Genetic evidence that the non-homologous end-joining repair pathway is involved in LINE retrotransposition

Abstract

Long interspersed elements (LINEs) are transposable elements that proliferate within eukaryotic genomes, having a large impact on eukaryotic genome evolution. LINEs mobilize via a process called retrotransposition. Although the role of the LINE-encoded protein(s) in retrotransposition has been extensively investigated, the participation of host-encoded factors in retrotransposition remains unclear. To address this issue, we examined retrotransposition frequencies of two structurally different LINEs--zebrafish ZfL2-2 and human L1--in knockout chicken DT40 cell lines deficient in genes involved in the non-homologous end-joining (NHEJ) repair of DNA and in human HeLa cells treated with a drug that inhibits NHEJ. Deficiencies of NHEJ proteins decreased retrotransposition frequencies of both LINEs in these cells, suggesting that NHEJ is involved in LINE retrotransposition. More precise characterization of ZfL2-2 insertions in DT40 cells permitted us to consider the possibility of dual roles for NHEJ in LINE retrotransposition, namely to ensure efficient integration of LINEs and to restrict their full-length formation.

Figure 1. Retrotransposition frequencies (RFs) in DT40 cell lines.

(A) Schematic of zebrafish ZfL2-2 and human L1. ZfL2-2 encodes only one ORF, but L1 encodes two ORFs. (B) RFs of ZfL2-2 in six different DT40 cell lines. (C) RFs of L1 in six different DT40 cell lines. (B, C) Means and standard deviations of RFs. WT, wild-type DT40 cell line. Ku70^−/−, Ku70-deficient DT40 cell line. Art^−/−, Artemis-deficient DT40 cell line. LigIV^−/−, DNA ligase IV-deficient DT40 cell line. Rad18^−/−, Rad18-deficient DT40 cell line. SHIP1^−/−, hematopoietic-restricted SH2-containing inositol 5′-phosphatase-1-deficient DT40 cell line. An asterisk indicates P<0.01 by two-tailed Student's t-test. For ZfL2-2: WT vs Ku70^−/−, P = 1.7×10⁻⁸; WT vs Art^−/−, P = 2.8×10⁻⁴; WT vs LigIV^−/−, P = 1.2×10⁻⁶. For L1: WT vs Ku70^−/−, P = 1.1×10⁻⁴; WT vs Art^−/−, P = 6.6×10⁻⁵; WT vs LigIV^−/−, P = 5.7×10⁻³. (D) Ku70 complementation assay. The control expression vector, pAneo, or the Ku70 expression vector, chicken Ku70/pAneo, was transiently transfected into the WT, Ku70^−/−, and LigIV^−/− DT40 cell lines (exogKu − or +, respectively). Using these transiently transfected cell lines, the ZfL2-2 retrotransposition assay was performed. Mean values (with standard deviations) of the ZfL2-2 RFs are shown. Transcription of the exogenous (cloned) and/or endogenous Ku70 gene was detected by RT-PCR (middle). Transcription of the β-actin gene was also detected by RT-PCR as a control (bottom). The asterisk indicates P<1×10⁻⁴ by two-tailed Student's t-test (for Ku70^−/− cells, P = 4.7×10⁻⁵).

Figure 2. Effect of LINE expression on DT40 cell viability.

DT40 cells were co-transfected with pEGFP-FLAG-1 and one of the LINE expression vectors (pBZ2-5, p131.11, pJM102/L1.3, or pJM102/L1.3 H230A) by electroporation (see Tracing of EGFP-positive cells in the Materials and Methods section). After transfection, the cells were monitored for 8 days. (A) ZfL2-2 expression in DT40 cells. The relative proportion of EGFP-expressing cells (left), the geometric mean of the EGFP fluorescence intensity (FI) (middle) and the median of the EGFP FI (right) calculated using the values 3 days after electroporation as the standard are indicated (the raw data are shown in Figures S5 and S6). DT40 WT, wild-type DT40 cell line. DT40 Ku70^−/−, Ku70-deficient DT40 cell line. ZfL2-2 WT, wild-type ZfL2-2 element. ZfL2-2 ENm, endonuclease-mutated ZfL2-2 element. Two independent experiments were performed (upper and lower panels). (B) L1 expression in DT40 cells. The relative proportion of EGFP-expressing cells (left), the geometric mean of the EGFP FI (middle) and the median of the EGFP FI (right) calculated using the values 3 days after electroporation as the standard are indicated (the raw data are shown in Figures S9 and S10). DT40 WT, wild-type DT40 cell line. DT40 Ku70^−/−, Ku70-deficient DT40 cell line. L1 WT, wild-type L1 element. L1 ENm, endonuclease-mutated L1 elements. Two independent experiments were performed (upper and lower panels).

Figure 3. Characterization of ZfL2-2 insertions in DT40 cells.

Abbreviations are as defined for Figure 1. (A) ZfL2-2 insertions isolated from DT40 cells. The top diagram shows the full-length ZfL2-2 element containing the mneoI₄₀₀/ColE1 cassette (Cassette) in the 3′ UTR. ORF, open reading frame. Each horizontal line represents one of the 26, 25, 24 or 27 ZfL2-2 insertions isolated from the various DT40 cell lines. Blue lines represent insertions with a 5′ truncation. Red lines represent full-length insertions. The dashed line in Art^−/− indicates a deletion. (B) A box-and-whisker plot shows the median (red line), the first and third quartiles, and the upper and lower limits of the length of insertions indicated in (A). P values less than 0.05 are indicated (Mann-Whitney U test). (C) Full-length vs. truncated elements. The ZfL2-2 insertions in (A) were categorized by the absence (Full) or presence (Truncated) of a 5′ truncation. The number of insertions identified is indicated inside each bar. P values less than 0.05 are indicated (two-sided Fisher's exact test). (D) Target site alterations. The ZfL2-2 insertions shown in (A) were categorized with regard to target site alterations. The number of insertions identified is indicated inside each bar. L-TST, long target site truncation (>20 bp). S-TST, short target site truncation (≤20 bp). BEJ, blunt end joining. S-TSD, short target site duplication (≤20 bp). L-TSD, long target site duplication (>20 bp). (E) Short target site alterations. The ZfL2-2 insertions with short target site alterations in (D) were compared. The number of insertions identified is indicated inside each bar. Abbreviations and definitions are as for panel D. P values less than 0.05 are indicated (Wilcoxon Rank Sum test).

Figure 4. Retrotransposition assay in HeLa cells with NU7026.

(A) Survival rate of HeLa cells treated with NU7026 in the presence or absence of etoposide. HeLa cells treated with these agents for 2 h were plated on a 100-mm plate. Three independent experiments were performed, and the means with standard deviations are shown. (B) The result of the retrotransposition assay in HeLa cells treated with NU7026. Retrotransposition frequency (RF) values are relative to those measured in the absence of NU7026. Two independent experiments were performed, and the means with standard deviations are shown.

Figure 5. A model for ZfL2-2 integration.

See Discussion for explanation of the model. TSD, target site duplication. BEJ, blunt end joining. TST, target site truncation. Blue lines denote chromosomal DNA that is duplicated in TSD. Green lines denote chromosomal DNA that is truncated in TST.