LINE-1 retrotransposons contribute to mouse PV interneuron development.

Retrotransposons are mobile DNA sequences duplicated via transcription and reverse transcription of an RNA intermediate. Cis-regulatory elements encoded by retrotransposons can also promote the transcription of adjacent genes. Somatic LINE-1 (L1) retrotransposon insertions have been detected in mammalian neurons. It is, however, unclear whether L1 sequences are mobile in only some neuronal lineages or therein promote neurodevelopmental gene expression. Here we report programmed L1 activation by SOX6, a transcription factor critical for parvalbumin (PV) interneuron development. Mouse PV interneurons permit L1 mobilization in vitro and in vivo, harbor unmethylated L1 promoters and express full-length L1 mRNAs and proteins. Using nanopore long-read sequencing, we identify unmethylated L1s proximal to PV interneuron genes, including a novel L1 promoter-driven Caps2 transcript isoform that enhances neuron morphological complexity in vitro. These data highlight the contribution made by L1 cis-regulatory elements to PV interneuron development and transcriptome diversity, uncovered due to L1 mobility in this milieu.

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.

Locus-resolution analysis of L1 regulation and retrotransposition potential in mouse embryonic development.

Mice harbor ∼2800 intact copies of the retrotransposon Long Interspersed Element 1 (L1). The in vivo retrotransposition capacity of an L1 copy is defined by both its sequence integrity and epigenetic status, including DNA methylation of the monomeric units constituting young mouse L1 promoters. Locus-specific L1 methylation dynamics during development may therefore elucidate and explain spatiotemporal niches of endogenous retrotransposition but remain unresolved. Here, we interrogate the retrotransposition efficiency and epigenetic fate of source (donor) L1s, identified as mobile in vivo. We show that promoter monomer loss consistently attenuates the relative retrotransposition potential of their offspring (daughter) L1 insertions. We also observe that most donor/daughter L1 pairs are efficiently methylated upon differentiation in vivo and in vitro. We use Oxford Nanopore Technologies (ONT) long-read sequencing to resolve L1 methylation genome-wide and at individual L1 loci, revealing a distinctive "smile" pattern in methylation levels across the L1 promoter region. Using Pacific Biosciences (PacBio) SMRT sequencing of L1 5' RACE products, we then examine DNA methylation dynamics at the mouse L1 promoter in parallel with transcription start site (TSS) distribution at locus-specific resolution. Together, our results offer a novel perspective on the interplay between epigenetic repression, L1 evolution, and genome stability.

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.

Retrotransposon instability dominates the acquired mutation landscape of mouse induced pluripotent stem cells.

Induced pluripotent stem cells (iPSCs) can in principle differentiate into any cell of the body, and have revolutionized biomedical research and regenerative medicine. Unlike their human counterparts, mouse iPSCs (miPSCs) are reported to silence transposable elements and prevent transposable element-mediated mutagenesis. Here we apply short-read or Oxford Nanopore Technologies long-read genome sequencing to 38 bulk miPSC lines reprogrammed from 10 parental cell types, and 18 single-cell miPSC clones. While single nucleotide variants and structural variants restricted to miPSCs are rare, we find 83 de novo transposable element insertions, including examples intronic to Brca1 and Dmd. LINE-1 retrotransposons are profoundly hypomethylated in miPSCs, beyond other transposable elements and the genome overall, and harbor alternative protein-coding gene promoters. We show that treatment with the LINE-1 inhibitor lamivudine does not hinder reprogramming and efficiently blocks endogenous retrotransposition, as detected by long-read genome sequencing. These experiments reveal the complete spectrum and potential significance of mutations acquired by miPSCs.

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.

No evidence of human genome integration of SARS-CoV-2 found by long-read DNA sequencing.

A recent study proposed that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) hijacks the LINE-1 (L1) retrotransposition machinery to integrate into the DNA of infected cells. If confirmed, this finding could have significant clinical implications. Here, we apply deep (>50×) long-read Oxford Nanopore Technologies (ONT) sequencing to HEK293T cells infected with SARS-CoV-2 and do not find the virus integrated into the genome. By examining ONT data from separate HEK293T cultivars, we completely resolve 78 L1 insertions arising in vitro in the absence of L1 overexpression systems. ONT sequencing applied to hepatitis B virus (HBV)-positive liver cancer tissues located a single HBV insertion. These experiments demonstrate reliable resolution of retrotransposon and exogenous virus insertions by ONT sequencing. That we find no evidence of SARS-CoV-2 integration suggests that such events are, at most, extremely rare in vivo and therefore are unlikely to drive oncogenesis or explain post-recovery detection of the virus.

A sensitive and affordable multiplex RT-qPCR assay for SARS-CoV-2 detection.

With the ongoing COVID-19 (Coronavirus Disease 2019) pandemic, caused by the novel coronavirus SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus 2), there is a need for sensitive, specific, and affordable diagnostic tests to identify infected individuals, not all of whom are symptomatic. The most sensitive test involves the detection of viral RNA using RT-qPCR (quantitative reverse transcription PCR), with many commercial kits now available for this purpose. However, these are expensive, and supply of such kits in sufficient numbers cannot always be guaranteed. We therefore developed a multiplex assay using well-established SARS-CoV-2 targets alongside a human cellular control (RPP30) and a viral spike-in control (Phocine Herpes Virus 1 [PhHV-1]), which monitor sample quality and nucleic acid extraction efficiency, respectively. Here, we establish that this test performs as well as widely used commercial assays, but at substantially reduced cost. Furthermore, we demonstrate >1,000-fold variability in material routinely collected by combined nose and throat swabbing and establish a statistically significant correlation between the detected level of human and SARS-CoV-2 nucleic acids. The inclusion of the human control probe in our assay therefore provides a quantitative measure of sample quality that could help reduce false-negative rates. We demonstrate the feasibility of establishing a robust RT-qPCR assay at approximately 10% of the cost of equivalent commercial assays, which could benefit low-resource environments and make high-volume testing affordable.

Nanopore Sequencing Enables Comprehensive Transposable Element Epigenomic Profiling.

Transposable elements (TEs) drive genome evolution and are a notable source of pathogenesis, including cancer. While CpG methylation regulates TE activity, the locus-specific methylation landscape of mobile human TEs has to date proven largely inaccessible. Here, we apply new computational tools and long-read nanopore sequencing to directly infer CpG methylation of novel and extant TE insertions in hippocampus, heart, and liver, as well as paired tumor and non-tumor liver. As opposed to an indiscriminate stochastic process, we find pronounced demethylation of young long interspersed element 1 (LINE-1) retrotransposons in cancer, often distinct to the adjacent genome and other TEs. SINE-VNTR-Alu (SVA) retrotransposons, including their internal tandem repeat-associated CpG island, are near-universally methylated. We encounter allele-specific TE methylation and demethylation of aberrantly expressed young LINE-1s in normal tissues. Finally, we recover the complete sequences of tumor-specific LINE-1 insertions and their retrotransposition hallmarks, demonstrating how long-read sequencing can simultaneously survey the epigenome and detect somatic TE mobilization.

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.

Dynamic Methylation of an L1 Transduction Family during Reprogramming and Neurodifferentiation.

The retrotransposon LINE-1 (L1) is a significant source of endogenous mutagenesis in humans. In each individual genome, a few retrotransposition-competent L1s (RC-L1s) can generate new heritable L1 insertions in the early embryo, primordial germ line, and germ cells. L1 retrotransposition can also occur in the neuronal lineage and cause somatic mosaicism. Although DNA methylation mediates L1 promoter repression, the temporal pattern of methylation applied to individual RC-L1s during neurogenesis is unclear. Here, we identified a de novo L1 insertion in a human induced pluripotent stem cell (hiPSC) line via retrotransposon capture sequencing (RC-seq). The L1 insertion was full-length and carried 5' and 3' transductions. The corresponding donor RC-L1 was part of a large and recently active L1 transduction family and was highly mobile in a cultured-cell L1 retrotransposition reporter assay. Notably, we observed distinct and dynamic DNA methylation profiles for the de novo L1 and members of its extended transduction family during neuronal differentiation. These experiments reveal how a de novo L1 insertion in a pluripotent stem cell is rapidly recognized and repressed, albeit incompletely, by the host genome during neurodifferentiation, while retaining potential for further retrotransposition.

L1 Retrotransposon Heterogeneity in Ovarian Tumor Cell Evolution.

LINE-1 (L1) retrotransposons are a source of insertional mutagenesis in tumor cells. However, the clinical significance of L1 mobilization during tumorigenesis remains unclear. Here, we applied retrotransposon capture sequencing (RC-seq) to multiple single-cell clones isolated from five ovarian cancer cell lines and HeLa cells and detected endogenous L1 retrotransposition in vitro. We then applied RC-seq to ovarian tumor and matched blood samples from 19 patients and identified 88 tumor-specific L1 insertions. In one tumor, an intronic de novo L1 insertion supplied a novel cis-enhancer to the putative chemoresistance gene STC1. Notably, the tumor subclone carrying the STC1 L1 mutation increased in prevalence after chemotherapy, further increasing STC1 expression. We also identified hypomethylated donor L1s responsible for new L1 insertions in tumors and cultivated cancer cells. These congruent in vitro and in vivo results highlight L1 insertional mutagenesis as a common component of ovarian tumorigenesis and cancer genome heterogeneity.

L1 retrotransposition is a common feature of mammalian hepatocarcinogenesis.

The retrotransposon Long Interspersed Element 1 (LINE-1 or L1) is a continuing source of germline and somatic mutagenesis in mammals. Deregulated L1 activity is a hallmark of cancer, and L1 mutagenesis has been described in numerous human malignancies. We previously employed retrotransposon capture sequencing (RC-seq) to analyze hepatocellular carcinoma (HCC) samples from patients infected with hepatitis B or hepatitis C virus and identified L1 variants responsible for activating oncogenic pathways. Here, we have applied RC-seq and whole-genome sequencing (WGS) to an Abcb4 (Mdr2)-/- mouse model of hepatic carcinogenesis and demonstrated for the first time that L1 mobilization occurs in murine tumors. In 12 HCC nodules obtained from 10 animals, we validated four somatic L1 insertions by PCR and capillary sequencing, including TF subfamily elements, and one GF subfamily example. One of the TF insertions carried a 3' transduction, allowing us to identify its donor L1 and to demonstrate that this full-length TF element retained retrotransposition capacity in cultured cancer cells. Using RC-seq, we also identified eight tumor-specific L1 insertions from 25 HCC patients with a history of alcohol abuse. Finally, we used RC-seq and WGS to identify three tumor-specific L1 insertions among 10 intra-hepatic cholangiocarcinoma (ICC) patients, including one insertion traced to a donor L1 on Chromosome 22 known to be highly active in other cancers. This study reveals L1 mobilization as a common feature of hepatocarcinogenesis in mammals, demonstrating that the phenomenon is not restricted to human viral HCC etiologies and is encountered in murine liver tumors.

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.

Retrotransposon Capture Sequencing (RC-Seq): A Targeted, High-Throughput Approach to Resolve Somatic L1 Retrotransposition in Humans.

Mobile genetic elements (MGEs) are of critical importance in genomics and developmental biology. Polymorphic and somatic MGE insertions have the potential to impact the phenotype of an individual, depending on their genomic locations and functional consequences. However, the identification of polymorphic and somatic insertions among the plethora of copies residing in the genome presents a formidable technical challenge. Whole genome sequencing has the potential to address this problem; however, its efficacy depends on the abundance of cells carrying the new insertion. Robust detection of somatic insertions present in only a subset of cells within a given sample can also be prohibitively expensive due to a requirement for high sequencing depth. Here, we describe retrotransposon capture sequencing (RC-seq), a sequence capture approach in which Illumina libraries are enriched for fragments containing the 5' and 3' termini of specific MGEs. RC-seq allows the detection of known polymorphic insertions present in an individual, as well as the identification of rare or private germline insertions not previously described. Furthermore, RC-seq can be used to detect and characterize somatic insertions, providing a valuable tool to elucidate the extent and characteristics of MGE activity in healthy tissues and in various disease states.

Ubiquitous L1 mosaicism in hippocampal neurons.

Somatic LINE-1 (L1) retrotransposition during neurogenesis is a potential source of genotypic variation among neurons. As a neurogenic niche, the hippocampus supports pronounced L1 activity. However, the basal parameters and biological impact of L1-driven mosaicism remain unclear. Here, we performed single-cell retrotransposon capture sequencing (RC-seq) on individual human hippocampal neurons and glia, as well as cortical neurons. An estimated 13.7 somatic L1 insertions occurred per hippocampal neuron and carried the sequence hallmarks of target-primed reverse transcription. Notably, hippocampal neuron L1 insertions were specifically enriched in transcribed neuronal stem cell enhancers and hippocampus genes, increasing their probability of functional relevance. In addition, bias against intronic L1 insertions sense oriented relative to their host gene was observed, perhaps indicating moderate selection against this configuration in vivo. These experiments demonstrate pervasive L1 mosaicism at genomic loci expressed in hippocampal neurons.

Glucose conjugation of anti-HIV-1 oligonucleotides containing unmethylated CpG motifs reduces their immunostimulatory activity.

Antisense oligodeoxynucleotides (ODNs) are short synthetic DNA polymers complementary to a target RNA sequence. They are commonly designed to halt a biological event, such as translation or splicing. ODNs are potentially useful therapeutic agents for the treatment of different human diseases. Carbohydrate-ODN conjugates have been reported to improve the cell-specific delivery of ODNs through receptor mediated endocytosis. We tested the anti-HIV activity and biochemical properties of the 5'-end glucose-conjugated GEM 91 ODN targeting the initiation codon of the gag gene of HIV-1 RNA in cell-based assays. The conjugation of a glucose residue significantly reduces the immunostimulatory effect without diminishing its potent anti-HIV-1 activity. No significant effects were observed in either ODN stability in serum, in vitro degradation of antisense DNA-RNA hybrids by RNase H, cell toxicity, cellular uptake and ability to interfere with genomic HIV-1 dimerisation.

Efficient HIV-1 inhibition by a 16 nt-long RNA aptamer designed by combining in vitro selection and in silico optimisation strategies.

The human immunodeficiency virus type-1 (HIV-1) genome contains multiple, highly conserved structural RNA domains that play key roles in essential viral processes. Interference with the function of these RNA domains either by disrupting their structures or by blocking their interaction with viral or cellular factors may seriously compromise HIV-1 viability. RNA aptamers are amongst the most promising synthetic molecules able to interact with structural domains of viral genomes. However, aptamer shortening up to their minimal active domain is usually necessary for scaling up production, what requires very time-consuming, trial-and-error approaches. Here we report on the in vitro selection of 64 nt-long specific aptamers against the complete 5'-untranslated region of HIV-1 genome, which inhibit more than 75% of HIV-1 production in a human cell line. The analysis of the selected sequences and structures allowed for the identification of a highly conserved 16 nt-long stem-loop motif containing a common 8 nt-long apical loop. Based on this result, an in silico designed 16 nt-long RNA aptamer, termed RNApt16, was synthesized, with sequence 5'-CCCCGGCAAGGAGGGG-3'. The HIV-1 inhibition efficiency of such an aptamer was close to 85%, thus constituting the shortest RNA molecule so far described that efficiently interferes with HIV-1 replication.

HIV RNA dimerisation interference by antisense oligonucleotides targeted to the 5' UTR structural elements.

The HIV-1 genome consists of two identical RNA molecules non-covalently linked by their 5' unstranslatable regions (5' UTR). The high level of sequence and structural conservation of this region correlates with its important functional involvement in the viral cycle, making it an attractive target for antiviral treatments based on antisense technology. Ten unmodified DNA antisense oligonucleotides (ODNs) targeted against different conserved structural elements within the 5' UTR were assayed for their capacity to interfere with HIV-1 RNA dimerisation, inhibit gene expression, and prevent virus production in cell cultures. The results show that, in addition to the well-characterised dimerisation initiation site (DIS), targeting of the AUG-containing structural element may reflect its direct role in HIV-1 genomic RNA dimerisation in vitro. Similarly, blocking the 3' end sequences of the stem-loop domain containing the primer biding site interferes with RNA dimerisation. Targeting the apical portion of the TAR element, however, appears to promote dimerisation. ODNs targeted against the conserved polyadenylation signal [Poly(A)], the primer binding site (PBS), the major splicing donor (SD) or the major packaging signal (Psi), and AUG-containing structural elements led to a highly efficient inhibition of HIV-1 gene expression and virus production in cell culture. Together, these results support the idea that ODNs possess great potential as molecular tools for the functional characterisation of viral RNA structural domains. Moreover, the targeting of these domains leads to the potent inhibition of viral replication, underscoring the potential of conserved structural RNA elements as antiviral targets.

Identification of an hepatitis delta virus-like ribozyme at the mRNA 5'-end of the L1Tc retrotransposon from Trypanosoma cruzi.

L1Tc is a non-LTR LINE element from Trypanosoma cruzi that encodes its transposition machinery and bears an internal promoter. Herewith, we report the identification of an in vitro active hepatitis delta virus-like ribozyme located in the first 77 nt at the 5'-end of the L1Tc mRNA (L1TcRz). The data presented show that L1TcRz has a co-transcriptional function. Using gel-purified uncleaved RNA transcripts, the data presented indicate that the kinetics of the self-cleaving, in a magnesium-dependent reaction, fits to a two-phase decay curve. The cleavage point identified by primer extension takes place at +1 position of the element. The hydroxyl nature of the 5'-end of the 3'-fragment generated by the cleavage activity of L1TcRz was confirmed. Since we have previously described that the 77-nt long fragment located at the 5'-end of L1Tc has promoter activity, the existence of a ribozyme in L1Tc makes this element to be the first described non-LTR retroelement that has an internal promoter-ribozyme dual function. The L1Tc nucleotides located downstream of the ribozyme catalytic motif appear to inhibit its activity. This inhibition may be influenced by the existence of a specific L1Tc RNA conformation that is recognized by RNase P.