| Human Molecular Genetics | Pages |
The BCR gene recombines preferentially with Aluelements in complex BCR-ABL translocations of chronic myeloid leukaemia
Introduction
Results
Isolation and chromosomal localization of 3[prime]BCR recombination sites
Identification of repeat sequences
Recurring sequence motifs
Coding regions around breakpoint sites
Discussion
Materials And Methods
Patients
Probes
Southern blot analysis
Construction and screening of genomic libraries
DNA sequencing
Sequence analysis
Fluorescent in situ hybridization (FISH)
Acknowledgements
References
The BCR gene recombines preferentially with Aluelements in complex BCR-ABL translocations of chronic myeloid leukaemia
Chronic myeloid leukaemia (CML) develops when two genes, BCR on chromosome 22 and ABL on chromosome 9, recombine to form a hybrid BCR-ABL gene with leukaemogenic properties. The mechanism which underlies this recombination is unknown, but additional chromosome sites may be involved to form complex BCR-ABL rearrangements. The majority of breakpoints in BCR occur within a 5 kb major breakpoint cluster region, M-Bcr. Here, we show that the 3[prime] part of M-Bcr recombined within, or immediately adjacent to, Alu elements at the additional sites in all five complex BCR-ABL rearrangements that have been examined so far. This is a new finding which suggests that Alu sequences have an affinity for the BCR-ABL recombination process in complex rearrangements, and provides additional evidence for the association of these elements with somatic rearrangements which cause human leukaemia. We further show that sequence motifs similar to IgH switch pentamers and consensus binding sites of the lymphoid-associated Translin protein are present on one or more participating strands at 3[prime]M-Bcr recombination sites. Motifs similar to Translin-binding sites were also identified within the Alu consensus. Expressed sequences mapped close to the breakpoint sites on other chromosomes in three of the five cases examined.
INTRODUCTION
Chronic myeloid leukaemia (CML) is a clonal myeloproliferative disease which arises following somatic gene rearrangement in a pluripotent bone marrow stem cell. In ~90% of patients, simple two-way exchange between BCR and ABL occurs, and results in the cytogenetically obvious chromosomal translocation, t(9;22)(q34;q11), the small derivative chromosome 22 product of which is well known as the Philadelphia or Ph chromosome (1-3). In other patients, BCR-ABL gene association arises from complex rearrangements which may clearly involve one or more chromosomes in addition to 9 and 22, or which may be masked by an apparently normal karyotype (4 and refs therein,5).
The precise molecular mechanism which underlies BCR-ABL recombination is unknown. Coding regions of both ABL and BCR extend across large genomic domains of ~225 kb on 9q34 and ~152 kb on 22q11, respectively (6). In CML, the translocation breakpoints on chromosome 22 occur almost exclusively within a tightly confined region (M-Bcr) which encompasses exons 12-15 of the BCR gene (6,7). Breakpoints on chromosome 9 occur widely within a >200 kb region extending from sites 5[prime] of the entire ABL gene to exon 2 of ABL, although there is evidence of clustering at three sites (6,8,9). Sequence analysis of BCR-ABL and/or ABL-BCR recombination products from 21 standard t(9;22) rearrangements of CML (6,10) and our analysis of M-Bcr (7) has provided some indication that Alu repeatelements may be important to the recombination process. There is a single Aluelement in M-Bcr, and >70% of breakpoints occur within a 3 kb region encompassing that element (7,11). Alusequences are also overrepresented at sites of recombination in the ABL gene compared with their overall incidence throughout the genome (10).
Aluelements are the most abundant class of dispersed repeat sequences in the human genome. An estimated one million copies, distributed non-randomly and favouring G-band light regions of chromosomes, comprise ~10% of DNA in human cells. They are 280 bp in length, and consist of two similar monomers that have sequence homology to the likely source gene, 7SL RNA. Alu elements are retrotransposable, and several Alusubfamilies mobilized from different source genes at different evolutionary times can be recognized on the basis of their sequence divergence and diagnostic base changes (12). Alu-mediated gene mutation underlies several important constitutional diseases of man, including rare cases of familially inherited breast and colon cancer. Different mechanisms for these mutations include recombination between homologous or non-homologous regions of Aluelements at different locations within a gene, or on the same or different chromosomes. Coding regions of genes may also be disrupted by transpositional insertion of actively transcribed younger Alu elements (13-16).
In all complex BCR-ABL rearrangements, the 5[prime] part of BCR recombines with ABL to form the 5[prime]BCR-ABL fusion gene. However, the 3[prime] part of BCR,which unites with the 5[prime] ABL remnant in the standard t(9;22)(q34;q11), usually recombines with one of the additional chromosomes in the complex translocations, or with part of chromosome 9 outside the ABL gene (17-20). Detailed sequence analysis of those chromosomal domains which break and rejoin with 3[prime]M-Bcr can therefore provide useful clues to the mechanism of BCR-ABL recombination which leads to CML. Few cases have been reported. In one complex t(9;22;11)(q34;q11;q13), 3[prime]M-Bcrrecombined with a site immediately adjacent to an Aluelement in the transcribed domain of the glutathione S-transferase Pi class gene(GSTP1) on chromosome 11q13 (19). We have also sequenced through a 3[prime]M-Bcr recombination site on 11q13 for a different CML patient and showed that Aluelements mapped within 500 bp either side of the 11q13 breakpoint (20). We have now extended our analysis of the 11q13 recombination products in that rearrangement, and also sequenced through 3[prime]M-Bcr recombination sites involved in complex BCR-ABL rearrangements of a further three patients. In this report, we show that Aluelements are present at breakpoint sites on the additional chromosomal regions in all five cases that have been examined so far.
RESULTS
Chromosomal breakpoints and genomic recombination products of the complex rearrangements, including ABL and BCR relocations described in previous publications of our four cases, are illustrated in Figure 1. a-d. Leukaemic cells of two patients showed complex Ph translocations, and two had apparently normal karyotypes which masked complex rearrangements. The 3[prime] part of M-Bcrhad recombined with a chromosomal region other than 5[prime]ABL in all four patients. We have cloned and determined the DNA sequence surrounding the four 3[prime]M-Bcr junction sites: 7q11.23/3[prime]M-Bcr, 9q34/3[prime]M-Bcr, 22q11.2/3[prime]M-Bcrand11q13/3[prime]M-Bcr, from cases Nos 1-4 respectively, and of germline fragments which span the corresponding breakpoint sites on chromosomal bands 7q11.23, 9q34, 22q11.2 and 11q13. In addition, we have compared our findings with published sequence data of the complex t(9;22;11)(q34;q11;q13) BCR-ABL rearrangement reported by Koduru et al. (19). Because the 11q13 breakpoint in their case disrupted a non-coding site in the transcriptional domain of GSTP1, we refer to the GSTP1-3[prime]M-Bcr junction and GSTP1 germline fragments to distinguish them from the 11q13/3[prime]M-Bcr junction and 11q13 germline fragments of our case No. 4.
Figure 1. Partial karyotypes in ideogrammatic format showing predicted chromosome and gene rearrangements of the four different complex BCR-ABL rearrangements of CML cases Nos 1-4. Normal chromosomes are shown on the left of each homologue pair, and derivative chromosomes to the right. Brackets mark the breakpoint junctions cloned and sequenced in this study.(a) t(7;9;22)(q11.23;ABL;BCR) of case No. 1. The 3[prime] part of BCR joins with chromosome 7 within the disrupted band q11.23. (b) dir ins (22;9)(22pter->5[prime]BCR::3[prime]ABL->?9q34::3[prime]BCR->22qter) of Ph-negative case No. 2. The 3[prime] part of the ABL gene and a segment of 9q34 telomeric from it is inserted between the divided 5[prime] and 3[prime] parts of the BCR gene to form the characteristic 5[prime]BCR-3[prime]ABL fusion gene. The 3[prime] part of BCR joins with a site in chromosome band 9q34 an unknown distance telomeric from the ABL gene (17,56). (c)Complex hidden rearrangement t(9;22;?)(9pter->5[prime]ABL::?9q34; 22pter->22q11.2::3[prime]BCR->22qter; ?::22q11.2->5[prime]BCR::3[prime]ABL->9q34::?) of Ph-negative case No. 3. Unpublished data and results presented in this report provide good evidence that the 3[prime] part of BCR is joined to a site within IGL in band 22q11.2, ~500 kb proximal to the BCR gene. The 5[prime] BCR-3[prime]ABL fusion gene has inserted at an as yet unknown chromosomal site. (d) The complex `masked' Ph rearrangement of case No. 4: t(9;22;11)(9pter->5[prime]ABL::?9q34; 22pter-> 5[prime]BCR::3[prime]ABL->?9q34::11q13->11qter; 11pter->q13::3[prime]BCR(22qter). The 3[prime] part of BCR joins to chromosome 11 within the disrupted band 11q13. To isolate 3[prime]BCR breakpoint fragments, genomic bacteriophage libraries prepared from leukaemic DNA were screened with a 3[prime]M-Bcr probe. Germline BCR fragments derived from the normal chromosome 22 homologue were identified and eliminated from selection after hybridization with a 5[prime]M-Bcr probe. Remaining positives were purified, restriction mapped and the breakpoint sites identified. Genomic inserts of all breakpoint phage DNAs showed homology with the 3[prime] end of M-Bcr. Divergence from germline BCR towards the 5[prime] end identified the site of the breakpoint junction for individual patients (Fig. 2b, d, f and h). Authenticity of cloned and mapped breakpoint fragments was confirmed by hybridization of probes isolated from the non-BCR region immediately 5[prime] of the breakpoint to Southern-blotted leukaemic DNA. In all four cases, rearranged BglII, BamHI, HindIII and EcoRI fragment sizes were consistent with the restriction maps (Fig. 3). The same 5[prime] non-BCR probes were used to screen further genomic bacteriophage libraries and isolate germline fragments which spanned the recombination sites on the additional chromosomes. All purified germline fragments overlapped with non-BCR regions of the corresponding junction fragments, and diverged towards the 3[prime] end (compare Fig. 2b, d, f and h with c, e, g and i, respectively). None showed homology with BCR, and informative germline BglII, BamHI, HindIII or EcoRI allele sizes detected on Southern blots with probes from the four different chromosomal regions [Fig. 3c, e and i; (19)] were consistent in size with restriction maps of the cloned germline fragments (Fig. 2b, d, f and h). Figure 2. (a)Partialrestriction map of the BCR gene region which encompasses M-Bcr. Breakpoint sites in M-Bcr of cases Nos 1-4 are indicated by arrowheads above the map. M-Bcr-5[prime] and M-Bcr-3[prime] probes used to isolate and select the 3[prime]M-Bcr breakpoint clones are indicated by solid bars beneath the map. Locations of BCR exons 12-16 are marked by grey boxes. (b-i) Restriction maps of 3[prime]M-Bcr junction fragments and germline fragments corresponding to other chromosomal sites which break and rejoin with M-Bcr for cases Nos 1 (b andc), 2 (dand e), 3 (f and g) and 4 (h and i). Breakpoint sites are indicated by arrowheads above each map, and different colors correspond to different chromosomal origins as depicted in Figure 1. The asterisk in (f)marks insertion of a 64 bp inverted complementary M-Bcr sequence at the breakpoint site (see Fig. 5c). Clone designations (top) and chromosomal origin (bottom) are indicated to the left of each map. Solid bars beneath the breakpoint maps (b), (d), (f) and (h) mark probes used to isolate the corresponding germline fragments (c), (e), (g) and (i). The 7q11.23 probe was a BglII-BalI fragment. Dotted lines mark sequenced regions, major features of which are summarized in Figure 4a-i. Restriction sites are abbreviated: Bg, BglII; B, BamHI; H, HindIII; E, EcoRI; X, XbaI. Figure 3. Southern blot studies of DNA extracted from leukaemic cells of CML patients Nos 1-3 and digested with restriction enzymes BglII (Bg), BamHI (B), HindIII (H) or EcoRI (E). Shown are germline (closed arrows) and rearranged (open arrows) fragment sizes detected after sequential hybridization of the same filter with the M-Bcr-3[prime] probe (a,d and g) and with probes adjacent to the breakpoint sites in 7q11.23 (case No. 1) (b), 9q34 (case No. 2) (e) and 22q11.2 (case No. 3) (h) (see Fig. 2 for probe locations). `Control' panels (c, f and i) show germline fragment patterns for the indicated probes. The expected ~9 kb germline 7q11.23 BamHI fragment of case No. 1 was not resolved because of difficulty in suppressing repeats in the high molecular weight region. Germline and breakpoint M-Bcr or 9q34 fragments were not resolvable in EcoRI digests of case No. 2 because of their large size, and for similar reasons, the BglII and BamHI digests of case No. 3 were not informative in resolving germline or rearranged 22q11.2 fragments. The 22q11.2 probe used identifies an insertion polymorphism in non-leukemic control DNA, and asterisks mark germline alleles (Benjes et al., in preparation). The control DNA (i) was heterozygous for the germline alleles. Note the co-migration of rearranged BglII, BamHI and EcoRI fragments in case No. 1; rearranged BglII fragments in case No. 2; and rearranged EcoRI fragments in case No. 3. Molecular weight size standards shown on the left were derived from HindIII-digested [lambda] DNA. Similar studies of 3[prime]M-Bcr and germline 11q13 fragment sizes for case No. 4 were reported previously (20). The chromosomal location of the bacteriophage clones which spanned the four different germline chromosomal sites involved in 3[prime]M-Bcr recombination was determined using fluorescent in situ hybridization (FISH). Specific signals were detected on chromosome band 7q11.23 (case No. 1), 9q34 (case No. 2), 22q11.2 (case No. 3) and 11q13 (case No. 4) (data not shown). Sequences of 3[prime]M-Bcr junction fragments and germline fragments which spanned the four recombination sites were submitted to the repeat identification program CENSOR. A summary of the location and kinds of repeat elements relative to the breakpoint sites in M-Bcr and on the recombinant chromosomes is presented in Figure 4. . For case No. 1, the 3[prime] part of the disrupted Alu-Szof M-Bcr recombined with the 3[prime] part of an Alu-Jo on 7q11.23. The recombination was non-homologous, with breaks at different positions in each of the Aluelements relative to the consensus Alusequence (Fig. 4a-c). The Alu-Jo was one of nine complete or partial Alu elements clustered within the 4051 bp of available sequence surrounding the 7q11.23 breakpoint region (Fig. 4c). For case No. 2, a break occurred5[prime] of the Alu-Szelement in M-Bcr, and within the BamHI-XhoI fragment (Fig. 4a). The 3[prime] side of M-Bcr had recombined with the 5[prime] end of an Alu-Y element on 9q34, one of a tightly linked cluster of three Aluelements which mapped in reverse orientation across the breakpoint region (Fig. 4d and e). The break in M-Bcr of case No. 3 occurred just downstream of the 3[prime] HindIII site, within a truncated LINE-related element, LIMB7 (Fig. 4a). In this case, 3[prime]M-Bcr recombined with the 3[prime]-terminal end of an Alu-Sp element (Fig. 4f and g). CENSOR also identified an MER4A (human medium reiteration frequency) repeat, apparently invaded by a truncated LINE element, L1MA5, immediately upstream of and in the opposite orientation to the Alu-S (Fig. 4g). A 874 bp region immediately 3[prime] of the disrupted Alu-S on the germline 22q11 sequence showed homology (mostly within the range 60-70%) with many human and other primate [alpha]-satellite sequences. The breakpoint in M-Bcr of case No. 4 occurred downstream from the L1MB7 repeat, and within the 3[prime] HindIII-BglII fragment (Fig. 4a). 3[prime]M-Bcr recombined with a site in band 11q13 which was apparently repeat free (Fig. 4h and i). However, ongoing molecular studies of the reciprocal 9q34/11q13 breakpoint junction of this complex rearrangement (Fig. 1d) have identified a deletion on the participating 11q13 strand. This deletion probably occurred during the recombination process, and disrupts a more 3[prime] Alu-Szelement at the 9q34 recombination site (Fig. 4i and j).We also resubmitted the GSTPI/3[prime]M-Bcr junction and germline GSTPI sequences of the t(9;22;11) described by Koduru et al. (19) to the updated CENSOR database. In this case, the break in M-Bcr occurred immediately downstream of a truncated human endogenous retrovirus-related element, HERVKC4(Fig. 4a), and 3[prime]M-Bcr recombined with the 5[prime] end of an Alu-Spqxzg in GSTPI. Truncated sequences of a second Alu-Spqxzg and an MSTA repeat mapped close to and upstream of the GSTP1 breakpoint site (Fig. 4a, k and l). In two cases, 3[prime]M-Bcr joined with chromosomes 7q11.23 (case No. 1) and 22q11.2 (case No. 3) at almost identical sites within the right monomer of an Alu, towards the 3[prime] end of the element (Fig. 4b and g). DOTPLOT analysis did not identify any new significant repeat features around the breakpoint sites (data not shown). Figure 4. (a) Summary of repeat element features in the 3[prime] part of M-Bcr. Breakpoint sites of cases 1-4 are indicated by arrowheads above the map. (b-i) Expanded maps showing repeat element features of sequenced 3[prime]M-Bcr breakpoints and corresponding germline regions of cases Nos 1 (b and c), 2 (d and e), 3 (f and g) and 4 (h and i), respectively. Breakpoint sites are indicated by arrowheads above each map, and different colours correspond to different chromosomal origins as depicted in Figures 1 and 2. The asterisk in (f) marks insertion of a 64 bp inverted complementary M-Bcr sequence at the breakpoint site (see Fig. 5c). Locations of the sequenced regions shown here are indicated by dotted underlines in the restriction maps of Figure 2a-i. (j) Preliminary sequence features of a 2.38 kb reciprocal 9q34/11q13 breakpoint fragment cloned from leukaemic cells of case No. 4 (see Fig. 1d, and data not shown). Note that 9q34 sequences are joined to a site within an Alu-Sz on 11q13 (open arrowhead in i) which maps ~625 bp downstream from the site which joined with 3[prime]M-Bcr (closed arrowhead in i). The intervening 11q13 sequences are presumed to have been lost. (k and l)Update of the repeat element content of the 3[prime]M-Bcr-GSTPI breakpoint and GSTPI germline sequences reported by Koduru et al. (19). Different repeat element families identifed by CENSOR (59) are designated by symbols as in the key. Arrowheads mark the direction 5[prime]->3[prime] of the different repeat elements. The subfamily origin of MERs interrupted by Alus is shown at the 3[prime] end of each MER (f, g and j). Restriction enzyme sites marked above the maps are intended as guides only, and not all sites are shown. Abbreviations are as follows: Bg, BglII; B, BamHI; H, HindIII; E, EcoRI; X, XbaI; Xh, XhoI; P, PstI; Pv, PvuII, S, SstI. To identify sequence patterns which might disclose a common site-specific mechanism for DNA breakage and rejoining, we performed exhaustive computer-based pairwise and pile-up comparisons of 40 bp of sequence spanning the breakpoint sites in M-Bcr, 7q11.23, 9q34, 22q11.2, 11q13 and GSTP1. Initial comparisons revealed limited clusters of 2-4 bp homology when M-Bcr sequences were aligned directly against the recombination sites on chromosomes 7q11.23 (case No. 1), 9q34 (case No. 2), 11q13 (case No. 4) and GSTP1 (19) (Fig. 5and data not shown). The 22q11.2-3[prime]BCR junction of case No. 3 was complicated by a 64 bp inversion of complementary M-Bcr sequences (Fig. 5). In this case, a tight cluster of 7 bp homology (5[prime]-CCTGGGC-3[prime]) was detected between sequences which spanned the 22q11.2 germline sequence and sequences in M-Bcr located 3[prime] and downstream of the 64 bp sequence (Fig. 5). A direct repeat, 5[prime]-TCTTT-3[prime], flanked the breakpoint sites of the inverted segment in M-Bcr. Sequence comparisons using the CLUSTALW and BOXSHADE programs identified a pentameric motif 5[prime]-CCCAG-3[prime] which was consistently present at, or near to, the 3[prime]M-Bcr recombination sites on chromosomes 9q34, 22q11, 11q13 and GSTP1, and a 1 bp mismatch to that motif at two sites across the breakpoint on 7q11.23 (Fig. 5). In addition, two short tetramers, or their complements, were common within 20 bp either side of all breakpoints in M-Bcr: 5[prime]-CAGG-3[prime] and 5[prime]-GTGG-3[prime] (Fig. 5). All of these motifs show similarities to parts of a number of recombination-prone sequences, including prokaryotic Chi ([chi]) (5[prime]-GCTGGTGG-3[prime]), human hypervariable minisatellites, various DNA polymerase frameshift hotspots, and the 26 bp recombinogenic Alu core (21,22). However, we could find no regular association of any of those recombination-prone patterns with the 3[prime]M-Bcr breakpoint sites we have characterized. Figure 5. Alignment of 100 bp sequence encompassing 3[prime]M-Bcr junction fragments, recombination sites in germline 3[prime]M-Bcr, and in regions extending across the 3[prime]M-Bcr recombination sites on chromosomes 7q11.23 (case No. 1) (a), 9q34 (case No. 2) (b), 22q11.2 (case No. 3)(c) and 11q13 (case No. 4) (d). The 64 bp inverted complementary M-Bcr sequence inserted between the breakpoint site proximal to BCR in chromosome band 22q11.2 and 3[prime]M-Bcr sequences in normal sense is shown in the inset region of (c). Bases marked in bold flank breakpoint sites. The CAGG and GTGG tetramers (or complements) common within 20 bp of all 3[prime]M-Bcrbreakpoints are marked in red. Pentameric CCCAG (CTGGG) motifs common within 20 bp of 3[prime]M-Bcr recombination sites on other chromosomes are underlined. The closely related CCCTG and CCGGG pentamers at the breakpoint site in 7q11.23 are marked by dotted underlining in (a). Boxed regions correspond to Translin-related DNA-binding sequences (>80% similarity). In some cases, these domains overlap. Sequence features are also shown across the GSTPI, BCR and ABL germline and breakpoint regions of the complex t(9;22;11) previously characterized by Koduru et al. (19)(e). The consistent sequence patterns we detected are similar to the pentamers, GAGCT, GGGCT and GGGGT, involved in class switch recombination at the immunoglobulin heavy chain locus (IgH) (23,24). They are also contained within the consensus DNA-binding sequences of Translin protein (25). Translin binds specifically to consensus sequences at breakpoint junctions of chromosomal translocations found in many kinds of lymphoid malignancies which lack recognizable heptamer-nonamer V-D-J recombinase signals. Two kinds of consensus sequences were identified: (i) (A,C)TGCAG-N(0->4 bp)-GCCC(A/T)(G/C)(G/C)-(A/T); or (ii) a tandem repeat of GCCC(A/T)(G/C)(G/C)(A/T), with a gap of a few nucleotides separating each repeat (25). Computer-based FINDPATTERNS searches identified >80% similarity (maximum of 3 bp mismatch to consensus) to the full-length Translin DNA-binding consensus sequences across the breakpoints on M-Bcr and/or at the recombination sites on other chromosomes in all five of the cases we examined (Fig. 5). BLASTN searches of repeat-free regions indicated that 3[prime]M-Bcr had recombined with sequences upstream and within the IGL locus on chromosome 22 in case No. 3 (Benjes et al., in preparation). A 64 bp stretch at the 3[prime] end of the germline 7q11.23 sequence of case No. 1 also showed 100% homology with nucleotide sequences of several anonymous human cDNA fragments in DBEST (BLASTN; locus nos H95168, AA479537, H17584, AA340567, AA160751, W51926, AA220220, AA130689, N29217 and N26362), raising the likelihood that the breakpoint at 7q11.23 has occurred within the coding domain, or close to the coding domain, of an actively transcribed gene. Repeat-free sequences of the germline 9q34 and 11q13 fragments of cases Nos 2 and 4, respectively, showed no significant homology when compared against expressed sequences in the databases.
Isolation and chromosomal localization of 3[prime]BCR recombination sites
Identification of repeat sequences
Recurring sequence motifs
Coding regions around breakpoint sites
DISCUSSION
In contrast to many lymphoid leukaemias, the basis of the specific molecular recombinations which lead to myeloid leukaemias is unknown. Our research supports earlier indications that Alu sequences may have a role in BCR-ABL recombination which initiates CML. There is a single Alu-Sz in M-Bcr around which most breakpoints occur (7,11). However, our results show that this Alu-Sz is not always close to sites of recombination with Alu elements on the additional chromosomes. Consistent with sequence findings in standard t(9;22) BCR-ABL rearrangements, these observations exclude both homologous and non-homologous recombination between Alu elements as a likely common mechanism generating complex BCR-ABL rearrangements.
We have shown that Alu sequences at the additional sites nonetheless appear to have an affinity for the BCR-ABL recombination process. Mutual attraction of Alu elements is an obvious explanation, and need not always be exact. Alu elements may hold chromosome regions near each other so that recombination is more likely to occur in their vicinity (26). The mechanisms which determine chromosome geography in the interphase nucleus remain controversial (27-29), but a potential role for Alu elements as structural modifiers of chromatin organization is recognized (30). It is relevant that, according to recently reported two-dimensional FISH studies of interphase lymphocytes, the BCR and ABL genes map closer together more often than expected by chance, providing the first evidence that physical juxtaposition of chromosomal parts may influence the frequency of t(9;22) recombination (31). The preferred involvement of sites in chromosome bands 3p21, 6p21, 7p22, 11q13, 12p13, 17q21, 17q25 and 19q13 in complex BCR-ABL translocations (32) may reflect a tendency for non-random nuclear compartmentalization to juxtapose these sites with 9q34 (ABL) and 22q11 (BCR). These preferred regions show a high density of Alu repeat elements and high transcriptional activity (33,34).
Transcriptionally active chromatin is more open in structure, has a high number of matrix attachment regions (MARs) and is prone to recombine more frequently than inactive chromatin (35-38). The 3[prime]M-Bcr recombination site disrupted coding regions of a gene in two of the five complex rearrangements examined [IGL, case No. 3; GSTPI,Koduru et al. (19)], and expressed sequence tag (EST) homology suggests coding regions of an actively transcribed gene close to the breakpoint in our case No. 1. Furthermore, although most Alu elements are silent in somatic cells, in two of our cases an Alu element of the evolutionarily younger Alu-Y subgroup mapped close to (case No. 1) or directly at (case No. 2) the breakpoint sites. These did not have features diagnostic of the actively retroposing Alu-Ya or -Yb subfamilies, but may yet belong to an active young family outside of the known subgroups (J. Jurka, personal communication). The transcriptional status in normal bone marrow stem cells of BCR, ABL and other genes so far described in complex rearrangements is presently unknown, but it is noteworthy that ABL, GSTP1 and Alu elements allshow increased expression in response to cell stress agents such as viral infection, chemicals or irradiation (19,39-42).
There are three other reported examples of the involvement of Alu elements in genomic rearrangements which lead to leukaemia. The first relates to BCR-ABL rearrangements found in a subgroup of patients diagnosed with acute leukaemia. Breakpoints occur within one of at least two minor breakpoint cluster regions, m-Bcr-1 and m-Bcr-2, in the large first intron of BCR (reviewed in ref. 43). Reciprocal 5[prime]m-Bcr-3[prime]ABL and 5[prime]ABL-3[prime]m-Bcr breakpoints of five patients have been isolated and sequenced to date. Four showed the presence of an Alu element either at, or within 70 bp of, the breakpoint on one or both participating BCR and ABL strands (6,43).
The second example is the 100 kb ALL-1 gene on 11q23 which is rearranged in a high proportion of infant and adult acute leukaemias (44). The ALL-1 gene has an 8 kb breakpoint cluster region which contains eight Alu repeats and may recombine with one of at least 25 different chromosome bands (44). Sequence analysis showed breaks within Alu elements of the ALL-1 breakpoint cluster region in five of 10 patients but, in contrast to our analysis of M-Bcr recombination sites, none showed breaks within Alu elements on the partner chromosomes (45). Instability of this breakpoint cluster region, especially its proneness to recombine with a wide range of other chromosomal sites, is attributed to localized chromatin structure and the high density of Aluelements rather than to recombination-prone features of particular elements within it (45,46). Tandem duplication of part of the coding segment of the ALL-1 gene, including the breakpoint cluster region, has also been described in acute leukaemia patients who present with a normal karyotype or trisomy 11 as the sole aberration in their leukaemic cells (47,48). Alu-mediated homologous recombination occurred in two of three cases for whom sequence information was available, and an Alu was disrupted at one of the breakpoint sites for the remaining case (49). A specific deletion found in ~5% of T-cell acute lymphoid leukaemias (ALLs) is a further indication that Alu elements in the ALL-1 breakpoint cluster region underly its instabilty. In three cases analysed, a 1634 bp deletion, including exon 8, occurred between a palindromic sequence 19 bp downstream of exon 7 (TGATCA) and a second palindrome unique to the first Alu element of intron 8 (CTCGAG) (50).
Analysis of 3q21 breakpoint sites in t(3;3)(q21;q26) acute myeloid leukaemia provides the third example. Recombination of different Alu repeats,located 30-40 kb downstream of the Ribophorin I gene in band 3q21, with non-Alu sequences upstream of the EVII gene in band 3q26, was identified in each of two cases analysed(51). Both of the Alu breakpoints occurred in the left arm, at a highly conserved site between the A and B boxes of the putative RNA polymerase III promotor which maps within the 26 bp recombinogenic core (22).
Breakpoints on the additional chromosome mapped within the recombinogenic core of the Alu consensus in one of our complex translocations (case No. 2), but not in the remaining three or in the case of Koduru et al. (19). Instead, almost identical tetrameric or pentameric motifs, or their complements, were mapped within 20 bp either side of all of the breakpoint sites in M-Bcr (CAGG and GTGG), and on the other involved chromosomes (CCCAG) (Fig. 5). These motifs suggest an enzyme- or protein-mediated recombination mechanism, and are similar to switch pentamers which flank IgH constant regions and the tetramer CTGG which is cleaved preferentially by the lymphoid-specific endonuclease, Endo-SR (23,24,52). However, it is unlikely that erroneous IgH switch activity underlies the complex BCR recombinations. Errors of switch recombination characteristically are associated with mature B-cell malignancies (53,54), and the transforming BCR-ABL mutation arises in a very early blood cell progenitor for which there is no evidence of active switch recombination processes. Furthermore, the pentamers of switch regions are usually repeated over several kilobases of DNA (23). We identified a high frequency of switch-like sequences (1 bp mismatch) across a 40 bp domain spanning the breakpoint sites in M-Bcr and on the other involved chromosomes, but no higher incidence or particular clustering compared with their distribution over the rest of the BCR gene or relevant sequenced regions (data not shown).
Sequence patterns at breakpoint sites in M-Bcr and its recombination partners also showed >80% similarity to the consensus DNA-binding sites of the Translin protein. This protein is detected in the cytoplasm of many cell types, but only cells of lymphoid lineage, specifically with rearranged Ig and TCR loci, showed a nuclear localization (25). Translin-binding sequences were identified previously at m-Bcr breakpoint junctions of three cases of BCR-ABL rearrangement-positive ALL (25). Our preliminary searches suggest that they may also be associated with BCR-ABL rearrangements of CML (unpublished data). Because of the high frequency of involvement of Alu elements at sites of 3[prime]M-Bcr recombination, we also searched specifically for Translin-related DNA-binding sequences within the Alu consensus (obtainable from CENSOR). This analysis revealed surprisingly close homology at several sites (data not shown), and indicates that the Translin protein theoretically could bind to one of the most ubiquitous repeat elements in the genome. A better understanding of Translin function, including the mechanisms which regulate its expression and determine its DNA-binding specificity, is required to interpret the significance of these associations.
MATERIALS AND METHODS
Patients
Peripheral blood or bone marrow samples from four CML patients showed different, cytogenetically complex Ph translocations and rearrangement of M-Bcr in Southern blots. Case history, karyotype and some molecular features were described previously: case No. 1 (case 3 in ref. 55), case No. 2 (case 1 in ref. 56; also in ref. 17), case No. 3 (case 2 in ref. 56) and case No. 4 (case 4 in ref. 55; also in ref. 20).
Probes
Probes used in this study included M-Bcr-5[prime], a 2 kb BglII-HindIII fragment, and M-Bcr-3[prime], a 1.2 kb HindIII-BglII fragment (Fig. 2); a 479 bp BglII-BalI fragment which maps upstream of the 7q11.23 breakpoint in case No. 1 (Fig. 2); a 400 bp BamHI fragment which maps upstream of the 9q34 breakpoint in case No. 2 (Fig. 2); and a 526 bp EcoRI-XbaI fragment from upstream of the breakpoint site in 22q11.2 of case No. 3 (Fig. 2).
Southern blot analysis
High molecular weight DNA isolated from leukaemic peripheral blood or bone marrow cells was digested with restriction enzymes, electrophoresed, Southern blotted onto Hybond N+ membrane and hybridized using methods previously described (17). Repeat sequences in probes were CotI suppressed (Gibco BRL, Life Technologies Inc., Gaithersburg, MD).
Construction and screening of genomic libraries
For cases Nos 1, 2 and 4, aliquots of leukaemic cell DNA were partially restricted with MboI to a modal size of 9-23 kb, alkaline phosphatased to prevent self-ligation (57), then ligated to the BamHI sites of [lambda]DASH and packaged in vitro with the Gigapak Gold system (Stratagene, La Jolla, CA). For case No. 3, EcoRI-restricted leukaemic DNA was ligated to EcoRI-digested arms of [lambda]gt10. A previously described partial Sau3A genomic library in [lambda]2001 (58) was used to isolate germline fragments extending across the site with which BCR recombined on chromosome bands 9q34 (case No. 2), 22q11 (case No. 3) and 11q13 (case No. 4) (20). The germline fragment which spanned the breakpoint site in 7q11.23 (case No. 1) was purified from the normal chromosome 7 homologue in the patients leukaemic DNA library. Phage libraries on nylon membranes were screened by hybridization with radioactive probes using described methods (17).
DNA sequencing
DNA subcloned into Bluescript (Stratagene) was prepared for double-stranded sequencing by nested deletions (Pharmacia LKB Biotechnology, Uppsala, Sweden) and manually sequenced using universal T3 and T7 primers with either Sequenase v2.0 (US Biochemical Corp., OH) or an f-mol cycle sequencing kit (Promega). Fluorescent cycle sequencing reactions, including direct sequencing of PCR products, were performed using Thermosequenase (Amersham, Buckinghamshire, UK) with universal (M13 forward, M13 reverse, T3, T7) or custom IRD41-labelled primers (MWG Biotech, Germany), and run on a LI-COR 4000L automated DNA sequencer.
Sequence analysis
Consensus sequences derived from overlapping fragments using Staden's sequence assembly program (SAP) or the Wisconsin Genetics Computer Group (GCG) sequencing package were compared with the most recent sequence databases using BLASTN (http://www.ncbi.nlm.nih.gov/BLAST/) (September 19, 1997). CENSOR (http://charon.lpi.org/~server/censor.html) (Release 6.2.1; August 1997) was used to identify repeat elements. To identify common sequence, 40 bp across the different germline M-Bcr breakpoint sites or the five recombination sites on other chromosomes (7q11.23, 9q34, 22q11, 11q13, GSTPI) were aligned using the software program CLUSTALW (http://alfredo.wustl.edu/msa/clustal_old.cgi). All possible pairwise combinations of these fragments were also compared to obtain maximum information. Output from CLUSTALW was submitted to BOXSHADE, a program for pretty printing and consensus derivation of multiply aligned sequences (http://ulrec3.unil.ch/software/BOX_form.html). The GCG program FINDPATTERNS was used to search for Translin, switch, heptamer, nonamer and other cited recombinogenic motifs. Putative coding domains were identified by extracting repeat-free regions from the CENSOR asap output file for each of the four germline fragments, and comparing them, in turn, against the non-redundant (NR) nucleotide sequence and expressed sequence tag (DBEST) databases (September 19, 1997). Sequence accession numbers are as follows: AFO45527, AFO45528, AFO45529, AFO45530, AFO45531, AFO45532, AFO45533 and AFO45605.
Fluorescent in situ hybridization (FISH)
Human metaphase cells were prepared from phytohaemagglutinin (PHA)-stimulated blood of a normal male donor using routine culture and cell synchronization procedures. Probes were biotinylated, and hybridization, washing and probe detection procedures were as described (20). Probes used for FISH were: [lambda]A/6.2.1 (case No. 1, germline 7q11.23 breakpoint region), [lambda]Be/8.2.1 (case No. 2, germine 9q34 breakpoint region), [lambda]Bi/11.1.1 (case No. 3, germline 22q11.2 breakpoint region) and [lambda]K/9.1.1.1 (case No.4, germline 11q13 breakpoint region).
ACKNOWLEDGEMENTS
We thank A.E. Reeve and L.J. Millow for making clone pLM-5 available for this study; P. Rodley for assistance in confirming the restriction maps of [lambda] A1.7.1.1 and [lambda]A6.2.1, and P.E. Crossen, M.A. Kennedy and J. Jurka for helpful comments. We are especially grateful to P.H. Fitzgerald for his encouragement throughout the project and criticisms of the final manuscript. This work was generously supported by The Cancer Society of New Zealand, Robert McClelland Cancer Research Trust, Health Research Council of New Zealand, New Zealand Lottery Grants Board (Health) and the Canterbury Medical Research Foundation. C.M. is a Senior Travis Trust Cancer Research Fellow.
REFERENCES
This page is run by Oxford University Press, Great Clarendon Street, Oxford OX2 6DP, as part of the OUP Journals
Comments and feedback: www-admin{at}oup.co.uk
Last modification: 4 Apr 1998
Copyright© Oxford University Press, 1998.
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  E. M. Seitz and S. C. Kowalczykowski Human Rad51 protein displays enhanced homologous pairing of DNA sequences resembling those at genetically unstable loci Nucleic Acids Res., May 24, 2006; 34(10): 2847 - 2852. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. M. Weinstock, B. Elliott, and M. Jasin A model of oncogenic rearrangements: differences between chromosomal translocation mechanisms and simple double-strand break repair Blood, January 15, 2006; 107(2): 777 - 780. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N Haruki, K S Kawaguchi, S Eichenberger, P P Massion, A Gonzalez, A F Gazdar, J D Minna, D P Carbone, and T P Dang Cloned fusion product from a rare t(15;19)(q13.2;p13.1) inhibit S phase in vitro J. Med. Genet., July 1, 2005; 42(7): 558 - 564. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Libura, D. J. Slater, C. A. Felix, and C. Richardson Therapy-related acute myeloid leukemia-like MLL rearrangements are induced by etoposide in primary human CD34+ cells and remain stable after clonal expansion Blood, March 1, 2005; 105(5): 2124 - 2131. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
W Borozdin, D Boehm, M Leipoldt, C Wilhelm, W Reardon, J Clayton-Smith, K Becker, H Muhlendyck, R Winter, O Giray, et al. SALL4 deletions are a common cause of Okihiro and acro-renal-ocular syndromes and confirm haploinsufficiency as the pathogenic mechanism J. Med. Genet., September 1, 2004; 41(9): e113 - e113. [Full Text] [PDF]
|
|
|
|
|
|
E. Kolomietz, J. Al-Maghrabi, S. Brennan, J. Karaskova, S. Minkin, J. Lipton, and J. A. Squire Primary chromosomal rearrangements of leukemia are frequently accompanied by extensive submicroscopic deletions and may lead to altered prognosis Blood, June 1, 2001; 97(11): 3581 - 3588. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A.J.R. Bishop, B. Kosaras, N. Carls, R.L. Sidman, and R.H. Schiestl Susceptibility of proliferating cells to benzo[a]pyrene-induced homologous recombination in mice Carcinogenesis, April 1, 2001; 22(4): 641 - 649. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. Laurent, M. Talpaz, H. Kantarjian, and R. Kurzrock The BCR Gene and Philadelphia Chromosome-positive Leukemogenesis Cancer Res., March 1, 2001; 61(6): 2343 - 2355. [Full Text]
|
|
|
|
 |
|
 C. A. Mundt, I. C. Nicholson, X. Zou, A. V. Popov, C. Ayling, and M. Bruggemann Novel Control Motif Cluster in the IgH {{delta}}-{{gamma}}3 Interval Exhibits B Cell-Specific Enhancer Function in Early Development J. Immunol., March 1, 2001; 166(5): 3315 - 3323. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 I. Panagopoulos, T. Fioretos, M. Isaksson, U. Samuelsson, R. Billstrom, B. Strombeck, F. Mitelman, and B. Johansson Fusion of the MORF and CBP genes in acute myeloid leukemia with the t(10;16)(q22;p13) Hum. Mol. Genet., February 1, 2001; 10(4): 395 - 404. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Richardson and M. Jasin Coupled Homologous and Nonhomologous Repair of a Double-Strand Break Preserves Genomic Integrity in Mammalian Cells Mol. Cell. Biol., December 1, 2000; 20(23): 9068 - 9075. [Abstract] [Full Text]
|
|
|
|
|
|
A. J.R. Bishop and R. H. Schiestl Homologous recombination as a mechanism for genome rearrangements: environmental and genetic effects Hum. Mol. Genet., October 1, 2000; 9(16): 2427 - 2334. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. S. Hill, N. J. Foot, T. L. Chaplin, and B. D. Young The most frequent constitutional translocation in humans, the t(11;22)(q23;q11) is due to a highly specific Alu-mediated recombination Hum. Mol. Genet., June 12, 2000; 9(10): 1525 - 1532. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. W. Touchman, Y. Anikster, N. L. Dietrich, V. V. B. Maduro, G. McDowell, V. Shotelersuk, G. G. Bouffard, S. M. Beckstrom-Sternberg, W. A. Gahl, and E. D. Green The Genomic Region Encompassing the Nephropathic Cystinosis Gene (CTNS): Complete Sequencing of a 200-kb Segment and Discovery of a Novel Gene within the Common Cystinosis-Causing Deletion Genome Res., February 1, 2000; 10(2): 165 - 173. [Abstract] [Full Text]
|
|
|
|
|
|
L. Edelmann, R. K. Pandita, E. Spiteri, B. Funke, R. Goldberg, N. Palanisamy, R. S. K. Chaganti, E. Magenis, R. J. Shprintzen, and B. E. Morrow A common molecular basis for rearrangement disorders on chromosome 22q11 Hum. Mol. Genet., July 1, 1999; 8(7): 1157 - 1167. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Neves, C. Ramos, M. G. da Silva, A. Parreira, and L. Parreira The Nuclear Topography of ABL, BCR, PML, and RARalpha Genes: Evidence for Gene Proximity in Specific Phases of the Cell Cycle and Stages of Hematopoietic Differentiation Blood, February 15, 1999; 93(4): 1197 - 1207. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Richardson, M. E. Moynahan, and M. Jasin Double-strand break repair by interchromosomal recombination: suppression of chromosomal translocations Genes & Dev., December 15, 1998; 12(24): 3831 - 3842. [Abstract] [Full Text]
|
|
|
|
 |
|
 V. M. Chennathukuzhi, Y. Kurihara, J. D. Bray, and N. B. Hecht Trax (Translin-associated Factor X), a Primarily Cytoplasmic Protein, Inhibits the Binding of TB-RBP (Translin) to RNA J. Biol. Chem., April 13, 2001; 276(16): 13256 - 13263. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||