Chromosomal Translocation Mechanisms at Intronic Alu Elements in Mammalian Cells

Molecular Cell, Vol. 17, 885–894, March 18, 2005, Copyright ©2005 by Elsevier Inc. DOI 10.1016/j.molcel.2005.02.028

Chromosomal Translocation Mechanisms at Intronic Alu Elements in Mammalian Cells Beth Elliott,1 Christine Richardson,1,2 and Maria Jasin1,* 1 Molecular Biology Program Memorial Sloan-Kettering Cancer Center 1275 York Avenue New York, New York 10021

Summary Repetitive elements comprise nearly half of the human genome. Chromosomal rearrangements involving these elements occur in somatic and germline cells and are causative for many diseases. To begin to understand the molecular mechanisms leading to these rearrangements in mammalian cells, we developed an intron-based system to specifically induce chromosomal translocations at Alu elements, the most numerous family of repetitive elements in humans. With this system, we found that when doublestrand breaks (DSBs) were introduced adjacent to identical Alu elements, translocations occurred at high frequency and predominantly arose from repair by the single-strand annealing (SSA) pathway (85%). With diverged Alu elements, translocation frequency was unaltered, yet pathway usage shifted such that nonhomologous end joining (NHEJ) predominated as the translocation pathway (93%). These results emphasize the fluidity of mammalian DSB repair pathway usage. The intron-based system is highly adaptable to addressing a number of issues regarding molecular mechanisms of genomic rearrangements in mammalian cells. Introduction Genomic rearrangements are characteristic of tumor cells, and specific genomic rearrangements are responsible for many inherited diseases, yet genetic systems to study their etiology at the molecular level have been limited. Repetitive elements, which comprise at least 45% the human genome (Lander et al., 2001), present ample opportunity for genomic rearrangements (Deininger et al., 2003). Alu elements make up the largest family of repetitive elements, numbering approximately one million copies and comprising an estimated 11% of the genome (Lander et al., 2001). In germline cells, Alu-Alu intrachromosomal recombination has been implicated in the etiology of several inherited diseases, including some cancers (Deininger and Batzer, 1999; Kolomietz et al., 2002). In somatic cells, recombination between Alu elements has also been documented, with the most intensively studied example involving the MLL gene (Hess, 2004). Recombination between intronic Alu elements, leading to par*Correspondence: [email protected] 2 Present address: Institute for Cancer Genetics, Columbia University, 1150 St. Nicholas Ave., New York, New York 10032.

Technique

tial duplication of MLL, has been demonstrated in cases of acute myeloid leukemia (AML) in which patients lack cytogenetic defects (or have trisomy 11) (Caligiuri et al., 1994; Schichman et al., 1994; So et al., 1997; Strout et al., 1998). The involvement of Alu elements in MLL partial duplications is not limited to AluAlu recombination, as breakpoint junctions between an Alu element and non-Alu sequences have also been identified (Strout et al., 1998). Alu-Alu recombination has also been reported in the generation of a reciprocal translocation present in tumor DNA (Onno et al., 1992). However, in most reciprocal translocations, Alu elements are either joined to non-Alu sequences or fused to other Alu elements or simply occur within the vicinity of breakpoint junctions (Kolomietz et al., 2002). Genomic rearrangements arise from breakage and misrepair, especially of DSBs. Multiple pathways of repair have been demonstrated in mammalian cells for the repair of a single chromosomal DSB (Liang et al., 1998). The two primary pathways are NHEJ, involving little or no homology, and conservative homologous recombination (HR) (van Gent et al., 2001). A third pathway, SSA, can also occur at homologous sequences near a DSB. Unlike HR, which involves strand invasion, SSA involves the annealing of DNA strands formed after resection at the DSB (Pâques and Haber, 1999). We set out to investigate the involvement of DSB repair pathways in genomic rearrangements in mammalian cells, specifically chromosomal translocations. Previously, we showed that translocations could be formed during the repair of DSBs in mouse ES cells (Richardson and Jasin, 2000). Translocations were a fraction of the recovered repair products in this system, and each translocation had the same overall structure due to constraints in the substrate design. We devised, therefore, a strategy that would allow us to specifically select translocations in order to determine which mechanism of DSB repair predominates as the translocation pathway. This strategy involves an intron-based substrate design that is adaptable to addressing a number of issues regarding mechanisms of genomic rearrangements in mammalian cells. Results Translocation Substrates: Breakpoint Junctions within an Intron Key to our translocation substrate design is an introncontaining neomycin phosphotransferase gene (neo): the DSBs occur within a split intron of the neo gene to allow the selection of a variety of breakpoint junctions in the formation of the derivative (der) chromosomes (Figure 1A). For example, translocations involving NHEJ and/or SSA can be selected. Another important aspect of the design is that an Alu element is incorporated adjacent to each of the DSB sites. A second set of repeats, derived from the puromycin resistance gene (puro), is also incorporated adjacent to the DSB sites but on the opposite side from the Alu elements (brackets, Figure 1A).

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Figure 1. Translocation Substrates and Possible Reciprocal Translocation Outcomes (A) Translocation substrates on chromosomes (chrs.) 17 and 14 are based on a split neo intron design with an Alu element from the MLL gene (blue box) inserted 3# and 5# of the splice donor and splice acceptor portions of the neo gene (neoSD and SAneo, respectively). DSBs generated by the I-SceI endonuclease followed by interchromosomal repair can potentially generate translocation chromosomes. Repair by SSA at the identical Alu elements or puro sequences (brackets) will delete one of the repeats, whereas NHEJ could fuse the Alu elements or puro sequences, as shown. A neo+ gene is formed on der(17) by either pathway, whereas on der(14), a puro+ gene is formed by SSA and a puro− gene is formed by NHEJ. By using SSA and/or NHEJ, four combinations of reciprocal translocations are possible. The total size of the intron after precise NHEJ is approximately 1 kb. (B) Gene targeting of the chrs. 17 and 14 at the Pim1 and Rb loci to create targeted alleles with the translocation substrates (p5 and pF, respectively). The chr. 17 substrate was targeted first for the creation of the p5 cell line. The chr. 14 substrate was subsequently targeted to the Rb locus in the p5 cell line to create the p5pF cell lines #5, #6, and #18. Vertical black bars are exons 1–4 for the Pim1 locus and exon 20 for the Rb locus. Abbreviations: HII, HincII and Pst, PstI.

The two translocation substrates were targeted to loci on chromosomes 17 and 14 in mouse ES cells (Figure 1). The chromosome 17 substrate, termed p5, consists of the following four components: a 5# neo fragment with a splice donor site (neoSD), intronic sequences including an Alu element, an I-SceI endonuclease cleavage site for DSB formation, and a 3# puro fragment (3#puro) (Figure 1A). The Alu element, derived from intron 1 of the MLL gene, has been demonstrated to participate in some of the partial tandem duplications of MLL found in patients with AML (Hess, 2004). By using a linked hygromycin resistance gene (hyg), we targeted this translocation substrate to the Pim1 locus on chromosome 17 in ES cells to create the p5 allele (Figure 1B). The chromosome 14 substrate, termed pF, consists of a 5# puro fragment (5#puro), an I-SceI site, the same MLL intron 1 Alu element followed by additional intronic sequences, and a 3# neo fragment with a splice acceptor site (SAneo) (Figure 1A). The two Alu elements share 290 bp of identity, whereas the 5#puro and 3#puro share 265 bp of identity (brackets, Figure 1A). By using a linked HPRT mini-gene, the pF translocation substrate

was introduced into the p5 cell line by targeting to an intron in the Rb locus on chromosome 14 to create the pF allele (Figure 1B). HPRT+ colonies were selected, and three independent clones that were correctly targeted (Figure 1B) were chosen for use in the translocation assays.

Table 1. DSB-Induced Translocation Frequencya neo+ Colonies (×10−5)b Cell Lines

No DNA

DSB-Induced (pCBASce)

p5pF Hom Alu Het Alu

<0.01 <0.01 <0.01

2.6 ± 1.9 5.0 ± 4.0 2.7 ± 1.4

a

For the p5pF and Het Alu cell lines, three independent clones were examined for each. For the Hom Alu cell lines, two independent clones were examined. Two experiments were done on each cell line, except for one cell line of p5pF, Hom Alu, and Het Alu. b The frequency of neo+ colonies was determined by dividing the number of neo+ colonies by the number of electroporated cells (2 × 107), correcting for 50% viability after electroporation.

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Figure 2. DSBs Induce Reciprocal Translocations (A) Reciprocal translocations are observed in neo+ clones derived from the p5pF cell lines after DSB induction. Parental clones have two normal chrs. 17 (red) and 14 (green), and neo+ clones have one normal chr. 17 and chr. 14 and two derivative chromosomes generated by a reciprocal translocation, i.e., der(17) and der(14). (B) The DSB repair pathway used in the formation of the derivative chromosomes is determined by Southern and PCR analyses. Fragment sizes from Southern (top arrows in each panel) and PCR (bottom arrows) analyses are indicated. Whereas SSA gives a unique product, NHEJ can occur by precise ligation or can result in deletion (del.) or insertion (ins.) of nucleotides at the breakpoint junction so as to decrease or increase the size of the Southern or PCR fragments, respectively (see also Supplemental Data). A conservative HR event would give rise to one derivative chromosome that is identical to that derived from SSA, i.e., with one Alu element or puro repeat, whereas the reciprocal derivative chromosome would have the remaining repeat segments, either puro-Alu-puro or Alu-puro-Alu, respectively. Because this was not observed in any of the neo+ clones, it is not diagrammed. Abbreviations: RI, EcoRI; HII, HincII; and H3, HindIII. (C and D) Southern (C) and PCR (D) analyses of neo+ clones. Parental p5pF cell lines do not give PCR products (see [D], left), because the primers for each pair are located on separate chromosomes. See also Supplemental Data.

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Figure 3. Translocations by an SSA Mechanism Predominate at Identical Alu Elements (A) Classes of reciprocal translocation outcomes obtained in neo+ clones from the p5pF and Rev48 cell lines after I-SceI expression. These two cell lines differ in that the p5pF cell line has the I-SceI sites in the same relative orientation on chrs. 17 and 14, whereas for the Rev48 cell line, the I-SceI sites are in opposite orientation. Thus, in the formation of translocation chromosomes, intact I-SceI overhangs at the DNA ends have the potential to be precisely ligated in the p5pF cell line, but not in the Rev48 cell line. (B) Sequence analysis of breakpoint junctions from der(17) and der(14) from p5pF neo+ clones. The boxed nucleotides show the breakpoint sequences on both strands after I-SceI cleavage. Breakpoint junction sequences were obtained for each of the derivative chromosomes except in four cases, which are indicated in parenthesis. The number of nucleotides deleted (del.) from the top strand of each end are indicated, as well as the number or sequence of nucleotides inserted (ins.). Nucleotides that could have been derived from the I-SceI overhang on the bottom strand are in bold. The four base I-SceI overhangs are underlined with thin lines in the three junctions derived from precise ligation (asterisks); microhomologies which occur at three other junctions are underlined with thick lines. (C) Local derivation of the insertion on der(14) of clone 18G-3. The insertion is derived from sequences 5# and 3# of the chr. 17 DSB (thick and thin black bars, respectively). This includes 173 bp of the Alu element, partial I-SceI sequences, and 3#puro sequence, in addition to 9 bp of a (TAn)2 insertion. Spaces between thick bars represent sequences from chr. 17 that were not contiguous. The number of inserted nucleotides is indicated in bp below each insertion. The dotted line indicates 31 bp of originally noncontiguous sequence that was repeated in the junction.

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To induce translocations, the three p5pF cell lines were electroporated with the I-SceI endonuclease expression vector, and neo+ colonies were selected. Each of the p5pF cell lines gave similar numbers of neo+ colonies after I-SceI expression, resulting in an average frequency of 2.6 ± 1.9 × 10−5 (Table 1). In the absence of I-SceI expression, spontaneously arising neo+ colonies were not detected (<10−7), indicating that neo+ colonies arose from DSB repair. Fluorescence in situ hybridization (FISH) demonstrated that the neo+ clones carried the two derivative chromosomes, i.e., der(14) and der(17), expected from a reciprocal translocation (Figure 2A). The neo+ clones also had one intact chromosome 14 and chromosome 17, as expected, as the translocation substrates are present on only one chromosome 14 and chromosome 17 in the parental p5pF cell lines. SSA Predominates as the Translocation Pathway with Identical Alu Elements To identify the translocation pathway(s), 47 neo+ clones from the p5pF cell lines were analyzed by Southern blotting and PCR (Figures 2B–2D; see Supplemental Data available online with this article). The predominant class of clones was found to have both derivatives chromosomes formed by SSA (Class 1, 81%; Figure 3A). Considering the individual derivative chromosomes, 40 (85%) of the der(17) chromosomes were derived from SSA of the identical Alu elements, with the remaining seven (15%) derived from NHEJ (Figure 3A). Similarly, 39 (83%) of the der(14) chromosomes were derived from SSA at the puro repeat, whereas only eight (17%) were derived from NHEJ. Thus, SSA is the preferred translocation pathway for generating either derivative chromosome in the p5pF cell lines. Translocations associated with deletions that extend beyond the intronic sequences into the neo coding sequences would preclude the formation of an intact neo gene. We therefore took advantage of the ability to select der(14) translocations with puromycin in order to determine if translocations selected in this way would arise in a similar manner as when neo+ clones were selected. Of 54 puro+ clones, we found that 52 clones (96%) were also neo+ (data not shown). Thus, neo+ selection appears to capture most of the translocations associated with DSB induction on chromosomes 14 and 17. As before, der(17) primarily arose by SSA of the Alu elements (data not shown). Because the 18 bp I-SceI sites in the translocation substrates are in the same relative orientation on both chromosomes 14 and 17, it is formally possible that a derivative chromosome could be formed by precise ligation of the I-SceI cohesive overhangs but then undergo another DSB that is repaired by intrachromosomal SSA. To verify that SSA is the initial repair event leading to the translocations, we constructed another cell line, termed Rev48, in which the I-SceI sites are in opposite relative orientations on chromosomes 14 and 17. In this cell line, the p5 allele (Figure 1B) is present as before on chromosome 17, but the pF allele on chromosome 14 was modified to contain the I-SceI site in reverse orientation (see Supplemental Data). I-SceI endonuclease was expressed in the Rev48 cell line and 20 neo+ clones were analyzed at the molecular level to

determine the translocation pathway. From these clones, 18 (80%) had der(17) chromosomes generated by SSA and all 20 had der(14) generated by SSA (Figure 3A). Thus, the bias toward SSA at the translocation breakpoint junctions is not the result of a secondary DSB repair event. Translocation Breakpoint Junctions Derived from NHEJ A total of 15 of the 47 translocation breakpoint junctions from the p5pF neo+ clones were formed by NHEJ. These junctions were analyzed in more detail. Three of the breakpoint junctions had a restored I-SceI site (asterisk, Figure 3B) from precise ligation of the I-SceI overhangs (thin underline, Figure 3B). Small deletions and/or insertions, i.e., involving <22 bp, were found in seven junctions. In one junction, a larger insertion (224 bp) was found that had a complex origin (Figure 3C). The insertion was “locally derived,” i.e., derived from nearby sequences, as has been seen in translocation breakpoint junctions in a number of cancers (ZucmanRossi et al., 1998). Larger insertions of approximately 0.9 kb, and 0.7 kb occurred at two der(17) junctions, as determined by Southern analysis. For one der(14) junction, no signal was obtained by Southern blot analysis, indicating that the puro probe sequence was deleted (data not shown). For another der(14) junction, a more complex Southern hybridzation pattern was observed, indicating that the puro gene sequences were rearranged and/or deleted. We also sequenced five of the SSA products (as indicated in Figure 3B) and verified that no mutations were introduced into the repeats during SSA. In summary, from 47 neo+ clones, 15 of the 94 breakpoint junctions were derived from NHEJ, the majority (10 of 15 junctions) of which had little, if any, degradation or other alteration to the ends (0–21 bp); the remaining breakpoint junctions involved more extensive end modifications. Heterology between the Alu Elements Has Little Effect on Translocation Frequency Alu elements in human genomes are frequently quite divergent from each other, with a range of divergence from the consensus Alu element of between 2% and 30% (Smit, 1996). We therefore asked if the substitution of a heterologous Alu element in one of the translocation substrates would affect the translocation frequency and/or translocation pathway. For this, we substituted the MLL intron 1 Alu element in the chromosome 14 translocation substrate with an Alu element from intron 6 of the MLL gene, which is within the major breakpoint cluster region (see Figure S1). The two Alu elements, which are both from the Alu Sx subfamily, are 20% divergent from each other, although there are regions of up to 25 bp of complete identity. We paired these particular Alu elements because somatic recombination events between them have been found in patients with AML (So et al., 1997; Strout et al., 1998) (Figure S1). To construct cell lines containing the heterologous Alu element, termed the Het Alu cell lines, the chromosome 14 targeting vector (Figure 1B, Figure S1A) was modified to contain the intron 6 Alu element and then

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Figure 4. Translocations by an NHEJ Mechanism Predominate at Heterologous Alu Elements (A) Classes of reciprocal translocation outcomes obtained in neo+ clones from the Het Alu cell lines after I-SceI expression. The same class designation is used as described in Figure 3A, except that “SSA” indicates formation of an intact Alu element that could be consistent with an SSA mechanism. Blue and red boxes indicate MLL intron 1 and intron 6 Alu element-derived sequences, respectively. (B) Sequence analysis of der(17) breakpoint junctions derived from NHEJ from the Het Alu neo+ clones. The 69 der(17) junctions were divided into three groups based on the amount of degradation from the DNA ends prior to joining (<100, 100–400, and >400 bp), and four junctions from each group were sequenced. The boxed nucleotides show the breakpoint sequences on both strands after I-SceI cleavage. The number of nucleotides deleted (del.) from the top strand of each end are indicated, as well as the number or sequence of nucleotides inserted (ins.). Microhomologies are underlined. (C) Structure of the breakpoint junctions containing intact or nearly intact Alu elements. In five clones, an intact Alu element of 290 bp is found at the breakpoint junction. This hybrid Alu element consists of sequences from the MLL intron 1 Alu element (blue box) and the MLL intron 6 Alu element (red box), as well as microhomology between the two Alu elements (white box; the length of microhomology is indicated). A similar overall structure is found in three breakpoint junctions derived from NHEJ, although the fused Alu element is somewhat smaller or larger than an intact element because the junction does not occur at the same position in both Alu elements. (D) Breakpoint junctions that restore an intact or nearly intact Alu element. The positions of the five breakpoint junctions that restore an intact

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targeted to chromosome 14 in the p5 cell line. Three clones that were correctly targeted were used in the subsequent analysis. Because this modification led to some sequence changes near the I-SceI site, we recloned the intron 1 Alu element into the chromosome 14 targeting vector in an identical manner to create two “Hom Alu” cell lines in which the intron 1 Alu element is on both chromosomes 17 and 14 (Figure S1A). In the Hom Alu and Het Alu parental cell lines, the I-SceI site on chromosome 14 is in the opposite orientation relative to the I-SceI site on chromosome 17, as with the Rev48 cell line. The I-SceI expression vector was electroporated into the Hom Alu and Het Alu cell lines, and neo+ colonies were selected. The Hom Alu cell lines had an average frequency of 5.0 ± 4.0 × 10−5 of neo+ colonies, and the Het Alu cell lines had an average frequency of neo+ colonies of 2.7 ± 1.4 × 10−5 (p = 0.27; Table 1). By using similar analyses as described for the p5pF cell lines, the neo+ clones from the Hom Alu and Het Alu cell lines were found to contain reciprocal chromosomal translocations (see below; data not shown). Thus, the substitution of a highly diverged Alu element has little or no effect on the overall translocation frequency. NHEJ Predominates as the Translocation Pathway When Heterology Is Present at the DNA Ends We analyzed a number of neo+ clones from the Hom Alu cell lines and found that, as with the p5pF and Rev48 cell lines, SSA predominated as the translocation pathway for both derivative chromosomes (data not shown). We next characterized 74 neo+ clones derived from the Het Alu cell lines. In contrast to cell lines with identical Alu elements in which >80% of neo+ clones were in Class 1 (SSA/SSA), 92% of neo+ clones derived from the Het Alu cell lines fell into Class 4 (NHEJ/SSA) (Figure 4A). Thus, although heterology at the chromosome ends did not substantially affect the translocation frequency, it dramatically shifted translocation pathway usage from SSA to NHEJ in the formation of the der(17) chromosome. Formation of the reciprocal der(14) chromosome by SSA was not affected by this shift in pathway usage. To further characterize the der(17) breakpoint junctions derived from NHEJ, we analyzed PCR products from the neo+ gene. The 69 clones with breakpoint junctions derived from NHEJ were classified into three groups depending on the estimated amount of degradation from the DNA ends prior to NHEJ (Figure 4B) as deduced from the size of the neo PCR products (data not shown). The majority of neo+ clones (55 clones; 80%) had less than 100 bp deleted at one or both DSBs; an additional six clones (9%) had between 100 and 400 bp deleted. Thus, in most of the clones, NHEJ led to fusion of the two heterologous Alu elements, either in their entirety or between portions of the ele-

ments. In some cases, the fusion formed a nearly unitlength Alu element (Figure 4C). The remaining eight clones (11%) had greater than 400 bp deleted, leading to complete loss of one of the Alu elements (Figure 4B). In these cases, the deletion approached the intron/ exon border of the neo gene on one side. Junctions with various deletion lengths were sequenced (Figure 4B). In some junctions, the deletions were nearly symmetrical around the DSBs, as in clone 1B-21 in which 129 and 143 bp were deleted from the chromosome 17 and chromosome 14 ends, respectively. In several other junctions, the deletions were highly asymmetrical, as in clone 1B-15 in which 425 and 1 bp were deleted from the chromosome 17 and chromosome 14 ends, respectively. Microhomology of 1–5 bp was present at all of the junctions that did not contain an insertion. Positions of microhomology are indicated for two of the fused Alu elements that formed a nearly unit-length Alu element (clones 1B-5 and 1B21; Figures 4C and 4D). In addition to the clones with bona fide NHEJ junctions, five clones had events that led to the formation of a single, intact Alu element at the breakpoint junction, which would be consistent with an SSA event (hence, “SSA” in Figure 4A). In these intact Alu elements, the 5# portion was derived from the intron 1 Alu element, and the 3# portion was derived from the intron 6 Alu element (Figure 4C). This was very similar to the fused Alu elements arising from NHEJ, except that these five junctions were “in register,” i.e, at the same position in both Alu elements, rather than being offset (Figure 4D). Microhomology was present at each of the five junctions. In two clones, the microhomology was short, i.e., 3 and 7 bp (clones 1A-1 and 1A-10, respectively; Figures 4C and 4D). In the other three clones (clones 1A-8, 2B-3, and 2B-17), the junction occurred within the longest stretch of near identity between the two Alu elements at the position of the breakpoint junction found in an AML patient (patient 20; Figure 4C). Within this stretch, 32 of 33 nucleotides are identical: the junctions of clones 2B-17 and 2B-3 occurred 5# and 3# of the single nucleotide polymorphism that exists between the two Alu elements in this stretch, respectively, whereas the junction for clone 1A-8 contained a G/T to A mutation at this polymorphic site. Discussion In this report, we investigated translocation pathway choice in mammalian cells. In particular, we examined the role of Alu elements, because these repetitive elements comprise a large portion of the human genome. By using an intron-based translocation system, we found that when identical Alu elements are present at DNA ends, SSA predominated as the translocation pathway, such that 85% of the derivative chromosomes

hybrid Alu element are shown in brackets below the aligned sequences. Three of these junctions occur at a position where 32 of 33 bp are identical between the two Alu elements and at which the breakpoint junction (boxed sequence) occurs for AML patient 20 (So et al., 1997). The breakpoint junction for patient 300 (Strout et al., 1998) is also boxed. The position of two junctions from clones with fused Alu elements that are not in register are also indicated; the point of fusion with respect to the intron 1 Alu and intron 6 Alu is shown above and below the line, respectively.

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had a single Alu element at the breakpoint junction. When diverged Alu elements are present at DNA ends, translocation frequency was not substantially altered; however, there was a dramatic shift to the NHEJ pathway, such that 93% of the derivative chromosomes had breakpoint junctions involving little or no sequence identity. Our translocation system provides a good model for oncogenic translocations, which frequently have breakpoint junctions within intronic sequences and often in the vicinity of Alu elements (Deininger and Batzer, 1999; Greaves and Wiemels, 2003; Kolomietz et al., 2002). A yeast intron-based translocation system has also been recently developed (Yu and Gabriel, 2004), which will be important for phylogenetic comparisons. Although intron-based systems impose some constraints, our results suggest that the majority of translocation chromosomes are recovered with neo+ gene selection in our system. The intron-based system, therefore, provides the flexibility necessary to recover a variety of breakpoint junctions. Translocation Pathway Choice Our results provide an explanation for why most oncogenic translocation junctions rarely involve recombination between repetitive elements: sequence divergence is sufficient to shift homology-based repair events to nonhomologous repair events. It seems remarkable that the translocation frequency in our experiments was not substantially altered in cell lines containing heterologous Alu elements compared with cell lines containing identical elements, even though the DSB repair pathway dramatically shifted. We do not expect the SSA events on der(14) to somehow “drive” the recovery of der(17) events by NHEJ, because we find that translocations can occur in the absence of nearby homology at a similar frequency to the events described here (D. Weinstock, B.E., and M.J., unpublished data). A priori it could have been predicted that cellular factors would control repair pathway usage, as has been seen in yeast (Frank-Vaillant and Marcand, 2002; Karathanasis and Wilson, 2002). Our results, therefore, highlight the potential for overlapping use of repair pathways at a DSB in mammalian cells and emphasize how readily cells can shift from one pathway to another, indicating the “fluidity” of DSB repair. Competition and collaboration of DSB repair pathways have been noted previously in other studies in vertebrate cells; however, these other studies have utilized DSB repair mutants which are defective for one or more pathways of repair (e.g., Couedel et al. [2004], Mills et al. [2004], Stark et al. [2004], and Takata et al. [1998]). Factors that are thought to influence the choice of DSB repair pathway include cell cycle stage, cell type, growth conditions, and age of cells. This control of DSB repair pathway usage would have made it seem likely that cells would “choose” a pathway prior to sensing the degree of sequence homology at the DNA ends and then begin processing the DNA ends in a manner appropriate to the chosen pathway. Based on studies in yeast, the initial processing steps at a DSB differ for SSA and NHEJ. In SSA, DNA ends are resected in a 5# to 3# direction to produce 3# single-stranded tails that

then anneal to each other (Pâques and Haber, 1999). On the other hand, NHEJ efficiently utilizes intact DNA ends and is suppressed by 5# to 3# end resection (Frank-Vaillant and Marcand, 2002; Ira et al., 2004). As a result, when SSA is impaired in yeast by the removal of repeat sequences or by rad52 mutation, NHEJ is not enhanced (Karathanasis and Wilson, 2002). For SSA events in our cell lines containing identical Alu elements, w290 nucleotides would need to be resected from each end in order to reveal complementary strands for annealing. By contrast, for NHEJ in our cell lines containing heterologous Alu elements, a need for extensive end modification would seem to be abrogated for most events, given the limited end modifications observed at the breakpoint junctions. A major difference between NHEJ in yeast and mammalian cells is the ability of mammalian cells to efficiently join a number of DNA end structures (Smith et al., 2001). Mammalian cells may be able to either fill in resected ends or, alternatively, make use of resected DNA ends for NHEJ in a “micro-SSA” type of NHEJ reaction involving only a few bps of sequence identity. However, distinct repair factors are expected to be recruited for micro-SSA events involving a few bps and for “macroSSA” events involving 290 bp, because in yeast, the genetic requirement for these events differ (Kramer et al., 1994). Therefore, these events are not expected to be equivalent. Given the major class of reciprocal translocations in the Het Alu cell lines (i.e., class 4), as well as previous results (Richardson and Jasin, 2000), our results imply that NHEJ and SSA are available to repair broken chromosome ends at the same stage(s) of the cell cycle. In yeast, strand resection is cell cycle regulated, being much reduced in G1 cells (Ira et al., 2004); presumably, therefore, SSA would also be reduced in G1 cells. In vertebrate cells, NHEJ is considered to be the preferred pathway in the G1 phase of the cell cycle, whereas HR, which like SSA requires strand resection, is most active in late S/G2 (Takata et al., 1998). Recent work, however, has emphasized that NHEJ is not restricted to the G1 stage (Couedel et al., 2004; Mills et al., 2004; Rothkamm et al., 2003). Thus, both NHEJ and SSA are presumably active in late S/G2, suggesting that our translocations may be occurring at this point in the cell cycle. It should be noted that translocations are thought to be only a small portion of total DSB repair events in our cell lines. Translocations are recovered at a frequency of approximately 3–5 × 10−5, whereas intrachromosomal NHEJ and HR events are presumed to occur at >10−2, based on results from other I-SceI substrates in ES cells (Moynahan et al., 2001). Alu Elements and Genomic Rearrangements Our experiments raise the question as to the mechanism of Alu-Alu recombination that gives rise to disease-causing alleles. At least in somatic cells, HR would not appear to be a favored mechanism to generate chromosomal rearrangements, given previously published reports (Richardson and Jasin, 2000; Richardson et al., 1998). Moreover, conservative HR events would be dependent on RAD51, yet we find that expression of a dominant negative RAD51 protein (Stark

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et al., 2004) does not reduce the recovery of translocations (B.E. and M.J., unpublished observations). The five Het Alu clones in which intact, unit-length Alu elements were generated by translocation (Figure 4C) are similar to the SSA products from cell lines containing identical elements. However, it is not certain that these junctions are derived from SSA. Annealing of the strands from the two heterologous Alu elements would produce multiple mismatches along the lengths of the Alu elements and would have to escape heteroduplex rejection. Presumably, such an annealed product would be a substrate for mismatch repair (Sugawara et al., 2004), which might be expected to produce an Alu element containing patches from each Alu element, rather than the hybrid element we observed with a single crossover position. It is notable, however, that we obtained fused Alu elements from NHEJ that were not in register but which came close in size to restoring an intact Alu element (Figure 4C). In each case, the breakpoint junction for the fused elements occurred at a region of microhomology, such that the structure of these fused elements is strikingly similar to the intact Alu elements. These observations suggest the possibility that apparent Alu-Alu recombination events between heterologous elements may arise in some instances from microhomology-mediated NHEJ in which the two Alu elements are in register with each other. This is especially attractive given that NHEJ is very efficient in mammalian cells and that sequence divergence significantly suppresses SSA and HR. Microhomology-mediated NHEJ has the potential to give rise to a wide range of events—deletions, duplications (if between sister chromatids), and translocations. The intron-based system we have developed in this report will allow us to further explore the genetic requirements of these events. Experimental Procedures

line with this article at http://www.molecule.org/cgi/content/full/17/6/ 885/DC1/.

Acknowledgments We thank Margaret Leversha at the Molecular Cytogenetics Core Facility (MSKCC) for performing FISH experiments and Michael Backlund and David Weinstock for assistance and discussions. This project was supported by the Dorothy Rodbell Cohen Foundation (B.E.), National Science Foundation 0346354, and National Institutes of Health GM54668 (M.J.). Received: December 4, 2004 Revised: February 4, 2005 Accepted: February 24, 2005 Published: March 17, 2005 References Caligiuri, M.A., Schichman, S.A., Strout, M.P., Mrozek, K., Baer, M.R., Frankel, S.R., Barcos, M., Herzig, G.P., Croce, C.M., and Bloomfield, C.D. (1994). Molecular rearrangement of the ALL-1 gene in acute myeloid leukemia without cytogenetic evidence of 11q23 chromosomal translocations. Cancer Res. 54, 370–373. Couedel, C., Mills, K.D., Barchi, M., Shen, L., Olshen, A., Johnson, R.D., Nussenzweig, A., Essers, J., Kanaar, R., Li, G.C., et al. (2004). Collaboration of homologous recombination and nonhomologous end-joining factors for the survival and integrity of mice and cells. Genes Dev. 18, 1293–1304. Deininger, P.L., and Batzer, M.A. (1999). Alu repeats and human disease. Mol. Genet. Metab. 67, 183–193. Deininger, P.L., Moran, J.V., Batzer, M.A., and Kazazian, H.H., Jr. (2003). Mobile elements and mammalian genome evolution. Curr. Opin. Genet. Dev. 13, 651–658. Frank-Vaillant, M., and Marcand, S. (2002). Transient stability of DNA ends allows nonhomologous end joining to precede homologous recombination. Mol. Cell 10, 1189–1199. Greaves, M.F., and Wiemels, J. (2003). Origins of chromosome translocations in childhood leukaemia. Nat. Rev. Cancer 3, 639– 649. Hess, J.L. (2004). MLL: a histone methyltransferase disrupted in leukemia. Trends Mol. Med. 10, 500–507.

DNA Manipulations and Cell Line Constructions The first intron from the adenovirus (Ad1) major late transcription unit was previously used in a mouse lacZ recombination reporter (Moynahan et al., 1996) and was adapted for use here in the neo gene of pMC1neo. The resulting construct was verified to give rise to G418R colonies upon transfection into mammalian cells. This p-i-neo construct was used in subsequent steps to derive neoSD and SAneo. The Pim1 and Rb targeting constructs have been described previously (Richardson et al., 1998). For details on cloning, see Supplemental Data.

Ira, G., Pellicioli, A., Balijja, A., Wang, X., Fiorani, S., Carotenuto, W., Liberi, G., Bressan, D., Wan, L., Hollingsworth, N.M., et al. (2004). DNA end resection, homologous recombination and DNA damage checkpoint activation require CDK1. Nature 431, 1011– 1017.

Translocation Analysis For translocation experiments, 2 × 107 cells in 1 ml of phosphatebuffered saline were electroporated with 20–25 µg of the I-SceI expression vector pCBASce (Richardson et al., 1998) in 0.4 cm electrode-gap cuvette (250 V, 960 µF). Electroporated cells were aliquoted into 2–5 10 cm diameter dishes. Colonies were selected in ES media with a drug 20–24 hr after electroporation and were grown in selection media for 10–14 days before colony counts (or after colony expansion): G418 (200 ␮g/ml), hygromycin (150 ␮g/ml), puromycin (1.6 ␮g/ml), or HAT (1× Sigma H0262). For analyses of neo+ clones, see Supplemental Data.

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Supplemental Data Supplemental Data include Supplemental Experimental Procedures, Supplemental References, and one figure and are available on-

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