Genomic Deletions Created upon LINE-1 Retrotransposition Nicolas Gilbert, Sheila Lutz-Prigge, John V. Moran  Cell  Volume 110, Issue 3, Pages 315-325 (August 2002) DOI: 10.1016/S0092-8674(02)00828-0

Figure 1 An Overview of the L1 Recovery System (A) Rationale of the assay. The 3′UTR of a human RC-L1 was tagged with a reporter cassette designed to detect retrotransposition events. The gray rectangles indicate L1 ORF1 and L1 ORF2, respectively, and the relative positions of the endonuclease (EN), reverse transcriptase (RT), and cysteine-rich domain (C) are indicated. The positions of the L1 promoter (P) and the SV40 late polyadenylation signal (pA) also are indicated. The light blue rectangle indicates the mneoI gene and the lavender rectangle indicates the ColE1 bacterial origin of replication. The relative positions of the prokaryotic/eukaryotic promoter (P′) and the thymidine kinase polyadenylation signal (A′) required for the expression of the reporter gene also are shown. The mneoI gene is interrupted by an intron (γ-globin intron 2, indicated by the orange line) in the opposite transcriptional orientation. SD and SA indicate the splice donor and splice acceptor sites. This arrangement ensures that a functional Neo transcript will only be translated following L1 retrotransposition. The putative structure of a resultant retrotransposition event that confers G418-resistance (G418R) to HeLa cells is shown at the bottom of the figure. The red lines indicate flanking genomic DNA. (B) Flowchart of the rescue procedure. Genomic DNA was extracted from cell lines derived from either a single G418R focus or small pools of G418R foci and the L1s were recovered using the protocol described (see Experimental Procedures). (C) Results of RT-PCR analyses. RT-PCR was performed to monitor the presence of the spliced mneoI transcript. Molecular size standards (1 kb plus ladder; Gibco/BRL) are indicated in Lane M. The sizes of the unspliced mneoI product (Lane 8) and the respective cDNA products also are indicated. RT-PCR products from cells transfected with pJM101/L1.3, pK7/L1.3mneoI/ColE1, pK7/L1.3mneoI400/ColE1, pCEP4/L1.3mneoI/ColE1, or pCEP4/L1.3mneoI400/ColE1 are indicated in Lanes 1–5, respectively. RT-PCR products from untransfected HeLa cells and no RNA control are indicated in Lanes 6 and 7, respectively. (D) Results of the retrotransposition assay. L1 retrotransposition was assayed using the transient retrotransposition assay (Wei et al., 2000). The top panel displays the results from mock-transfected HeLa cells (No DNA) or HeLa cells (either 2 × 103, 2 × 104, or 2 × 105) transfected with pJM101/L1.3 (Sassaman et al., 1997). The bottom panel displays the results from HeLa cells (2 × 105) transfected with pK7/L1.3mneoI/ColE1, pK7/L1.3mneoI400/ColE1, pCEP4/L1.3mneoI/ColE1, and pCEP4/L1.3mneoI400/ColE1, respectively. The relative retrotransposition efficiency of each of the constructs (relative to pJM101/L1.3, a previously characterized RC-L1 [Sassaman et al., 1997]). The standard deviation is indicated. Cell 2002 110, 315-325DOI: (10.1016/S0092-8674(02)00828-0)

Figure 2 Recovered Events Demonstrate L1 Structural Hallmarks (A) Size distributions of retrotransposition events from pK7/L1.3mneoI/ColE1. A schematic of a full-length L1 after retrotransposition from pK7/L1.3mneoI/ColE1 is indicated. The relative positions of the L1 5′UTR, L1 ORF1, L1 ORF2, the retrotransposed mneoI/ColE1 gene, and the SV40 pA signal are indicated. Lines below the figure indicate the lengths of the retrotransposed L1s. Bold black lines are used to indicate the relative sizes and positions of L1s that have undergone either an inversion/deletion or inversion/duplication. The names of the rescued clones are indicated at the right of the figure. Clones derived from the pK7/L1.3mneoI400/ColE1 vectors are denoted with a subscript 400. The estimated size of the L1 poly (A) tail, and the relative positions of the BglII (Bg), HindIII (H), BclI (Bc), and BamHI (Ba) restriction sites used in the rescue procedure also are indicated. Notably, 2329 bp must be retrotransposed to deliver the entire mneoI/ColE1 cassette. The lengths of the chimeric L1s can only be estimated (see Figure 6A for a complete description). The gray shading indicates their respective 5′ ends. (B) Size distributions of retrotransposition events from pCEP4/L1mneoI/ColE1. A schematic of a full-length L1 after retrotransposition pCEP4/L1mneoI/ColE1 is indicated. Labels are the same as in Figure 3A; however, three possible polyadenylation signals exist (SV40pA1 (pA1), L1pA (pA2), and SV40pA2 (pA3), respectively). Thus, 2328, 2474, or 2676 bp must be retrotransposed to deliver the entire mneoI/ColE1 cassette. Cell 2002 110, 315-325DOI: (10.1016/S0092-8674(02)00828-0)

Figure 3 L1s Predominantly Integrate at an L1 EN Cleavage Site Names of the rescued clones are indicated on the left. The “gap” in the center of the alignment indicates the bottom strand cleavage site. Black shading indicates nucleotides in agreement with the degenerate consensus L1 EN cleavage site sequence, which is indicated above the alignment (3′-A/TTTT-5′). Gray shading indicates stretches of pyrimidines or purines that are 5′ or 3′ of the bottom strand cleavage site, respectively. The sizes of the TSDs or deletions are indicated at the right of the figure. Underlining indicates nucleotides present in the TSD. The sequences of the long TSDs are available upon request. The asterisk indicates an L1 insertion (4HindIIIL1.3) that lacks a poly (A) tail. This L1 sequence also contains two ambiguous nucleotides (GT) that can be derived from either the L1 or the preintegration site. Thus, there are three possible cleavage sites (5′-GCTG/C, 5′-AGCT/G, or 5′-GAGC/T). The integration site of 1HindIIIL1.2 could not be determined (see text for details). Cell 2002 110, 315-325DOI: (10.1016/S0092-8674(02)00828-0)

Figure 4 L1 Retrotransposition Can Result in Interstitial Deletions (A) Schematic representation of the pre- and postintegration sequences. The structures of the preintegration site present in the HGWD and the postintegration site of 5BglIIL1.3 are indicated. Black lines indicate sequences present in both the pre- and postintegration sites. The thick gray lines indicate sequences that are present only in the preintegration site. The horizontal arrows indicate the relative positions of PCR primers. The vertical arrow indicates the L1 EN cleavage site, where integration began. The rectangles indicate the structure of the 2.9 kb retrotransposed L1mneoI/ColE1. (B) PCR analyses. PCR reactions confirmed that at least 24 kb of DNA was deleted upon L1 retrotransposition. PCR primers are indicated above the gel. M indicates 1 kb plus molecular weight markers (Gibco/BRL). P indicates PCR products from SLP5BglII DNA, which contains the 5BglIIL1.3 insertion. H indicates PCR products from naı̈ve HeLa cell DNA. O indicates PCR with no DNA. As an additional control, we demonstrated that PCR reactions using the 5′ flank/173NEOAS primer pair only amplified the expected sized product in SLP5BglII DNA. Sequencing of this product demonstrated that it was identical to the analogous segment in 5BglIIL1.3. Cell 2002 110, 315-325DOI: (10.1016/S0092-8674(02)00828-0)

Figure 5 L1 Retrotransposition Can Result in Chimeric L1s (A) Schematic representation of three chimeric L1s. The structures of 3HindIIIL1.3, 4HindIIIL1.3, and 1HindIIIL1.2 are indicated by the rectangles. Different shading indicates the junction between the endogenous L1 (gray rectangle) and the retrotransposed L1 (white rectangle). Lollipops indicate the relative positions of nucleotide sequence changes that differentiate the endogenous L1 from the retrotransposed L1mneoI/ColE1. The white lollipop indicates the position of a nucleotide specific to L1.3mneoI/ColE1 (see description in 5E). There are 23 nucleotide differences in a 374 bp segment between 1HindIIIL1.2 and pL1.2mneoI/ColE1. Arrows indicate the relative position of PCR primers. (B) Schematic representation of the pre- and postintegration sequences of 3HindIIIL1.3. The structures of the preintegration and the postintegration site of 3HindIIIL1.3 are shown. Labels are as in Figure 5A and the relative positions of the restriction sites are indicated. Also indicated are sequences deleted upon retrotransposition (3114 bp; the 5′ end of the deletion size was derived from the position of the first nucleotide polymorphism that exists between 3HindIIIL1.3 and pL1.3mneoI/ColE1). The sizes of the predicted PCR products generated from the 5′ flank and 3′ flank primer pairs for both the preintegration (3997 bp) and postintegration (4790 bp) site and the relative positions of the 5′ and 3′ probes used in Southern blot analysis also are indicated. Primer 3HindIIIint was used to determine whether tandemly integrated L1s were present. (C) PCR analysis. Amplification of the pre-and postintegration sites confirmed that 3.1 kb of DNA was deleted upon the creation of the chimeric L1. PCR primers are indicated above the gel and their relative location is shown in Figure 5B. M indicates 1 kb plus molecular weight markers (Gibco/BRL). P1 and P2 indicate PCR products from the DNA of two different clonal cell lines containing 3HindIIIL1.3. H1 and H2 indicate PCR products generated either from HeLa cell DNA or from 50HindIIIL1.3 DNA, which contains a different (i.e., irrelevant) retrotransposed L1 sequence. O indicates PCR with no DNA. (D) Southern analysis. Genomic DNA was isolated from naı̈ve HeLa cells (H1) or from the two clonal cell lines containing the 3HindIIIL1.3 insertion (P1 and P2). 10 μg of DNA was restricted with HindIII and was subjected to Southern blot analysis using either the 1.2 kb 5′ probe or the 581 bp 3′ probe. Lane C contains DNA from the 3HindIIIL1.3 rescued clone, which was linearized with HindIII. It serves as both a size and hybridization control. The sizes of the respected fragments are indicated. (E) Nucleotide sequence at the junction of 3HindIIIL1.3. Black shading indicates nucleotides diagnostic for the endogenous L1, whereas the gray shading indicates nucleotides diagnostic for the retrotransposed L1. The asterisk indicates an engineered sequence change in pL1.3mneoI/ColE1, which removes a BamHI site. SNP typing showed that the endogenous L1 contains the BamH1 site (data not shown). L1 nucleotides are numbered according to a reference L1 (L1.2; Dombroski et al., 1991). Underlining indicates the TSD flanking the endogenous L1. Cell 2002 110, 315-325DOI: (10.1016/S0092-8674(02)00828-0)

Figure 6 Models for L1 Integration (A) Target site alterations created upon L1 retrotransposition. A model proposing how L1 retrotransposition results in various target site alterations (i.e., TSDs, blunt insertions, or small deletions) is shown. Each event is initiated by L1 EN cleavage of the bottom strand followed by L1 cDNA synthesis (represented by the orange arrow). The position of top strand cleavage can create a 3′ overhang (left), no overhang (i.e., a blunt end; middle), or a 5′ overhang (right). The proposed intermediate can be recognized as a substrate by the host repair machinery resulting in 5′-3′ exonucleolytic processing. Plus (+) strand L1 cDNA synthesis by either L1 RT or host enzymes followed by recombinational repair would lead to the final product. This model accounts for the generation of TSDs (indicated by the light blue rectangles on the left side), conservative insertions (middle) or small deletions (right side). (B) Generation of other target site alterations. Modifications of the above model explain how retrotransposition can result in the formation of chimeric L1s, large deletions, or long TSDs. Details are provided in the text. L1 cDNA is indicated in orange. TSDs are indicated by the light blue rectangles. The black rectangle in the left panel indicates an endogenous L1. ENDO indicates a proposed substrate for a 3′ processing endonuclease. The pink lines in the center panel indicate endogenous DNA sequences located upstream of the L1 integration site. The red lines in the right panel indicate endogenous DNA sequences located downstream of the L1 integration site. A gap is used to indicate the position of a double-strand break or single-strand nick. The dark blue arrow indicates the initiation site of second (+) strand cDNA synthesis. Cell 2002 110, 315-325DOI: (10.1016/S0092-8674(02)00828-0)