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Table 1 A comparison of the methods and findings from three studies

From: Cell division promotes efficient retrotransposition in a stable L1 reporter cell line

 

Kubo et al.[8]

Shi et al.[9]

This study

L1 vector

Embedded in a helper-dependent adenovirus

Episomal plasmid

Embedded in an SB DNA transposon

(1) Promoter

Mouse phosphoglycerate kinase-1

A native human L1 promoter (5′UTR)

Bi-directional tetracycline-inducible promoter

(2) ORFs

Human L1 RP

Human L1 LRE3

Synthetic mouse L1 ORFeus

(3) Reporter

EGFP

EGFP

Fluc

Gene delivery

Adenoviral transduction

Transient transfection with nucleofector

Stably integrated by SB100X

L1 expression detection

Co-expressed β-gal

L1 RNA (RT-PCR)

Co-expressed Rluc; L1 ORF1p

Cells for cell-cycle arrest assay

Human glioma (Gli36)

Human fetal lung fibroblast (IMR-90); human cervical carcinoma (HeLa)

HeLa Tet-ORFeus stable cell line

Cell-cycle arrest experiments and observed effects on retrotransposition

(i) G0 arrest ➜ complete inhibitiona;

(i) G1, S, G2, or M arrest ➜ strong inhibitionc

(i) S, or S+G2/M arrest ➜ strong inhibition;

(ii) G1/S arrest ➜ partial inhibitionb

(ii) Cell-cycle synchronized cells ➜reduced retrotransposition if cells divided one fewer cycle

Conclusion(s) regarding to the role of cell division

L1 retrotransposition can occur in non-dividing cells

Cell division is required for L1 retrotransposition; L1 transcription is the limiting step

Cell division promotes efficient L1 retrotransposition; the inhibitory effect of cell-cycle arrest on retrotransposition cannot be explained by reduced L1 transcription alone

Role of active nuclear import

L1 RNP can be actively imported into the nucleus

Not discussed

An active nuclear import mechanism is a possible explanation for residual retrotransposition in cell-cycle arrested cells

  1. aCompared with cells resuming cycling, G0 arrested Gli36 cells showed a 16-fold reduction in the fraction of GFP-positive cells (54-fold if normalized to co-expressed β-gal; see Figure five B in ref[8]).
  2. bCompared with cycling cells, G1/S arrested Gli36 cells showed a three-fold reduction in the fraction of GFP-positive cells (six-fold if normalized to co-expressed β-gal; see Figure four A in ref[8]).
  3. cFold reduction in retrotransposition was not stated in the main text; up to 40-fold inhibition in retrotransposition could be discerned from raw data (see Figure four A and B in ref[9]; data were not normalized to L1 RNA levels, which were reduced by approximately 10- to 20-fold in most conditions (see Figure five B in ref[9]). However, the dynamic range of observed L1 retrotransposition activity (from 0 to 40 GFP-positive cells per 10,000 cells analyzed by flow cytometry) does not allow the authors to discern additional layers of regulation for retrotransposition.
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