Functional characterization of piggyBat from the bat Myotis lucifugus unveils an active mammalian DNA transposon

Abstract

A revelation of the genomic age has been the contributions of the mobile DNA segments called transposable elements to chromosome structure, function, and evolution in virtually all organisms. Substantial fractions of vertebrate genomes derive from transposable elements, being dominated by retroelements that move via RNA intermediates. Although many of these elements have been inactivated by mutation, several active retroelements remain. Vertebrate genomes also contain substantial quantities and a high diversity of cut-and-paste DNA transposons, but no active representative of this class has been identified in mammals. Here we show that a cut-and-paste element called piggyBat, which has recently invaded the genome of the little brown bat (Myotis lucifugus) and is a member of the piggyBac superfamily, is active in its native form in transposition assays in bat and human cultured cells, as well as in the yeast Saccharomyces cerevisiae. Our study suggests that some DNA transposons are still actively shaping some mammalian genomes and reveals an unprecedented opportunity to study the mechanism, regulation, and genomic impact of cut-and-paste transposition in a natural mammalian host.

Fig. 1.

piggyBac-like element demography versus other TEs in the M. lucifugus genome assembly. Percentage of divergence from consensus sequences are from RepeatMasker (47) (Methods) and corrected as in ref. , i.e., corrected %divergence = −300/4 × ln(1 − %divergence × 4/300). MITEs, nonautonomous elements. “All other TEs” corresponds to all sequences masked in the genome as transposable elements minus all piggyBac-like elements.

Fig. 2.

Insect piggyBac vs. bat piggyBat transposition in HeLa and M. lucifugus cells in culture. Cells as indicated were cotransfected with a transposon donor plasmid containing either a piggyBac or piggyBat element containing a Blasticidin resistance gene and another plasmid with the cognate transposase. After 2 d, cells were treated with Blasticidin and selection continued for 18 d. Following dilution as indicated, living cells were stained with methylene blue. (A) Transposition in HeLa cells. The relative average frequencies of element transposition in five independent experiments. (B) Transposition in M. lucifugus fibroblast cells.

Fig. 3.

Comparison of the target site selectivity of insect piggyBac and bat piggyBat in humans. (A) Patterns of the 190,000 mapped insect piggyBac insertions in HCT116 cells (18) and 98,800 bat piggyBat insertions in HeLa cells show a nonuniform distribution of insertions across the genome. (B) Regions with a higher local density of TTAA sites showed an increased preference for insect piggyBac insertions in HCT116 cells (18) and bat piggyBat insertions in HeLa cells to insert into that region. (C) Insect piggyBac insertions in HCT116 cells (18) and bat piggyBat insertions in HeLa cells are enriched in regions of open chromatin as assayed by DNase I sensitivity in terms of enrichment of insertions over the number of TTAA sites available for insertion in those regions. (D) Distrubtions of insect piggyBac insertions in HCT116 cells (18) and bat piggyBat insertions in HeLa cells in defined genomic regions.

Fig. 4.

piggyBat transposition in yeast. (A) Excision. piggyBat excision from a URA3 allele was assayed by measuring reversion of uracil auxotropy to prototrophy. (B) Integration. In the parental strain, piggyBat is present on a plasmid that also carries URA3. Integration was assayed by measuring the number of cells that retained the G418^R marker in the piggyBat element when the parental plasmid is excluded by treatment of the cells with 5-FOA, which is toxic to Ura⁺ cells. (C) Reintegration. In the parental strain, a piggyBat element carrying G418^R disrupts URA3 such that the cells are Ura⁻. Excision is selected for by isolating cells that revert to Ura⁺, and reintegration is followed by measuring cells that continue to be G418^R.