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. 2010 Aug 5;466(7307):769-73.
doi: 10.1038/nature09209.

Epigenetic silencing of engineered L1 retrotransposition events in human embryonic carcinoma cells

Jose L Garcia-Perez  1 , Maria Morell, Joshua O Scheys, Deanna A Kulpa, Santiago Morell, Christoph C Carter, Gary D Hammer, Kathleen L Collins, K Sue O'Shea, Pablo Menendez, John V Moran
Affiliations

Epigenetic silencing of engineered L1 retrotransposition events in human embryonic carcinoma cells

Jose L Garcia-Perez et al. Nature. .

Abstract

Long interspersed element-1 (LINE-1 or L1) retrotransposition continues to affect human genome evolution. L1s can retrotranspose in the germline, during early development and in select somatic cells; however, the host response to L1 retrotransposition remains largely unexplored. Here we show that reporter genes introduced into the genome of various human embryonic carcinoma-derived cell lines (ECs) by L1 retrotransposition are rapidly and efficiently silenced either during or immediately after their integration. Treating ECs with histone deacetylase inhibitors rapidly reverses this silencing, and chromatin immunoprecipitation experiments revealed that reactivation of the reporter gene was correlated with changes in chromatin status at the L1 integration site. Under our assay conditions, rapid silencing was also observed when reporter genes were delivered into ECs by mouse L1s and a zebrafish LINE-2 element, but not when similar reporter genes were delivered into ECs by Moloney murine leukaemia virus or human immunodeficiency virus, suggesting that these integration events are silenced by distinct mechanisms. Finally, we demonstrate that subjecting ECs to culture conditions that promote differentiation attenuates the silencing of reporter genes delivered by L1 retrotransposition, but that differentiation, in itself, is not sufficient to reactivate previously silenced reporter genes. Thus, our data indicate that ECs differ from many differentiated cells in their ability to silence reporter genes delivered by L1 retrotransposition.

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Figures

Figure 1
Figure 1. L1 expression and retrotransposition in EC cells
(a) ECs express endogenous L1 ORF1p. The ribosomal S6 protein is a loading control. MW=molecular weight standards. (b) Results of the retrotransposition assay in HeLa and EC cells. G418-resistant foci that expressed the retrotransposed NEO reporter gene were stained for visualization. (c) PCR assay for intron removal (retrotransposition) in both HeLa and PA-1 cells. LRE3=a retrotransposition-competent L1. JM111=a retrotransposition-defective L1. MW= 1 kb molecular weight ladder.
Figure 2
Figure 2. Engineered L1 retrotransposition events are efficiently silenced in EC cells
(a) A cartoon of an L1 and the experimental rationale (top). Cells were transfected with an RC-L1 (kpLRE3/mEGFPI, top two panels; cpLRE3/mEGFPI, bottom panel) and were untreated (left panel) or treated with TSA (right panel). The percentage of EGFP-positive cells and standard deviation (n=3) is indicated. Hoechst staining (blue) highlights the nuclei of cells. P=experiments where puromycin was used to select for the episomal L1 expression plasmid. (b) Retroviral insertions are not efficiently silenced in PA-1 cells (see methods). The graphs indicate the percentage of EGFP-positive cells and the standard deviation (n=3).
Figure 3
Figure 3. Analyses of L1 silencing in a clonal (pk-5) cell line
(a) The cartoon indicates the chromosomal location of a silenced L1-retro-EGFP event in a clonal PA-1 cell line. L1-retro-EGFP expression can be reactivated by TSA treatment. (b) Southern blot analysis reveals that pk-5 cells contain a single L1-retro-EGFP event. Genomic DNA was digested with HindIII and the blot was probed with an α-32P radiolabeled EGFP probe. MW=molecular weight standards (kb). (c) Withdrawal of TSA (bottom panels) results in the reestablishment of L1-retro-EGFP silencing. (d) ChIP analysis on naïve and TSA treated pk-5 cells using AcH4 and diMeH3 antibodies. qPCR revealed the enrichment (AcH4) or depletion (diMeH3) of the retrotransposed EGFP sequences in the TSA treated pk-5 cells. The input cycle threshold (Ct) was designated as 1 and used to calculate fold change differences. Samples were run in triplicate from the same experiment. The standard deviation (SD, n=3) is indicated in the graph.
Figure 4
Figure 4. Analysis of L1 silencing in differentiating cells
(a) The cartoon shows the experimental rationale. Silencing was efficient in PA-1 cells grown in medium containing 10% FBS (white rectangles), but was attenuated in DM (gray rectangles). Red highlighted rectangles indicate experiments with TSA treatment. (b) Differentiation is not sufficient to derepress L1 silencing (details are provided in the text). In a & b, graphs indicate the percentage of EGFP-positive cells and the standard deviation (n=3). (c) A model for the initiation and maintenance of L1 silencing in EC cells (details are provided in the text).
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