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.

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'.

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.

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.

Study of Transposable Elements and Their Genomic Impact.

Transposable elements (TEs) have been considered traditionally as junk DNA, i.e., DNA sequences that despite representing a high proportion of genomes had no evident cellular functions. However, over the last decades, it has become undeniable that not only TE-derived DNA sequences have (and had) a fundamental role during genome evolution, but also TEs have important implications in the origin and evolution of many genomic disorders. This concise review provides a brief overview of the different types of TEs that can be found in genomes, as well as a list of techniques and methods used to study their impact and mobilization. Some of these techniques will be covered in detail in this Method Book.

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.

Wiping DNA methylation: Wip1 regulates genomic fluidity on cancer.

Wip1 phosphatase plays an important role in cancer by inactivating p53 and INK4a/ARF pathways. In this issue of Cancer Cell, Filipponi and colleagues further connect the oncogenic role of Wip1 with heterochromatin dynamics, transposable element expression, and a mutation-prone environment that may enhance heterogeneity and ultimately contribute to tumor evolution.

Endogenous retrotransposition activates oncogenic pathways in hepatocellular carcinoma.

LINE-1 (L1) retrotransposons are mobile genetic elements comprising ~17% of the human genome. New L1 insertions can profoundly alter gene function and cause disease, though their significance in cancer remains unclear. Here, we applied enhanced retrotransposon capture sequencing (RC-seq) to 19 hepatocellular carcinoma (HCC) genomes and elucidated two archetypal L1-mediated mechanisms enabling tumorigenesis. In the first example, 4/19 (21.1%) donors presented germline retrotransposition events in the tumor suppressor mutated in colorectal cancers (MCC). MCC expression was ablated in each case, enabling oncogenic ß-catenin/Wnt signaling. In the second example, suppression of tumorigenicity 18 (ST18) was activated by a tumor-specific L1 insertion. Experimental assays confirmed that the L1 interrupted a negative feedback loop by blocking ST18 repression of its enhancer. ST18 was also frequently amplified in HCC nodules from Mdr2(-/-) mice, supporting its assignment as a candidate liver oncogene. These proof-of-principle results substantiate L1-mediated retrotransposition as an important etiological factor in HCC.

Analysis of LINE-1 expression in human pluripotent cells.

Half of the human genome is composed of repeated DNA, and some types are mobile within our genome (transposons and retrotransposons). Despite their abundance, only a small fraction of them are currently active in our genome (Long Interspersed Element-1 (LINE-1), Alu, and SVA elements). LINE-1 or L1 elements are a family of active non-LTR retrotransposons, the ongoing mobilization of which still impacts our genome. As selfish DNA elements, L1 activity is more prominent in early human development, where new insertions would be transmitted to the progeny. Here, we describe the conventional methods aimed to determine the expression level of LINE-1 elements in pluripotent human cells.

Reprogramming somatic cells into iPS cells activates LINE-1 retroelement mobility.

Long interspersed element-1 (LINE-1 or L1) retrotransposons account for nearly 17% of human genomic DNA and represent a major evolutionary force that has reshaped the structure and function of the human genome. However, questions remain concerning both the frequency and the developmental timing of L1 retrotransposition in vivo and whether the mobility of these retroelements commonly results in insertional and post-insertional mechanisms of genomic injury. Cells exhibiting high rates of L1 retrotransposition might be especially at risk for such injury. We assessed L1 mRNA expression and L1 retrotransposition in two biologically relevant cell types, human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs), as well as in control parental human dermal fibroblasts (HDFs). Full-length L1 mRNA and the L1 open reading frame 1-encoded protein (ORF1p) were readily detected in hESCs and iPSCs, but not in HDFs. Sequencing analysis proved the expression of human-specific L1 element mRNAs in iPSCs. Bisulfite sequencing revealed that the increased L1 expression observed in iPSCs correlates with an overall decrease in CpG methylation in the L1 promoter region. Finally, retrotransposition of an engineered human L1 element was ~10-fold more efficient in iPSCs than in parental HDFs. These findings indicate that somatic cell reprogramming is associated with marked increases in L1 expression and perhaps increases in endogenous L1 retrotransposition, which could potentially impact the genomic integrity of the resultant iPSCs.

An epi [c] genetic battle: LINE-1 retrotransposons and intragenomic conflict in humans.

The ongoing activity of the human retrotransposon Long Interspersed Element 1 (LINE-1 or L1) continues to impact the human genome in various ways. Throughout evolution, mammalian and primate genomes have been under selection to generate strategies to reduce the activity of selfish DNA like L1. Similarly, selfish DNA has evolved to elude these containment systems. This intragenomic conflict has left many inactive versions of LINEs and other Transposable Elements (TEs) littering the human genome, which together account for roughly half of our DNA. Here, we survey the distinct mechanisms operating in the human genome that seem to reduce the mobility of L1s. In addition, we discuss recent findings that strongly suggest epigenetic mechanisms specifically regulate L1 activity in pluripotent human cells.

Enrichment of human ESC-derived multipotent mesenchymal stem cells with immunosuppressive and anti-inflammatory properties capable to protect against experimental inflammatory bowel disease.

Human ESCs provide access to the earliest stages of human development and may serve as an unlimited source of functional cells for future cell therapies. The optimization of methods directing the differentiation of human embryonic stem cells (hESCs) into tissue-specific precursors becomes crucial. We report an efficient enrichment of mesenchymal stem cells (MSCs) from hESCs through specific inhibition of SMAD-2/3 signaling. Human ESC-derived MSCs (hESC-MSCs) emerged as a population of fibroblastoid cells expressing a MSC phenotype: CD73+ CD90+ CD105+ CD44+ CD166+ CD45- CD34- CD14- CD19- human leucocyte antigen-DR (HLA-DR)-. After 28 days of SMAD-2/3 inhibition, hESC cultures were enriched (>42%) in multipotent MSCs. CD73+CD90+ hESC-MSCs were fluorescence activated cell sorting (FACS)-isolated and long-term cultures were established and maintained for many passages displaying a faster growth than somatic tissue-derived MSCs while maintaining MSC morphology and phenotype. They displayed osteogenic, adipogenic, and chondrocytic differentiation potential and exhibited potent immunosuppressive and anti-inflammatory properties in vitro and in vivo, where hESC-MSCs were capable of protecting against an experimental model of inflammatory bowel disease. Interestingly, the efficient enrichment of hESCs into MSCs through inhibition of SMAD-2/3 signaling was not reproducible with distinct induced pluripotent stem cell lines. Our findings provide mechanistic insights into the differentiation of hESCs into immunosuppressive and anti-inflammatory multipotent MSCs with potential future clinical applications.

Epigenetic control of retrotransposon expression in human embryonic stem cells.

Long interspersed element 1s (LINE-1s or L1s) are a family of non-long-terminal-repeat retrotransposons that predominate in the human genome. Active LINE-1 elements encode proteins required for their mobilization. L1-encoded proteins also act in trans to mobilize short interspersed elements (SINEs), such as Alu elements. L1 and Alu insertions have been implicated in many human diseases, and their retrotransposition provides an ongoing source of human genetic diversity. L1/Alu elements are expected to ensure their transmission to subsequent generations by retrotransposing in germ cells or during early embryonic development. Here, we determined that several subfamilies of Alu elements are expressed in undifferentiated human embryonic stem cells (hESCs) and that most expressed Alu elements are active elements. We also exploited expression from the L1 antisense promoter to map expressed elements in hESCs. Remarkably, we found that expressed Alu elements are enriched in the youngest subfamily, Y, and that expressed L1s are mostly located within genes, suggesting an epigenetic control of retrotransposon expression in hESCs. Together, these data suggest that distinct subsets of active L1/Alu elements are expressed in hESCs and that the degree of somatic mosaicism attributable to L1 insertions during early development may be higher than previously anticipated.

DNA transposons: nature and applications in genomics.

Repeated DNA makes up a large fraction of a typical mammalian genome, and some repetitive elements are able to move within the genome (transposons and retrotransposons). DNA transposons move from one genomic location to another by a cut-and-paste mechanism. They are powerful forces of genetic change and have played a significant role in the evolution of many genomes. As genetic tools, DNA transposons can be used to introduce a piece of foreign DNA into a genome. Indeed, they have been used for transgenesis and insertional mutagenesis in different organisms, since these elements are not generally dependent on host factors to mediate their mobility. Thus, DNA transposons are useful tools to analyze the regulatory genome, study embryonic development, identify genes and pathways implicated in disease or pathogenesis of pathogens, and even contribute to gene therapy. In this review, we will describe the nature of these elements and discuss recent advances in this field of research, as well as our evolving knowledge of the DNA transposons most widely used in these studies.

iPSC lines that do not silence the expression of the ectopic reprogramming factors may display enhanced propensity to genomic instability.

Here, we provide data suggesting that the absence of silencing of the ectopic reprogramming factors used to reprogram somatic cells to induced pluripotent stem cells (iPSCs) may predispose iPSCs to genomic instability. We encourage stem cell scientists to undertake an extensive characterization and standardization of much larger cohorts of iPSC lines in order to set up rigorous criteria to define safe and stable bona fide iPSCs.

Transposition of Mboumar-9: identification of a new naturally active mariner-family transposon.

Although mariner transposons are widespread in animal genomes, the vast majority harbor multiple inactivating mutations and only two naturally occurring elements are known to be active. Previously, we discovered a mariner-family transposon, Mboumar, in the satellite DNA of the ant Messor bouvieri. Several copies of the transposon contain a full-length open reading frame, including Mboumar-9, which has 64% nucleotide identity to Mos1 of Drosophila mauritiana. To determine whether Mboumar is currently active, we expressed and purified the Mboumar-9 transposase and demonstrate that it is able to catalyze the movement of a transposon from one plasmid to another in a genetic in vitro hop assay. The efficiency is comparable to that of the well-characterized mariner transposon Mos1. Transposon insertions were precise and were flanked by TA duplications, a hallmark of mariner transposition. Mboumar has been proposed to have a role in the evolution and maintenance of satellite DNA in M. bouvieri and its activity provides a means to examine the involvement of the transposon in the genome dynamics of this organism.

Detection of a mariner-like element and a miniature inverted-repeat transposable element (MITE) associated with the heterochromatin from ants of the genus Messor and their possible involvement for satellite DNA evolution.

The satellite DNA of ants Messor bouvieri, M. barbarus and M. structor, studied in a previous paper, is organized as tandemly repeated 79-bp monomers in the three species showing high sequence similarity. In the present paper, a mariner-like element (Mboumar) and a new MITE (miniature inverted-repeat transposable element) called IRE-130, inserted into satellite DNA from M. bouvieri, are analyzed. The study of Mboumar element, of its transcription and the putative transposase that it would encode, suggests that it could be an active element. Mboumar elements inserted into IRE-130 elements have also been detected. It is the first time, to our knowledge, that a MITE has been described in Hymenoptera and it is also the first time that a mariner-like element inserted into a MITE has been detected. A mariner-like element, inserted into satellite DNA from M. structor and in M. barbarus, also has been found. The results seem to indicate that transposition events have participated in the satellite DNA mobilization and evolution.

Characterization and evolutionary dynamics of a complex family of satellite DNA in the leaf beetle Chrysolina carnifex (Coleoptera, Chrysomelidae).

The present study characterizes the complex satellite DNA from the specialized phytophagous beetle species Chrysolina carnifex. The satellite DNA is formed by six monomer types, partially homologous but having diverged enough to be separate on the phylogenetic trees, since each monomer type is located on a different branch, having statistically significant bootstrap values. Its analysis suggests a common evolutionary origin of all monomers from the same 211-bp sequence mainly by means of base-substitution mutations evolutionarily fixed to each monomer type and duplications and/or deletions of pre-existing segments in the 211-bp sequence. The analysis of the sequences and Southern hybridizations suggest that the monomers are organized in three types of repeats: monomers (211-bp) and higher-order repeats in the form of dimers (477-bp) or even trimers (633-bp). These repetitive units are not isolated from others, and do not present the pattern characteristic for the regular tandem arrangement of satellite DNA. In-situ hybridization with biotinylated probes corresponding to the three types of repeats showed the pericentromeric location of these sequences in all meiotic bivalents, coinciding with the heterochromatic blocks revealed by C-banding, indicating in addition that each type of repeat is neither isolated from others nor located in specific chromosomes but rather that they are intermixed in the heterochromatic regions. The presence of this repetitive DNA in C. haemoptera, C. bankii and C. americana was also tested by Southern analysis. The results show that this satellite DNA sequence is specific to the C. carnifex genome but has not been found in three other species of Chrysolina occupying similar or different host plants.