Characterisation of retrotransposon insertion polymorphisms in whole genome sequencing data from individuals with amyotrophic lateral sclerosis.

The genetics of an individual is a crucial factor in understanding the risk of developing the neurodegenerative disease amyotrophic lateral sclerosis (ALS). There is still a large proportion of the heritability of ALS, particularly in sporadic cases, to be understood. Among others, active transposable elements drive inter-individual variability, and in humans long interspersed element 1 (LINE1, L1), Alu and SINE-VNTR-Alu (SVA) retrotransposons are a source of polymorphic insertions in the population. We undertook a pilot study to characterise the landscape of non-reference retrotransposon insertion polymorphisms (non-ref RIPs) in 15 control and 15 ALS individuals' whole genomes from Project MinE, an international project to identify potential genetic causes of ALS. The combination of two bioinformatics tools (mobile element locator tool (MELT) and TEBreak) identified on average 1250 Alu, 232 L1 and 77 SVA non-ref RIPs per genome across the 30 analysed. Further PCR validation of individual polymorphic retrotransposon insertions showed a similar level of accuracy for MELT and TEBreak. Our preliminary study did not identify a specific RIP or a significant difference in the total number of non-ref RIPs in ALS compared to control genomes. The use of multiple bioinformatic tools improved the accuracy of non-ref RIP detection and our study highlights the potential importance of studying these elements further in ALS.

Alu complementary DNA is enriched in atrophic macular degeneration and triggers retinal pigmented epithelium toxicity via cytosolic innate immunity.

Long interspersed nuclear element-1 (L1)­mediated reverse transcription (RT) of Alu RNA into cytoplasmic Alu complementary DNA (cDNA) has been implicated in retinal pigmented epithelium (RPE) degeneration. The mechanism of Alu cDNA­induced cytotoxicity and its relevance to human disease are unknown. Here we report that Alu cDNA is highly enriched in the RPE of human eyes with geographic atrophy, an untreatable form of age-related macular degeneration. We demonstrate that the DNA sensor cGAS engages Alu cDNA to induce cytosolic mitochondrial DNA escape, which amplifies cGAS activation, triggering RPE degeneration via the inflammasome. The L1-extinct rice rat was resistant to Alu RNA­induced Alu cDNA synthesis and RPE degeneration, which were enabled upon L1-RT overexpression. Nucleoside RT inhibitors (NRTIs), which inhibit both L1-RT and inflammasome activity, and NRTI derivatives (Kamuvudines) that inhibit inflammasome, but not RT, both block Alu cDNA toxicity, identifying inflammasome activation as the terminal effector of RPE degeneration.

Hepatitis C virus infection restricts human LINE-1 retrotransposition in hepatoma cells.

LINE-1 (L1) retrotransposons are autonomous transposable elements that can affect gene expression and genome integrity. Potential consequences of exogenous viral infections for L1 activity have not been studied to date. Here, we report that hepatitis C virus (HCV) infection causes a significant increase of endogenous L1-encoded ORF1 protein (L1ORF1p) levels and translocation of L1ORF1p to HCV assembly sites at lipid droplets. HCV replication interferes with retrotransposition of engineered L1 reporter elements, which correlates with HCV RNA-induced formation of stress granules and can be partially rescued by knockdown of the stress granule protein G3BP1. Upon HCV infection, L1ORF1p localizes to stress granules, associates with HCV core in an RNA-dependent manner and translocates to lipid droplets. While HCV infection has a negative effect on L1 mobilization, L1ORF1p neither restricts nor promotes HCV infection. In summary, our data demonstrate that HCV infection causes an increase of endogenous L1 protein levels and that the observed restriction of retrotransposition of engineered L1 reporter elements is caused by sequestration of L1ORF1p in HCV-induced stress granules.

Frequency and methylation status of selected retrotransposition competent L1 loci in amyotrophic lateral sclerosis.

Long interspersed element-1 (LINE-1/L1) is the only autonomous transposable element in the human genome that currently mobilises in both germline and somatic tissues. Recent studies have identified correlations between altered retrotransposon expression and the fatal neurodegenerative disease amyotrophic lateral sclerosis (ALS) in a subset of patients. The risk of an individual developing ALS is dependent on an interaction of genetic variants and subsequent modifiers during life. These modifiers could include environmental factors, which can lead to epigenetic and genomic changes, such as somatic mutations, occurring in the neuronal cells that degenerate as the disease develops. There are more than 1 million L1 copies in the human genome today, but only 80-100 L1 loci in the reference genome are considered to be retrotransposition-competent (RC) and an even smaller number of these RC-L1s loci are highly active. We hypothesise that RC-L1s could affect normal cellular function through their mutagenic potential conferred by their ability to retrotranspose in neuronal cells and through DNA damage caused by the endonuclease activity of the L1-encoded ORF2 protein. To investigate whether either an increase in the genomic burden of RC-L1s or epigenetic changes to RC-L1s altering their expression, could play a role in disease development, we chose a set of seven well characterised genomic RC-L1 loci that were reported earlier to be highly active in a cellular L1 retrotransposition reporter assay or serve as major source elements for germline and/or somatic retrotransposition events. Analysis of the insertion allele frequency of five polymorphic RC-L1s, out of the set of seven, for their presence or absence, did not identify an increased number individually or when combined in individuals with the disease. However, we did identify reduced levels of methylation of RC-L1s in the motor cortex of those individuals with both familial and sporadic ALS compared to control brains. The changes to the regulation of the loci encompassing these RC-L1s demonstrated tissue specificity and could be related to the disease process.

Loop 1 of APOBEC3C Regulates its Antiviral Activity against HIV-1.

APOBEC3 deaminases (A3s) provide mammals with an anti-retroviral barrier by catalyzing dC-to-dU deamination on viral ssDNA. Within primates, A3s have undergone a complex evolution via gene duplications, fusions, arms race, and selection. Human APOBEC3C (hA3C) efficiently restricts the replication of viral infectivity factor (vif)-deficient Simian immunodeficiency virus (SIVΔvif), but for unknown reasons, it inhibits HIV-1Δvif only weakly. In catarrhines (Old World monkeys and apes), the A3C loop 1 displays the conserved amino acid pair WE, while the corresponding consensus sequence in A3F and A3D is the largely divergent pair RK, which is also the inferred ancestral sequence for the last common ancestor of A3C and of the C-terminal domains of A3D and A3F in primates. Here, we report that modifying the WE residues in hA3C loop 1 to RK leads to stronger interactions with substrate ssDNA, facilitating catalytic function, which results in a drastic increase in both deamination activity and in the ability to restrict HIV-1 and LINE-1 replication. Conversely, the modification hA3F_WE resulted only in a marginal decrease in HIV-1Δvif inhibition. We propose that the two series of ancestral gene duplications that generated A3C, A3D-CTD and A3F-CTD allowed neo/subfunctionalization: A3F-CTD maintained the ancestral RK residues in loop 1, while diversifying selection resulted in the RK â†’ WE modification in Old World anthropoids' A3C, possibly allowing for novel substrate specificity and function.

The impact of transposable element activity on therapeutically relevant human stem cells.

Human stem cells harbor significant potential for basic and clinical translational research as well as regenerative medicine. Currently ~ 3000 adult and ~ 30 pluripotent stem cell-based, interventional clinical trials are ongoing worldwide, and numbers are increasing continuously. Although stem cells are promising cell sources to treat a wide range of human diseases, there are also concerns regarding potential risks associated with their clinical use, including genomic instability and tumorigenesis concerns. Thus, a deeper understanding of the factors and molecular mechanisms contributing to stem cell genome stability are a prerequisite to harnessing their therapeutic potential for degenerative diseases. Chemical and physical factors are known to influence the stability of stem cell genomes, together with random mutations and Copy Number Variants (CNVs) that accumulated in cultured human stem cells. Here we review the activity of endogenous transposable elements (TEs) in human multipotent and pluripotent stem cells, and the consequences of their mobility for genomic integrity and host gene expression. We describe transcriptional and post-transcriptional mechanisms antagonizing the spread of TEs in the human genome, and highlight those that are more prevalent in multipotent and pluripotent stem cells. Notably, TEs do not only represent a source of mutations/CNVs in genomes, but are also often harnessed as tools to engineer the stem cell genome; thus, we also describe and discuss the most widely applied transposon-based tools and highlight the most relevant areas of their biomedical applications in stem cells. Taken together, this review will contribute to the assessment of the risk that endogenous TE activity and the application of genetically engineered TEs constitute for the biosafety of stem cells to be used for substitutive and regenerative cell therapies.

Retrotransposons in the development and progression of amyotrophic lateral sclerosis.

Endogenous retrotransposon sequences constitute approximately 42% of the human genome, and mobilisation of retrotransposons has resulted in rearrangements, duplications, deletions, novel transcripts and the introduction of new regulatory domains throughout the human genome. Both germline and somatic de novo retrotransposition events have been involved in a range of human diseases, and there is emerging evidence for the modulation of retrotransposon activity during the development of specific diseases. Particularly, there is unequivocal consensus that endogenous retrotransposition can occur in neuronal lineages. This review addresses our current knowledge of the different mechanisms through which retrotransposons might influence the development of and predisposition to amyotrophic lateral sclerosis.

Author Correction: Reprogramming triggers endogenous L1 and Alu retrotransposition in human induced pluripotent stem cells.

This Article contains an error in the author affiliations. The correct affiliation for author Ruchi Shukla is 'MRC Human Genetics Unit, Institute of Genetics and Molecular Medicine, University of Edinburgh, Crewe Road, Edinburgh, EH4 2XU, UK', and is not 'Mater Research Institute - University of Queensland, TRI Building, Woolloongabba QLD 4102, Australia'.

APOBEC3B Activity Is Prevalent in Urothelial Carcinoma Cells and Only Slightly Affected by LINE-1 Expression.

The most common mutational signature in urothelial carcinoma (UC), the most common type of urinary bladder cancer is assumed to be caused by the misdirected activity of APOBEC3 (A3) cytidine deaminases, especially A3A or A3B, which are known to normally restrict the propagation of exogenous viruses and endogenous retroelements such as LINE-1 (L1). The involvement of A3 proteins in urothelial carcinogenesis is unexpected because, to date, UC is thought to be caused by chemical carcinogens rather than viral activity. Therefore, we explored the relationship between A3 expression and L1 activity, which is generally upregulated in UC. We found that UC cell lines highly express A3B and in some cases A3G, but not A3A, and exhibit corresponding cytidine deamination activity in vitro. While we observed evidence suggesting that L1 expression has a weak positive effect on A3B and A3G expression and A3B promoter activity, neither efficient siRNA-mediated knockdown nor overexpression of functional L1 elements affected catalytic activity of A3 proteins consistently. However, L1 knockdown diminished proliferation of a UC cell line exhibiting robust endogenous L1 expression, but had little impact on a cell line with low L1 expression levels. Our results indicate that UC cells express A3B at levels exceeding A3A levels by far, making A3B the prime candidate for causing genomic mutations. Our data provide evidence that L1 activation constitutes only a minor and negligible factor involved in induction or upregulation of endogenous A3 expression in UC.

Report of the international conference on manufacturing and testing of pluripotent stem cells.

Sessions included an overview of past cell therapy (CT) conferences sponsored by the International Alliance for Biological Standardization (IABS). The sessions highlighted challenges in the field of human pluripotent stem cells (hPSCs) and also addressed specific points on manufacturing, bioanalytics and comparability, tumorigenicity testing, storage, and shipping. Panel discussions complemented the presentations. The conference concluded that a range of new standardization groups is emerging that could help the field, but ways must be found to ensure that these efforts are coordinated. In addition, there are opportunities for regulatory convergence starting with a gap analysis of existing guidelines to determine what might be missing and what issues might be creating divergence. More specific global regulatory guidance, preferably from WHO, would be welcome. IABS and the California Institute for Regenerative Medicine (CIRM) will explore with stakeholders the development of a practical and innovative road map to support early CT product (CTP) developers.

Endogenous LINE-1 (Long Interspersed Nuclear Element-1) Reverse Transcriptase Activity in Platelets Controls Translational Events Through RNA-DNA Hybrids.

OBJECTIVE: One source of endogenous reverse transcriptase (eRT) activity in nucleated cells is the LINE-1/L1 (long interspersed nuclear element-1), a non-LTR retrotransposon that is implicated in the regulation of gene expression. Nevertheless, the presence and function of eRT activity and LINE-1 in human platelets, an anucleate cell, has not previously been determined. APPROACH AND RESULTS: We demonstrate that human and murine platelets possess robust eRT activity and identify the source as being LINE-1 ribonucleoprotein particles. Inhibition of eRT in vitro in isolated platelets from healthy individuals or in people with HIV treated with RT inhibitors enhanced global protein synthesis and platelet activation. If HIV patients were treated with reverse transcriptase inhibitor, we found that platelets from these patients had increased basal activation. We next discovered that eRT activity in platelets controlled the generation of RNA-DNA hybrids, which serve as translational repressors. Inhibition of platelet eRT lifted this RNA-DNA hybrid-induced translational block and was sufficient to increase protein expression of target RNAs identified by RNA-DNA hybrid immunoprecipitation. CONCLUSIONS: Thus, we provide the first evidence that platelets possess L1-encoded eRT activity. We also demonstrate that platelet eRT activity regulates platelet hyperreactivity and thrombosis and controls RNA-DNA hybrid formation and identify that RNA-DNA hybrids function as a novel translational control mechanism in human platelets.

The Flow of the Gibbon LAVA Element Is Facilitated by the LINE-1 Retrotransposition Machinery.

LINE-Alu-VNTR-Alu-like (LAVA) elements comprise a family of non-autonomous, composite, non-LTR retrotransposons specific to gibbons and may have played a role in the evolution of this lineage. A full-length LAVA element consists of portions of repeats found in most primate genomes: CT-rich, Alu-like, and VNTR regions from the SVA retrotransposon, and portions of the AluSz and L1ME5 elements. To evaluate whether the gibbon genome currently harbors functional LAVA elements capable of mobilization by the endogenous LINE-1 (L1) protein machinery and which LAVA components are important for retrotransposition, we established a trans-mobilization assay in HeLa cells. Specifically, we tested if a full-length member of the older LAVA subfamily C that was isolated from the gibbon genome and named LAVAC, or its components, can be mobilized in the presence of the human L1 protein machinery. We show that L1 proteins mobilize the LAVAC element at frequencies exceeding processed pseudogene formation and human SVAE retrotransposition by > 100-fold and ≥3-fold, respectively. We find that only the SVA-derived portions confer activity, and truncation of the 3' L1ME5 portion increases retrotransposition rates by at least 100%. Tagged de novo insertions integrated into intronic regions in cell culture, recapitulating findings in the gibbon genome. Finally, we present alternative models for the rise of the LAVA retrotransposon in the gibbon lineage.

APOBEC4 Enhances the Replication of HIV-1.

APOBEC4 (A4) is a member of the AID/APOBEC family of cytidine deaminases. In this study we found a high mRNA expression of A4 in human testis. In contrast, there were only low levels of A4 mRNA detectable in 293T, HeLa, Jurkat or A3.01 cells. Ectopic expression of A4 in HeLa cells resulted in mostly cytoplasmic localization of the protein. To test whether A4 has antiviral activity similar to that of proteins of the APOBEC3 (A3) subfamily, A4 was co-expressed in 293T cells with wild type HIV-1 and HIV-1 luciferase reporter viruses. We found that A4 did not inhibit the replication of HIV-1 but instead enhanced the production of HIV-1 in a dose-dependent manner and seemed to act on the viral LTR. A4 did not show detectable cytidine deamination activity in vitro and weakly interacted with single-stranded DNA. The presence of A4 in virus producer cells enhanced HIV-1 replication by transiently transfected A4 or stably expressed A4 in HIV-susceptible cells. APOBEC4 was capable of similarly enhancing transcription from a broad spectrum of promoters, regardless of whether they were viral or mammalian. We hypothesize that A4 may have a natural role in modulating host promoters or endogenous LTR promoters.

CD30 Receptor-Targeted Lentiviral Vectors for Human Induced Pluripotent Stem Cell-Specific Gene Modification.

Cultures of induced pluripotent stem cells (iPSCs) often contain cells of varying grades of pluripotency. We present novel lentiviral vectors targeted to the surface receptor CD30 (CD30-LV) to transfer genes into iPSCs that are truly pluripotent as demonstrated by marker gene expression. We demonstrate that CD30 expression is restricted to SSEA4(high) cells of human iPSC cultures and a human embryonic stem cell line. When CD30-LV was added to iPSCs during routine cultivation, efficient and exclusive transduction of cells positive for the pluripotency marker Oct-4 was achieved, while retaining their pluripotency. When added during the reprogramming process, CD30-LV solely transduced cells that became fully reprogrammed iPSCs as confirmed by co-expression of endogenous Nanog and the reporter gene. Thus, CD30-LV may serve as novel tool for the selective gene transfer into PSCs with broad applications in basic and therapeutic research.

The Engineered SVA Trans-mobilization Assay.

Mammalian genomes harbor autonomous retrotransposons coding for the proteins required for their own mobilization, and nonautonomous retrotransposons, such as the human SVA element, which are transcribed but do not have any coding capacity. Mobilization of nonautonomous retrotransposons depends on the recruitment of the protein machinery encoded by autonomous retrotransposons. Here, we summarize the experimental details of SVA trans-mobilization assays which address multiple questions regarding the biology of both nonautonomous SVA elements and autonomous LINE-1 (L1) retrotransposons. The assay evaluates if and to what extent a noncoding SVA element is mobilized in trans by the L1-encoded protein machinery, the structural organization of the resulting marked de novo insertions, if they mimic endogenous SVA insertions and what the roles of individual domains of the nonautonomous retrotransposon for SVA mobilization are. Furthermore, the highly sensitive trans-mobilization assay can be used to verify the presence of otherwise barely detectable endogenously expressed functional L1 proteins via their marked SVA trans-mobilizing activity.

Reprogramming triggers endogenous L1 and Alu retrotransposition in human induced pluripotent stem cells.

Human induced pluripotent stem cells (hiPSCs) are capable of unlimited proliferation and can differentiate in vitro to generate derivatives of the three primary germ layers. Genetic and epigenetic abnormalities have been reported by Wissing and colleagues to occur during hiPSC derivation, including mobilization of engineered LINE-1 (L1) retrotransposons. However, incidence and functional impact of endogenous retrotransposition in hiPSCs are yet to be established. Here we apply retrotransposon capture sequencing to eight hiPSC lines and three human embryonic stem cell (hESC) lines, revealing endogenous L1, Alu and SINE-VNTR-Alu (SVA) mobilization during reprogramming and pluripotent stem cell cultivation. Surprisingly, 4/7 de novo L1 insertions are full length and 6/11 retrotransposition events occurred in protein-coding genes expressed in pluripotent stem cells. We further demonstrate that an intronic L1 insertion in the CADPS2 gene is acquired during hiPSC cultivation and disrupts CADPS2 expression. These experiments elucidate endogenous retrotransposition, and its potential consequences, in hiPSCs and hESCs.

Enhanced expression of LINE-1-encoded ORF2 protein in early stages of colon and prostate transformation.

LINE-1 (L1) retrotransposons are a source of endogenous reverse transcriptase (RT) activity, which is expressed as part of the L1-encoded ORF2 protein (L1-ORF2p). L1 elements are highly expressed in many cancer types, while being silenced in most differentiated somatic tissues. We previously found that RT inhibition reduces cell proliferation and promotes differentiation in neoplastic cells, indicating that high endogenous RT activity promotes cancer growth. Here we investigate the expression of L1-ORF2p in several human types of cancer.We have developed a highly specific monoclonal antibody (mAb chA1-L1) to study ORF2p expression and localization in human cancer cells and tissues.We uncover new evidence for high levels of L1-ORF2p in transformed cell lines and staged epithelial cancer tissues (colon, prostate, lung and breast) while no or only basal ORF2p expression was detected in non-transformed cells. An in-depth analysis of colon and prostate tissues shows ORF2p expression in preneoplastic stages, namely transitional mucosa and prostate intraepithelial neoplasia (PIN), respectively.Our results show that L1-ORF2p is overexpressed in tumor and in preneoplastic colon and prostate tissues; this latter finding suggests that ORF2p could be considered as a potential early diagnostic biomarker.

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.

Primate-specific endogenous retrovirus-driven transcription defines naive-like stem cells.

Naive embryonic stem cells hold great promise for research and therapeutics as they have broad and robust developmental potential. While such cells are readily derived from mouse blastocysts it has not been possible to isolate human equivalents easily, although human naive-like cells have been artificially generated (rather than extracted) by coercion of human primed embryonic stem cells by modifying culture conditions or through transgenic modification. Here we show that a sub-population within cultures of human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) manifests key properties of naive state cells. These naive-like cells can be genetically tagged, and are associated with elevated transcription of HERVH, a primate-specific endogenous retrovirus. HERVH elements provide functional binding sites for a combination of naive pluripotency transcription factors, including LBP9, recently recognized as relevant to naivety in mice. LBP9-HERVH drives hESC-specific alternative and chimaeric transcripts, including pluripotency-modulating long non-coding RNAs. Disruption of LBP9, HERVH and HERVH-derived transcripts compromises self-renewal. These observations define HERVH expression as a hallmark of naive-like hESCs, and establish novel primate-specific transcriptional circuitry regulating pluripotency.

Human LINE-1 restriction by APOBEC3C is deaminase independent and mediated by an ORF1p interaction that affects LINE reverse transcriptase activity.

LINE-1 (L1) retrotransposons are mobile genetic elements whose extensive proliferation resulted in the generation of ≈ 34% of the human genome. They have been shown to be a cause of single-gene diseases. Moreover, L1-encoded endonuclease can elicit double-strand breaks that may lead to genomic instability. Mammalian cells adopted strategies restricting mobility and deleterious consequences of uncontrolled retrotransposition. The human APOBEC3 protein family of polynucleotide cytidine deaminases contributes to intracellular defense against retroelements. APOBEC3 members inhibit L1 retrotransposition by 35-99%. However, genomic L1 retrotransposition events that occurred in the presence of L1-restricting APOBEC3 proteins are devoid of detectable G-to-A hypermutations, suggesting one or multiple deaminase-independent L1 restricting mechanisms. We set out to uncover the mechanism of APOBEC3C (A3C)-mediated L1 inhibition and found that it is deaminase independent, requires an intact dimerization site and the RNA-binding pocket mutation R122A abolishes L1 restriction by A3C. Density gradient centrifugation of L1 ribonucleoprotein particles, subcellular co-localization of L1-ORF1p and A3C and co-immunoprecipitation experiments indicate that an RNA-dependent physical interaction between L1 ORF1p and A3C dimers is essential for L1 restriction. Furthermore, we demonstrate that the amount of L1 complementary DNA synthesized by L1 reverse transcriptase is reduced by ≈ 50% if overexpressed A3C is present.