Nucleotide-resolution mapping of topoisomerase-mediated and apoptotic DNA strand scissions at or near an MLL translocation hotspot

Abstract

The emergence of therapy-related acute myeloid leukemia (t-AML) has been associated with DNA topoisomerase II (TOP2)-targeted drug treatments and chromosomal translocations frequently involving the MLL, or ALL-1, gene. Two distinct mechanisms have been implicated as potential triggers of t-AML translocations: TOP2-mediated DNA cleavage and apoptotic higher-order chromatin fragmentation. Assessment of the role of TOP2 in this process has been hampered by a lack of techniques allowing in vivo mapping of TOP2-mediated DNA cleavage at nucleotide resolution in single-copy genes. A novel method, extension ligation-mediated polymerase chain reaction (ELMPCR), was used here for mapping topoisomerase-mediated DNA strand breaks and apoptotic DNA cleavage across a translocation-prone region of MLL in human cells. We report the first genomic map integrating translocation breakpoints and topoisomerase I, TOP2, and apoptotic DNA cleavage sites at nucleotide resolution across an MLL region harboring a t-AML translocation hotspot. This hotspot is flanked by a TOP2 cleavage site and is localized at one extremity of a minor apoptotic cleavage region, where multiple single- and double-strand breaks were induced by caspase-activated apoptotic nucleases. This cleavage pattern was in sharp contrast to that observed approximately 200 bp downstream in the exon 12 region, which displayed much stronger apoptotic cleavage but where no double-strand breaks were detected and no t-AML-associated breakpoints were reported. The localization and remarkable clustering of the t-AML breakpoints cannot be explained simply by the DNA cleavage patterns but might result from potential interactions between TOP2 poisoning, apoptotic DNA cleavage, and DNA repair attempts at specific sites of higher-order chromatin structure in apoptosis-evading cells. ELMPCR provides a new tool for investigating the role of DNA topoisomerases in fundamental genetic processes and translocations associated with cancer treatments involving topoisomerase-targeted drugs.

Figure 1.

Distinctive features of ELMPCR compared with those of conventional LMPCR. LMPCR requires a 5′-phosphate (P) at the 5′ end of a genomic DNA break for linker ligation, in contrast to ELMPCR, which involves ligation of an antilinker (with a sequence complementary to the LMPCR linker) to the 3′-hydroxyl(OH) end of a primer 1 extension product.

Figure 2.

Comparison of efficiency of antilinkers terminated by a 3′-phosphate-deoxycytidine (3′P-dC) or a 2′,3′-dideoxycytidine (2′,3′-ddC) in ELMPCR detection of topoisomerase-mediated DNA cleavage. P6 cells were treated for 30 min or 6 h with 25 μM etoposide (VP-16), and CEM cells were treated with CPT for the same times. ELMPCR data are from duplicate cell treatments, by use of set 9 primers for noncoding-strand cleavage analysis. Cleavage sites are 6801, 6823/6824, and 6920/6921 (from bottom to top) for TOP1 and 6909/6910 for TOP2. The apoptotic cleavage region (Ap) extends downward from 6816 (see fig. 9 map). Lanes A, G, and T/C (T+C) are Maxam and Gilbert DNA sequencing ladders.

Figure 3.

Linear relationship between ELMPCR signal intensity and the extent of TOP2-mediated DNA cleavage. ELMPCR was performed with a 1.6-μg DNA sample composed of 0.2, 0.4., 0.8, or 1.6 μg of cellular DNA isolated from confluent primary human fibroblasts exposed to 50 μM etoposide for 1 h, to which was added 1.4, 1.2, 0.8, or 0 μg of DNA, respectively, from unexposed control fibroblasts. Average ELMPCR signal intensities of duplicates measured at TOP2 cleavage site 6909/6910 were detected as described in figure 7.

Figure 4.

Distinction of apoptotic versus TOP2-mediated DNA cleavage by combined use of ELMPCR and LMPCR. Human P6 lymphoblastoid cells were treated or were not treated (controls) with VP-16 or with CD95 antibody (Ab) to induce apoptosis. Part of the cells was then heat shocked (HS) at 60°C for 15 min to reverse topoisomerase-mediated DNA cleavage before DNA extraction. ELMPCR and LMPCR data are from duplicate cell cultures, by use of set 1 primers for noncoding-strand analysis (A) and set 2 primers for the coding-strand analysis (B). The TOP2 cleavage site is at 6909/6910, and the apoptotic cleavage region (Ap) extends upstream of 6816 on the noncoding strand (A) and downstream of 6816 on the coding strand (B) (see fig. 9). The asterisk (*) in panel B indicates the expected position for a reciprocal TOP2 cleavage, which was not detected, opposite to the TOP2 cleavage site identified on the coding strand. TOP2 cleavage positions indicate 5′ nucleotides covalently linked to the enzyme. Lanes G and A are Maxam and Gilbert DNA sequencing ladders. The ELMPCR and LMPCR exposure intensities were normalized to DNA sequencing ladders.

Figure 5.

Colocalization of etoposide- and teniposide-induced TOP2 DNA cleavage. CEM cells were treated for 30 min with etoposide (VP-16 [100 μM]) or teniposide (VM-26 [10 μM]) to induce stabilization of TOP2 cleavage complexes. Part of the cells was then heated shocked (HS), to reverse TOP2-mediated cleavage before DNA extraction. ELMPCR data were obtained with cellular DNA isolated from duplicate cell treatments, by use of set 1 primers for the lower strand and set 10 primers for the upper strand. TOP2 cleavage sites are at 6909/6910 on the lower strand (top panel) and 6918/6919 on the upper strand (bottom panel) (see fig. 9 map). Lanes G and A are Maxam and Gilbert DNA sequencing ladders.

Figure 6.

Caspase-dependent nucleolytic cleavage, distinguished from TOP1-mediated DNA cleavage. CEM cells were treated for 30 min or 6 h with 25 μM CPT in the absence or presence of the general caspase inhibitor zVAD-fmk (50 μM). Part of the cells was then heat shocked (HS) or was further incubated for 30 min without drug, to reverse TOP1-mediated DNA cleavage before DNA extraction. ELMPCR data were from duplicate cell treatments, by use of set 7 primers for noncoding-strand cleavage analysis. TOP1 cleavage sites are at 6626/6627, 6801, and 6823/6824 (from bottom to top), and apoptotic cleavage regions (Ap) extend from 6585–6633 (bottom) and 6773–6816 (top) (see fig. 9 map). Lanes G and A are Maxam and Gilbert DNA sequencing ladders.

Figure 7.

ELMPCR detection of TOP1- and TOP2-mediated DNA cleavage in primary human fibroblasts. Confluent fibroblast cultures were treated or were not treated (controls) with VP-16 (50 μM) or CPT (10 μM) for 1 h. Part of the cells was then heat shocked (HS), to reverse topoisomerase-mediated DNA cleavage. ELMPCR data were from duplicate cell cultures, by use of set 1 primers for the noncoding strand (A) and set 2 primers for the coding strand (B). A stronger exposure of the bottom part of panel B is shown below it, to enlarge the signal of a weak TOP2-mediated cleavage. Cleavage sites are at 6909/6910 for TOP2 and 6822/6823/6824 and 6920/6921 for TOP1 on the noncoding strand and at 6891/6892 and 6811/6812 for TOP1 on the coding strand (see fig. 9 map). TOP2 and TOP1 cleavage positions indicate the nucleotides covalently linked to the enzyme (5′-linked TOP2 and 3′-linked TOP1). Lanes G and A are Maxam and Gilbert DNA sequencing ladders.

Figure 8.

TOP2-mediated DNA cleavage in CEM and TOP2α-mutant CEM/VM-1 cells. Cells were treated with 25 μM or 100 μM (indicated by larger plus sign [+]) VP-16 or 5 μM CPT for 30 min. ELMPCR was performed with equal amounts of DNA isolated from duplicate drug-treated cell cultures or from untreated cultures including 0.6% CEM DNA cleaved to completion by restriction endonuclease ApoI. A and B, ELMPCR with primer extension (ext.) performed with Pfu exo⁻ (P) or Sequenase (S) or PCR amplification after Sequenase-mediated filling of 3′-recessed termini (with no heat denaturation and no primer 1 extension) and antilinker ligation (B), by use of set 1 primers (A) or set 10 primers (B). In panel C, set 1 primers were used. The noncoding-strand cleavage sites for TOP2, TOP1, and ApoI are 6909/6910, 6920/6921, and 6897, respectively. No reciprocal TOP2 cleavage was detected on the coding strand (TOP2* at 6906/6907).

Figure 9.

Nucleotide-resolution map of topoisomerase DNA cleavage sites, apoptotic cleavage hotspots, and translocation breakpoints. The map integrates the results of multiple ELMPCR and LMPCR analyses performed as described in figures 4, 7, 8, and 10, by use of all sets of primers listed in table 1. The coding strand is the upper strand. Blue arrows, Etoposide-induced TOP2 cleavage sites. Green arrows, CPT-induced TOP1 cleavage sites. Hotspots for apoptotic cleavage are shown underlined in red (thick line indicates stronger cleavage). Red arrows, Predominant cleavage sites (thick arrows indicate stronger cleavage sites). Asterisks (*) indicate blunt DSBs. The black box defines a major translocation hotspot, which includes nine therapy-related translocation breakpoints associated with multidrug treatments involving TOP2-targeted drugs,^,,, and the breakpoint reported for a cell line (SN-1) derived from a patient with T-cell acute lymphoblastic leukemia with no history of previous treatment. Vertical marks show additional translocation breakpoints reported in the literature,^,,, with each color corresponding to a different patient, except for all orange breakpoints, which resulted from Fas-mediated apoptosis in vitro.^, Duplicate marks of same color reflect uncertainties about the exact MLL breakpoint location because of local overlapping of DNA sequences at the MLL-partner junction or because of sequence gaps between derivative MLL and derivative partner breakpoints.

Figure 10.

Colocalization of apoptotic and topoisomerase-mediated DNA cleavages at a major MLL translocation hotspot. CEM cells were treated with etoposide (100 μM), CPT (5 μM), or CD95 antibody (Ab) for the time indicated. ELMPCR data were from duplicate cell cultures, by use of set 7 primers for noncoding-strand cleavage analysis (A) and set 4 primers for coding-strand analysis (B). The upper panel in A shows a lower exposure of the top portion of the main panel. Cleavage sites are 6822/23, 6801, and 6626/27 for TOP1 on the noncoding strand (A) and 6588/89 for TOP1 and 6583/84 for TOP2 on the coding strand (B). C, Detection of blunt DSBs by direct PCR amplification after linker or antilinker ligation (no heat denaturation and no primer 1 extension), as compared with LMPCR (left) or ELMPCR (right), respectively. The apoptotic cleavage regions (Ap) extend from 6816–6773 (top) and 6633–6585 (bottom) on the noncoding strand (A), and from 6633–6595 on the coding strand (B); an asterisk (*) indicate cleavages forming a major blunt DSB at 6602/6603. The TH side boxes locate the translocation hotspot (fig. 9). Lanes G and A are Maxam and Gilbert DNA sequencing ladders. HS = heat shocked.