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

Gladstone Institute of Virology and Immunology, University of California, San Francisco, CA, USA.
Human Molecular Genetics (Impact Factor: 6.39). 01/2012; 21(1):208-18. DOI: 10.1093/hmg/ddr455
Source: PubMed


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.

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    • "The expression of TE proteins and their modes of mobility are dependent upon the stage of cell development. While L1 retrotransposon activity is increased in embryonic stem cells (Garcia perez et al., 2007), it is inhibited in fully differentiated cells (Wissing et al., 2012). Interestingly, inhibition of L1 RT enzyme resulted in an arrest in embryo development (Spadafora, 2015). "
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    ABSTRACT: Mobile DNA elements transposable elements (TEs) are genomic sequences capable of moving themselves independently into different parts of the genome. Viral invasion of eukaryotic genomes is assumed to be the main source of TEs. Selfish transposition of these elements could be a serious threat to the host cell, as they can insert themselves into the middle of coding genes and/or induce genomic instability. In response, through million years of evolution, cells have come up with various mechanisms such as genomic imprinting, DNA methylation, heterochromatin formation, and RNA interference to deactivate them. Interestingly, these processes have also greatly contributed to important cellular functions involved in cell differentiation, development, and differential gene expression. Propagation of TE copies during the course of evolution have resulted in increasing the genome size and providing proper space and flexibility in shaping the genome by creating new genes and establishing essential cellular structures such as heterochromatin, centromere, and telomeres. Yet, these elements are mostly labeled for playing a role in pathogenesis of human diseases. In this study, we attempt to introduce TEs as factors necessary for making us human rather than just selfish sequences or obligatory guests invading our DNA.
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    • "Our results are consistent with those of a previous study where L1 mobility was detected in iPSCs using an exogenous L1 reporter but are in contrast to those of two other studies which showed stable number of repetitive sequences such as L1 in human or mouse iPSCs by whole genome sequencing [29]–[31]. However, detection of possible copy number variation of repetitive sequences by whole genome sequencing has limitations [32]. "
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    ABSTRACT: Long interspersed element-1 (LINE-1 or L1) retrotransposition induces insertional mutations that can result in diseases. It was recently shown that the copy number of L1 and other retroelements is stable in induced pluripotent stem cells (iPSCs). However, by using an engineered reporter construct over-expressing L1, another study suggests that reprogramming activates L1 mobility in iPSCs. Given the potential of human iPSCs in therapeutic applications, it is important to clarify whether these cells harbor somatic insertions resulting from endogenous L1 retrotransposition. Here, we verified L1 expression during and after reprogramming as well as potential somatic insertions driven by the most active human endogenous L1 subfamily (L1Hs). Our results indicate that L1 over-expression is initiated during the reprogramming process and is subsequently sustained in isolated clones. To detect potential somatic insertions in iPSCs caused by L1Hs retotransposition, we used a novel sequencing strategy. As opposed to conventional sequencing direction, we sequenced from the 3' end of L1Hs to the genomic DNA, thus enabling the direct detection of the polyA tail signature of retrotransposition for verification of true insertions. Deep coverage sequencing thus allowed us to detect seven potential somatic insertions with low read counts from two iPSC clones. Negative PCR amplification in parental cells, presence of a polyA tail and absence from seven L1 germline insertion databases highly suggested true somatic insertions in iPSCs. Furthermore, these insertions could not be detected in iPSCs by PCR, likely due to low abundance. We conclude that L1Hs retrotransposes at low levels in iPSCs and therefore warrants careful analyses for genotoxic effects.
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    • "Indeed, it is thought that the wave of hypomethylation taking place during early embryogenesis might represent " a window of opportunity " for L1 to accumulate new copies [78]. Consistent with this, human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) are characterized by the hypomethylation of LINE-1 promoters [54,78–80]; notably, both hESCs and hiPSCs are characterized by overexpressing L1-RNPs [54] [80]. However, there is no definitive proof of ongoing endogenous LINE-1/Alu/SVA retrotransposition in these cell types. "
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    ABSTRACT: Transposable Elements are pieces of DNA able to mobilize from one location to another within genomes. Although they constitute more than 50% of the human genome, they have been classified as selfish DNA, with the only mission to spread within genomes and generate more copies of themselves that will ensure their presence over generations. Despite their remarkable prevalence, only a minor group of transposable elements remain active in the human genome and can sporadically be associated with the generation of a genetic disorder due to their ongoing mobility. Most of the transposable elements identified in the human genome corresponded to fixed insertions that no longer move in genomes. As selfish DNA, transposable element insertions accumulate in cell types where genetic information can be passed to the next generation. Indeed, work from different laboratories has demonstrated that the main heritable load of TE accumulation in humans occurs during early embryogenesis. Thus, active transposable elements have a clear impact on our pluripotent genome. However, recent findings suggest that the main proportion of fixed non-mobile transposable elements might also have emerging roles in cellular plasticity. In this concise review, we provide an overview of the impact of currently active transposable elements in our pluripotent genome and further discuss new roles of transposable elements (active or not) in regulating pluripotency. This article is part of a Special Issue entitled: Stress as a fundamental theme in cell plasticity.
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