Testing the palindromic target site model for DNA transposon insertion using the Drosophila melanogaster P-element

Abstract

Understanding the molecular mechanisms that influence transposable element target site preferences is a fundamental challenge in functional and evolutionary genomics. Large-scale transposon insertion projects provide excellent material to study target site preferences in the absence of confounding effects of post-insertion evolutionary change. Growing evidence from a wide variety of prokaryotes and eukaryotes indicates that DNA transposons recognize staggered-cut palindromic target site motifs (TSMs). Here, we use over 10 000 accurately mapped P-element insertions in the Drosophila melanogaster genome to test predictions of the staggered-cut palindromic target site model for DNA transposon insertion. We provide evidence that the P-element targets a 14-bp palindromic motif that can be identified at the primary sequence level, which predicts the local spacing, hotspots and strand orientation of P-element insertions. Intriguingly, we find that the although P-element destroys the complete 14-bp target site upon insertion, the terminal three nucleotides of the P-element inverted repeats complement and restore the original TSM, suggesting a mechanistic link between transposon target sites and their terminal inverted repeats. Finally, we discuss how the staggered-cut palindromic target site model can be used to assess the accuracy of genome mappings for annotated P-element insertions.

Figure 1.

The P-element targets a 14-bp palindromic TSM. (A) Sequence logo depicting the relative base usage for a 51 bp window centered around 10 221 P-element insertion sites. The insertion site on the positive strand is just before position zero, and the insertion site on the negative strand is just after position seven. Insertions on the minus strand have been reverse complemented before being included in the alignment. The Y-axis is in bit (log base 2) units of the usage of bases in the motif relative to the random expectation of equal frequency. (B) Table of base usage in the 14-bp TSM and χ² statistics testing the null hypothesis that base usage at each position of the motif is random under the genome-wide background base composition in D. melanogaster. All positions deviate significantly from random base usage (3 df, P < 2.2 × 10⁻¹⁶ for all 14 motif positions).

Figure 2.

Nonrandom local spacing reveals two types of P-element insertion hotspot. (A) Distances, in base pairs (bp), between all consecutive P-element insertions in the genome. (B) Distances between consecutive P-element insertions on the same strand (+/+ or −/−), showing same-strand hotspots at a distance of 0 bp. (C and D) Distances between consecutive P-element insertions on opposite strands (+/− or −/+), showing opposite-strand hotspots at a distance of 8 bp. Note that the x-axis has been truncated at 50 bp in all three panels for clarity.

Figure 3.

The 14-bp palindromic TSM discriminates P-element insertion sites, hotspots and background DNA. Shown are the distributions of log-likelihood scores of the 14-bp palindromic TSM relative to random background base composition for nontarget site background DNA, nonhotspot target sites with one insertion and hotspot target sites with more than one insertion. See main text for details on different types of hotspots.

Figure 4.

Model of P-element sequences in the context of the palindromic target site. Genomic sequences are shown in black, P-element sequences are shown in blue and cut sites for transposase activity are shown as black arrowheads. (A) The terminal three nucleotides of the P-element inverted repeats restore and complement the optimal target sequences flanking the TSD. Specifically, the terminal 3 bp flanking the TSD at the 5′ (ATR…) and 3′ (…WAT) end of the TSM are complementary to the terminal 3 bp of the 3′ (…ATG) and 5′ (CAT…) ends, respectively, of the P-element TIRs. Note that this occurs on both ends of the P-element regardless of whether the 5′ or 3′ insertion site is used and the resulting orientation of the P-element insertion. (B) TSMs in the P-element terminal repeat and the target site flank the 17-bp staggered cut sites for donor excision. Shown also are the positions of binding sites for transposase and the IRBP. Only the 3′ terminus of the P-element is shown for clarity, and a similar configuration exists in inverted orientation at the 5′ terminus.