Chromosomal Translocation Mechanisms at Intronic Alu Elements in Mammalian Cells  Beth Elliott, Christine Richardson, Maria Jasin  Molecular Cell  Volume 17, Issue 6, Pages 885-894 (March 2005) DOI: 10.1016/j.molcel.2005.02.028 Copyright © 2005 Elsevier Inc. Terms and Conditions

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. Molecular Cell 2005 17, 885-894DOI: (10.1016/j.molcel.2005.02.028) Copyright © 2005 Elsevier Inc. Terms and Conditions

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. Molecular Cell 2005 17, 885-894DOI: (10.1016/j.molcel.2005.02.028) Copyright © 2005 Elsevier Inc. Terms and Conditions

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. Molecular Cell 2005 17, 885-894DOI: (10.1016/j.molcel.2005.02.028) Copyright © 2005 Elsevier Inc. Terms and Conditions

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 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. Molecular Cell 2005 17, 885-894DOI: (10.1016/j.molcel.2005.02.028) Copyright © 2005 Elsevier Inc. Terms and Conditions