Human Molecular Genetics, 2000, Vol. 9, No. 11 1671-1679
© 2000 Oxford University Press
DNA structural properties of AF9 are similar to MLL and could act as recombination hot spots resulting in MLL/AF9 translocations and leukemogenesis
University of Chicago Department of Medicine, Section of Hematology Oncology, 5841 South Maryland Avenue, MC2115, Chicago, IL 60637-1470, USA, 1University of Oklahoma, Department of Chemistry and Biochemistry, 620 Parrington Oval, Room 311, Norman, OK 73019, USA and 2Loyola University Chicago, Cardinal Bernardin Cancer Center, 2160 South First Avenue, Maywood, IL 60153, USA
Received 20 March 2000; Revised and Accepted 2 May 2000.
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ABSTRACT |
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The human AF9 gene at 9p22 is one of the most common fusion partner genes with the MLL gene at 11q23, resulting in the t(9;11)(p22;q23). The MLL–AF9 fusion gene is associated with de novo acute myelogenous leukemia (AML), rarely with acute lymphocytic leukemia (ALL) and with therapy related leukemia (t-AML). The AF9 gene is >100 kb and two patient breakpoint cluster regions (BCRs) have been identified; BCR1 is within intron 4, previously called site A, whereas BCR2 or site B spans introns 7 and 8. Patient breakpoint locations were determined previously by RT–PCR and by genomic DNA cloning. In this study, we defined the exon–intron boundaries and identified several different structural elements in AF9 including a co-localizing in vivo DNA topo II cleavage site and an in vitro DNase I hypersensitive (DNase 1 HS) site in intron 7 in BCR2. Reversibility experiments demonstrated a religation of the topo II cleavage sites. The location of the in vivo topo II cleavage site was confirmed in vitro using a topo II cleavage assay. In addition, two scaffold associated regions (SARs) are located centromeric to the topo II and DNase I HS cleavage sites and border both patient breakpoint regions: SAR1 is located in intron 4, whereas SAR2 encompasses parts of exons 5–7. This study demonstrates that the patient breakpoint regions of AF9 share the same structural elements as the MLL BCR. We describe a DNA breakage and repair model for non-homologous recombination between MLL and its partner genes, particularly AF9.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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The MLL gene (1) [also called ALL-1 (2), Htrx (3) or HRX (4)] which maps to human chromosome 11 band q23 (11q23) is involved in chromosome translocations in ~15% of patients with acute myelogenous leukemia (AML) and acute lymphocytic leukemia (ALL). MLL recombines with ~40 different chromosomal regions (5–9), as well as with itself in a partial MLL gene duplication of exons 2–6 or 2–8 with or without trisomy 11 (10–12). The most common 11q23 translocations involving MLL are the t(9;11), t(6;11) and t(11;19) detected in AML, and the t(4;11) and t(11;19) found primarily in ALL patients (5–7). Eighty percent of ALL and AML in infants under 1 year of age show MLL rearrangements (13–15). Chemotherapeutic agents, such as the epipodophyllotoxins such as VP16 and natural bioflavonoids found in certain foods and dietary supplements, target cellular DNA topoisomerase II (topo II) and trap topo II in a DNA-cleavable complex (16–18). Depending on the treatment schedule and the total dosage of drugs, particularly the topo II inhibitors, between 1 and 15% of cancer patients develop therapy related leukemia (t-AML) or therapy related myelodysplastic syndrome (t-MDS) and rarely therapy related acute lymphocytic leukemia (t-ALL) many of which involve the MLL gene (19–22). Thus, topo II inhibitor compounds have been implicated in particular MLL gene translocations including the t(9;11) in t-AML and in infant de novo AML and also the t(4;11) in de novo ALL and t-ALL (13,18–25).
AF9 is the partner gene of MLL involved in 9;11 translocations (also called LTG9 or MLLT3), which maps to 9p22 (26–28). The AF9 protein is highly homologous (56% amino acid identity) to the gene product of ENL located on 19p13.3, which is another common partner gene with MLL in translocations involving ALL and rarely, AML (4,29). At present, the function of most of these specific partner genes is unknown. Multiple in vitro and in vivo systems have demonstrated that the MLL–AF9 fusion gene plays a critical role in stem cell development and leukemogenesis (30–32). For example, chimeric mice with a knock-in Mll–AF9 fusion gene developed AML or, rarely, ALL, similar to patients with the t(9;11), indicating that the Mll–AF9 fusion in mice leads to aberrant hematopoietic cell proliferation (30,31). Additional in vitro studies using MLL–AF9-transformed myeloid progenitor cells demonstrated an inhibition of specific Hox genes, which led to the hypothesis that the MLL–AF9 fusion protein could inhibit hematopoietic cell differentiation and lead to leukemogenesis (32). Therefore, the MLL–AF9 fusion gene formed after gene breakage and rearrangement of these partner genes must provide the cell with a proliferative advantage which then leads to leukemia.
Although the MLL gene spans ~120 kb (12,18,33), virtually all MLL patient translocation breakpoints occur in an 8.3 kb BamHI fragment, the breakpoint cluster region (BCR) (6,7). The critical question therefore is, why does MLL breakage occur in such a restricted region? In previous studies, we and others identified structural features in the MLL BCR that may play a role in determining the location of DNA breaks and therefore of translocation breakpoints (18,34–36). For example, in the MLL BCR, an in vivo topo II cleavage site and a DNase I hypersensitive (DNase 1 HS) site co-localize near or within exon 9 between nucleotides 6800 and 7000, within a high affinity scaffold associated region (SAR) (34–36). We and others have observed that the majority of de novo leukemia breakpoints map to the 5' half of the 8.3 kb MLL BCR, which contains a series of Alu repeat sequences. In contrast, therapy related and de novo infant leukemia t(4;11) DNA breakpoints occur more frequently in the 3' half of the MLL BCR, within the strong SAR (34,37), supporting the proposal that a similar recombination mechanism is involved in both. In contrast, MLL self-fusion events of de novo leukemia patients often involve Alu–Alu mediated homologous recombination (10–12,34).
SARs (also called MARs) are AT-rich DNA regions of variable size, which define the chromosomal bases of interphase and metaphase chromatin loops in the DNA scaffold–loop model of chromosomes (38,39). SARs are found at specific regions of the genome, for example, in the non-transcribed flanking regions or within transcribed intronic regions of genes where many associate with regulatory elements (34,40–45), at telomeric repeats and within centromeric alpha satellite DNA (46–48). It has been proposed that SARs, because of their DNA unwinding properties, facilitate the entry of transcription, replication or chromosome condensation protein factors to target sequences (38,40,49). SARs also play a key role as cis elements of chromosome dynamics and as initiation sites for chromosome condensation (50). In addition, either the mouse immunoglobulin kappa (Ig
) light chain or the IgM (Igµ) heavy chain gene enhancers along with their associated SARs have been implicated in immunoglobulin gene hypermutation (51), induction of extended DNA accessibility for transcription (52) and also with de-methylation, to generate an extended domain of accessible chromatin (53).
Scaffold proteins such as DNA topo II and scaffold protein II (ScII) bind preferentially to the metaphase scaffold as well as being essential for chromosome condensation (39,54). In addition, topo II is essential in transcription and replication events (55). Multiple in vitro and few in vivo topo II cleavage sites have been characterized, most of which are located within SARs (56–60). Topo II has also been implicated in illegitimate recombination events, for example, at the mouse Ig gene intronic SAR, and also at the MLL BCR SAR (34,57).
DNase I HS sites represent open and accessible regions of DNA as defined by their susceptibility to DNase I enzyme cleavage. Some DNase I HS sites represent nucleosomal DNA where the conformation is changed due to the binding of specific proteins to target DNA sequences, whereas others represent open DNA regions due to intrinsic DNA structural features. Thus, many DNase I HS sites are associated with transcriptional regulatory DNA elements at gene boundaries near or within genes; some of these sites also co-localize with SARs and/or topo II sites (34–36,40,43,57,58,61,62).
In this study, we investigated whether the various structural elements, topo II induced DNA cleavage sites, DNase I HS sites and SARs, previously identified in the MLL BCR, were also a common feature in the AF9 gene and whether they were associated with the AF9 BCRs seen in patients.
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An in vivo topo II cleavage site and a DNase I HS site co-localize within AF9 intron 7 in the AF9 BCR2
Figure 1A represents a map of the AF9 gene encompassing >100 kb including the location of 10 exons and nine introns (Table 1). Genomic sequence information for the region including exon 3 is not yet available. Using BV173 cells, we analyzed a 70 kb region spanning exons 4 –10 for both in vivo topo II DNA cleavage sites after treatment of cells with VP16, Doxorubicin (Dox) or for DNase I HS sites after treatment of isolated cell nuclei with DNase I. The 70 kb genomic region encompasses all of the published AML and ALL patient breakpoints identified using RT–PCR and/or genomic sequencing (Fig. 1A and B) (25–28,63,64). Hybridization of probe 1 to DNA isolated from VP16, Dox and DNase I treated cells and nuclei, respectively, and then restricted with BamHI, revealed a new 3.0 kb DNA fragment, in addition to the germline 12.7 kb BamHI fragment [Figs 1 and 2 (data not shown)]. We also observed three additional lower molecular weight bands that were weaker topo II-induced DNA cleavage fragments (2.9, 2.6 and 2.3 kb) in VP16 treated cells. These topo II cleavage sites were observed using VP16 concentrations in a range of 5–100 µM as well as with 1–5 µM Dox. However, these 2.3, 2.6 and 2.9 kb DNA cleavage fragments were not detected in DNase I treated nuclei (Fig. 2), and not with the in-nuclei topo II cleavage assay (Fig. 4).
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Two different in vivo drug reversibility experiments were performed to investigate drug–topo II interactions (65,66) and to assess whether the AF9 BCR cleavage could be reversed (Fig. 3). BV173 cells were incubated with 25 and 50 µM VP16 for 6 h, then the cells were washed and immediately incubated for 15 min at 60°C in the absence of proteinase K and SDS. Alternatively, after the 6 h incubation the drugs were removed and the cells were incubated in drug-free medium for an additional 2 h. After DNA isolation and Southern blotting, no AF9 cleavage was detected using either method (Fig. 3), indicating the repair of VP16-induced DNA breakage. Topo II–DNA cleavable complexes have the unusual property of being unstable at high temperatures (60–65°C), thus causing rapid reversal of the cleavage reaction (65).
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Different cell lines were used in this study to confirm both the in vivo topo II and DNase I DNA cleavage sites (Table 2). All cell lines demonstrated the same strong AF9 DNase I HS site. As expected, the VP16 resistant cell line K562 (18,35,36,67) showed no in vivo topo II cleavage, but did show the DNase I cleavage site. In addition, the MM6 t(9;11) cell line demonstrated the same in vivo topo II and in vitro DNase I cleavage sites as seen in the BV173 cells, including the three weaker topo II sites.
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In summary, a single co-localizing in vivo topo II and in vitro DNase I cleavage site and three weaker in vivo topo II DNA cleavage sites are all located between AF9 exons 7 and 8 in the AF9 BCR2. No additional in vivo topo II or DNase I cleavage sites were present in the remaining 70 kb of AF9 analyzed.
Purified topo II induces specific in vitro DNA cleavage at the AF9 in vivo topo II site in intron 7
We confirmed the presence and location of the AF9 in vivo VP16-induced topo II cleavage site using an in-nuclei topo II DNA cleavage assay. Incubating isolated BV173 nuclei with purified topo II enzyme in the presence of VP16 for 5 min at 30°C, we observed a 3.0 kb cleavage fragment similar to our in vivo topo II studies (Fig. 4). However, using these in vitro conditions we did not observe the three weaker topo II cleavage sites that were seen in the in vivo assay. Thus, these results confirm that topo II causes DNA cleavage within the same AF9 gene location as identified in vivo using topo II inhibitors.
Two high affinity SARs border both AF9 BCRs
In addition to topo II and DNase I cleavage sites, we analyzed 61 kb of the AF9 gene for scaffold protein bound DNA fragments, another structural element, also present in the MLL BCR. We identified two high affinity SARs within the analyzed AF9 region, a 6.2 kb SAR in intron 4 (SAR1) and a 4.6 kb SAR encompassing parts of introns 5–7 (SAR2). Probes 2 and 6 identified parts of the high affinity scaffold bound fragments of SAR2 (3.6 kb EcoRI), and SAR1 (2.0 kb EcoRI), respectively (Figs 1B and 5). The remaining 15 012 bp between SAR1 and SAR2 and the DNA region centromeric of SAR1 ending ~3.0 kb 3' of exon 4 all showed non-scaffold binding; for example, probe 4 identified a non-scaffold DNA fragment (4.0 kb HindIII) centromeric to SAR2 [Figs 1A and B and 5 (data not shown)]. In addition, we analyzed the DNA sequence of the AF9 SARs and the MLL telomeric BCR SAR for similarities. Interestingly, the only sequence similarities found were AT-homopolymers (A/T tracts). AF9 SAR1 and SAR2 showed a total of 143 and 100 A/T tracts > 4 bp in length, respectively, and the MLL BCR telomeric SAR had 69 A/T tracts > 4 bp. Both the AF9 SAR1 and the MLL telomeric SAR also exhibited A/T tracts > 8 bp in length as well as single A/T tracts of 22 bp.
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Relationship of leukemia patient breakpoints to the AF9 chromatin structure
Analyzing nine known de novo AML, ALL and t-AML breakpoints from the literature in relation to the AF9 structural elements we observed the following. (i) Two cloned de novo patient breakpoints, one infant AML and one ALL breakpoint mapped <1 kb telomeric to the in vivo topo II and in vitro DNase I cleavage sites in BCR2 and 4.5 kb telomeric from SAR2 (Fig. 1B) (27,63). In addition, three ALL breakpoints also mapped in BCR2 as determined by RT–PCR analysis (26,28,64). (ii) Four t-AML and two de novo AML patient breakpoints in BCR1 are bordered by SAR1 and SAR2 (Fig. 1B) (24,25).
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DISCUSSION |
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Based on the results described in this report, we show that the breakpoint regions of AF9 share the same structural elements found in the MLL BCR (Fig. 1B and C). Two different elements map within the AF9 BCR2 in intron 7, namely: one in vivo and in vitro topo II cleavage site and one in vitro DNase I HS cleavage site that co-localize, plus three additional weaker in vivo topo II sites. In addition, two SAR elements border both sides of BCR1 and one side of BCR2; SAR1 is located in intron 4 and SAR2 encompasses parts of exons 5–7. Our data therefore suggest that the similarities in structure at the breakpoints of these two translocation partner genes result in illegitimate recombination events. This then leads to translocations and ultimately, because of the selective advantage of the fusion gene, to leukemia.
The in vivo topo II and in vitro DNase I HS sites are the only cleavage sites in 70 kb of AF9 and in the entire MLL gene (~120 kb) (Figs 1 and 2 and unpublished results) (18,35). The DNase I HS sites for both genes are constitutively accessible in various cell types, for example, hematopoietic cell lines representing progenitor T/B cells, myeloid and monocytic cells and also normal lung fibroblasts (Table 2) (35). The AF9 in vivo topo II cleavage sites were detected in progenitor T/B, and monocytic cells (Table 2). The MLL in vivo topo II cleavage site has also been demonstrated in multiple cell lines, including the T/B progenitor cell line, BV173 (18,35,36).
In contrast to MLL, which exhibits only one in vivo topo II cleavage site, multiple topo II cleavage sites, as shown for the AF9 BCR, have also been detected in other genes, especially in or adjacent to SARs (40,57,58). AF9 showed three additional weaker in vivo topo II cleavage sites just centromeric to the single strong topo II site (Figs 2 and 3). These cleavage sites were not detected using the in-nuclei topo II DNA cleavage assay or by DNase I treatment of nuclei (Fig. 4). The differences between the topo II and the DNase I HS cleavage sites may be due to the in vivo (cells) versus in vitro (nuclei) methods used. Similar differences have been demonstrated for in vivo versus in-nuclei topo II cleavage assays in Drosophila (67).
Using either low concentrations of VP16 (5–50 µM) or brief treatment of nuclei isolated from logarithmically grown cells with purified topo II in the presence of VP16, or with DNase I, we identified specific AF9 and MLL DNA cleavages using non-apoptotic conditions (18). In addition, both the AF9 and MLL BCR topo II cleavage sites were reversible due to religation (Fig. 3). Moreover, we found that no AF9 or MLL cleavage occurred in VP16 resistant K562 cells (18,35,36,68). Therefore, we conclude that topo II is responsible for cleaving specifically at the open and accessible region of DNA in AF9 as well as in MLL and that these DNA cleavages are not due to the apoptotic nucleases in cells committed to apoptosis (18,69).
Relating AF9 patient DNA breakpoints to the AF9 structural elements, we found that some breakpoints map <1 kb telomeric to the topo II and DNase I cleavage sites in BCR2, whereas others map several kb centromeric in BCR1 (Fig. 1B). These breakpoint regions are bordered by two SARs, which are separated by 15 kb; SAR2 maps 3.5 kb centromeric to the topo II and DNase I cleavage sites in BCR2 and SAR1 is positioned at the centromeric end of BCR1. In contrast to the AF9 SARs, the MLL high affinity telomeric SAR is located in the same region as the in vivo topo II and in vitro DNase I cleavage sites, in the region of many t-AML and infant leukemia breakpoints (Fig. 1C) (7,34,35,37). However, as in the AF9 gene, the remaining de novo AML and ALL patient DNA breakpoints of MLL are clustered in the centromeric portion of the BCR between the two SARs (Fig. 1B and C). For the AF9 gene, more genomic breakpoints need to be mapped in order to determine their relationship to these structural elements in the AF9 BCRs.
Previous studies have shown that non-homologous chromosomal recombination was mediated especially by SARs and topo II. SARs were found to be hotspots of topo II binding and cleavage (38,57,58). SARs have been further implicated in illegitimate recombination events including translocations in plasmacytomas involving the mouse Ig-chain gene SAR, in deletions of the rabbit Ig2 chain gene, and the human interferon (IFN) gene complex (44,57). Interestingly, in the latter example, SAR/SAR or SAR/LINE elements were identified at two glioma deletion breakpoint junctions. In addition, it was proposed that the IFN SAR A/T tracts could mediate recombination leading to deletions in the short arm of human chromosome 9 (44). Both the AF9 and MLL SARs are similar in size; 6.2 kb and 4.6 kb for the AF9 SAR1 and SAR2, respectively, and 4.5 kb and 5.5 kb for the centromeric and telomeric MLL SARs. In addition, particularly for AF9 SAR2 and the MLL BCR telomeric SAR, 100 and 62 A/T tracts were identified, respectively. Both of these SARs also share a 22 bp A/T tract, which could mediate DNA alignment together with other A/T tracts. In addition to A/T tracts DNA unwinding elements or nucleation sites with the ATATAT motif have the capability of stably unwinding DNA (70,71). We detected five ATATAT motifs in each AF9 SAR1, SAR2 and four in the MLL SAR. We propose that together with A/T tracts, the DNA unwinding elements help facilitate SAR–SAR alignment through DNA hybridization.
Topo II has been involved in mediating illegitimate recombination in vitro and in vivo. Using phage in vitro systems, purified calf thymus or Drosophila topo II have the intrinsic ability to mediate illegitimate recombination through so called topo II subunit exchange (72–74). An interchromosomal reciprocal exchange has also been demonstrated for the eukaryotic aprt gene by topo II inhibitors in vivo (75). In contrast to a reciprocal chromosomal exchange, which occurs specifically at topo II cleavage sites, we found that many leukemia patient breakpoints involving MLL and AF9 were located 100 to several 1000 bp away from the in vivo topo II DNA cleavage site (Fig. 1B). We therefore propose for MLL–AF9 (as well as for other partner genes of MLL) a refined non-homologous chromosome translocation (NHCT)-model, in which an initial topo II cleavage occurs with subsequent DNA repair. This process is mediated by the alignment of the MLL and AF9 SARs due to similar A/T tract distribution along with scaffold protein interaction. This recombination mechanism is independent of Alu–Alu homologous recombination, which has been described for some leukemia patient breakpoints, particularly MLL partial duplications (10–12). In our model, the first NHCT event is initiation of DNA damage and breakage in the accessible DNA region at the MLL and AF9 in vivo topo II DNA cleavage sites. Following DNA cleavage, a complex repair mechanism occurs involving exonucleolytic activity extending away from the initial DNA cleavage sites. At the same time, both homologues may be used to repair gene sequences up to the location of the recombination junction. After DNA strand invasion occurs and the recombination junction is ligated, the MLL and AF9 genes become fused. It is also possible that during NHCT, topo II associates with the DNA repair protein complex as it moves along the DNA. In this circumstance a subunit exchange of topo II dimers between MLL and AF9 is possible, which could lead to chromosome translocation or gene fusion. Interestingly, it has been demonstrated that the structure specific endonuclease co-localizes with topo II and the DNA repair enzyme DNA polymerase ß (76).
Our proposed NHCR-model could explain: (i) Why many non-Alu-Alu patient DNA breakpoints are located one hundred to several thousand base pairs away from the MLL and AF9 in vivo topo II cleavage sites; (ii) Why topo II and recombination consensus sites are not found at many published patient DNA breakpoint junctions in AF9, MLL and also for example, AF4 in t(4;11) (77); (iii) Why large (~25%) and small deletions, duplications and inversions were found at MLL, AF9 and more recently at AF4 patient DNA breakpoint junctions (7,64,77). In conclusion, it will be important to examine other MLL partner and also non-partner genes to determine whether similar structural elements are present in order to further unravel the molecular events involving NHCR resulting in MLL gene translocations leading to acute leukemia.
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MATERIALS AND METHODS |
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Cell lines
All suspension and attached cell lines were maintained in RPMI 1640, or DMEM, with fetal calf serum 10% (Life Technologies, Rockville, MD). Specific cell lines studied were the chronic myelogenous leukemia (CML) BV173 (35) (undifferentiated stem cells) and K562 (erythroleukemia) cells both carrying the t(9;22)(q34;q11), and the AML Mono Mac 6 cell line carrying a t(9;11)(p22;q23) with an MLL–AF9 gene fusion (35). Additional cell lines studied were: Jurkat (T-cell leukemia), and a normal lung fibroblast cell line 22133 (ATCC).
In vivo topo II cleavage of DNA
The in vivo topo II DNA cleavage assay was according to Strissel et al. (35). Briefly, cells were grown exponentially, and then treated for 6 or 16 h with the non-intercalating topo II inhibitor etoposide VP16 in a range of concentrations from 5 to 100 µM (Sigma), or with Dox in a range of 1–5 µM to produce endogenous topo II-cleaved complexes. Cells were then lysed and the DNA was isolated using a previously described method to trap cleaved topo II DNA (35).
Topo II induced DNA cleavage reversibility reaction
BV173 cells were incubated with 25 µM and 50 µM VP16 for 6 h and the topo II induced DNA cleavage was reversed by two different methods. After drug treatment the cells were either centrifuged and then incubated with 100 mM KCl, 40 mM Tris pH 7.5, 0.5 mM EDTA pH 8.0 and 3.0 mM MgCl2 for 15 min at 60°C (65), or washed and incubated with new media for an additional 2 h (66). The reactions were then stopped with 1% SDS and 300 µg/ml proteinase K and genomic DNA was purified and then analyzed.
Isolation of nuclei used for cleavage in nuclei for topo II, in vitro for DNase I and for SAR assays
For each hematopoetic cell line, nuclei were isolated according to Mirkovitch et al. (41), as modified by Strissel et al. (34,46). Nuclei were frozen in a solution containing 50% glycerol (34,46) at –20°C for up to 4 months or at –70°C for up to 8 months. The in-nuclei topoisomerase II cleavage assay was according to Kas and Laemmli (58) with modifications. Briefly, 1.5 A260 of isolated BV173 whole nuclei were incubated in a buffer containing 20 mM KCl, 70 mM NaCl, 10 mM MgCl2, 2.5 mM CaCl2, 0.05 mM spermine, 0.125 mM spermidine, 0.5 mM DTT, 0.2 mM PMSF, 0.1% digitonin and 100 U/ml aprotinin for 10 min at 30°C. Four units of purified human topo II (TopoGen, Columbus, OH) were added and then were incubated for 15 min at 30°C. Finally, 1.5 mM ATP and 50 µM VP16 (Sigma) were incubated with the nuclei for an additional 5 min at 30°C. The reaction was stopped with 1% SDS and 15 mM EDTA then 400 µg/ml proteinase K was added for 1 h at 37°C. For DNase I treatment of nuclei, we used the methods of Kas and Laemmli (58) as modified by Strissel et al. (35). Methods according to Mirkovitch et al. (41) and Strissel et al. (34,46) were used for isolation of SAR and non-SAR fractions. BV173 whole nuclei were treated with 2,5-di-iodosalicylate to obtain isolated scaffolds, which were then incubated for 3–5 h at 37°C with 1000 U/ml restriction enzymes (Roche, Indianapolis, IN).
DNA purification, Southern blot and DNA probe hybridizations for mapping of structural elements
For all assays described above, the DNA was extracted and purified with phenol, phenol/chloroform, then precipitated with isopropanol in the presence of 0.7 M ammonium acetate, and then solubilized. For in vivo topo II cleavage and in vitro DNase I samples, the DNA was digested with 5 U of restriction enzymes (Roche). Approximately 15 µg of restricted DNA (determined by optical density readings) was electrophoresed on 0.8% agarose gels. For SAR assays, equal amounts (µg) of SAR and non-SAR DNA per restricted sample were electrophoresed. Using standard conditions for Southern blotting, without acid depurination, the DNA was transferred by electroblotting in 12 mM Tris, 6 mM Na acetate, and 0.3 mM EDTA (pH 7.5) buffer to GeneScreen Plus (NEN, Boston, MA) positively charged nylon membranes. Hybridization of DNA probes to Southern blots was performed according to standard protocols, with 50% formamide at 42°C, and washes at room temperature (1x SSC, 1% SDS), then at 60°C for 1 h (0.5 x SSC, 1% SDS).
DNA clones and probe isolation
Figure 1A and B show a map of the AF9 gene and the location of all the AF9 probes used for this study. The AF9 cosmid clones, 34a5 (accession number AC002052), 232g8 (AC002469), 92f5 (AC002053), C48 (AC000007) and 213a7 (AC002050) were sequenced according to Bodenteich et al. (78) (Table 1). PCR amplified products from cosmid DNA were purified from agarose gels after gel electrophoresis and were radiolabeled for use as probes. Primers top forward (TF) and bottom reverse (BR) chosen for AF9 PCR amplification corresponded to non-repetitive regions and are listed in the centromeric to telomeric orientation according to their cosmid position. C48 probe 1 32547–33122, C48 probe 2 29194–29767, C48 probe 3 21262–21782, C48 probe 4 23389–22579, C48 probe 5 14509–15197, C48 probe 6 6413–6933, C48 probe 7 3353–3903 92f5 probe 8 25177–25945, 92f5 probe 9 20590–21451, 92f5 probe 10 14431–15183 and 92f5 probe 11 8497–9380. All of the probes were hybridized to SAR and non-SAR fractions. For analysis of topo II/DNase I HS cleavage products, probes 1, 2, 5, 6, 7, 8 and 9 were used primarily for indirect end labeling analysis (35). DNA alignments and analysis between the AF9 and MLL SARs were performed using MacVector 6.5.
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ACKNOWLEDGEMENTS |
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We would like to thank Susanne Borgers for expert technical assistance. This work was supported in part by the G. Harold and Leila Mathers Charitable Foundation (P.L.S., R.S.) and by the National Cancer Institute, CA-42557 (J.D.R.) and CA-40046 (J.D.R., N.Z.L.).
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FOOTNOTES |
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+ These authors contributed equally to this work
§ To whom correspondence should be addressed. Tel: +1 773 834 1539; Fax: +1 773 702 3163; Email: pstrisse@medicine.bsd.uchicago.edu
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