LINE-1 retrotransposition activity in human genomes

Abstract

Highly active (i.e., "hot") long interspersed element-1 (LINE-1 or L1) sequences comprise the bulk of retrotransposition activity in the human genome; however, the abundance of hot L1s in the human population remains largely unexplored. Here, we used a fosmid-based, paired-end DNA sequencing strategy to identify 68 full-length L1s that are differentially present among individuals but are absent from the human genome reference sequence. The majority of these L1s were highly active in a cultured cell retrotransposition assay. Genotyping 26 elements revealed that two L1s are only found in Africa and that two more are absent from the H952 subset of the Human Genome Diversity Panel. Therefore, these results suggest that hot L1s are more abundant in the human population than previously appreciated, and that ongoing L1 retrotransposition continues to be a major source of interindividual genetic variation.

Figure 1. A strategy for identifying dimorphic L1Hs elements in individual human genomes

In silico comparison of the fosmid end sequences (red squares) from individual genomic libraries (blue horizontal line) and the HGR (pink horizontal line) enables the detection of fosmids that may contain insertions or deletions with respect to the HGR (see dashed lines). Insertion fosmids were screened by allele specific oligonucleotide hybridization to detect characters that are present in the 5′ UTR of newer L1 elements (one discriminating character utilized, a deletion of the G residue at bp 74 in recent L1s, is indicated in maroon). Putative L1Hs-containing fosmids were analyzed by Southern blotting with a 5′ UTR probe (blue arrow). A representative digest and Southern blot is shown. The ~6kb band is diagnostic for the full-length L1. The additional hybridizing band (~1.3kb band liberated from the L1 5′ flank in this Southern blot example) serves to distinguish individual fosmids. ATLAS and/or DNA sequencing confirmed the presence of a dimorphic, full-length L1Hs insertion.

Figure 2. L1Hs activity in 6 human genomes

(a) Cloning strategy: All but one L1Hs element were cloned directly from fosmids using AccI sites in their 5′ UTR and 3′ UTRs, respectively (red vertical lines; see Extended Experimental Procedures). The L1s then were ligated into vectors that either contain or lack a CMV promoter (black rectangle). Both vectors contain the mneoI retrotransposition indicator cassette (light blue) in the L1 3′ UTR. This cassette allows for detection of retrotransposition events in a cell culture retrotransposition assay. SD=splice donor. SA=splice acceptor. Active elements confer G418 resistance to HeLa cells, whereas defective elements, as illustrated by the RT mutant control (RT- L1), do not. (b) Representative G418-resistant foci for the 20 elements from the Yoruban library, ABC10: Nine of these elements were highly active (large suns to the left of assay image), and two more retained a low level of activity (small suns). One element (#3-5, red box) is a ‘hot’ pre-Ta L1 (#3-5 was tested in a pBluescript backbone (5′UTR+); all others were tested in a pCEP4 (CMV+/5′UTR+)) backbone (Extended Experimental Procedures). Table S1 displays retrotransposition efficiencies for each L1 identified in this study. Figure S1 provides details on the EN-deficient element #3-24. (c) The 68 distinct L1Hs elements identified in this study and their positions in the genome: Red vertical lines and text represent ‘hot’ or highly active elements. Orange vertical lines with black text represent low-level activity elements. Blue vertical lines with black text represent dead or inactive elements. The black line indicates the one untested element (#2-42). Ideograms were adapted from UCSC genome browser: http://genome.ucsc.edu (Kent et al., 2002).

Figure 3. Allele frequencies of L1Hs alleles in the population

(a) Genotyping assays: L1s were queried in panels of individuals for their absence (solid grey lines), or presence (red line). Genotyping of 26 elements in the three panels allowed the discovery of population restricted or potentially ‘private’ L1Hs elements. The expected amplicon sizes are diagrammed for element #3-24. (b) Pedigrees showing the inheritance of two elements typed in the ABC10 trio: Genotyping gels show the heritability of #3-31 (African specific) and #3-24 (absent from the HGDP). E and F at the top of the gel image indicate PCR results for empty and filled sites. M, F, and C at the bottom of the image indicate lanes for the mother, father, and child of the trio. (c) Example data sheet for the G248 element #1-5: Empty site: insertion site in the HGR. EN cleavage site: the endonucleolytic cleavage site used by L1 EN to initiate retrotransposition. pA length: the approximate L1 poly (A) tail length; 3′ transductions and interrupted poly (A) tails also are annotated. TSD length: the length of the target site duplication flanking the L1Hs element (underlined lettering). Table S2 contains data sheets for each L1 in this study. Table S3 contains L1Hs insertion locations with respect to genes. Figure S2 displays a non-canonical L1Hs insertion and documents a possible sequence anomaly in the HGR.

Figure 4. An estimate of the number of active L1Hs elements in an individual (ABC13) genome

(a) In silico genotyping: The last library in our study, ABC13, was examined in silico (see text) for the presence of insertion fosmids mapping to the location of L1Hs elements found in other individuals. Element 3–17 is used as an example. All blue lines represent insertion fosmids in the genomes of the 8 individuals on the HGSV track (http://hgsv.washington.edu/) of the UCSC genome browser (http://genome.ucsc.edu) (Kent et al., 2002). The ABC7, 8, and 14 libraries were not investigated in this study. (b) PCR validation: The elements identified in silico were genotyped using the scheme shown in Figure 3 to validate the predictions from the HGSV track of the UCSC browser. Element 3–17 is used to illustrate the genotyping. ABC10 and ABC13 are heterozygous with respect to the L1Hs insertion. ABC11 lacks the L1Hs insertion. Table S4 displays genotyping results for all elements in this study.

Figure 5. Phylogenetic tree of the L1Hs elements identified in this study

The tree is a single neighbor-joining tree (with branch lengths corrected using the Kimura 2 parameter model of nucleotide substitution) with 68 full-length elements from our study. The numbers at particular nodes indicate the number of times that node was observed in 1000 bootstrap replicates of the dataset. Only bootstrap values exceeding 70% are shown. The brackets at the right side indicate previously described ‘transduction subfamilies’ (L1_RP (labeled RP in the Figure) & LRE3) and distinct L1Hs ‘subfamilies’ currently capable of amplifying in human genomes (I–V) (Goodier et al., 2000; Pickeral et al., 2000). Those subfamilies are highlighted in the same color to show their clustering on the tree. Retrotransposition activity (% relative to L1.3) as well as allele frequency (e.g., AF= 0.012), if determined, is appended to the sequence identifiers. Element #11-17 contains ACG characters in its 3′ UTR, which are diagnostic for pre-Ta L1s; however, the element clusters with the Ta0 subfamily. The tree and age estimates use sequences indicated in the Supplemental Information.

Fig. 6. Multiple source loci model for continued L1Hs activity

An element (source locus) that is both active and in a conducive genomic environment can retrotranspose. Shown here is an example of a progenitor element that can be associated with subsequent members of a ‘family’ through the use of interrupted poly (A) tails and/or 3′ transduced sequence (3′ red arrow and line). Distinct elements are marked by distinguishing TSDs specific for their new integration site (different colored horizontal arrows). There are many of these ‘families’ active in human genomes, such as L1_RP, LRE3, and the 5 ‘families’ noted in Figure 5. Although host processes (lightning bolt) may inactivate some older elements, some of their descendents may retain the ability to retrotranspose and could harbor the 3′ transduction/interrupted poly (A) tail.