Reprogramming of the HepG2 genome by long interspersed nuclear element‐1

Abstract

Long Interspersed Nuclear Element-1 (LINE-1 or L1) is an autonomous, mobile element within the human genome that transposes via a "copy and paste" mechanism and relies upon L1-encoded endonuclease and reverse transcriptase (RT) activities to compromise genome integrity. L1 has been implicated in various forms of cancer, but its role in the regulation of the oncogenic phenotype is not understood. The present studies were conducted to evaluate mechanisms of genetic regulatory control in HepG2 cells by human L1, or a D702Y mutant deficient in RT activity, and their influence on cellular phenotype. Forced expression of synthetic L1 ORF1p and ORF2p was associated with formation of cytoplasmic foci and minor association with the nuclear compartment. While de novo L1 mobilizations were only identified in cells expressing wild type L1, and were absent in the D702Y mutant, changes in gene expression profiles involved RT dependent as well as RT independent mechanisms. Synthetic L1 altered the expression of 24 in silico predicted genetic targets; ten of which showed RT-dependence, ten RT-independence, and four reciprocal regulatory control by both wild type and RT mutant. Of five targets examined, only VCAM1 and PTPRB colocalized with newly retrotransposed wild type L1. Biological discretization to partition patterns of gene expression into unique frequencies identified adhesion, inflammation, and cellular metabolism as key processes targeted for molecular interference with disruption of epithelial-to-mesenchymal programming seen irrespective of the RT phenotype. These findings establish L1 as a key regulator of genome plasticity and EMT via mechanisms independent of RT activity.

LINE-1 in cancer: multifaceted functions and potential clinical implications

“…An excellent example of this comes from the participation of L1 in X chromosome inactivation during embryo development. 64 In a HepG2 cell line, 65 ectopic L1 expression resulted in detectable expression changes of 24 genes, with half of them being retrotransposition-independent.…”

De novo LINE - 1 retrotransposition in HepG2 cells preferentially targets gene poor regions of chromosome 13

“…Cloning of vectors pB001 CTR (vector backbone or CTR), pB016 MUT (Aspartate (D) to Tyrosine (Y) mutant at position 702 (D702Y)) and pB015 WT (Wildtype or WT) were done as described in Bojang et al 2013 [30]. These vectors were used to generate stable transfected HepG2 cell lines used to monitor retrotransposition activity of L1 RP .…”

“…The integration of L1 sequences near genes can modulate their expression, induce alternative splicing, reshuffle the genome causing inter-individual genetic variations and/or lead to epigenetic dysregulation at the insertion site [25, 26, 27]. Within this context, we recently showed that forced expression of L1 RP , an active L1 isolated from exon 1 of the retinitis pigmentosa gene (RP) of a patient with X-linked retinitis pigmentosa [28, 29], modulates genetic networks involved in the regulation of inflammation, adhesion and cellular metabolism in HepG2 cells [30]. The HePG2 cell line is frequently used because it retains high functional activity of liver-specific genes [31, 32].…”

“…Both wildtype and RTdomain mutant (i.e. D702Y) of L1 RP induce epithelial to mesenchymal transition (EMT) in HepG2 cells, confirming a biological role for L1 RP that does not involve retrotransposition [30]. …”

“…Analysis of L1 retrotransposition in HepG2 cells by fluorescence in-situ hybridization (FISH), an approach previously used to localize genomic consensus sequences of L1 ORF2 [30], coupled with DNA sequencing, established a strong preference for insertion into gene-poor regions of chromosome 13. These findings establish for the first time that L1 insertions are not entirely random events as originally proposed, and raise questions about the biology and molecular mechanism involved in the regulation of L1 retrotransposition.…”