Mechanisms of MLL gene rearrangement: site-specific DNA

Published by Oxford University Press Human Molecular Genetics, 2001, Vol. 10, No. 22 2481–2491

Mechanisms of MLL gene rearrangement: site-specific DNA cleavage within the breakpoint cluster region is independent of chromosomal contextMartin Stanulla2,4, Pradheepkumar Chhalliyil2, Junjie Wang2, Sheila N. Jani-Sait3 and Peter D. Aplan1,*

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

The MLL gene at chromosome band 11q23 is specif-ically cleaved at a unique site within its breakpointcluster region (bcr) during the higher order chromatinfragmentation associated with apoptosis. We nowshow that the same specific DNA cleavage event canbe detected in an exogenous MLL bcr fragment thatis integrated into the genome outside of its normalchromosomal context, as well as in an extrachromo-somal episome containing an MLL bcr fragment. Wealso show that episomal or randomly integratedcopies of the MLL bcr behave similar to the endo-genous MLL bcr when tested in a scaffold-associatedregion (SAR) assay. Furthermore, an episomal murineMLL bcr introduced into human cells is cleaved at thesame site as the endogenous murine MLL bcr; thisepisomal murine MLL bcr also functions as a SAR inhuman cells. We conclude that both nuclear DNAscaffold attachment as well as site-specific DNAcleavage can be directed by sequences containedwithin the MLL bcr, and that it is feasible to studythese events using episomal shuttle vectors.

INTRODUCTION

One of the most frequently observed genetic alterations inpatients with acute leukemia is chromosomal translocationinvolving the MLL gene (also called ALL-1, HRX or Htrx) (1–4).The MLL gene is homologous to the Drosophila trithoraxgene, which is involved in pattern development during embryo-genesis (1–4). Interestingly, the translocation breakpointswithin the MLL gene almost always fall within a limited regionof 8.3 kb, referred to as the MLL breakpoint cluster region (bcr)(5). MLL translocations result in the generation of fusionproteins that retain the MLL N-terminus, including both an A-Thook domain and a region similar to mammalian DNA methyl-transferase (6).

The precise molecular mechanisms that cause MLL rearrange-ments remain largely unknown. Homologous recombinationbetween Alu repeat sequences (7) and inappropriate V(D)J

recombinase activity (8) have been proposed as potentialcauses of MLL gene rearrangements. More recently, extensiveanalysis of t(4;11) breakpoints involving MLL has demon-strated nucleotide sequence features such as small regions ofdeletion, duplication and inversion that strongly implicate non-homologous end joining (NHEJ) of broken DNA (9,10) in thegeneration of many of these translocations. Additionally, ofparticular clinical importance, treatment of a primary malignancywith topoisomerase II (topo II) poisons, such as doxorubicin oretoposide (VP-16), is associated with development of secondary,or therapy-related acute myelogenous leukemias (t-AML) thatoften display MLL gene rearrangements (11,12). Furthermore,a majority of infants with acute leukemia have MLL generearrangements; it has been speculated that infant leukemiasmay be due to in utero exposure to genotoxic agents (13).These clinical findings, along with the observation that treat-ment of peripheral blood lymphocytes with etoposide in vitrocan induce chromosomal rearrangements (14), stronglysuggest that treatment of mammalian cells with topo II poisonscan cause MLL gene rearrangements. We have previouslydescribed a specific site within the MLL bcr that is sensitive toDNA double strand cleavage induced by topo II poisons, andproposed that this site-specific cleavage may be an initiatingevent for MLL chromosomal translocations caused by treat-ment with topo II poisons (15,16). While this site has not yetbeen precisely mapped, it can be localized to a region of ∼75 bpwithin MLL exon 9 (17) [or exon 12 using the more recentnumbering proposed by Nilson et al. (18)]. Interestingly, asimilar cleavage site has recently been identified within theAF9 gene, which is a frequent translocation partner of MLL(19). Subsequent experiments have shown that site-specificcleavage within the MLL bcr could also be induced by apoptosis(16) or DNAse I treatment (20), suggesting that this cleavagesite might represent a genomic region which is uniquelysusceptible to DNA double-strand breaks.

AT-rich DNA sequences that are preferentially associatedwith nuclear scaffold proteins are referred to as scaffold-associated regions or SARs (21). SARs [also called matrixattachment/associated regions (MARs)] are thought to repre-sent the bases, or attachment sites, of chromosomal loops(reviewed in 22). MLL breakpoints in patients with t-AML

*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: [email protected]

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have been shown to cluster near a high affinity SAR (23) thatencompasses the MLL cleavage site described above (15,16,20).In addition, topo II is one of the major protein components of thenuclear scaffold and can be detected at chromosomal DNASARs (24–26). Taken together, these findings suggest a poten-tial relationship between SARs and preferred topo II bindingsequences in the production of illegitimate recombinationevents, such as chromosomal translocations, within the MLLbcr. Supporting such a hypothesis, a SAR in the murineimmunoglobulin κ locus is preferentially cleaved by topo II andthis cleavage occurs in close proximity (14 bp) to a translocationbreakpoint (27).

To better understand the specific DNA double-strandcleavage event within the MLL gene, and the relationship ofthis cleavage to chromosomal context, we introduced DNAfragments containing portions of the human and murine MLLbcr into human cell lines. We then evaluated the ability of theexogenous MLL bcr sequences (either stably integrated into thegenome or stably maintained within the cells as an episome) toserve as SARs and preferential cleavage sites.

RESULTS

Site-specific DNA cleavage within a stably integrated MLL bcr fragment

To determine if site-specific DNA cleavage could be inducedwithin an MLL bcr that was introduced into the genome outside

of its normal chromosomal context, an 8.3 kb genomicfragment encompassing the MLL bcr (5) was cloned into thepRC-CMV vector, linearized, and transfected into the Jurkatcell line. Pure clones were selected by limiting dilution in thepresence of 800 µg/ml G418; those that had integrated one ortwo copies of MLL were chosen for further analysis (Fig. 1A).Etoposide treatment of these clones demonstrated specificcleavage of the transfected copy of MLL using an indirect end-labeling assay (16). In this assay, genomic DNA is extractedfrom treated cells, digested with a restriction enzyme, size-fractionated, transferred to nitrocellulose membranes, andhybridized to a radiolabeled DNA probe. To detect specificMLL bcr cleavage, we used an MLL cDNA probe (0.2HB)containing exon 9, 10 and 11 sequences, as previouslydescribed (15,16). Figure 1B shows a novel 4.0 kb SstI restric-tion fragment is produced by etoposide treatment of parentalJurkat cells and a novel fragment of 1.5 kb is produced in theJurkat clones. In each case, one end of the novel fragment isproduced by restriction enzyme cleavage, whereas the otherend of the fragment is produced by site-specific DNA doublestrand cleavage site following etoposide treatment. Since oneend of the fragment is fixed by the restriction enzyme, the siteof DNA cleavage induced by etoposide treatment can bemapped by comparison with germline or transfected frag-ments, 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

Figure 1. Site-specific MLL cleavage within a single-copy MLL integrant. (A) Single-copy integration of the MLL bcr. HindIII-digested genomic DNA from paren-tal Jurkat cells and individual clones was hybridized to the 0.2HB probe. The endogenous 15 kb MLL fragment is indicated. Several clones show integration of asingle copy of the exogenous MLL. (B) Site-specific cleavage within the transfected MLL bcr. Jurkat cells and selected clones were treated (V) with 10 µM etopo-side or vehicle alone (C) for 8 h. Genomic DNA was digested with SstI and analyzed by indirect end-labeling to the 0.2 HB MLL probe. The endogenous andtransfected copies of MLL are indicated with one or two asterisks, respectively. The novel fragments produced by site-specific cleavage are indicated with arrows.In some control lanes (J9, J18, J20) a faint band is seen migrating slightly above the cleavage-induced fragment. This finding was not consistently detected andmight represent cross-hybridization to a related gene. Size standards are in kb. (C) Restriction maps of endogenous and tranfected copies of the MLL bcr. B,BamHI; R, EcoRI; S, SstI. The cleavage site is indicated with a downward arrow.

Human Molecular Genetics, 2001, Vol. 10, No. 22 2483

transfected copy of MLL is consistently 5-fold less intense thanthat from the endogenous MLL; this is not unexpected sinceFISH experiments showed that the parental Jurkat cells areaneuploid 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 requiredto support MLL cleavage, an EBV-based episomal vectorcontaining the MLL bcr and a selectable marker (hygromycin)was generated by cloning a genomic DNA fragment encom-passing the MLL bcr into the EBV-based pMEP4 vector(28,29). This construct was introduced into Jurkat cells byelectroporation. After selection with hygromycin, 10 inde-pendent clones carrying the episome were isolated; threeclones (JC-1, JC-12 and JM-5) were chosen for further study.Southern blot analysis of undigested genomic DNA using aprobe isolated from pMEP4 vector sequences (nucleotides10 084–10 303) demonstrated a band at ∼9 kb (Fig. 2A),indicating that this vector had not become integrated intochromosomal DNA but was instead a type I closed extrachromo-somal circle. In addition, transformation of Escherichia coliwith genomic DNA isolated from the JC-1 clone yielded 37 ± 16transformants per microgram of DNA, whereas genomic DNAisolated from Jurkat cells or Jurkat/5.5 cells (a Jurkat clonewith a tandem array of 220 integrated copies of an AmpR MLLplasmid; P.D.Aplan and M.Stanulla, unpublished data) yieldedzero transformants per microgram of genomic DNA (Table 1).Taken together, these results indicate that the pMEPMLLplasmid was maintained as an episome in the JC-1 clone. Analysisof 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 was12.6–15.1 (mean 14.2) times as intense as that from the germlineMLL. Since Jurkat cells have five copies of MLL, we estimatethat the JC-1 clone has approximately 70 copies of thepMEPMLL episome per cell.

To determine whether site-specific cleavage within the MLLbcr can also be induced within this MLL episome, we treatedthe JC-1 clone with etoposide. Genomic DNA was harvested,digested with BamHI or HindIII, and assayed for site-specificMLL bcr cleavage using indirect end-labeling. Figure 2Cshows novel fragments induced by etoposide in both the Jurkatand JC-1 lanes. Although one cannot discriminate betweencleavage of the endogenous MLL bcr and the episomal MLLbcr with a BamHI digest, since the fragments are of identicalsize, HindIII-digested DNA from etoposide treated JC-1 cellsproduced discrete fragments of 2.2 or 1.5 kb, derived from theendogenous or episomal MLL, respectively. Figure 3 shows anidentical pattern of site-specific cleavage within the episomalMLL bcr in three independent clones (JC-1, JC-12 and JM-5),demonstrating that the result seen with the JC-1 clone was notunique to this particular clone. From these experiments, weconclude that site-specific cleavage of the MLL bcr can begenerated within MLL bcr sequences placed on an episome,demonstrating that site-specific MLL bcr cleavage is notrestricted to chromosomal DNA.

The JC-1 clone was treated with either etoposide or a non-genotoxic apoptotic stimuli (C2 ceramide) (30,31) to determineif site-specific cleavage of the episomal MLL was part of ageneralized apoptotic pathway. Genomic DNA was harvested

Figure 2. An episomal vector containing MLL bcr sequences undergoes site-specific cleavage. (A) Hybridization of undigested genomic DNA from JC-1,JC-12 and JM-5 clones to a probe from the pMEPMLL vector (0.2APA). Lane1, Jurkat; lane 2, JC-1; lane 3, JC-12; lane 4, JM-5. The hybridizing episome isindicated with an asterisk; size standards are in kb. (B) Restriction map of theMLL bcr (upper) and the pMEPMLL episomal vector (lower). The MLL bcr isbracketed, exons are shown as solid boxes. The cleavage site is indicated by adownward arrow. The 0.2HB cDNA probe containing exon 9–11 sequences isshown, restriction enzyme sites are: B, BamHI; H, HindIII. The 8.3 kb BamHIfragment was cloned into the BamHI site of pMEP4 to generate thepMEPMLL episomal vector. The position of oriP in the vector is indicated.(C) Parental Jurkat cells and JC-1 cells were treated with either vehicle alone(C) or 10 µM etoposide (VP) for 8 h. Genomic DNA digested with eitherBamHI or HindIII was analyzed by indirect end-labeling with the 0.2HB MLLcDNA probe. The fragments induced by etoposide treatment are indicated byarrows; size standards are in kb. The germline and episomal MLL fragmentsare 8.3 kb on a BamHI digest, and either 15 or 19 kb, respectively, on a HindIIIdigest. The size of the induced fragment is 1.5 kb with a BamHI digest (endo-genous or episomal MLL bcr) and either 2.2 kb (endogenous MLL bcr, seemap) or 1.5 kb (episomal MLL bcr, see map) with a HindIII digest.


and assayed for site-specific MLL bcr cleavage; apoptotic celldeath was verified by detection of oligonucleosomal laddersand nuclear fragmentation (Fig. 3). Specific DNA cleavagewithin the episomal MLL bcr induced by non-genotoxic stimuliof apoptotic cell death was detected by indirect end-labeling(Fig. 3). To determine whether site-specific cleavage of MLLepisomes was unique to Jurkat cells, we introduced thepMEPMLL vector into CEM cells, and selected stable trans-fectants as described above for Jurkat cells. Etoposide-inducedcleavage of the episomal MLL resident in CEM cells wascomparable to that seen in Jurkat cells (Fig. 4 and data notshown).

To determine if the entire 8.3 kb MLL bcr was required tosupport site-specific MLL cleavage, we cloned a 540 bp fragment

encompassing 200+ nucleotides on either side of the MLLcleavage site into the pMEP4 vector. This plasmid (p328; Fig. 4)was introduced into CEM cells and stable transfectants wereisolated and treated with etoposide. Figure 4 demonstrates thatthis vector was cleaved at the same site within MLL exon 9 aswas the endogenous MLL bcr, although the cleavage was lessdramatic than that seen with episomes containing the 8.3 kbMLL bcr fragment.

We used exonuclease III to generate deletion mutants (plas-mids p510 and p512, containing 2984 and 2360 nucleotides ofthe MLL bcr sequence, respectively; Fig. 4B) that had smallersegments of the 8.3 kb MLL bcr contained within the pMEP4vector. The plasmids were transfected into CEM cells; stableclones were isolated and treated with etoposide to induce MLLcleavage. Cleavage of the endogenous MLL bcr was compar-able among the four clones and the parental CEM cell line(16–43% using 30 µM etoposide). Cleavage of the episomalMLL bcr also was comparable for the clones containing thep327, p510 and p512 episomes (14–19% using 30 µM etopo-side). However, although the C328A clone, containing only540 bp of the MLL bcr sequence supported site-specific MLLcleavage, the specific cleavage (1%) was not as extensive asthat seen using episomes with the larger MLL fragments. Weconclude that episomes containing 2.4 or 3.0 kb of the MLL bcrsequence are almost as effective as those containing 8.3 kb ofthe MLL bcr sequence in directing site-specific MLL cleavage,and clearly more effective than those containing a minimalportion of the MLL bcr (clone C328A).

Table 1. Transformation of E.coli with genomic DNA from JC-1 cells

aResults represent the mean ± SD of four experiments.bThe Jurkat/5.5 clone has approximately 220 copies of an AmpR MLL plasmidintegrated in a tandem array.cSupercoiled plasmid, only 1 ng used for transformation.

DNA Transformants per microgram of genomic DNAa

Jurkat 0

Jurkat/5.5b 0

JC-1 37 ± 16

pMEPMLLc TNTC

Figure 3. Cleavage of the episomal MLL induced by apoptosis. (A) Etoposide or C2 ceramide was used to induce apoptosis in JC-1 cells. Genomic DNA wasdigested with HindIII and analyzed by indirect end-labeling with the 0.2HB probe. The 1.5 kb fragment indicating site-specific cleavage of the episome is detectedin all three treated samples. Size standards are in kb. (B) Oligonucleosomal ladders present in undigested genomic DNA from etoposide (VP) or C2-ceramide(C2) treated JC-1 cells; the control (C) in both panels is genomic DNA from JC-1 cells treated with vehicle alone. (C) Nuclear fragmentation of JC-1 cells treatedwith etoposide (VP) or C2 ceramide (C2) compared to cells treated with vehicle alone (C) JC-1 cells. (D) Three independent clones harboring the pMEPMLLepisomal vector (JC-1, JC-12, JM-5) were treated for 8 h with either 10 µM etoposide (VP) or vehicle (C) alone and analyzed by indirect end-labeling as describedin Figure 2. Etoposide-induced fragments of 2.2 kb (endogenous MLL bcr) or 1.5 kb (episomal MLL bcr) are seen in the VP lanes of all three clones. Size standardsare in kb.


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 highaffinity SAR (23). This co-localization is consistent with amodel in which higher order chromatin fragmentation duringthe early stages of apoptosis occurs as a specific event at SARs,where DNA loops are anchored to the nuclear scaffold (32). Todetermine if the scaffold association of the transfectedepisomal MLL bcr was similar to that of the germline MLL bcr,we conducted SAR assays. Nuclear scaffolds were prepared byLIS extraction (21) of nuclei from both parental Jurkat cellsand the JC-1 cells, which contained an episomal MLL bcr.Scaffolds were then digested with BamHI, and equal amountsof DNA from pellet (scaffold-associated) and supernatant(non-scaffold-associated) fractions were analyzed by Southernblot hybridization to the 0.7 kb MLL cDNA probe that encom-passes the entire MLL bcr (Fig. 5). Similar to previouslyreported results (23), the 8.3 kb BamHI fragment encom-passing the MLL bcr was enriched in the pellet fraction fromJurkat cells (Fig. 5A). An identical pattern of enrichment isseen in the JC-1 cells. Fragments derived from the episomalMLL co-migrate and cannot be distinguished from thosederived from the endogenous MLL with absolute certainty.However, since the JC-1 clone contains approximately 14times as many copies of the episomal MLL as the endogenous

MLL (note the much more intense signal from JC-1 lanes thanJurkat lanes in Fig. 5A, also see Figs 2 and 3), it seems reason-able to assume that 13/14 or >90% of the signal on the auto-radiograph is derived from the episomal MLL. Thus, it seemshighly likely that both the endogenous and the episomal copiesof the MLL bcr were associated with the nuclear scaffold.

We used a BamHI/EcoRI double digest to refine themapping of the MLL SAR (Fig. 5). Three fragments (4.6, 2.7and 1.0 kb) were generated. The 2.7 and 4.6 kb fragments wereenriched in the pellet fraction, suggesting preferential bindingof these fragments to nuclear scaffold proteins. In contrast, the1.0 kb fragment, representing the most centromeric portion ofthe MLL bcr, was equally distributed between the pellet andsupernatant fractions. These results are again similar to previ-ously reported experiments (23) that mapped a ‘high affinity’SAR to a region including the 4.6 and 2.7 kb fragments, and aweak or ‘low affinity’ SAR to a region encompassing the1.0 kb fragment. Similar to Figure 5A, the hybridizationpattern for JC-1 cells is identical and more intense than that ofthe parental Jurkat cells, indicating that episomal copies ofMLL behaved similar to the chromosomal MLL. To exclude thepossibility that larger DNA fragments were preferentiallyprecipitated in the pellet fraction based solely on fragment size,and not scaffold binding, we rehybridized the blots to either anMLL exon 34 probe, which lies outside of the previouslymapped SAR (23), or a hygromycin probe. A 4 kb exon 34

Figure 4. Cleavage of episomes containing 0.5–3.3 kb of MLL bcr sequence. (A) The p328 plasmid (diagram in B), was stably transfected into CEM cells. Thetransfectants were treated for 0, 8 or 24 h with 30 µM etoposide. Genomic DNA was harvested and analyzed by indirect end-labeling to the HYG probe (B). AnEcoRI digest produced a 2.0 kb fragment (indicated with an arrow) and PstI digest produced a 1.9 kb fragment (indicated with an arrow). The site of MLL cleavagein the p328 episome maps to the previously identified site near MLL exon 9. Size standards are in kb. (B) Map of the endogenous MLL (End) and p327, p512, p510and p328 episomal vectors. The thick black line indicates MLL sequences; thin line indicates plasmid sequences. The downward arrow indicates the site of MLLcleavage and the location of the HYG probe is indicated. Restriction sites: B, BamHI; K, KpnI; P, PstI; the KpnI site in parentheses was destroyed during the nesteddeletion process. (C) Stable transfectants harboring either the p327, p510, p512 or p328 vectors (C27H, C10B, C12D or C328A, respectively) were treated with10 or 30 µM etoposide for 24 h. MLL cleavage was assayed by indirect end-labeling using a PstI digest and the 0.2 HB probe. The endogenous and episomal MLLcleavage products are indicated with a closed circle and asterisk, respectively. The percent of endogenous (End) and episomal (Epi) alleles cleaved (estimated bydividing the intensity of the cleaved allele by the intensity of cleaved allele plus germline allele; all intensities adjusted for background) is indicated below the lane.The episomal band from C328A is not visualized by indirect end-labeling with the 0.2HB probe since there are only 66 bp of complementary exon 9 sequencecontained on the 0.2HB probe, and the C328A clone undergoes minimal cleavage. Size standards are in kb. (D) The blot in (C) was rehybridized to the HYG probe.Specific cleaved episomal fragments are indicated with an asterisk. The percent of episomal alleles cleaved, as detected by the HYG probe, is indicated below thelanes.


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 preferen-tially located in the supernatant fraction. To determine whetherMLL bcr sequences randomly integrated into the genomewould likewise function as SARs, we assayed clones with asingle copy of the MLL bcr. Figure 6 shows that a transfected

MLL bcr behaves similar to the chromosomal MLL in thisLIS-based SAR assay. In sum, these results are consistent withthe previously reported high-affinity SAR in the telomericportion of the MLL bcr (23), and demonstrate that the associa-tion of transfected copies of the MLL bcr with the nuclear scaf-fold 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 evolutionarilyconserved (33,34). For instance, Drosophila histone geneSARs compete with murine immunoglobulin κ SARs forbinding of nuclear scaffolds derived from murine MPC-11plasmacytoma cells (34). In addition, site-specific cleavagewithin the MLL bcr induced by apoptosis is conserved betweenspecies, as it can be observed within the murine MLL bcr (16).Since apoptotic site-specific DNA cleavage within the humanMLL bcr can be recapitulated with episomal vectors, wewished to determine if an episome containing the murine MLLbcr could be cleaved in human cells, and if it would also func-tion as a SAR. An episomal vector (Fig. 7) containing a 4 kbHindIII fragment from the murine MLL bcr was transfectedinto Jurkat cells by electroporation and clones selected withhygromycin. Site-specific cleavage of an episomal murineMLL introduced into human cells was detected by indirect end-labeling (Fig. 7); this cleavage site is identical to that seen inthe endogenous murine MLL bcr (16).

We also analyzed the behavior of the episomal murine MLLfragment using the LIS-based SAR assay described above. Inthis experiment, an endogenous 15 kb HindIII fragment

Figure 5. SAR assays of the germline and episomal MLL bcr. (A) Nucleiisolated from parental Jurkat and Jurkat cells harboring the pMEPMLLepisome (JC-1) were extracted with LIS, followed by digestion with BamHI.Genomic DNA from pellet (P) and supernatant (S) fractions was analyzed bySouthern blot hybridization to the 0.7B MLL cDNA probe. Size standards arein kb. (B) Genomic DNA from Jurkat and JC-1 cells was prepared andanalyzed by Southern blot hybridization to the 0.7B probe as in (A), except thatthe nuclear scaffolds were simultaneously digested with BamHI and EcoRI.The 4.6 and 2.7 kb fragments derived from both the endogenous (Jurkat) andepisomal (JC-1) copies of the MLL bcr are preferentially associated with thepellet fraction. Size standards are in kb. (C) The blot shown in (A) was strippedand rehybridized to an MLL exon 34 probe. (D) A duplicate of the blot shownin (B) was hybridized to the HYG probe (Fig. 4B). (E) Restriction map of theMLL bcr. The bcr is bracketed, exons are shown as solid boxes. The specificcleavage site is indicated with a downward arrow. Restriction enzyme sites:B, BamHI; E, EcoRI. The previously reported high affinity SAR (23) is shownas a black bar.

Figure 6. A randomly integrated MLL bcr behaves similar to the endogenousMLL in a SAR assay. (A) Nuclei from five independent clones (Fig. 2) contain-ing a single copy of the MLL bcr randomly integrated were analyzed as inFigure 8. The 4.4 kb (endogenous) and 3.5 kb (transfected) MLL bcr are indi-cated. The ratio of pellet to supernatant signal intensity for the endogenous andtransfected MLL bcr is shown below each pair of lanes. The slower migratingband in the J2 pellet lane is due to partially digested DNA. Ethidium bromidestaining showed that the J18 pellet lane was overloaded and the J9 pellet lanewas underloaded. (B) The blot in (A) was rehybridized to an MLL exon 34probe. The ratio of pellet to supernatant signal intensity for the endogenousand transfected MLL bcr is again shown below each pair of lanes, note that thesignal is now preferentially in the supernatant lanes.


encompassing the MLL bcr was enriched in the pellet fraction(Fig. 8); the 4.0 kb fragment representing the episomal MLLbcr was also enriched in the pellet fraction. As a control, anMLL exon 34 probe was hybridized to a 7 kb fragment whichwas equally distributed between the pellet and supernatant

fractions. We conclude that the murine MLL bcr, like thehuman MLL bcr, is preferentially associated with nuclear scaf-fold proteins.

DISCUSSION

Recurrent, non-random chromosomal translocations have beenrecognized in acute leukemia patients for several decades andare likely to be causal events leading to leukemic transform-ation (35,36). One of the genes most frequently translocated ina wide spectrum of acute leukemias is MLL. In contrast to most

Figure 7. Site-specific cleavage of an episomal murine MLL bcr in humancells. (A) Hybridization of undigested genomic DNA from Jurkat (lane 1) anda clone harboring the murine MLL bcr episome (clone muC-6, lane 2) to the0.2 HB MLL cDNA probe. The hybridizing episome is indicated with anarrow; size standards are in kb. (B) Indirect end-labeling of parental Jurkatcells and Jurkat cells harboring an episome containing the murine MLL bcr(clone J482C). Genomic DNA was isolated from Jurkat and J482C cells beforeand after an 8 h incubation with 10 µM etoposide and digested with XhoI orHindIII, as indicated. Indirect end-labeling was performed as described above,except a murine genomic probe (0.7PP) was used in place of the 0.2HB. Anarrow indicates the end-labeled fragments produced by cleavage of theepisomal murine MLL bcr. A cleavage product from the endogenous humanMLL bcr is not detected as the 0.7PP murine probe does not cross-hybridizewell to the endogenous human MLL under these conditions (note that theendogenous 15 kb germline MLL fragment is only faintly visible in the HindIIIlanes). (C) Map of the human MLL bcr (hu, upper part) and episomal vectorcontaining a portion of the murine MLL bcr (lower part). Exons are shown assolid boxes. Chromosomal orientation from centromere (cen) to telomere (tel)is as shown. The point of site-specific cleavage is indicated by a downwardarrow. The 4 kb HindIII fragment from the murine MLL bcr (mu) was clonedinto pMEP4 as shown. The murine genomic MLL probe is shown as an openbox labeled PP. Selected restriction sites are: H, HindIII; N, NotI; X, XhoI.

Figure 8. The murine MLL bcr episome acts as a SAR in human cells.(A) Nuclear scaffolds prepared from Jurkat cells harboring an episomecontaining the murine MLL bcr (clone muC-6) were digested with HindIII.Pellet (P) and supernatant (S) fractions were analyzed by Southern blot hybrid-ization to the 0.7B probe. Both the endogenous human fragment (15 kb) andthe episomal fragment (4.0 kb) are preferentially associated with the pelletfraction. (B) To rule out the possibility that the MLL bcr fragments preferen-tially precipitated in the pellet fraction based solely on size, the blot in (A) wasstripped and rehybridized to an MLL exon 34 probe, which hybridizes to a 7 kbfragment that is equally distributed in the pellet and supernatant fractions.(C) Restriction map of the endogenous human and episomal murine MLL bcr.


genes involved in chromosomal translocations, where a trans-forming gene has one or a few ‘partners’ (i.e. PML and RARα;BCR and ABL), MLL has been referred to as a ‘promiscuous’oncogene because of its involvement in translocations fusing atleast 30 different genes with MLL sequences (6,37). Despitethis wide range of translocation partners, MLL translocationbreakpoints almost invariably lie within an 8.3 kb bcr. Thisobservation suggests that this 8.3 kb region may be uniquelysusceptible to chromosomal translocations, which, in mostgeneral terms, can be regarded as DNA cleavage and religationevents; with the translocations being produced when thecleaved DNA becomes religated to a non-homologouschromosome.

We and others (15,20) have recently identified a site withinthe MLL bcr that is uniquely susceptible to DNA double-strandcleavage, both in vitro (16) and in vivo (15,20,38), in responseto topo II poisons, DNAse I or apoptosis. Furthermore, this sitefalls within a high affinity SAR (23). One hypothesis thataccounts for the observed colocalization of site-specificcleavage of the MLL bcr at a SAR proposes that the SAR is amarker for ‘accessible’ regions of the genome, which are pref-erentially cleaved in response to a variety of stimuli, includingDNAse I and apoptotic nucleases. In support of this model, ithas been suggested that the 50–100 kb fragments releasedduring the initial stages of apoptosis are due to DNA cleavageat SARs or the bases of chromosomal loops (32). We have nowshown that cloned fragments of the MLL bcr, stably introducedinto the nucleus, either integrated into chromosomal DNA oras an extrachromosomal episome, can still mediate site-specific DNA double-strand cleavage, and are preferentiallyattached to the nuclear scaffold.

Although we had predicted that an exogenous copy of theMLL bcr, integrated outside of its normal chromosomalcontext, would be able to mediate site-specific cleavage, wedid not anticipate that an episomal copy of the MLL bcr wouldbe able to support site-specific cleavage in an efficient fashion.However, an EBV-based episome containing MLL bcrsequences clearly directed site-specific cleavage within theMLL bcr in response to topo II poisons as well as any of theadditional apoptotic stimuli used. The sensitivity ofthe episomal MLL bcr to cleavage was comparable to thatof the endogenous MLL bcr (Figs 2–4), although most experi-ments showed that the episomal copy of the MLL bcr wassomewhat less sensitive to site-specific cleavage (Fig. 4 anddata not shown). Sequences critical for directing cleavage ofthe MLL bcr seem to be located within the telomeric 2.4 kb ofthe MLL bcr, as an episome containing only these sequenceswas cleaved nearly as efficiently as was the episomecontaining 8.3 kb of the MLL bcr (Fig. 4). Although anepisome which contained only 540 bp of the MLL bcr sequencewas able to direct specific MLL cleavage, the efficiency ofcleavage directed by this fragment was much reducedcompared to episomes which contained 2.4–8.3 kb of the MLLbcr sequence.

Randomly integrated or episomal copies of the MLL bcrbehaved in a similar fashion to the endogenous MLL bcr in aLIS-based SAR assay, suggesting that sequences within the8.3 kb MLL bcr fragment are sufficient to mediate attachmentof nuclear scaffold proteins as well as direct site-specific MLLbcr cleavage. Although the primary nucleotide sequence ofSARs 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, whiledisplaying only 60% nucleotide identity to the human MLL bcrover a 2 kb region encompassing MLL exons 9–11, also actedas a SAR and supported site-specific cleavage when intro-duced into human cells as an episome. Interestingly, we wereunable to detect site-specific cleavage within a well-describedSAR (the murine Igκ locus; data not shown) suggesting thatonly a subset of SARs can support site-specific cleavage.

The ability of episomal copies of the human and murineMLL bcr to bind to nuclear scaffold proteins, as well as directsite-specific DNA cleavage, demonstrates the feasibility ofusing episomes as shuttle vectors to study site-specific DNAcleavage associated with apoptosis, as well as SAR function.In addition, since stably integrated copies of the MLL bcr alsocan direct site-specific DNA cleavage, this property may beuseful as a technique for specifically cleaving chromosomalDNA. Finally, the ability of MLL bcr sequences to direct bothspecific DNA cleavage as well as the binding of nuclear scaf-fold proteins even when located outside of its normal chromo-somal context supports the hypothesis that this site-specificcleavage is influenced by nuclear scaffold proteins. The sensi-tivity of this site within the MLL bcr to double-strand DNAcleavage may help explain the frequent involvement of theMLL gene in chromosomal translocations associated withcancer.

MATERIALS AND METHODS

Vector construction, transfections and transformation

The 8.3 kb genomic BamHI fragment encompassing the humanMLL bcr was isolated from a placental lambda phage library(Stratagene, La Jolla, CA) and the 4 kb genomic HindIII frag-ment encompassing the 3′ portion of the murine MLL bcr wasobtained from a mouse liver lambda phage library. Relevantfragments were subsequently subcloned into pBluescript II(Stratagene). An episomal vector (pMEPMLL) containing the8.3 kb MLL bcr BamHI fragment was constructed by cloningthe 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 vectorcontaining the 8.3 kb MLL bcr BamH1 fragment cloned intothe pMEP4 vector in an orientation opposite to that of thepMEPMLL vector was named p327. Deletion mutants of thep327 plasmid were generated by exonuclease III digestion(Erase-A-Base; Promega, Madison, WI) following the manu-facturer’s recommended procedure. A 540 bp XbaI–ScaI frag-ment containing nucleotides 6602–7142 of the MLL bcr(accession no. HS04737) was cloned into the NotI and HindIIIsites of pMEP4 (the NotI and HindIII sites were obtained bypassage through pBluescript II); this vector was named p328.The episomal vector carrying the murine MLL bcr wasconstructed by releasing a 4 kb HindIII genomic fragmentencompassing murine MLL exons 9–11 from pBluescript withBamHI and XhoI. This fragment was then cloned into theunique BamHI and XhoI sites of pMEP4. The pRCMLL vectorused for integrating MLL into chromosomal DNA was gener-ated by cloning the 8.3 kb MLL bcr fragment into the HindIIIand NotI sites of pRC-CMV (Invitrogen). Transfections wereperformed by electroporation using a Gene Pulser II system


(Bio-Rad, Hercules, CA) according to the manufacturer’srecommendations. Individual clones were selected by limitingdilution in the presence of either 800 µg/ml G418 or 400 µg/mlhygromycin B. Competent MAX Efficiency STBL2 cells(Invitrogen) were transformed with 1 µg of genomic DNAisolated from selected clones, including the JC-1 clone and theJurkat/5.5 clone (a clone with approximately 220 copies of alarge tandem array of an AmpR MLL plasmid integrated intothe genome), following the manufacturer’s recommendedprotocol. Transformants were selected for AmpR.

Preparation and analysis of DNA

Genomic DNA was isolated using a salting-out extractionprocedure, as previously described (15). One to 10 µg ofgenomic DNA was digested with the indicated restrictionenzyme (Life Technologies, Gaithersburg, MD), size-fractionatedon 0.8% agarose gels containing 1.0 µg/ml ethidium bromide,photographed, denatured, neutralized and transferred to nitro-cellulose membranes (Schleicher & Schuell, Keene, NH).DNA was immobilized by UV crosslinking. The probes usedwere a 0.2 kb HhaI–BamHI human MLL cDNA fragment(probe 0.2HB, nucleotides 4207–4423 of GenBank accessionno. L04731), a 0.7 kb human MLL cDNA fragment (probe0.7B) (39), a 0.7 kb PvuII–PvuII (0.7PP) murine genomic frag-ment including MLL exon 10 and 11 sequences (16), a PCR-generated MLL exon 34 probe (40), a 0.5 kb EcoRI–SstII frag-ment of pMEP4 encoding hygromycin resistance (HYG) and aprobe 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 previ-ously (16). Probes were labeled with 32P by the randompriming technique using a Prime-It II kit (Stratagene)according to the manufacturer’s protocol, and hybridizedto Southern blots as previously described (15). Final washingconditions were 0.1% SDS/0.1× SSC (1× SSC = 0.15 mol/lNaCl and 0.015 mol/l sodium citrate) at 56°C for all probes.Analysis of oligonucleosomal DNA fragmentation wasperformed by gel electrophoresis of undigested genomic DNAusing 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 withT-cell acute lymphoblastic leukemia and maintained in RPMI1640 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 ofapoptosis, exponential growth phase cells were washed twicewith serum-free media, resuspended at a concentration of5 × 105 cells/ml in complete media, and incubated for a periodof 4–16 h with the indicated drug or vehicle alone, followed byisolation of genomic DNA for analysis. Cells were eithertreated with etoposide (VP16; 10 µM; 4–16 h unless otherwisenoted) or C2 ceramide (25 µM; 16 h). Etoposide and C2 cera-mide were dissolved in dimethyl sulfoxide (DMSO). All drugswere purchased from Sigma (St Louis, MO). Chromatinstaining of fragmented nuclei to verify apoptosis wasperformed as previously described (42). Immediately aftertreatment, cells were cyto-centrifuged onto glass slides, airdried, fixed for 30 min using a solution of 4% para-formaldehyde in phosphate buffered saline (PBS; pH 7.4) at

room temperature, washed in PBS (pH 7.4), incubated for2 min in permeabilization solution (0.1% Triton X-100, 0.1%sodium citrate) at 4°C, washed again with PBS (pH 7.1) andstained with 8 µg/ml bis-benzimide (Hoechst 33258; Sigma) inPBS (pH 7.1) for 15 min. The samples were then washed inPBS, mounted, coverslipped, analyzed and photographed(magnification 400×) by fluorescence microscopy using aNikon OPTIPHOT microscope with a UV-2A filter (NikonInc., Melville, NY).

Isolation of nuclei, preparation of nuclear scaffolds and in vivo SAR assay

Nuclei were isolated by sucrose gradient centrifugation (43).Approximately 1 × 108 cells were washed twice with ice-coldPBS 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 mMdithiothreiotol). The cells were transferred to a Douncehomogenizer and disrupted with several strokes of the B pestle.The homogenate was overlaid on 5 ml of buffer B (same as Abut with 0.88 M sucrose) and centrifuged for 10 min at 4°C and1000 g. Pellets were gently resuspended and washed in bufferC [37.5 mM Tris pH 7.5, 0.05 mM spermine, 0.125 mM sper-midine, 20 mM KCl, 0.5 mM EDTA–KOH pH 7.4, 0.01%digitonin, 1% thiodiglycol, 0.5 mM phenylmethylsulfonylfluoride (PMSF), 0.5 µg/ml leupeptin, 0.5 µg/ml pepstatin].Nuclei were used immediately for isolation of nuclear scaf-folds (21,44). Ten OD260 units of nuclei were mixed with anequal volume of buffer C, incubated for 10 min at 37°C andgently mixed with 7 ml of lithium salt buffer (5 mM HEPESpH 7.4, 0.25 mM spermidine, 2 mM EDTA–KOH pH 7.4,2 mM KCl, 0.01% digitonin, 15 mM lithium 3,5-diiodo-salicylate). After an incubation for 10 min at room temperature,nuclear scaffolds were pelleted for 10 min at 2500 g at 4°C andwashed 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 resus-pended in 2 ml of digestion buffer. For in vivo SAR assays,500 µl of nuclear scaffolds were incubated for 4 h with 250 Uof restriction enzyme at 37°C. Samples were then centrifugedfor 10 min at 3000 g. Supernatants were saved, pellets washedonce with digestion buffer and finally resuspended in TEbuffer (10 mM Tris pH 7.5, 1 mM EDTA). Pellet and super-natant fractions were adjusted to 160 mM NaCl, 0.5% SDS and0.2 mg/ml proteinase K, followed by an incubation for 16 h at50°C. After phenol/chloroform extraction and ethanol precipi-tation, 10 µg of DNA from both the pellet and supernatantfractions was analyzed by size-fractionation on 0.8% agarosegels and Southern transfer as described above.

Fluorescence in situ hybridization (FISH)

A YAC clone encompassing the entire MLL bcr was labeledwith digoxigenin and hybridized to metaphase spreads ofselected Jurkat transfectants as previously described (40).

ACKNOWLEDGEMENTS

We would like to thank Drs Michael Kuehl, Ilan Kirsch, TamasVarga and Leroy Liu for insightful discussions. This work wassupported in part by grants from the Roswell Park Alliance


Foundation and the NIH (CA73773). P.D.A. is a Scholar of theLeukemia Society of America. M.S. was the recipient of a‘Kind Philipp-Rückkehrstipendium’ through the ‘Stifterver-band der Deutschen Wissenschaft’, Essen, Germany, andsupported by the HILF program of the Medical School ofHannover, Germany.

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Mechanisms of MLL gene rearrangement: site-specific DNA