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

Abstract

Long interspersed nuclear elements (Line-1 or L1s) account for ~17% of the human genome. While the majority of human L1s are inactive, ~80–100 elements remain retrotransposition competent and mobilize through RNA intermediates to different locations within the genome. De novo insertions of L1s account for polymorphic variation of the human genome and disruption of target loci at their new location. In the present study, fluorescence in situ hybridization and DNA sequencing were used to characterize retrotransposition profiles of L1RP in cultured human HepG2 cells. While expression of synthetic L1RP was associated with full-length and truncated insertions throughout the entire genome, a strong preference for gene-poor regions, such as those found in chromosome 13 was observed for full-length insertions. These findings shed light into L1 targeting mechanisms within the human genome and question the putative randomness of L1 retrotransposition.

Analysis of LINE-1 Retrotransposition at the Single Nucleus Level

“…The approach can be used for both quantitative and qualitative evaluation of retrotransposition events and applied to interphase nuclei. This methodology boasts the accuracy of subsequent in-silico sequence alignment methods employed to determine ectopic L1 insertion sites 16 . Alignment of repetitive sequences can be ambiguous due to the formation of homopolymers, while repetitive sequences can be lost during primer amplification of template resulting in underrepresentation of repetitive sequences.…”

“…FISH methodology can overcome these problems because FISH probes can anneal to homopolymers and are unlikely to be biased against intact repeat sequences It remains unclear where young L1s prefer to insert and whether a targeting mechanism exists to direct these insertions within the genome. Using the above methodology we have shown that L1 inserts preferentially into gene poor regions of the genome 16 . Others have shown that the abundance of L1 sequences is increase in chromosomes with a lesser gene density 17,18 .…”

“…Although full length L1 insertions can be determined using this methodology, it will not discern new endogenous truncated insertions due to the number of truncated L1s within the genome. The accessibility of probe might also be restricted by increase heterochromatin formation 16,24 . However, the inclusion of LNA within FISH probes can be employed to enhance probe annealing.…”

“…For detailed descriptions of experiments in which the use of these vectors and the expression of L1 proteins and retrotransposition are characterized, please refer to published works 12,16 .…”

“…Generate stable clones expressing vector backbone (control) or retrotransposition competent L1 using standard selection methodologies. 12,16 While non-episomal reporters were used to correlate findings with studies of transcription, episomal vectors could also be used. Grow cells stably expressing control or L1 vector (1 x 10 6 cells per 10 cm plate, with adjustments in plate size made as needed) in complete growth media.…”