Human Molecular Genetics, 2001, Vol. 10, No. 22 2481-2491
© 2001 Oxford University Press
Mechanisms of MLL gene rearrangement: site-specific DNA cleavage within the breakpoint cluster region is independent of chromosomal context
1Genetics Branch, Center for Cancer Research, National Cancer Institute, Gaithersburg, MD, USA, 2Department of Cancer Genetics and 3Department of Clinical Cytogenetics, Roswell Park Cancer Institute, USA and 4Department of Pediatric Hematology and Oncology, Hannover Medical School, Hannover, Germany
Received May 25, 2001; Revised and Accepted August 22, 2001.
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
The MLL gene at chromosome band 11q23 is specifically cleaved at a unique site within its breakpoint cluster region (bcr) during the higher order chromatin fragmentation associated with apoptosis. We now show that the same specific DNA cleavage event can be detected in an exogenous MLL bcr fragment that is integrated into the genome outside of its normal chromosomal context, as well as in an extrachromosomal episome containing an MLL bcr fragment. We also show that episomal or randomly integrated copies of the MLL bcr behave similar to the endogenous MLL bcr when tested in a scaffold-associated region (SAR) assay. Furthermore, an episomal murine MLL bcr introduced into human cells is cleaved at the same site as the endogenous murine MLL bcr; this episomal murine MLL bcr also functions as a SAR in human cells. We conclude that both nuclear DNA scaffold attachment as well as site-specific DNA cleavage can be directed by sequences contained within the MLL bcr, and that it is feasible to study these events using episomal shuttle vectors.
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
One of the most frequently observed genetic alterations in patients with acute leukemia is chromosomal translocation involving the MLL gene (also called ALL-1, HRX or Htrx) (1–4). The MLL gene is homologous to the Drosophila trithorax gene, which is involved in pattern development during embryogenesis (1–4). Interestingly, the translocation breakpoints within the MLL gene almost always fall within a limited region of 8.3 kb, referred to as the MLL breakpoint cluster region (bcr) (5). MLL translocations result in the generation of fusion proteins that retain the MLL N-terminus, including both an A-T hook domain and a region similar to mammalian DNA methyltransferase (6).
The precise molecular mechanisms that cause MLL rearrangements remain largely unknown. Homologous recombination between Alu repeat sequences (7) and inappropriate V(D)J recombinase activity (8) have been proposed as potential causes of MLL gene rearrangements. More recently, extensive analysis of t(4;11) breakpoints involving MLL has demonstrated nucleotide sequence features such as small regions of deletion, duplication and inversion that strongly implicate non-homologous end joining (NHEJ) of broken DNA (9,10) in the generation of many of these translocations. Additionally, of particular clinical importance, treatment of a primary malignancy with topoisomerase II (topo II) poisons, such as doxorubicin or etoposide (VP-16), is associated with development of secondary, or therapy-related acute myelogenous leukemias (t-AML) that often display MLL gene rearrangements (11,12). Furthermore, a majority of infants with acute leukemia have MLL gene rearrangements; it has been speculated that infant leukemias may be due to in utero exposure to genotoxic agents (13). These clinical findings, along with the observation that treatment of peripheral blood lymphocytes with etoposide in vitro can induce chromosomal rearrangements (14), strongly suggest that treatment of mammalian cells with topo II poisons can cause MLL gene rearrangements. We have previously described a specific site within the MLL bcr that is sensitive to DNA double strand cleavage induced by topo II poisons, and proposed that this site-specific cleavage may be an initiating event for MLL chromosomal translocations caused by treatment with topo II poisons (15,16). While this site has not yet been precisely mapped, it can be localized to a region of 
75 bp within MLL exon 9 (17) [or exon 12 using the more recent numbering proposed by Nilson et al. (18)]. Interestingly, a similar cleavage site has recently been identified within the AF9 gene, which is a frequent translocation partner of MLL (19). Subsequent experiments have shown that site-specific cleavage within the MLL bcr could also be induced by apoptosis (16) or DNAse I treatment (20), suggesting that this cleavage site might represent a genomic region which is uniquely susceptible to DNA double-strand breaks.
AT-rich DNA sequences that are preferentially associated with nuclear scaffold proteins are referred to as scaffold-associated regions or SARs (21). SARs [also called matrix attachment/associated regions (MARs)] are thought to represent the bases, or attachment sites, of chromosomal loops (reviewed in 22). MLL breakpoints in patients with t-AML have been shown to cluster near a high affinity SAR (23) that encompasses the MLL cleavage site described above (15,16,20). In addition, topo II is one of the major protein components of the nuclear scaffold and can be detected at chromosomal DNA SARs (24–26). Taken together, these findings suggest a potential relationship between SARs and preferred topo II binding sequences in the production of illegitimate recombination events, such as chromosomal translocations, within the MLL bcr. Supporting such a hypothesis, a SAR in the murine immunoglobulin 
locus is preferentially cleaved by topo II and this cleavage occurs in close proximity (14 bp) to a translocation breakpoint (27).
To better understand the specific DNA double-strand cleavage event within the MLL gene, and the relationship of this cleavage to chromosomal context, we introduced DNA fragments containing portions of the human and murine MLL bcr into human cell lines. We then evaluated the ability of the exogenous MLL bcr sequences (either stably integrated into the genome or stably maintained within the cells as an episome) to serve as SARs and preferential cleavage sites.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
Site-specific DNA cleavage within a stably integrated MLL bcr fragment
To determine if site-specific DNA cleavage could be induced within an MLL bcr that was introduced into the genome outside of its normal chromosomal context, an 8.3 kb genomic fragment encompassing the MLL bcr (5) was cloned into the pRC-CMV vector, linearized, and transfected into the Jurkat cell line. Pure clones were selected by limiting dilution in the presence of 800 µg/ml G418; those that had integrated one or two copies of MLL were chosen for further analysis (Fig. 1A). Etoposide treatment of these clones demonstrated specific cleavage of the transfected copy of MLL using an indirect end-labeling assay (16). In this assay, genomic DNA is extracted from treated cells, digested with a restriction enzyme, size-fractionated, transferred to nitrocellulose membranes, and hybridized to a radiolabeled DNA probe. To detect specific MLL bcr cleavage, we used an MLL cDNA probe (0.2HB) containing exon 9, 10 and 11 sequences, as previously described (15,16). Figure 1B shows a novel 4.0 kb SstI restriction fragment is produced by etoposide treatment of parental Jurkat cells and a novel fragment of 1.5 kb is produced in the Jurkat clones. In each case, one end of the novel fragment is produced by restriction enzyme cleavage, whereas the other end of the fragment is produced by site-specific DNA double strand cleavage site following etoposide treatment. Since one end of the fragment is fixed by the restriction enzyme, the site of DNA cleavage induced by etoposide treatment can be mapped by comparison with germline or transfected fragments, respectively (15,16). In some cases (clones J9 and J18), the specific cleavage is obscured by non-specific cleavage; however, in the clones with higher copy number (J7 and J13) the specific cleavage is more obvious. The signal from the transfected copy of MLL is consistently 5-fold less intense than that from the endogenous MLL; this is not unexpected since FISH experiments showed that the parental Jurkat cells are aneuploid and contain five copies of MLL (data not shown).
|
Site-specific cleavage of episomal copies of the MLL bcr
To determine whether chromosomal integration was required to support MLL cleavage, an EBV-based episomal vector containing the MLL bcr and a selectable marker (hygromycin) was generated by cloning a genomic DNA fragment encompassing the MLL bcr into the EBV-based pMEP4 vector (28,29). This construct was introduced into Jurkat cells by electroporation. After selection with hygromycin, 10 independent clones carrying the episome were isolated; three clones (JC-1, JC-12 and JM-5) were chosen for further study. Southern blot analysis of undigested genomic DNA using a probe isolated from pMEP4 vector sequences (nucleotides 10 084–10 303) demonstrated a band at
9 kb (Fig. 2A), indicating that this vector had not become integrated into chromosomal DNA but was instead a type I closed extrachromosomal circle. In addition, transformation of Escherichia coli with genomic DNA isolated from the JC-1 clone yielded 37 ± 16 transformants per microgram of DNA, whereas genomic DNA isolated from Jurkat cells or Jurkat/5.5 cells (a Jurkat clone with a tandem array of 220 integrated copies of an AmpR MLL plasmid; P.D.Aplan and M.Stanulla, unpublished data) yielded zero transformants per microgram of genomic DNA (Table 1). Taken together, these results indicate that the pMEPMLL plasmid was maintained as an episome in the JC-1 clone. Analysis of HindIII digested DNA from four separate indirect end-labeling experiments (Figs 2 and 3 and data not shown) demonstrated that the intensity of the episomal MLL signal was 12.6–15.1 (mean 14.2) times as intense as that from the germline MLL. Since Jurkat cells have five copies of MLL, we estimate that the JC-1 clone has approximately 70 copies of the pMEPMLL episome per cell.
|
|
|
To determine whether site-specific cleavage within the MLL bcr can also be induced within this MLL episome, we treated the JC-1 clone with etoposide. Genomic DNA was harvested, digested with BamHI or HindIII, and assayed for site-specific MLL bcr cleavage using indirect end-labeling. Figure 2C shows novel fragments induced by etoposide in both the Jurkat and JC-1 lanes. Although one cannot discriminate between cleavage of the endogenous MLL bcr and the episomal MLL bcr with a BamHI digest, since the fragments are of identical size, HindIII-digested DNA from etoposide treated JC-1 cells produced discrete fragments of 2.2 or 1.5 kb, derived from the endogenous or episomal MLL, respectively. Figure 3 shows an identical pattern of site-specific cleavage within the episomal MLL bcr in three independent clones (JC-1, JC-12 and JM-5), demonstrating that the result seen with the JC-1 clone was not unique to this particular clone. From these experiments, we conclude that site-specific cleavage of the MLL bcr can be generated within MLL bcr sequences placed on an episome, demonstrating that site-specific MLL bcr cleavage is not restricted to chromosomal DNA.
The JC-1 clone was treated with either etoposide or a non-genotoxic apoptotic stimuli (C2 ceramide) (30,31) to determine if site-specific cleavage of the episomal MLL was part of a generalized apoptotic pathway. Genomic DNA was harvested and assayed for site-specific MLL bcr cleavage; apoptotic cell death was verified by detection of oligonucleosomal ladders and nuclear fragmentation (Fig. 3). Specific DNA cleavage within the episomal MLL bcr induced by non-genotoxic stimuli of apoptotic cell death was detected by indirect end-labeling (Fig. 3). To determine whether site-specific cleavage of MLL episomes was unique to Jurkat cells, we introduced the pMEPMLL vector into CEM cells, and selected stable transfectants as described above for Jurkat cells. Etoposide-induced cleavage of the episomal MLL resident in CEM cells was comparable to that seen in Jurkat cells (Fig. 4 and data not shown).
|
To determine if the entire 8.3 kb MLL bcr was required to support site-specific MLL cleavage, we cloned a 540 bp fragment encompassing 200+ nucleotides on either side of the MLL cleavage site into the pMEP4 vector. This plasmid (p328; Fig. 4) was introduced into CEM cells and stable transfectants were isolated and treated with etoposide. Figure 4 demonstrates that this vector was cleaved at the same site within MLL exon 9 as was the endogenous MLL bcr, although the cleavage was less dramatic than that seen with episomes containing the 8.3 kb MLL bcr fragment.
We used exonuclease III to generate deletion mutants (plasmids p510 and p512, containing 2984 and 2360 nucleotides of the MLL bcr sequence, respectively; Fig. 4B) that had smaller segments of the 8.3 kb MLL bcr contained within the pMEP4 vector. The plasmids were transfected into CEM cells; stable clones were isolated and treated with etoposide to induce MLL cleavage. Cleavage of the endogenous MLL bcr was comparable among the four clones and the parental CEM cell line (16–43% using 30 µM etoposide). Cleavage of the episomal MLL bcr also was comparable for the clones containing the p327, p510 and p512 episomes (14–19% using 30 µM etoposide). However, although the C328A clone, containing only 540 bp of the MLL bcr sequence supported site-specific MLL cleavage, the specific cleavage (1%) was not as extensive as that seen using episomes with the larger MLL fragments. We conclude that episomes containing 2.4 or 3.0 kb of the MLL bcr sequence are almost as effective as those containing 8.3 kb of the MLL bcr sequence in directing site-specific MLL cleavage, and clearly more effective than those containing a minimal portion of the MLL bcr (clone C328A).
Both germline and exogenous MLL sequences function as SARs in a lithium diiodosalicylate (LIS) assay
The site-specific MLL bcr cleavage maps to the center of a high affinity SAR (23). This co-localization is consistent with a model in which higher order chromatin fragmentation during the early stages of apoptosis occurs as a specific event at SARs, where DNA loops are anchored to the nuclear scaffold (32). To determine if the scaffold association of the transfected episomal MLL bcr was similar to that of the germline MLL bcr, we conducted SAR assays. Nuclear scaffolds were prepared by LIS extraction (21) of nuclei from both parental Jurkat cells and the JC-1 cells, which contained an episomal MLL bcr. Scaffolds were then digested with BamHI, and equal amounts of DNA from pellet (scaffold-associated) and supernatant (non-scaffold-associated) fractions were analyzed by Southern blot hybridization to the 0.7 kb MLL cDNA probe that encompasses the entire MLL bcr (Fig. 5). Similar to previously reported results (23), the 8.3 kb BamHI fragment encompassing the MLL bcr was enriched in the pellet fraction from Jurkat cells (Fig. 5A). An identical pattern of enrichment is seen in the JC-1 cells. Fragments derived from the episomal MLL co-migrate and cannot be distinguished from those derived from the endogenous MLL with absolute certainty. However, since the JC-1 clone contains approximately 14 times as many copies of the episomal MLL as the endogenous MLL (note the much more intense signal from JC-1 lanes than Jurkat lanes in Fig. 5A, also see Figs 2 and 3), it seems reasonable to assume that 13/14 or >90% of the signal on the autoradiograph is derived from the episomal MLL. Thus, it seems highly likely that both the endogenous and the episomal copies of the MLL bcr were associated with the nuclear scaffold.
|
We used a BamHI/EcoRI double digest to refine the mapping of the MLL SAR (Fig. 5). Three fragments (4.6, 2.7 and 1.0 kb) were generated. The 2.7 and 4.6 kb fragments were enriched in the pellet fraction, suggesting preferential binding of these fragments to nuclear scaffold proteins. In contrast, the 1.0 kb fragment, representing the most centromeric portion of the MLL bcr, was equally distributed between the pellet and supernatant fractions. These results are again similar to previously reported experiments (23) that mapped a ‘high affinity’ SAR to a region including the 4.6 and 2.7 kb fragments, and a weak or ‘low affinity’ SAR to a region encompassing the 1.0 kb fragment. Similar to Figure 5A, the hybridization pattern for JC-1 cells is identical and more intense than that of the parental Jurkat cells, indicating that episomal copies of MLL behaved similar to the chromosomal MLL. To exclude the possibility that larger DNA fragments were preferentially precipitated in the pellet fraction based solely on fragment size, and not scaffold binding, we rehybridized the blots to either an MLL exon 34 probe, which lies outside of the previously mapped SAR (23), or a hygromycin probe. A 4 kb exon 34 fragment (larger than the 2.7 kb fragment from the MLL bcr) was equally distributed in the pellet and supernatant fractions, and a 1.5 kb fragment from the episomal plasmid backbone (close in size to the 2.7 kb MLL bcr fragment) was preferentially located in the supernatant fraction. To determine whether MLL bcr sequences randomly integrated into the genome would likewise function as SARs, we assayed clones with a single copy of the MLL bcr. Figure 6 shows that a transfected MLL bcr behaves similar to the chromosomal MLL in this LIS-based SAR assay. In sum, these results are consistent with the previously reported high-affinity SAR in the telomeric portion of the MLL bcr (23), and demonstrate that the association of transfected copies of the MLL bcr with the nuclear scaffold is similar to that of the endogenous MLL bcr.
|
The murine MLL bcr also contains a specific cleavage site and functions as a SAR
Chromosomal loop anchorage sites appear to be evolutionarily conserved (33,34). For instance, Drosophila histone gene SARs compete with murine immunoglobulin
SARs for binding of nuclear scaffolds derived from murine MPC-11 plasmacytoma cells (34). In addition, site-specific cleavage within the MLL bcr induced by apoptosis is conserved between species, as it can be observed within the murine MLL bcr (16). Since apoptotic site-specific DNA cleavage within the human MLL bcr can be recapitulated with episomal vectors, we wished to determine if an episome containing the murine MLL bcr could be cleaved in human cells, and if it would also function as a SAR. An episomal vector (Fig. 7) containing a 4 kb HindIII fragment from the murine MLL bcr was transfected into Jurkat cells by electroporation and clones selected with hygromycin. Site-specific cleavage of an episomal murine MLL introduced into human cells was detected by indirect end-labeling (Fig. 7); this cleavage site is identical to that seen in the endogenous murine MLL bcr (16).
|
We also analyzed the behavior of the episomal murine MLL fragment using the LIS-based SAR assay described above. In this experiment, an endogenous 15 kb HindIII fragment encompassing the MLL bcr was enriched in the pellet fraction (Fig. 8); the 4.0 kb fragment representing the episomal MLL bcr was also enriched in the pellet fraction. As a control, an MLL exon 34 probe was hybridized to a 7 kb fragment which was equally distributed between the pellet and supernatant fractions. We conclude that the murine MLL bcr, like the human MLL bcr, is preferentially associated with nuclear scaffold proteins.
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
Recurrent, non-random chromosomal translocations have been recognized in acute leukemia patients for several decades and are likely to be causal events leading to leukemic transformation (35,36). One of the genes most frequently translocated in a wide spectrum of acute leukemias is MLL. In contrast to most genes involved in chromosomal translocations, where a transforming gene has one or a few ‘partners’ (i.e. PML and RAR
; BCR and ABL), MLL has been referred to as a ‘promiscuous’ oncogene because of its involvement in translocations fusing at least 30 different genes with MLL sequences (6,37). Despite this wide range of translocation partners, MLL translocation breakpoints almost invariably lie within an 8.3 kb bcr. This observation suggests that this 8.3 kb region may be uniquely susceptible to chromosomal translocations, which, in most general terms, can be regarded as DNA cleavage and religation events; with the translocations being produced when the cleaved DNA becomes religated to a non-homologous chromosome. We and others (15,20) have recently identified a site within the MLL bcr that is uniquely susceptible to DNA double-strand cleavage, both in vitro (16) and in vivo (15,20,38), in response to topo II poisons, DNAse I or apoptosis. Furthermore, this site falls within a high affinity SAR (23). One hypothesis that accounts for the observed colocalization of site-specific cleavage of the MLL bcr at a SAR proposes that the SAR is a marker for ‘accessible’ regions of the genome, which are preferentially cleaved in response to a variety of stimuli, including DNAse I and apoptotic nucleases. In support of this model, it has been suggested that the 50–100 kb fragments released during the initial stages of apoptosis are due to DNA cleavage at SARs or the bases of chromosomal loops (32). We have now shown that cloned fragments of the MLL bcr, stably introduced into the nucleus, either integrated into chromosomal DNA or as an extrachromosomal episome, can still mediate site-specific DNA double-strand cleavage, and are preferentially attached to the nuclear scaffold.
Although we had predicted that an exogenous copy of the MLL bcr, integrated outside of its normal chromosomal context, would be able to mediate site-specific cleavage, we did not anticipate that an episomal copy of the MLL bcr would be able to support site-specific cleavage in an efficient fashion. However, an EBV-based episome containing MLL bcr sequences clearly directed site-specific cleavage within the MLL bcr in response to topo II poisons as well as any of the additional apoptotic stimuli used. The sensitivity of the episomal MLL bcr to cleavage was comparable to that of the endogenous MLL bcr (Figs 2–4), although most experiments showed that the episomal copy of the MLL bcr was somewhat less sensitive to site-specific cleavage (Fig. 4 and data not shown). Sequences critical for directing cleavage of the MLL bcr seem to be located within the telomeric 2.4 kb of the MLL bcr, as an episome containing only these sequences was cleaved nearly as efficiently as was the episome containing 8.3 kb of the MLL bcr (Fig. 4). Although an episome which contained only 540 bp of the MLL bcr sequence was able to direct specific MLL cleavage, the efficiency of cleavage directed by this fragment was much reduced compared to episomes which contained 2.4–8.3 kb of the MLL bcr sequence.
Randomly integrated or episomal copies of the MLL bcr behaved in a similar fashion to the endogenous MLL bcr in a LIS-based SAR assay, suggesting that sequences within the 8.3 kb MLL bcr fragment are sufficient to mediate attachment of nuclear scaffold proteins as well as direct site-specific MLL bcr cleavage. Although the primary nucleotide sequence of SARs is generally not well-conserved between species, SAR function is generally thought to be conserved between species (34). Consistent with this concept, the murine MLL bcr, while displaying only 60% nucleotide identity to the human MLL bcr over a 2 kb region encompassing MLL exons 9–11, also acted as a SAR and supported site-specific cleavage when introduced into human cells as an episome. Interestingly, we were unable to detect site-specific cleavage within a well-described SAR (the murine Ig locus; data not shown) suggesting that only a subset of SARs can support site-specific cleavage.
The ability of episomal copies of the human and murine MLL bcr to bind to nuclear scaffold proteins, as well as direct site-specific DNA cleavage, demonstrates the feasibility of using episomes as shuttle vectors to study site-specific DNA cleavage associated with apoptosis, as well as SAR function. In addition, since stably integrated copies of the MLL bcr also can direct site-specific DNA cleavage, this property may be useful as a technique for specifically cleaving chromosomal DNA. Finally, the ability of MLL bcr sequences to direct both specific DNA cleavage as well as the binding of nuclear scaffold proteins even when located outside of its normal chromosomal context supports the hypothesis that this site-specific cleavage is influenced by nuclear scaffold proteins. The sensitivity of this site within the MLL bcr to double-strand DNA cleavage may help explain the frequent involvement of the MLL gene in chromosomal translocations associated with cancer.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
Vector construction, transfections and transformation
The 8.3 kb genomic BamHI fragment encompassing the human MLL bcr was isolated from a placental lambda phage library (Stratagene, La Jolla, CA) and the 4 kb genomic HindIII fragment encompassing the 3' portion of the murine MLL bcr was obtained from a mouse liver lambda phage library. Relevant fragments were subsequently subcloned into pBluescript II (Stratagene). An episomal vector (pMEPMLL) containing the 8.3 kb MLL bcr BamHI fragment was constructed by cloning the fragment into the BamHI site of pMEP4 (Invitrogen, Carlsbad, CA). The pMEP4 vector contains the EBV oriP, EBNA-1 and a hygromycin resistance cassette. A vector containing the 8.3 kb MLL bcr BamH1 fragment cloned into the pMEP4 vector in an orientation opposite to that of the pMEPMLL vector was named p327. Deletion mutants of the p327 plasmid were generated by exonuclease III digestion (Erase-A-Base; Promega, Madison, WI) following the manufacturer’s recommended procedure. A 540 bp XbaI–ScaI fragment containing nucleotides 6602–7142 of the MLL bcr (accession no. HS04737) was cloned into the NotI and HindIII sites of pMEP4 (the NotI and HindIII sites were obtained by passage through pBluescript II); this vector was named p328. The episomal vector carrying the murine MLL bcr was constructed by releasing a 4 kb HindIII genomic fragment encompassing murine MLL exons 9–11 from pBluescript with BamHI and XhoI. This fragment was then cloned into the unique BamHI and XhoI sites of pMEP4. The pRCMLL vector used for integrating MLL into chromosomal DNA was generated by cloning the 8.3 kb MLL bcr fragment into the HindIII and NotI sites of pRC-CMV (Invitrogen). Transfections were performed by electroporation using a Gene Pulser II system (Bio-Rad, Hercules, CA) according to the manufacturer’s recommendations. Individual clones were selected by limiting dilution in the presence of either 800 µg/ml G418 or 400 µg/ml hygromycin B. Competent MAX Efficiency STBL2 cells (Invitrogen) were transformed with 1 µg of genomic DNA isolated from selected clones, including the JC-1 clone and the Jurkat/5.5 clone (a clone with approximately 220 copies of a large tandem array of an AmpR MLL plasmid integrated into the genome), following the manufacturer’s recommended protocol. Transformants were selected for AmpR.
Preparation and analysis of DNA
Genomic DNA was isolated using a salting-out extraction procedure, as previously described (15). One to 10 µg of genomic DNA was digested with the indicated restriction enzyme (Life Technologies, Gaithersburg, MD), size-fractionated on 0.8% agarose gels containing 1.0 µg/ml ethidium bromide, photographed, denatured, neutralized and transferred to nitrocellulose membranes (Schleicher & Schuell, Keene, NH). DNA was immobilized by UV crosslinking. The probes used were a 0.2 kb HhaI–BamHI human MLL cDNA fragment (probe 0.2HB, nucleotides 4207–4423 of GenBank accession no. L04731), a 0.7 kb human MLL cDNA fragment (probe 0.7B) (39), a 0.7 kb PvuII–PvuII (0.7PP) murine genomic fragment including MLL exon 10 and 11 sequences (16), a PCR-generated MLL exon 34 probe (40), a 0.5 kb EcoRI–SstII fragment of pMEP4 encoding hygromycin resistance (HYG) and a probe from nucleotides 10084–10303 of the pMEP4 vector (0.2APA). Modified indirect end-labeling of cleavage sites (41) within the MLL locus was performed as described previously (16). Probes were labeled with 32P by the random priming technique using a Prime-It II kit (Stratagene) according to the manufacturer’s protocol, and hybridized to Southern blots as previously described (15). Final washing conditions were 0.1% SDS/0.1x SSC (1x SSC = 0.15 mol/l NaCl and 0.015 mol/l sodium citrate) at 56°C for all probes. Analysis of oligonucleosomal DNA fragmentation was performed by gel electrophoresis of undigested genomic DNA using agarose gels containing 1.0 µg/ml ethidium bromide.
Cell culture and induction of apoptosis
The Jurkat and CEM cell lines were derived from patients with T-cell acute lymphoblastic leukemia and maintained in RPMI 1640 supplemented with 10% fetal bovine serum, L-glutamine (2 mM), penicillin (100 U/ml) and streptomycin (100 mg/ml) (all Life Technologies, Grand Island, NY). For induction of apoptosis, exponential growth phase cells were washed twice with serum-free media, resuspended at a concentration of 5 x 105 cells/ml in complete media, and incubated for a period of 4–16 h with the indicated drug or vehicle alone, followed by isolation of genomic DNA for analysis. Cells were either treated with etoposide (VP16; 10 µM; 4–16 h unless otherwise noted) or C2 ceramide (25 µM; 16 h). Etoposide and C2 ceramide were dissolved in dimethyl sulfoxide (DMSO). All drugs were purchased from Sigma (St Louis, MO). Chromatin staining of fragmented nuclei to verify apoptosis was performed as previously described (42). Immediately after treatment, cells were cyto-centrifuged onto glass slides, air dried, fixed for 30 min using a solution of 4% paraformaldehyde in phosphate buffered saline (PBS; pH 7.4) at room temperature, washed in PBS (pH 7.4), incubated for 2 min in permeabilization solution (0.1% Triton X-100, 0.1% sodium citrate) at 4°C, washed again with PBS (pH 7.1) and stained with 8 µg/ml bis-benzimide (Hoechst 33258; Sigma) in PBS (pH 7.1) for 15 min. The samples were then washed in PBS, mounted, coverslipped, analyzed and photographed (magnification 400x) by fluorescence microscopy using a Nikon OPTIPHOT microscope with a UV-2A filter (Nikon Inc., Melville, NY).
Isolation of nuclei, preparation of nuclear scaffolds and in vivo SAR assay
Nuclei were isolated by sucrose gradient centrifugation (43). Approximately 1 x 108 cells were washed twice with ice-cold PBS and resuspended in 10 ml of buffer A (0.3 M sucrose, 10 mM Tris pH 7.5, 5 mM MgCl2, 0.4% Nonidet-P40, 0.5 mM dithiothreiotol). The cells were transferred to a Dounce homogenizer and disrupted with several strokes of the B pestle. The homogenate was overlaid on 5 ml of buffer B (same as A but with 0.88 M sucrose) and centrifuged for 10 min at 4°C and 1000 g. Pellets were gently resuspended and washed in buffer C [37.5 mM Tris pH 7.5, 0.05 mM spermine, 0.125 mM spermidine, 20 mM KCl, 0.5 mM EDTA–KOH pH 7.4, 0.01% digitonin, 1% thiodiglycol, 0.5 mM phenylmethylsulfonyl fluoride (PMSF), 0.5 µg/ml leupeptin, 0.5 µg/ml pepstatin]. Nuclei were used immediately for isolation of nuclear scaffolds (21,44). Ten OD260 units of nuclei were mixed with an equal volume of buffer C, incubated for 10 min at 37°C and gently mixed with 7 ml of lithium salt buffer (5 mM HEPES pH 7.4, 0.25 mM spermidine, 2 mM EDTA–KOH pH 7.4, 2 mM KCl, 0.01% digitonin, 15 mM lithium 3,5-diiodosalicylate). After an incubation for 10 min at room temperature, nuclear scaffolds were pelleted for 10 min at 2500 g at 4°C and washed five times in digestion buffer (20 mM Tris pH 7.4, 0.05 mM spermine, 0.125 mM spermidine, 20 mM KCl, 10 mM MgCl2, 70 mM NaCl, 0.01% digitonin, 0.1 mM PMSF, 0.5 µg/ml leupeptin, 0.5 µg/ml pepstatin) before being resuspended in 2 ml of digestion buffer. For in vivo SAR assays, 500 µl of nuclear scaffolds were incubated for 4 h with 250 U of restriction enzyme at 37°C. Samples were then centrifuged for 10 min at 3000 g. Supernatants were saved, pellets washed once with digestion buffer and finally resuspended in TE buffer (10 mM Tris pH 7.5, 1 mM EDTA). Pellet and supernatant fractions were adjusted to 160 mM NaCl, 0.5% SDS and 0.2 mg/ml proteinase K, followed by an incubation for 16 h at 50°C. After phenol/chloroform extraction and ethanol precipitation, 10 µg of DNA from both the pellet and supernatant fractions was analyzed by size-fractionation on 0.8% agarose gels and Southern transfer as described above.
Fluorescence in situ hybridization (FISH)
A YAC clone encompassing the entire MLL bcr was labeled with digoxigenin and hybridized to metaphase spreads of selected Jurkat transfectants as previously described (40).
|
ACKNOWLEDGEMENTS |
|---|
We would like to thank Drs Michael Kuehl, Ilan Kirsch, Tamas Varga and Leroy Liu for insightful discussions. This work was supported in part by grants from the Roswell Park Alliance Foundation and the NIH (CA73773). P.D.A. is a Scholar of the Leukemia Society of America. M.S. was the recipient of a ‘Kind Philipp-Rückkehrstipendium’ through the ‘Stifterverband der Deutschen Wissenschaft’, Essen, Germany, and supported by the HILF program of the Medical School of Hannover, Germany.
|
FOOTNOTES |
|---|
+ To whom correspondence should be addressed at: National Cancer Institute, Advanced Technology Center, 8717 Grovemont Circle, Gaithersburg, MD 20877, USA. Tel: +1 301 435 5005; Fax: +1 301 402 3134; Email: aplanp@mail.nih.gov
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
1 Ziemin-van der Poel, S., McCabe, N.R., Gill, H.J., Espinosa, R., Patel, Y., Harden, A., Rubinelli, P., Smith, S.D., LeBeau, M.M., Rowley, J.D. et al. (1991) Identification of a gene, MLL, that spans the breakpoint in 11q23 translocations associated with human leukemias. Proc. Natl Acad. Sci. USA, 88, 10735–10739.
2 Tkachuk, D.C., Kohler, S. and Cleary, M.L. (1992) Involvement of a homolog of Drosophila trithorax by 11q23 chromosomal translocations in acute leukemias. Cell, 71, 691–700.[ISI][Medline]
3 Gu, Y., Nakamura, T., Alder, H., Prasad, R., Canaani, O., Cimino, G., Croce, C.M. and Canaani, E. (1992) The t(4;11) chromosome translocation of human acute leukemias fuses the ALL-1 gene, related to Drosophila trithorax, to the AF-4 gene. Cell, 71, 701–708.[ISI][Medline]
4 Djabali, M., Selleri, L., Parry, P., Bower, M., Young, B. and Evans, G.A. (1992) A trithorax-like gene is interrupted by chromosome 11q23 translocations in acute leukaemias. Nat. Genet., 2, 113–118.[ISI][Medline]
5 Thirman, M.J., Gill, H.J., Burnett, R.C., Mbangkollo, D., McCabe, N.R., Kobayashi, H., Ziemin-van der Poel, S., Kaneko, Y., Morgan, R., Sandberg, A.A. et al. (1993) Rearrangement of the MLL gene in acute lymphoblastic and acute myeloid leukemias with 11q23 chromosomal translocations. New Engl. J. Med., 329, 909–914.
6 Rubnitz, J.E., Behm, F.G. and Downing, J.R. (1996) 11q23 rearrangements in acute leukemia. Leukemia, 10, 74–82.[ISI][Medline]
7 Strout, M.P., Marcucci, G., Bloomfield, C.D. and Caligiuri, M.A. (1998) The partial tandem duplication of ALL1 (MLL) is consistently generated by Alu-mediated homologous recombination in acute myeloid leukemia. Proc. Natl Acad. Sci. USA, 95, 2390–2395.
8 Gu, Y., Cimino, G., Alder, H., Nakamura, T., Prasad, R., Canaani, O., Moir, D.T., Jones, C., Nowell, P.C., Croce, C.M. et al. (1992) The (4;11)(q21;q23) chromosome translocations in acute leukemias involve the VDJ recombinase. Proc. Natl Acad. Sci. USA, 89, 10464–10468.
9 Reichel, M., Gillert, E., Nilson, I., Siegler, G., Greil, J., Fey, G.H. and Marschalek, R. (1998) Fine structure of translocation breakpoints in leukemic blasts with chromosomal translocation t(4;11): the DNA damage-repair model of translocation. Oncogene, 17, 3035–3044.[ISI][Medline]
10 Gillert, E., Leis, T., Repp, R., Reichel, M., Hosch, A., Breitenlohner, I., Angermüller, S., Borkhardt, A., Harbott, J., Lampert, F. et al. (1999) A DNA damage repair mechanism is involved in the origin of chromosomal translocations t(4;11) in primary leukemic cells. Oncogene, 18, 4663–4671.[ISI][Medline]
11 Winick, N.J., McKenna, R.W., Shuster, J.J., Schneider, N.R., Borowitz, M.J., Bowman, W.P., Jacaruso, D., Kamen, B.A. and Buchanan, G.R. (1993) Secondary acute myeloid leukemia in children with acute lymphoblastic leukemia treated with etoposide. J. Clin. Oncol., 11, 209–217.
12 Super, H.J., McCabe, N.R., Thirman, M.J., Larson, R.A., Le Beau, M.M., Pedersen-Bjergaard, J., Philip, P., Diaz, M.O. and Rowley, J.D. (1993) Rearrangements of the MLL gene in therapy-related acute myeloid leukemia in patients previously treated with agents targeting DNA-topoisomerase II. Blood, 82, 3705–3711.
13 Cimino, G., Rapanotti, M.C., Biondi, A., Elia, L., Lo Coco, F., Price, C., Rossi, V., Rivolta, A., Canaani, E., Croce, C.M. et al. (1997) Infant acute leukemias show the same biased distribution of ALL1 gene breaks as topoisomerase II related secondary acute leukemias. Cancer Res., 57, 2879–2883.
14 Maraschin, J., Dutrillaux, B. and Aurias, A. (1990) Chromosome aberrations induced by etoposide (VP-16) are not random. Int. J. Cancer, 46, 808–812.[ISI][Medline]
15 Aplan, P.D., Chervinsky, D.S., Stanulla, M. and Burhans, W.C. (1996) Site-specific DNA cleavage within the MLL breakpoint cluster region induced by topoisomerase II inhibitors. Blood, 87, 2649–2658.
16 Stanulla, M., Wang, J., Chervinsky, D.S., Thandla, S. and Aplan, P.D. (1997) DNA cleavage within the MLL breakpoint cluster region is a specific event which occurs as part of higher-order chromatin fragmentation during the initial stages of apoptosis. Mol. Cell. Biol., 17, 4070–4079.[Abstract]
17 Rasio, D., Schichman, S.A., Negrini, M., Canaani, E. and Croce, C. (1996) Complete exon structure of the ALL1 gene. Cancer Res., 56, 1766–1769.
18 Nilson, I., Lochner, K., Siegler, G., Greil, J., Beck, J.D., Fey, G.H. and Marschalek, R. (1996) Exon/intron structure of the human ALL-1 (MLL) gene involved in translocations to chromosomal region 11q23 and acute leukemias. Br. J. Hematol., 93, 966–972.[ISI][Medline]
19 Strissel, P.L., Strick, R., Tomek, R.J., Roe, B.A., Rowley, J.D. and Zeleznik-Le, N.J. (2000) DNA structural properties of AF9 are similar to MLL and could act as recombination hot spots resulting in MLL/AF9 translocations and leukemogenesis. Hum. Mol. Genet., 9, 1671–1679.
20 Strissel, P.L., Strick, R., Rowley, J.D. and Zeleznik-Le, N.J. (1998) An in vivo topoisomerase II cleavage site and a DNase I hypersensitive site colocalize near exon 9 in the MLL breakpoint cluster region. Blood, 92, 3793–3803.
21 Mirkovitch, J., Mirault, M.E. and Laemmli, U.K. (1984) Organization of the higher-order chromatin loop: specific DNA attachment sites on nuclear scaffold. Cell, 39, 223–232.[ISI][Medline]
22 Berezney, R., Mortillaro, M.J., Ma, H., Wei, X. and Samarabandu, J. (1995) The nuclear matrix: a structural milieu for genomic function. Int. Rev. Cytol., 162A, 1–65.
23 Broeker, P.L., Super, H.G., Thirman, M.J., Pomykala, H., Yonebayashi, Y., Tanabe, S., Zeleznik-Le, N. and Rowley, J.D. (1996) Distribution of 11q23 breakpoints within the MLL breakpoint cluster region in de novo acute leukemia and in treatment-related acute myeloid leukemia: correlation with scaffold attachment regions and topoisomerase II consensus binding sites. Blood, 87, 1912–1922.
24 Borgnetto, M.E., Zunino, F., Tinelli, S., Kas, E. and Capranico, G. (1996) Drug-specific sites of topoisomerase II DNA cleavage in Drosophila chromatin: heterogeneous localization and reversibility. Cancer Res., 56, 1855–1862.
25 Kas, E., Poljak, L., Adachi, Y. and Laemmli, U.K. (1993) A model for chromatin opening: stimulation of topoisomerase II and restriction enzyme cleavage of chromatin by distamycin. EMBO J., 12, 115–126.[ISI][Medline]
26 Laemmli, U.K., Kas, E., Poljak, L. and Adachi, Y. (1992) Scaffold-associated regions: cis-acting determinants of chromatin structural loops and functional domains. Curr. Opin. Genet. Dev., 2, 275–285.[Medline]
27 Sperry, A.O., Blasquez, V.C. and Garrard, W.T. (1989) Dysfunction of chromosomal loop attachment sites: illegitimate recombination linked to matrix association regions and topoisomerase II. Proc. Natl Acad. Sci. USA, 86, 5497–5501.
28 Yates, J.L. and Guan, N. (1991) Epstein–Barr virus-derived plasmids replicate only once per cell cycle and are not amplified after entry into cells. J. Virol., 65, 483–488.
29 Yates, J.L., Warren, N. and Sugden, B. (1985) Stable replication of plasmids derived from Epstein–Barr virus in various mammalian cells. Nature, 313, 812–815.[Medline]
30 Cifone, M.G., De Maria, R., Roncaioli, P., Rippo, M.R., Azuma, M., Lanier, L.L., Santoni, A. and Testi, R. (1994) Apoptotic signaling through CD95 (Fas/Apo-1) activates an acidic sphingomyelinase. J. Exp. Med., 180, 1547–1552.
31 Beere, H.M., Chresta, C.M., Alejo-Herberg, A., Skladanowski, A., Dive, C., Larsen, A.K. and Hickman, J.A. (1995) Investigation of the mechanism of higher order chromatin fragmentation observed in drug-induced apoptosis. Mol. Pharmacol., 47, 986–996.[Abstract]
32 Gromova, I.L., Nielsen, O.F. and Razin, S.V. (1995) Long-range fragmentation of the eukaryotic genome by exogenous and endogenous nucleases proceeds in a specific fashion via preferential DNA cleavage at matrix attachment sites. J. Biol. Chem., 270, 18685–18690.
33 Cockerill, P.N. and Garrard, W.T. (1986) Chromosomal loop anchorage sites appear to be evolutionarily conserved. FEBS Lett., 204, 5–7.[ISI][Medline]
34 Cockerill, P.N. and Garrard, W.T. (1986) Chromosomal loop anchorage of the kappa immunoglobulin gene occurs next to the enhancer in a region containing topoisomerase II sites. Cell, 44, 273–282.[ISI][Medline]
35 Rowley, J.D. (1998) The critical role of chromosome translocations in human leukemias. Annu. Rev. Genet., 32, 495–519.[ISI][Medline]
36 Rabbitts, T.H. (1994) Chromosomal translocations in human cancer. Nature, 372, 143–149.[Medline]
37 Bernard, O.A. and Berger, R. (1995) Molecular basis of 11q23 rearrangements in hematopoietic malignant proliferations. Genes Chromosomes Cancer, 13, 75–85.[ISI][Medline]
38 Strick, R., Strissel, P.L., Borgers, S., Smith, S.L. and Rowley, J.D. (2000) Dietary bioflavonoids induce cleavage in the MLL gene and may contribute to infant leukemia. Proc. Natl Acad. Sci. USA, 97, 4790–4795.
39 McCabe, N.R., Burnett, R.C., Gill, H.J., Thirman, M.J., Mbangkollo, D., Kipiniak, M., van Melle, E., Ziemin-van der Poel, S., Rowley, J.D. and Diaz, M.O. (1992) Cloning of cDNAs of the MLL gene that detect DNA rearrangements and altered RNA transcripts in human leukemic cells with 11q23 translocations. Proc. Natl Acad. Sci. USA, 89, 11794–11798.
40 Felix, C.A., Megonigal, M.D., Chervinsky, D.S., Leonard, D.G., Tsuchida, N., Kakati, S., Block, A.M., Fisher, J., Grossi, M., Salhany, K.I. et al. (1998) Association of germline p53 mutation with MLL segmental jumping translocation in treatment-related leukemia. Blood, 91, 4451–4456.
41 Nedospasov, S.A. and Georgiev, G.P. (1980) Non-random cleavage of SV40 DNA in the compact minichromosome and free in solution by micrococcal nuclease. Biochem. Biophys. Res. Commun., 92, 532–539.[ISI][Medline]
42 Oberhammer, F., Wilson, J.W., Dive, C., Morris, I.D., Hickman, J.A., Wakeling, A.E., Walker, P.R. and Sikorska, M. (1993) Apoptotic death in epithelial cells: cleavage of DNA to 300 and/or 50 kb fragments prior to or in the absence of internucleosomal fragmentation. EMBO J., 12, 3679–3684.[ISI][Medline]
43 Schibler, U., Hagenbuchle, O., Wellauer, P.K. and Pittet, A.C. (1983) Two promoters of different strengths control the transcription of the mouse -amylase gene Amy-1a in the parotid gland and the liver. Cell, 33, 501–508.[ISI][Medline]
44 Hanson, R.D. and Ley, T.J. (1992) A-T-rich scaffold attachment regions flank the hematopoietic serine protease genes clustered on chromosome 14q11.2. Blood, 79, 610–618.
CiteULike 
Connotea 
Del.icio.us What's this?
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||