Vitamin C activates young LINE-1 elements in mouse embryonic stem cells via H3K9me3 demethylation.

BACKGROUND: Vitamin C (vitC) enhances the activity of 2-oxoglutarate-dependent dioxygenases, including TET enzymes, which catalyse DNA demethylation, and Jumonji-domain histone demethylases. The epigenetic remodelling promoted by vitC improves the efficiency of induced pluripotent stem cell derivation, and is required to attain a ground-state of pluripotency in embryonic stem cells (ESCs) that closely mimics the inner cell mass of the early blastocyst. However, genome-wide DNA and histone demethylation can lead to upregulation of transposable elements (TEs), and it is not known how vitC addition in culture media affects TE expression in pluripotent stem cells. RESULTS: Here we show that vitC increases the expression of several TE families, including evolutionarily young LINE-1 (L1) elements, in mouse ESCs. We find that TET activity is dispensable for L1 upregulation, and that instead it occurs largely as a result of H3K9me3 loss mediated by KDM4A/C histone demethylases. Despite increased L1 levels, we did not detect increased somatic insertion rates in vitC-treated cells. Notably, treatment of human ESCs with vitC also increases L1 protein levels, albeit through a distinct, post-transcriptional mechanism. CONCLUSION: VitC directly modulates the expression of mouse L1s and other TEs through epigenetic mechanisms, with potential for downstream effects related to the multiple emerging roles of L1s in cellular function.

A new human embryonic cell type associated with activity of young transposable elements allows definition of the inner cell mass.

There remains much that we do not understand about the earliest stages of human development. On a gross level, there is evidence for apoptosis, but the nature of the affected cell types is unknown. Perhaps most importantly, the inner cell mass (ICM), from which the foetus is derived and hence of interest in reproductive health and regenerative medicine, has proven hard to define. Here, we provide a multi-method analysis of the early human embryo to resolve these issues. Single-cell analysis (on multiple independent datasets), supported by embryo visualisation, uncovers a common previously uncharacterised class of cells lacking commitment markers that segregates after embryonic gene activation (EGA) and shortly after undergo apoptosis. The discovery of this cell type allows us to clearly define their viable ontogenetic sisters, these being the cells of the ICM. While ICM is characterised by the activity of an Old non-transposing endogenous retrovirus (HERVH) that acts to suppress Young transposable elements, the new cell type, by contrast, expresses transpositionally competent Young elements and DNA-damage response genes. As the Young elements are RetroElements and the cells are excluded from the developmental process, we dub these REject cells. With these and ICM being characterised by differential mobile element activities, the human embryo may be a "selection arena" in which one group of cells selectively die, while other less damaged cells persist.

LINE-1 Retrotransposition Assays in Embryonic Stem Cells.

The ongoing mobilization of active non-long terminal repeat (LTR) retrotransposons continues to impact the genomes of most mammals, including humans and rodents. Non-LTR retrotransposons mobilize using an intermediary RNA and a copy-and-paste mechanism termed retrotransposition. Non-LTR retrotransposons are subdivided into long and short interspersed elements (LINEs and SINEs, respectively), depending on their size and autonomy; while active class 1 LINEs (LINE-1s or L1s) encode the enzymatic machinery required to mobilize in cis, active SINEs use the enzymatic machinery of active LINE-1s to mobilize in trans. The mobilization mechanism used by LINE-1s/SINEs was exploited to develop ingenious plasmid-based retrotransposition assays in cultured cells, which typically exploit a reporter gene that can only be activated after a round of retrotransposition. Retrotransposition assays, in cis or in trans, are instrumental tools to study the biology of mammalian LINE-1s and SINEs. In fact, these and other biochemical/genetic assays were used to uncover that endogenous mammalian LINE-1s/SINEs naturally retrotranspose during early embryonic development. However, embryonic stem cells (ESCs) are typically used as a cellular model in these and other studies interrogating LINE-1/SINE expression/regulation during early embryogenesis. Thus, human and mouse ESCs represent an excellent model to understand how active retrotransposons are regulated and how their activity impacts the germline. Here, we describe robust and quantitative protocols to study human/mouse LINE-1 (in cis) and SINE (in trans) retrotransposition using (human and mice) ESCs. These protocols are designed to study the mobilization of active non-LTR retrotransposons in a cellular physiologically relevant context.

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.

Somatic retrotransposition in the developing rhesus macaque brain.

The retrotransposon LINE-1 (L1) is central to the recent evolutionary history of the human genome and continues to drive genetic diversity and germline pathogenesis. However, the spatiotemporal extent and biological significance of somatic L1 activity are poorly defined and are virtually unexplored in other primates. From a single L1 lineage active at the divergence of apes and Old World monkeys, successive L1 subfamilies have emerged in each descendant primate germline. As revealed by case studies, the presently active human L1 subfamily can also mobilize during embryonic and brain development in vivo. It is unknown whether nonhuman primate L1s can similarly generate somatic insertions in the brain. Here we applied approximately 40× single-cell whole-genome sequencing (scWGS), as well as retrotransposon capture sequencing (RC-seq), to 20 hippocampal neurons from two rhesus macaques (Macaca mulatta). In one animal, we detected and PCR-validated a somatic L1 insertion that generated target site duplications, carried a short 5' transduction, and was present in ∼7% of hippocampal neurons but absent from cerebellum and nonbrain tissues. The corresponding donor L1 allele was exceptionally mobile in vitro and was embedded in PRDM4, a gene expressed throughout development and in neural stem cells. Nanopore long-read methylome and RNA-seq transcriptome analyses indicated young retrotransposon subfamily activation in the early embryo, followed by repression in adult tissues. These data highlight endogenous macaque L1 retrotransposition potential, provide prototypical evidence of L1-mediated somatic mosaicism in a nonhuman primate, and allude to L1 mobility in the brain over the past 30 million years of human evolution.

LINE-1 transcription in round spermatids is associated with accretion of 5-carboxylcytosine in their open reading frames.

Chromatin of male and female gametes undergoes a number of reprogramming events during the transition from germ cell to embryonic developmental programs. Although the rearrangement of DNA methylation patterns occurring in the zygote has been extensively characterized, little is known about the dynamics of DNA modifications during spermatid maturation. Here, we demonstrate that the dynamics of 5-carboxylcytosine (5caC) correlate with active transcription of LINE-1 retroelements during murine spermiogenesis. We show that the open reading frames of active and evolutionary young LINE-1s are 5caC-enriched in round spermatids and 5caC is eliminated from LINE-1s and spermiogenesis-specific genes during spermatid maturation, being simultaneously retained at promoters and introns of developmental genes. Our results reveal an association of 5caC with activity of LINE-1 retrotransposons suggesting a potential direct role for this DNA modification in fine regulation of their transcription.

cGAS-mediated induction of type I interferon due to inborn errors of histone pre-mRNA processing.

Inappropriate stimulation or defective negative regulation of the type I interferon response can lead to autoinflammation. In genetically uncharacterized cases of the type I interferonopathy Aicardi-Goutières syndrome, we identified biallelic mutations in LSM11 and RNU7-1, which encode components of the replication-dependent histone pre-mRNA-processing complex. Mutations were associated with the misprocessing of canonical histone transcripts and a disturbance of linker histone stoichiometry. Additionally, we observed an altered distribution of nuclear cyclic guanosine monophosphate-adenosine monophosphate synthase (cGAS) and enhanced interferon signaling mediated by the cGAS-stimulator of interferon genes (STING) pathway in patient-derived fibroblasts. Finally, we established that chromatin without linker histone stimulates cyclic guanosine monophosphate-adenosine monophosphate (cGAMP) production in vitro more efficiently. We conclude that nuclear histones, as key constituents of chromatin, are essential in suppressing the immunogenicity of self-DNA.

sRNA/L1 retrotransposition: using siRNAs and miRNAs to expand the applications of the cell culture-based LINE-1 retrotransposition assay.

The cell culture-based retrotransposition reporter assay has been (and is) an essential tool for the study of vertebrate Long INterspersed Elements (LINEs). Developed more than 20 years ago, this assay has been instrumental in characterizing the role of LINE-encoded proteins in retrotransposition, understanding how ribonucleoprotein particles are formed, how host factors regulate LINE mobilization, etc. Moreover, variations of the conventional assay have been developed to investigate the biology of other currently active human retrotransposons, such as Alu and SVA. Here, we describe a protocol that allows combination of the conventional cell culture-based LINE-1 retrotransposition reporter assay with short interfering RNAs (siRNAs) and microRNA (miRNAs) mimics or inhibitors, which has allowed us to uncover specific miRNAs and host factors that regulate retrotransposition. The protocol described here is highly reproducible, quantitative, robust and flexible, and allows the study of several small RNA classes and various retrotransposons. To illustrate its utility, here we show that siRNAs to Fanconi anaemia proteins (FANC-A and FANC-C) and an inhibitor of miRNA-20 upregulate and downregulate human L1 retrotransposition, respectively. This article is part of a discussion meeting issue 'Crossroads between transposons and gene regulation'.

N6-methyladenosine regulates the stability of RNA:DNA hybrids in human cells.

R-loops are nucleic acid structures formed by an RNA:DNA hybrid and unpaired single-stranded DNA that represent a source of genomic instability in mammalian cells1-4. Here we show that N6-methyladenosine (m6A) modification, contributing to different aspects of messenger RNA metabolism5,6, is detectable on the majority of RNA:DNA hybrids in human pluripotent stem cells. We demonstrate that m6A-containing R-loops accumulate during G2/M and are depleted at G0/G1 phases of the cell cycle, and that the m6A reader promoting mRNA degradation, YTHDF2 (ref. 7), interacts with R-loop-enriched loci in dividing cells. Consequently, YTHDF2 knockout leads to increased R-loop levels, cell growth retardation and accumulation of γH2AX, a marker for DNA double-strand breaks, in mammalian cells. Our results suggest that m6A regulates accumulation of R-loops, implying a role for this modification in safeguarding genomic stability.

LINE-1 Evasion of Epigenetic Repression in Humans.

Epigenetic silencing defends against LINE-1 (L1) retrotransposition in mammalian cells. However, the mechanisms that repress young L1 families and how L1 escapes to cause somatic genome mosaicism in the brain remain unclear. Here we report that a conserved Yin Yang 1 (YY1) transcription factor binding site mediates L1 promoter DNA methylation in pluripotent and differentiated cells. By analyzing 24 hippocampal neurons with three distinct single-cell genomic approaches, we characterized and validated a somatic L1 insertion bearing a 3' transduction. The source (donor) L1 for this insertion was slightly 5' truncated, lacked the YY1 binding site, and was highly mobile when tested in vitro. Locus-specific bisulfite sequencing revealed that the donor L1 and other young L1s with mutated YY1 binding sites were hypomethylated in embryonic stem cells, during neurodifferentiation, and in liver and brain tissue. These results explain how L1 can evade repression and retrotranspose in the human body.

Synthesis and Characterization of Specific Reverse Transcriptase Inhibitors for Mammalian LINE-1 Retrotransposons.

Retrotransposons are a type of transposable element (TE) that have amplified to astonishing numbers in mammalian genomes, comprising more than a third of the human and mouse genomes. Long interspersed element class 1 (LINE-1 or L1) retrotransposons are abundant and currently active retroelements in the human and mouse genomes. Similarly, long terminal repeat (LTR)-containing retrotransposons are abundant in both genomes, although only active in mice. LTR- and LINE-1-retroelements use different mechanisms for retrotransposition, although both involve the reverse transcription of an intermediate retroelement-derived RNA. Retrotransposon activity continues to effect the germline and somatic genomes, generating interindividual variability over evolution and potentially influencing cancer and brain physiology, respectively. However, relatively little is known about the functional consequences of retrotransposition. In this study, we have synthesized and characterized reverse transcriptase inhibitors specific for mammalian LINE-1 retrotransposons, which might help deciphering the functional impact of retrotransposition in vivo.

Genome-wide de novo L1 Retrotransposition Connects Endonuclease Activity with Replication.

L1 retrotransposon-derived sequences comprise approximately 17% of the human genome. Darwinian selective pressures alter L1 genomic distributions during evolution, confounding the ability to determine initial L1 integration preferences. Here, we generated high-confidence datasets of greater than 88,000 engineered L1 insertions in human cell lines that act as proxies for cells that accommodate retrotransposition in vivo. Comparing these insertions to a null model, in which L1 endonuclease activity is the sole determinant dictating L1 integration preferences, demonstrated that L1 insertions are not significantly enriched in genes, transcribed regions, or open chromatin. By comparison, we provide compelling evidence that the L1 endonuclease disproportionately cleaves predominant lagging strand DNA replication templates, while lagging strand 3'-hydroxyl groups may prime endonuclease-independent L1 retrotransposition in a Fanconi anemia cell line. Thus, acquisition of an endonuclease domain, in conjunction with the ability to integrate into replicating DNA, allowed L1 to become an autonomous, interspersed retrotransposon.

Reconstitution of the Ataxia-Telangiectasia Cellular Phenotype With Lentiviral Vectors.

Ataxia-telangiectasia (A-T) is a complex disease arising from mutations in the ATM gene (Ataxia-Telangiectasia Mutated), which plays crucial roles in repairing double-strand DNA breaks (DSBs). Heterogeneous immunodeficiency, extreme radiosensitivity, frequent appearance of tumors and neurological degeneration are hallmarks of the disease, which carries high morbidity and mortality because only palliative treatments are currently available. Gene therapy was effective in animal models of the disease, but the large size of the ATM cDNA required the use of HSV-1 or HSV/AAV hybrid amplicon vectors, whose characteristics make them unlikely tools for treating A-T patients. Due to recent advances in vector packaging, production and biosafety, we developed a lentiviral vector containing the ATM cDNA and tested whether or not it could rescue cellular defects of A-T human mutant fibroblasts. Although the cargo capacity of lentiviral vectors is an inherent limitation in their use, and despite the large size of the transgene, we successfully transduced around 20% of ATM-mutant cells. ATM expression and phosphorylation assays indicated that the neoprotein was functional in transduced cells, further reinforced by their restored capacity to phosphorylate direct ATM substrates such as p53 and their capability to repair radiation-induced DSBs. In addition, transduced cells also restored cellular radiosensitivity and cell cycle abnormalities. Our results demonstrate that lentiviral vectors can be used to rescue the intrinsic cellular defects of ATM-mutant cells, which represent, in spite of their limitations, a proof-of-concept for A-T gene therapy.

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'.

Uridylation by TUT4/7 Restricts Retrotransposition of Human LINE-1s.

LINE-1 retrotransposition is tightly restricted by layers of regulatory control, with epigenetic pathways being the best characterized. Looking at post-transcriptional regulation, we now show that LINE-1 mRNA 3' ends are pervasively uridylated in various human cellular models and in mouse testes. TUT4 and TUT7 uridyltransferases catalyze the modification and function in cooperation with the helicase/RNPase MOV10 to counteract the RNA chaperone activity of the L1-ORF1p retrotransposon protein. Uridylation potently restricts LINE-1 retrotransposition by a multilayer mechanism depending on differential subcellular localization of the uridyltransferases. We propose that uridine residues added by TUT7 in the cytoplasm inhibit initiation of reverse transcription of LINE-1 mRNAs once they are reimported to the nucleus, whereas uridylation by TUT4, which is enriched in cytoplasmic foci, destabilizes mRNAs. These results provide a model for the post-transcriptional restriction of LINE-1, revealing a key physiological role for TUT4/7-mediated uridylation in maintaining genome stability.

RNase H2, mutated in Aicardi-Goutières syndrome, promotes LINE-1 retrotransposition.

Long INterspersed Element class 1 (LINE-1) elements are a type of abundant retrotransposons active in mammalian genomes. An average human genome contains ~100 retrotransposition-competent LINE-1s, whose activity is influenced by the combined action of cellular repressors and activators. TREX1, SAMHD1 and ADAR1 are known LINE-1 repressors and when mutated cause the autoinflammatory disorder Aicardi-Goutières syndrome (AGS). Mutations in RNase H2 are the most common cause of AGS, and its activity was proposed to similarly control LINE-1 retrotransposition. It has therefore been suggested that increased LINE-1 activity may be the cause of aberrant innate immune activation in AGS Here, we establish that, contrary to expectations, RNase H2 is required for efficient LINE-1 retrotransposition. As RNase H1 overexpression partially rescues the defect in RNase H2 null cells, we propose a model in which RNase H2 degrades the LINE-1 RNA after reverse transcription, allowing retrotransposition to be completed. This also explains how LINE-1 elements can retrotranspose efficiently without their own RNase H activity. Our findings appear to be at odds with LINE-1-derived nucleic acids driving autoinflammation in AGS.

L1 Mosaicism in Mammals: Extent, Effects, and Evolution.

The retrotransposon LINE-1 (long interspersed element 1, L1) is a transposable element that has extensively colonized the mammalian germline. L1 retrotransposition can also occur in somatic cells, causing genomic mosaicism, as well as in cancer. However, the extent of L1-driven mosaicism arising during ontogenesis is unclear. We discuss here recent experimental data which, at a minimum, fully substantiate L1 mosaicism in early embryonic development and neural cells, including post-mitotic neurons. We also consider the possible biological impact of somatic L1 insertions in neurons, the existence of donor L1s that are highly active ('hot') in specific spatiotemporal niches, and the evolutionary selection of donor L1s driving neuronal mosaicism.

Heritable L1 retrotransposition in the mouse primordial germline and early embryo.

LINE-1 (L1) retrotransposons are a noted source of genetic diversity and disease in mammals. To expand its genomic footprint, L1 must mobilize in cells that will contribute their genetic material to subsequent generations. Heritable L1 insertions may therefore arise in germ cells and in pluripotent embryonic cells, prior to germline specification, yet the frequency and predominant developmental timing of such events remain unclear. Here, we applied mouse retrotransposon capture sequencing (mRC-seq) and whole-genome sequencing (WGS) to pedigrees of C57BL/6J animals, and uncovered an L1 insertion rate of ≥1 event per eight births. We traced heritable L1 insertions to pluripotent embryonic cells and, strikingly, to early primordial germ cells (PGCs). New L1 insertions bore structural hallmarks of target-site primed reverse transcription (TPRT) and mobilized efficiently in a cultured cell retrotransposition assay. Together, our results highlight the rate and evolutionary impact of heritable L1 retrotransposition and reveal retrotransposition-mediated genomic diversification as a fundamental property of pluripotent embryonic cells in vivo.

Engineered LINE-1 retrotransposition in nondividing human neurons.

Half the human genome is made of transposable elements (TEs), whose ongoing activity continues to impact our genome. LINE-1 (or L1) is an autonomous non-LTR retrotransposon in the human genome, comprising 17% of its genomic mass and containing an average of 80-100 active L1s per average genome that provide a source of inter-individual variation. New LINE-1 insertions are thought to accumulate mostly during human embryogenesis. Surprisingly, the activity of L1s can further impact the somatic human brain genome. However, it is currently unknown whether L1 can retrotranspose in other somatic healthy tissues or if L1 mobilization is restricted to neuronal precursor cells (NPCs) in the human brain. Here, we took advantage of an engineered L1 retrotransposition assay to analyze L1 mobilization rates in human mesenchymal (MSCs) and hematopoietic (HSCs) somatic stem cells. Notably, we have observed that L1 expression and engineered retrotransposition is much lower in both MSCs and HSCs when compared to NPCs. Remarkably, we have further demonstrated for the first time that engineered L1s can retrotranspose efficiently in mature nondividing neuronal cells. Thus, these findings suggest that the degree of somatic mosaicism and the impact of L1 retrotransposition in the human brain is likely much higher than previously thought.