Analysis of LINE-1 Expression in Human Pluripotent Cells

Abstract

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.

LINE-1 retrotransposition impacts the genome of human pre-implantation embryos and extraembryonic tissues

“…Thus, only blastocysts with a clear and well-defined ICM were used in this study. Using RT-qPCR, we reproducibly observed abundant expression of full -length LINE-1 mRNAs in human blastocysts and hESCs (H9 line), which were used as a positive control for L1 expression (Figure 1b and S1) (Coufal et al, 2009;Garcia-Perez et al, 2007;Macia et al, 2017;Munoz-Lopez et al, 2012). Sequencing confirmed that 90% of detected L1 RNAs were derived from the L1Hs family, which encompasses all known human active L1s.…”

MOV10 RNA Helicase Is a Potent Inhibitor of Retrotransposition in Cells

“…Bisulfite analysis was performed exactly as described [90], [91] (Figure S3). The region amplified for analysis spanned 363-bp of the L1 5′UTR and contained 20 CpG dinucleotides.…”

L1 is dynamic during neurogenesis

“…Nonetheless, during reprogramming or expansion of hiPSCs in vitro, genetic and epigenetic aberrations can occur [119][120][121][122][123], including L1 mobilisation [1]. One explanation for L1 activity here is that during hiPSC reprogramming chromatin relaxation and epigenome-wide remodelling occur [124], providing a window of opportunity for L1 to retrotranspose [1,5,115] when L1 promoters are hypomethylated and undergo transcriptional activation.…”

“…Central to L1 epigenetic regulation is a CpG island present in the L1 5'UTR [178] that, via DNA methylation, can mediate L1 repression. L1 methylation has been investigated in various contexts, either looking genome-wide [4,5] or at specific L1 loci [6][7][8][9]. One key study investigated the genome-wide methylation state of the L1-Ta family in 3 hiPSC lines and 2 hESC lines, finding lower methylation in hiPSCs and in hESCs when compared to parental fibroblasts.…”

“…Crucially, L1 insertions bearing transductions have as yet not been identified in hiPSCs or embryonic stem cells (ESCs).Additionally, L1 promoters are methylated by the host genome to reduce their potential to initiate L1 mRNA transcription [3]. The genome-wide methylation state of L1 families, and specific L1 loci, has been analysed in ESCs and hiPSCs, and in neural stem cells (NSCs) [4][5][6][7][8][9][10]. However, the methylation state of individual L1 loci, including de novo L1 insertions and their progenitor elements, has to date not been reported.In the research described in this thesis, I identified a full-length de novo L1 insertion in a cultured hiPSC line.…”