASP are highly represented, but the extent to which this occurs is largely unknown. Using a genome-wide L1 chimera display (LCD) technique, we have isolated 4 L1 chimeric transcripts (LCTs) in the lung cancer genome. Then, we analyzed the structural characteristics of the 4 LCTs using bioinformatics tool. Expression patterns of the 4 LCTs were analyzed by reverse transcription polymerase chain reaction using normal and lung cancer tissues. LCT24 and LCT25 showed higher expression in lung cancer tissue than in normal tissue. These results suggest that the genome-wide LCD technique could be of great use for further study to find cancer markers via cancer-associated LCTs and to understand cancer biology.

On the lattice of subgroups of the lamplighter group

The techniques of modules and actions of groups on rooted trees are applied to study the subgroup structure and the lattice subgroup of lamplighter type groups of the form ℒn,p = (ℤ/pℤ)n ≀ ℤ for n ≥ 1 and p prime. We completely characterize scale invariant structures on ℒ1,2. We determine all points on the boundary of binary tree (on which ℒ1,p naturally acts in a self-similar manner) with trivial stabilizer. We prove the congruence subgroup property (CSP) and as a consequence show that the profinite completion [Formula: see text] of ℒ1,p is a self-similar group generated by finite automaton. We also describe the structure of portraits of elements of ℒ1,p and [Formula: see text] and show that ℒ1,p is not a sofic tree shift group in the terminology of [T. Ceccherini-Silberstein, M. Coornaert, F. Fiorenza and Z. Sunic, Cellular automata between sofic tree shifts, Theor. Comput. Sci.506 (2013) 79–101; A. Penland and Z. Sunic, Sofic tree shifts and self-similar groups, preprint].

Selection shaped the evolution of mouse androgen-binding protein (ABP) function and promoted the duplication of Abp genes.

In the present article, we summarize two aspects of our work on mouse ABP (androgen-binding protein): (i) the sexual selection function producing incipient reinforcement on the European house mouse hybrid zone, and (ii) the mechanism behind the dramatic expansion of the Abp gene region in the mouse genome. Selection unifies these two components, although the ways in which selection has acted differ. At the functional level, strong positive selection has acted on key sites on the surface of one face of the ABP dimer, possibly to influence binding to a receptor. A different kind of selection has apparently driven the recent and rapid expansion of the gene region, probably by increasing the amount of Abp transcript, in one or both of two ways. We have shown previously that groups of Abp genes behave as LCRs (low-copy repeats), duplicating as relatively large blocks of genes by NAHR (non-allelic homologous recombination). The second type of selection involves the close link between the accumulation of L1 elements and the expansion of the Abp gene family by NAHR. It is probably predicated on an initial selection for increased transcription of existing Abp genes and/or an increase in Abp gene number providing more transcriptional sites. Either or both could increase initial transcript production, a quantitative change similar to increasing the volume of a radio transmission. In closing, we also provide a note on Abp gene nomenclature.

Extensive transduction of nonrepetitive DNA mediated by L1 retrotransposition in cancer genomes

Long interspersed nuclear element-1 (L1) retrotransposons are mobile repetitive elements that are abundant in the human genome. L1 elements propagate through RNA intermediates. In the germ line, neighboring, nonrepetitive sequences are occasionally mobilized by the L1 machinery, a process called 3' transduction. Because 3' transductions are potentially mutagenic, we explored the extent to which they occur somatically during tumorigenesis. Studying cancer genomes from 244 patients, we found that tumors from 53% of the patients had somatic retrotranspositions, of which 24% were 3' transductions. Fingerprinting of donor L1s revealed that a handful of source L1 elements in a tumor can spawn from tens to hundreds of 3' transductions, which can themselves seed further retrotranspositions. The activity of individual L1 elements fluctuated during tumor evolution and correlated with L1 promoter hypomethylation. The 3' transductions disseminated genes, exons, and regulatory elements to new locations, most often to heterochromatic regions of the genome.

Processed pseudogene insertions in somatic cells.

Processed pseudogenes are copies of messenger RNAs that have been reverse transcribed into DNA and inserted into the genome using the enzymatic activities of active L1 elements. Processed pseudogenes generally lack introns, end in a 3’ poly A, and are flanked by target site duplications. Until recently, very few polymorphic processed pseudogenes had been discovered in mammalian genomes. Now several studies have found a number of polymorphic processed pseudogenes in humans. Moreover, processed pseudogenes can occur in somatic cells, including in various cancers and in early fetal development. One recent somatic insertion of a processed pseudogene has caused a Mendelian X-linked disease, chronic granulomatous disease.

Evolutionally dynamic L1 regulation in embryonic stem cells.

Mobile elements are important evolutionary forces that challenge genomic integrity. Long interspersed element-1 (L1, also known as LINE-1) is the only autonomous transposon still active in the human genome. It displays an unusual pattern of evolution, with, at any given time, a single active L1 lineage amplifying to thousands of copies before getting replaced by a new lineage, likely under pressure of host restriction factors, which act notably by silencing L1 expression during early embryogenesis. Here, we demonstrate that in human embryonic stem (hES) cells, KAP1 (KRAB [Krüppel-associated box domain]-associated protein 1), the master cofactor of KRAB-containing zinc finger proteins (KRAB-ZFPs) previously implicated in the restriction of endogenous retroviruses, represses a discrete subset of L1 lineages predicted to have entered the ancestral genome between 26.8 million and 7.6 million years ago. In mice, we documented a similar chronologically conditioned pattern, albeit with a much contracted time scale. We could further identify an L1-binding KRAB-ZFP, suggesting that this rapidly evolving protein family is more globally responsible for L1 recognition. KAP1 knockdown in hES cells induced the expression of KAP1-bound L1 elements, but their younger, human-specific counterparts (L1Hs) were unaffected. Instead, they were stimulated by depleting DNA methyltransferases, consistent with recent evidence demonstrating that the PIWI-piRNA (PIWI-interacting RNA) pathway regulates L1Hs in hES cells. Altogether, these data indicate that the early embryonic control of L1 is an evolutionarily dynamic process and support a model in which newly emerged lineages are first suppressed by DNA methylation-inducing small RNA-based mechanisms before KAP1-recruiting protein repressors are selected.

Regulation of L1 expression and retrotransposition by melatonin and its receptor: implications for cancer risk associated with light exposure at night.

Expression of long interspersed element-1 (L1) is upregulated in many human malignancies. L1 can introduce genomic instability via insertional mutagenesis and DNA double-strand breaks, both of which may promote cancer. Light exposure at night, a recently recognized carcinogen, is associated with an increased risk of cancer in shift workers. We report that melatonin receptor 1 inhibits mobilization of L1 in cultured cells through downregulation of L1 mRNA and ORF1 protein. The addition of melatonin receptor antagonists abolishes the MT1 effect on retrotransposition in a dose-dependent manner. Furthermore, melatonin-rich, but not melatonin-poor, human blood collected at different times during the circadian cycle suppresses endogenous L1 mRNA during in situ perfusion of tissue-isolated xenografts of human cancer. Supplementation of human blood with exogenous melatonin or melatonin receptor antagonist during the in situ perfusion establishes a receptor-mediated action of melatonin on L1 expression. Combined tissue culture and in vivo data support that environmental light exposure of the host regulates expression of L1 elements in tumors. Our data imply that light-induced suppression of melatonin production in shift workers may increase L1-induced genomic instability in their genomes and suggest a possible connection between L1 activity and increased incidence of cancer associated with circadian disruption.

Evaluating the extent of LINE-1 mobility following exposure to heavy metals in HepG2 cells.

The long interspersed elements-1 (LINE1 or L1 retrotransposon) constitute 17% of the human genome and retain mobility properties within the genome. At present, 80-100 human L1 elements are thought to be active in the genome. The mobilization of these active elements may be influenced upon exposure to the heavy metals. In the present study, we evaluated the association of aluminum, lead, and copper exposure with L1 retrotransposition in human hepatocellular carcinoma (HepG2) cell line. An in vitro retrotransposition assay using an enhanced green fluorescent protein (EGFP)-tagged L1RP cassette was established to track EGFP shining as the mark of retrotransposition. Following determination of noncytotoxic concentrations of these metals, pL1RP-EGFP-transfected HepG2 cells were subjected to long-term treatment. Flow cytometry analysis of cells treated with various concentrations of these metals along with quantitative real-time PCR was used to quantify L1 retrotransposition frequencies. Aluminum significantly increased L1 retrotransposition frequency, while no significant association was found concerning lead exposure and L1 retrotransposition. Copper treatment downregulated L1 retrotransposition as a result of EGFP-tagged L1RP expression. Our findings suggest that aluminum might have the potential to cause genomic instability by the enhancement of L1 mobilization. Thus, the risk of induced L1 retrotransposition should be considered during drug safety evaluation and risk assessments of exposure to toxic environmental agents. Further studies are needed for a more robust assay to evaluate any associations between long-term lead exposure and L1 mobility in cell culture assay.