The piggyBac transposon displays local and distant reintegration preferences and can cause mutations at noncanonical integration sites

Abstract

The DNA transposon piggyBac is widely used as a tool in mammalian experimental systems for transgenesis, mutagenesis, and genome engineering. We have characterized genome-wide insertion site preferences of piggyBac by sequencing a large set of integration sites arising from transposition from two separate genomic loci and a plasmid donor in mouse embryonic stem cells. We found that piggyBac preferentially integrates locally to the excision site when mobilized from a chromosomal location and identified other nonlocal regions of the genome with elevated insertion frequencies. piggyBac insertions were associated with expressed genes and markers of open chromatin structure and were excluded from heterochromatin. At the nucleotide level, piggyBac prefers to insert into TA-rich regions within a broader GC-rich context. We also found that piggyBac can insert into sites other than its known TTAA insertion site at a low frequency (2%). Such insertions introduce mismatches that are repaired with signatures of host cell repair pathways. Transposons could be mobilized from plasmids with the observed noncanonical flanking regions, indicating that piggyBac could generate point mutations in the genome.

Fig 1

Single-copy PB genomic mobilization system (A) and selection principle for PB reintegration (B). The genome coordinates in panel A are based on mouse genome assembly version NCBI37/mm9. Chr, chromosome; HAT^S, HAT susceptible; Puro^R, puromycin resistant; HAT^R, HAT resistant; Puro^S, puromycin susceptible.

Fig 2

Transposition controlled by nuclear PBase accumulation. (A) 4-OHT treatment time course design; (B) accumulation of mPBase-ERT2 protein in the nucleus after 4-OHT treatment; (C) mPBase-ERT2 persists in the nucleus after 4-OHT withdrawal; (D) number of colonies and PB integration sites obtained from multiple experiments.

Fig 3

Local hopping behavior of PB. (A) Histogram showing the difference in integrations observed from each donor locus on chromosomes 11 and X. Results for chromosome 1, which did not contain a PB donor locus, are shown for comparison. y axis, number of insertions from Hprt minus number from Gdf9 in 50-kb windows; red arrows, PB donor sites. (B) Percentage of local reintegrations. (C) Gdf9 local reintegration profile with chromatin interaction domain boundaries annotated from Hi-C data (red). Black bars, number of PB reintegrations; blue horizontal bars, locations of genes, with the height indicating the expression level represented by the RNA sequence (RNA-seq) read number on a log₁₀ scale.

Fig 4

PB integration characters. (A) Histogram showing number of PB insertions per 50-kb window (excluding regions with no PB insertions). (Inset) The region with the most insertions close to the mmu-miR-290∼295 cluster on chromosome 7. Red marks denote insertion sites. (B) Association of PB insertions and TTAA sites with genes. ***, P < 2.2 × 10⁻¹⁶; the comparison shown is for random TTAA versus the genome donor, although the comparison with the plasmid donor is also significant in all cases. (C and D) Associations with exons and introns, respectively. *, P < 0.001; ***, P < 2.2 × 10⁻¹⁶; n.s., not significant.

Fig 5

PB integration preferences for chromatin features. Association of PB insertions with chromatin features (A) and with expressed genes (B). PB insertions are enriched in early-replicating regions of the genome (C) and at binding sites of transcription factors regulating ES cell pluripotency (D). ***, P < 2.2 × 10⁻¹⁶; **, P < 10⁻¹², binomial test; the comparison shown is for random TTAA versus the genome donor.

Fig 6

Identification of non-TTAA integration sites and integration site sequence context. (A) Distribution of TTAA and non-TTAA insertion sites mapped. (B) Low-GC environment of PB insertion sites. Average percent AT contents are shown for each base surrounding the insertion site for TTAA and CTAA insertion sites.

Fig 7

PB can insert into non-TTAA sites. (A) Excision and integration mechanism of PB, based on in vitro study. Insertion into CTAA, as identified in read A, is used as an example. Two repair possibilities seen in read B (TTAA and TTAG, read 5′ to 3′) are shown. (B) Mismatch repair outcome for CTAA and ATAA insertions. Repair outcome of the predicted mismatches, written as transposon base/genome base. Sequences are given 5′ to 3′ as they appear in the sequencing read. T/T and A/A mismatches are generated upon ATAA insertion, whereas T/G and A/C are generated for CTAA insertion. (C) Influence of PB ITRs on the repair outcome. Mismatch, the observed mismatch adjacent to the specified PB ITR; repair to, number of repairs to A·T (transposon sequence) and G·C (genomic sequence); % host, percentage of events repaired to genomic sequence; P value, P value determined by Fisher's exact test for statistical significance of influence of PB ITRs over each type of mismatch repair outcome.

Fig 8

Mobilization and mismatch repair of PB transposons flanked by non-TTAA sites. (A) Plasmid mobilization of non-TTAA-flanked PB transposons with the indicated ends in wild-type cells. The predicted mismatches upon PB insertion into a TTAA site are shown below each plasmid sequence. Colonies were counted from separate low-density platings (n = 4). Error bars show SEMs. ns, not significant; *, **, and ***, P < 0.05, 0.01, and 0.001, respectively (two-tailed t test). (B) Repair outcome of mismatches caused by transposition of variant plasmids in wild-type (W.T.; plasmids NC2 to NC6) and Msh2 mutant (plasmid NC5 only) ES cells. Mismatches are written as transposon base/genome base. Mixed, number of mixed insertion site traces observed as shown in panel C, which are counted in the GC repair column. (C) Example of a sequencing trace (C and T coexist at position 11) showing two different repair outcomes mapping to the same genome position.

Fig 9

Schematic representation of PB genomic mobilization characteristics.

Fig 10

Models for PB integration-mediated mismatch repair. (A) Potential repair mechanisms to resolve mismatches generated by TTAA-flanking PB integration into non-TTAA sites; (B) PB cargo conformation may influence the mismatch repair outcome.