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INTRODUCTION |
V(D)J recombination is a physiological recombination of DNAs,
which occurs at the Ig and T cell receptor loci during the
differentiation of lymphoid cells (1, 2). These loci have recombination signal sequences (RSSs)1
consisting of a highly conserved heptamer sequence (consensus 5'-CACAGTG) and an AT-rich nonamer sequence (consensus 5'-ACAAAAACC). The recombination-activating gene (RAG) complex generates DNA double
strand breaks (DSBs) at pairs of RSSs in which one signal has a 12-bp
spacer between the heptamer and nonamer (12-signal) and the other
signal has a 23-bp spacer (23-signal) (1, 2). This is known as the
12/23 rule. Several nucleotides are inserted as junctional additions at
the junctions, and the broken DNA ends are rejoined by the
nonhomologous end joining pathway (1-3). The results of V(D)J
recombination are that coding junction products and, in some cases such
as the Ig 
locus, signal junction products are left in the human
genomic DNA (1). It has been suggested that recurrent chromosomal
aberrations in lymphoid malignancies, such as translocations and
inversions involving the Ig and T cell receptor loci, are caused by
illegitimate actions of the V(D)J recombination machinery at
"ectopic" RSSs, the sequences homologous to the consensus RSS at
non-Ig, non-T cell receptor loci (4-9). These chromosomal aberrations
cause the activation or fusion of several oncogenes leading to
leukemogenesis. In a large portion of these aberrations, insertions of
nucleotides were detected between the two joined germ line sequences.
In addition, recently, several DNA fragments containing the ectopic
RSSs were shown to have potential to undergo V(D)J recombination
in vivo (10-12). Thus, it was indicated that illegitimate
V(D)J recombination is a major cause for the occurrence of these
chromosomal aberrations.
Deletion of the chromosome 9p21 region is a crucial event for the
development of lymphoid leukemia irrespective of lineages, since
homozygous deletions of this region have been found in 20-50% of
lymphoid leukemia both of T- and B-lineages (13, 14). The chromosome
9p21 segment contains the 40-kb region encoding the p16/CDKN2A tumor suppressor gene and two other
related genes, p14/ARF and
p15/CDKN2B, all of which encode critical factors
for the regulation of cell cycle and/or apoptosis (15, 16). Previously, Cayuela et al. (17) characterized three sites of breakpoint clustering in T cell acute lymphoblastic leukemias (T-ALLs) in the
40-kb region. The three sites consisted of a major breakpoint cluster
site, MTS1bcr
, and two minor sites, MTS1bcr
and MTS2bcr1 (see
Fig. 1A). All three sites contained heptamer-RSS-like
sequences; therefore, it was suggested that illegitimate V(D)J
recombination is involved in the occurrence of 9p21 deletions in T-ALL.
However, in that study, structures of only a small number of
breakpoint-containing regions in leukemia cells only of T-lineage were
analyzed. In addition, the involvement of V(D)J recombination was
suggested only by the sequences of breakpoints and was not functionally assessed. Therefore, it still remains largely obscure whether illegitimate V(D)J recombination is a major cause for 9p21 deletions in
lymphoid leukemia.
In the present study, 30 breakpoints for 9p21 deletions were cloned in
15 human lymphoid leukemia cell lines consisting of nine T- and six
B-lineage lines. The breakpoints were distributed at 18 different sites
in the 9p21 segment. Seventeen (57%) of the breakpoints were mapped at
five breakpoint cluster sites, BCS-LL1 to LL5, each of <15-bp. One of
the breakpoint cluster sites, BCS-LL2, was the same as MTS1bcr
(17), whereas the remaining four sites were novel. Two
breakpoint clustering sites were located within the 40-kb region
encoding the CDKN2A, ARF, and CDKN2B
genes, whereas the remaining three were located >100 kb apart from the 40-kb region. Thus, the results further supported the previous finding
of breakpoint clustering for 9p21 deletions in lymphoid leukemia. All
five BCS-LLs and seven other breakpoint sites contained the CAC
trinucleotide, which is identical to the first three nucleotides of
heptamer-RSSs indispensable for V(D)J recombination. The sequences of
breakpoint junctions indicated that deletions in the majority (11 lines, 73%) of the cell lines were mediated by illegitimate V(D)J
recombination preferentially targeted at BCS-LLs, and such a
recombination occurred in both the T- and B-lineage cell lines. Thus,
we further performed an extrachromosomal V(D)J recombination assay to
functionally assess the V(D)J recombination potential of BCS-LLs. All
of the four BCS-LL fragments tested were shown to have the potential to
undergo V(D)J recombination as RSSs, whereas the efficiency and
specificity of the potential were different among the fragments. The
present results strongly indicated that illegitimate V(D)J
recombination, which was targeted at RSS-like sequences widely
distributed in the 9p21 segment, causes a major (>70%) fraction of
9p21 deletions in lymphoid leukemia of both T- and B-lineages. There
was no evidence of V(D)J recombination at the breakpoint junctions of
the remaining four (27%) cell lines, raising the possibility that 9p21
deletions in a small subset of lymphoid leukemia were caused by
mechanisms other than illegitimate V(D)J recombination.
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EXPERIMENTAL PROCEDURES |
Cell Lines--
High molecular weight DNA prepared from 42 human
lymphoid leukemia cell lines was used for the breakpoint analyses. The
cell lines consisted of 21 T-lymphoid lineage cell lines (MOLT-14, L-SAK, JURKAT, SKW-3, RPMI-8402, KAWAI, MOLT-16, ALL-SIL, H-SB2, CCRF-CEM, THP-6, P12/Ichikawa, MOLT-4, L-SMY, PEER, KOPT-K1, DND-41, HPB-ALL, KCMC-T, TALL-1, and KOPT-4) and 21 B-lymphoid lineage cell
lines (LC4-1, NALL-1, LAZ-221, NALM-6, NALM-16, NALM-17, NALM-26,
BAL-KH, KOPN-K, THP-7, HAL-01, HPB-NULL, UTP-L5, TAKEDA, KOPN-30,
KOPN-36, KOPN-55bi, JOK-1, BALM-1, BALM-9, and BALM-13). ALLs
classified as early pre-B ALL, pre-B ALL, or immature B ALL are
collectively described as B cell precursor ALL in this paper. Information on some of these cell lines was described previously (13,
18, 19). Information on other cell lines can be obtained upon request.
A human pre-B ALL cell line, Reh, and a human erythroleukemia cell
line, HEL, were used for the extrachromosomal V(D)J recombination assay. They were maintained in RPMI 1640 medium supplemented with 10%
fetal calf serum, 100 units of penicillin G/ml, 100 µg of streptomycin sulfate/ml. For the Reh cell line, 50 µM
mercaptoethanol was also added, as described previously (11).
Mapping of Breakpoints for 9p21 Deletions--
Sixteen loci in
the 9p21 region were first screened for homozygous deletions.
Information on microsatellite markers, D9S1748 and D9S1752, was
obtained from the Genome Database (available on the World Wide Web at
www.gdb.org/gdb/). The primer pairs used for the analysis of the
MTAP, p16/CDKN2A,
p15/CDKN2B, and p14/ARF loci were reported previously (20-22). The primer pairs for newly developed STSs (sequence-tagged sites) in the 9p21 regions were AC47
(5'-GACTAGGGAGGATACCTCAA-3' and 5'-TGGACTTAACATGTTGCCTCA-3'), C86 (5'-TGGCAAGTCAGATGGCAGAC-3' and 5'-GGAGCAAGTGGCATCTCACT-3'), CDKN2A/ARF-exon 1 (5'-TGCTCACCTCTGGTGCCAAAG-3' and
5'-CTCAGTAGCATCAGCACGAGG-3'), and CDKN2A/ARF-exon 3 (5'-GGATGTGCCACACATCTTTG-3' and 5'-ATGAAAACTACGAAAGCGGG-3'). A
number of additional STSs were developed to precisely map the locations
of deletion breakpoints in each cell line. Information of these STSs
can be obtained upon request. Fifty ng of genomic DNA was dissolved in
a total of 20 µl of PCR buffer containing 10 mM Tris-HCl
(pH 9.0), 50 mM KCl, 1.5 mM MgCl2,
250 µM deoxynucleotide triphosphate, 125 ng of each
primer, and 0.1 unit of Taq polymerase (Amersham
Biosciences). PCR amplification was performed for 35 cycles consisting
of denaturation at 94 °C for 60 s, annealing at 55 °C for
60 s, and extension at 72 °C for 90 s, except for the
CDKN2A/ARF-exon 1, CDKN2A/ARF-exon 2, CDKN2B-exon 1, and CDKN2B-exon
2. PCR conditions for these primers have already been described (21,
22). The amplified products were visualized on ethidium bromide-stained
agarose gels.
Cloning of Genomic DNA Fragments Containing Breakpoint
Junctions--
Breakpoints were cloned by the inverse PCR method (23)
or genomic PCR using primers that recognize sequences flanking distal and proximal breakpoints, respectively. The inverse PCR method had some
modifications as follows; briefly, DNA (1 µg) was digested with 10 units of restriction enzyme with 4- or 6-base pair recognition sites.
For circularization, the digested DNA was ligated in a 100-µl
reaction with 6 units of T4 DNA ligase (Promega, Madison, WI) and a
ligation buffer at 16 °C. After purification by using a QIA quick
spin PCR purification kit (Qiagen, Tokyo, Japan), circular products
were amplified by PCR using primers that recognize sequences located on
the known part of the rearranged fragment. PCR products were sequenced
directly or after they were subcloned into the pGEM-T easy vector by
using a pGEM-T easy vector system I kit (Promega). To determine DNA
sequences, a Big Dye Terminator cycle sequencing ready reaction kit
(PerkinElmer Life Sciences) and the ABI 310 DNA Sequence System were
used. Sequences of the DNA fragments obtained by the inverse PCR method
were confirmed by direct sequencing of the PCR products, which were
obtained by genomic PCR using the primers that recognize sequences
flanking distal and proximal breakpoints, respectively.
Construction and Analysis of a Contiguous Sequence at
9p21--
Sequences deposited in GenBankTM, AC000047,
AC000048, AC000049, AL359922, and AL449423, were processed by the SEQUENCHER program, version 3.1.1 (GENE CODES, Ann Arbor, MI). A
sequence of 291,432 bp with a single gap, whose size is unknown, at
position 238,144-238,145 was obtained. A genomic fragment of ~800 bp
containing the gap was amplified by PCR from the BAC clone DNA, 436N8,
which contains the CDKN2A, CDKN2B, and
ARF loci (22). The PCR products were subcloned into the
pGEM-T easy vector by using a pGEM-T easy vector system I kit (Promega)
and sequenced by using a Big Dye Terminator cycle sequencing ready
reaction kit (PerkinElmer Life Sciences) and the ABI 310 DNA Sequence
System. The length of the gapped region was 64 bp. Consequently, a
contiguous sequence of 291,496 bp, covering the MTAP,
CDKN2A, CDKN2B, and ARF loci, was
obtained. Sequence homology was examined by the BLAST2 program
(available on the World Wide Web at www.ncbi.nlm.nih.gov/blast/bl2seq/bl2.html).
Construction of Plasmids--
Plasmids for the extrachromosomal
V(D)J recombination assay were made by modifying the pGG49 plasmid
(24). The following oligomers phosphorylated at the 5'-ends were
supplied by Hokkaido System Science (Sapporo, Japan) and Amersham
Biosciences with SalI and BamHI complementary
ends to facilitate cloning: LL1-12F, 5'-TCGACCACAACAGTTCCTGGCCCATCTTTGCTCTGTCAG-3'; LL1-12R,
5'-TCGACTGACAGAGCAAAGATGGGCCAGGAACTGTTGTGG-3'; LL1-23F,
5'-GATCCCACAACAGTTCCTGGCCCATCTTTGCTCTGTCAGTAATTTTTGG-3'; LL1-23R,
5'-GATCCCAAAAATTACTGACAGAGCAAAGATGGGCCAGGAACTGTTGTGG-3'; LL2-12F,
5'-TCGACCACAGTAGGAAAGGTGTATTTCAAGCACACTTTG-3'; LL2-12R, 5'-TCGACAAAGTGTGCTTGAAATACACCTTTCCTACTGTGG-3'; LL2-23F,
5'-GATCCCACAGTAGGAAAGGTGTATTTCAAGCACACTTTCTTTCTCCTTG-3'; LL2-23R,
5'-GATCCAAGGAGAAAGAAAGTGTGCTTGAAATACACCTTTCCTACTGTGG-3'; LL3-12F,
5'-TCGACCACAGTACTTAGTTCCTTTTATGTTAGCAGTGTG-3'; LL3-12R, 5'-TCGACACACTGCTAACATAAAAGGAACTAAGTACTGTGG-3'; LL3-23F,
5'-GATCCCACAGTACTTAGTTCCTTTTATGTTAGCAGTGTCTTATGAGTGG-3'; LL3-23R,
5'-GATCCCACTCATAAGACACTGCTAACATAAAAGGAACTAAGTACTGTGG-3'; LL5- 12F, 5'-TCGACCACATCATTTTGTCTCCTTTACTTTTCCATAGGG-3'; LL5-12R, 5'-TCGACCCTATGGAAAAGTAAAGGAGACAAAATGATGTGG-3'; LL5-23F,
5'-GATCCCACATCATTTTGTCTCCTTTACTTTTCCATAGGCTGTTTTTTTG-3'; LL5-23R,
5'-GATCCAAAAAAACAGCCTATGGAAAAGTAAAGGAGACAAAATGATGTGG-3', Mutant
LL3- 12F, 5'-TCGACGACAGTACTTAGTTCCTTTTATGTTAGCAGTGTG-3'; and Mutant
LL3-12R, 5'-TCGACACACTGCTAACATAAAAGGAACTAAGTACTGTCG-3'. Oligomers LL1-12F and LL1-12R, LL1-23F and LL1-23R, LL2-12F and LL2-12R, LL2-23F and LL2-23R, LL3-12F and LL3-12R, LL3-23F and LL3-23R, LL5-12F and LL5-12R, LL5-23F and LL5-23R, and Mutant LL3-12F and Mutant LL3-12R were annealed, respectively, and the annealed (double-stranded) oligomers were cloned into the pGG49 vector
after removing the 12-signal (by SalI digestion), the
23-signal (by BamHI digestion), or both. The
LL1-12F/LL1-12R and LL1-23F/LL1-23R oligomers carrying the BCS-LL1
region were cloned into the SalI site and the
BamHI site of pGG49, respectively (pLL1-RSS and pRSS-LL1). The LL2-12F/LL2-12R and LL2-23F/LL2-23R oligomers carrying the BCS-LL2 region were cloned into the SalI site and the
BamHI site of pGG49, respectively (pLL2-RSS and pRSS-LL2).
The LL3-12F/LL3-12R and LL3-23F/LL3-23R oligomers carrying the
BCS-LL3 region were cloned into the SalI site and the
BamHI site of pGG49, respectively (pLL3-RSS and pRSS-LL3).
The LL5-12F/LL5-12R and LL5-23F/LL5-23R oligomers carrying the
BCS-LL5 region were cloned into the SalI site and the
BamHI site of pGG49, respectively (pLL5-RSS and pRSS-LL5). The Mutant LL3-12F/Mutant LL3-12R were cloned into the
SalI site of pGG49 (pLL3M-RSS). For pLL3-LL1,
LL3-12F/LL3-12R and LL1-23F/LL1-23R oligomers were cloned into
SalI site and the BamHI site of pGG49, respectively, after removing both the 12-signal (by SalI
digestion) and the 23-signal (by BamHI digestion).
Extrachromosomal V(D)J Recombination Assay--
The human
leukemia cell lines were grown logarithmically, transfected with the
appropriate plasmid substrates using the DEAE-dextran electroporation
method (25), and cultured for 48 h at 37 °C. Subsequently,
plasmid DNA was recovered from the cells and digested with
DpnI. DpnI cleaves plasmid that does not lose its
prokaryotic dam methylation pattern by replicating in eukaryotic cells.
Because only molecules replicated in eukaryotic cells remain undigested by DpnI and will transform the bacteria, after digestion,
they were electrotransformed into DH10B Escherichia coli
(Invitrogen). Bacterial transformants were plated on media containing
ampicillin alone (100 µg/ml) and on ampicillin and chloramphenicol
(100 and 33 µg/ml, respectively). In bacteria, all substrates confer
ampicillin resistance (designated as Ampr). Recombinants,
which resulted in the deletion of the prokaryotic transcription
terminator on the substrates, allow cat (chloramphenicol acetyltransferase) gene expression in bacteria and thus confer both
ampicillin and chloramphenicol resistance (designated as AmprCPr) (24). The ratio of
AmprCPr colonies to Ampr colonies
reflects the fraction of recovered substrates that underwent recombination leading to deletions of the prokaryotic transcription terminator on the substrates. The averaging of multiple different eukaryotic transfections of the same substrate was performed by dividing the sum of the numbers of AmprCPr
colonies from each transfection by the sum of the numbers of Ampr colonies. Thus, recombination frequency was expressed
as the weighted average (equal to
(
AmprCPr)/(Ampr)).
Recombinant Analyses--
Individual
AmprCPr colonies were picked up and were
subjected to PCR amplification using primers
5'-AGGCCCTTTCGTCTTCAAGA-3' and 5'-AACGGTGGTATATCCAGTGA-3',
which flank the SalI and BamHI sites in
pGG49, respectively. PCR was performed under the following conditions:
10 mM Tris-HCl, 50 mM KCl, 1.5 mM
MgCl2, 250 µM dNTP each, 0.5 units of
Taq polymerase (TaKaRa, Tokyo, Japan) in a volume of 20 µl. PCR amplification was performed for 35 cycles consisting of
denaturation at 95 °C for 60 s, annealing at 55 °C for
60 s, and extension at 72 °C for 60 s. PCR products were purified by a QIA quick spin PCR purification kit (Qiagen, Tokyo, Japan) and were directly sequenced by a Big Dye Terminater cycle sequencing kit and the ABI 310 DNA Sequence System (PerkinElmer Life Sciences).
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RESULTS |
Cloning of Breakpoint Junctions in Lymphoid
Leukemia--
Forty-two lymphoid leukemia cell lines (21 T-lineage and
21 B-lineage) were examined for homozygous deletions of the 9p21 region
using 16 STS markers mapped in the 9p21 region. The markers consisted
of 12 markers derived from exons of the CDKN2A,
CDKN2B, ARF, and MTAP
(methylthioadenosine phosphorylase) genes, two microsatellite markers
(D9S1748 and D9S1752), and two novel STS markers (AC47 and C86) and
were distributed in the 240-kb region in the 9p21 segment (STS in Fig.
1). Homozygous deletions at one or more
of the tested loci were detected in 31 cell lines (74%). Ten (32%) of
them showed deletion of all the 16 markers, indicating that the whole
240-kb region was deleted in these cell lines. Thus, they were not
further investigated in this study. The remaining 21 (68%) cell lines
showed retention of one or more markers among the 16 markers (Fig. 1).
In 12 of the 21 cell lines, some markers from both the telomeric and
centromeric regions were retained; therefore, interstitial deletions
occurred in the 240-kb region. Thus, both the distal (telomeric side)
and proximal (centromeric side) breakpoints were suggested to be
present in the 240-kb region (cases 1-12 in Fig. 1). In the remaining
nine cell lines, some of the 16 markers either from the telomeric or
centromeric regions were retained; therefore, either the distal or
proximal breakpoints were suggested to be present in the 240-kb region
(cases 13-21 in Fig. 1). In total, 33 breakpoints, which consisted of
17 distal and 16 proximal ones, were indicated to be present in the
240-kb region (Fig. 1).
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Fig. 1.
Mapping of breakpoints for 9p21
deletions. The locations of 16 STSs used for the initial screening
are shown. Twenty-four regions of 10 kb, A to X, are shown with the
number of breakpoints in each region. Regions of homozygous deletions
in 21 cases are shown with distal (red) and/or proximal
(blue) breakpoints, which were mapped at a 10-kb resolution.
One and two breakpoints were mapped to the A-B and I-K regions of
20 and 30 kb, respectively. Breakpoints clustered within a 50-bp
region in the C, T, and U regions are marked with asterisks.
1, H-SB2; 2, TAKEDA; 3, KOPN-55bi;
4, CCRF-CEM; 5, P12/Ichikawa; 6,
KAWAI; 7, HPB-NULL; 8, MOLT4; 9,
KOPN-30; 10, L-SMY; 11, KOPT-K1; 12,
PEER; 13, RPMI-8402; 14, HAL-01; 15,
THP-7; 16, ALL-SIL; 17, MOLT-16; 18,
NALM-16; 19, SKW-3; 20, KOPN-K; 21,
THP-6. Cases subjected to breakpoint cloning are in orange
or green (see "Results"). Breakpoints flanked by
RSS-like sequences are shaded.
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The distributions of these 33 breakpoints were further examined. The
240-kb region in 9p21 was divided into 24 regions of 10 kb, as shown in
Fig. 1, and the breakpoints were mapped within the regions of "A"
to "X" by using a number of STS markers prepared by the authors.
Thirty breakpoints were mapped into some of the 24 regions, whereas one
and two breakpoints were mapped to the A-B and I-K regions of 20 and 30 kb, respectively, since they were located in contiguous repetitive
sequences extending from the A to B and I to K regions (Fig. 1). The C,
T, and U regions contained 5, 10, and 5 breakpoints, respectively,
suggesting the clustering of breakpoints (Fig. 1). Meanwhile, other
10-kb regions contained less than five breakpoints. More precise
mapping of the breakpoints in the C, T, and U regions using STSs placed
at 50-bp intervals revealed that four breakpoints in region C, five in
T, and all five in U were clustered within a 50-bp region in the C, T,
and U regions, respectively (breakpoints with asterisks in
Fig. 1). The remaining breakpoints, one in C and five in T, were not
mapped within 50 bp of other breakpoints. The clustered 14 breakpoints
were derived from 11 cell lines (cases in orange, Fig. 1).
Therefore, genomic DNA fragments containing breakpoint junctions were
cloned from the 11 cell lines by inverse PCR or genomic PCR using sets
of primers recognizing the regions flanking the breakpoints. We also
cloned DNA fragments containing breakpoint junctions from four other
cell lines, KAWAI, HPB-NULL, KOPN-30, and THP-7, as representatives for
the cell lines with unclustered breakpoints (cases in green,
Fig. 1). In total, breakpoints were cloned from 15 cell lines
consisting of nine T- and six B-lineage ones.
The nucleotide sequences of these DNA fragments were compared with that
of the 9p21 segment. All of the deletions in the 15 cell lines
subjected to breakpoint cloning were simple interstitial ones (Fig. 1);
thus, the structures of 30 breakpoints at 15 junctions were defined.
Distal breakpoints for two cell lines, KOPN30 and PEER, were first
mapped to the K and T regions (cases 9 and 12, Fig. 1), respectively;
however, the breakpoints that had been joined with proximal breakpoints
by inverse PCR analysis were mapped to the regions more telomeric than
deduced (the J and D regions, respectively) (Fig. 1). Therefore, it was
indicated that these two cell lines have two different alleles with
deletions overlapping each other. Twenty-nine (97%) breakpoints
consisting of 14 proximal and 15 distal breakpoints were assigned to
the 250-kb region (the 240-kb segment analyzed plus an adjacent distal 10-kb segment) containing the CDKN2A, CDKN2B,
ARF, and MTAP genes, whereas the proximal
breakpoint of THP-7 was mapped to the region ~6.5 Mb proximal to the
C86 locus outside the 250-kb region (Fig. 1).
Presence of Multiple Breakpoint Cluster Sites in 9p21--
Four
and five breakpoints mapped within a 50-bp region in the C and U
regions, respectively, were clustered within a 10- and 14-bp interval,
respectively. We designated the breakpoint cluster sites in C and U as
BCS-LL (breakpoint cluster sites in
lymphoid leukemia) 1 and 3, respectively (Fig.
2, A and B). Five
breakpoints in the T region were clustered within a CA/GT tract in the
D9S1748 locus. This locus was the same as MTS1bcr, which had been
defined as a major breakpoint cluster site for primary T-ALLs in a
previous study (17). The length of the CA/GT tract in the D9S1748 locus is known to be highly polymorphic, and the corresponding noncancerous DNAs for these five cell lines were not available. Therefore, these
five breakpoints were assigned between the TACTGTG heptamer and the
T-G/G-T dinucleotides 3' to the TACTGTG heptamer so that this heptamer
sequence flanked the breakpoints (Fig. 2B). We designated this locus as BCS-LL2 to simplify the names of breakpoint cluster sites
in this study. BCS-LL1 was located in the intron 4 of MTAP, a neighboring gene of CDKN2A and ARF. BCS-LL2 was
located ~300 bp downstream of exon 1 of the ARF gene,
while BCS-LL3 was located in intron 1 of the CDKN2B
gene.
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Fig. 2.
Mapping of breakpoint cluster sites.
A, locations of breakpoint cluster sites. The locations of
breakpoint cluster sites defined in this and/or a previous study (17)
are shown with those of genes and their exons. B, nucleotide
sequences of five breakpoint cluster sites. Locations of breakpoints
are indicated by the arrows. Heptamer-RSS-like sequences are
boxed. Locations of the breakpoints in two cases of primary
T-ALL, which were previously identified (17), are marked with
asterisks.
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In addition, the presence of two other breakpoint cluster sites was
indicated. Distal breakpoints of two cell lines, KOPN-K and SKW-3, were
assigned to the same position of ~10 kb distal to the MTAP-exon 1 locus, indicating the presence of a breakpoint cluster site (Fig. 2,
A and B). The distal breakpoint of PEER was
located 3 bp proximal to the distal breakpoints of two primary T-ALLs,
which had been previously assigned (17), indicating the presence of
another site (Fig. 2, A and B). This site was located in intron 4 of the MTAP gene. We designated these
breakpoint cluster sites as BCS-LL4 and BCS-LL5, respectively.
In total, 17 (57%) of the 30 breakpoints identified in this study were
located at the five BCS-LLs (Fig. 2B), while the remaining 13 breakpoints were dispersed at 13 sites not being located within 50 bp of other breakpoints. Therefore, a total of 18 sites (5 BCS-LLs plus
13 other sites) were defined as containing breakpoints.
Sequence Analysis of the Breakpoint Junction Sites--
To
assess the involvement of illegitimate V(D)J recombination in 9p21
deletions, RSS-like sequences were examined at these 18 sites. For this
purpose, the 23-bp sequence flanking the breakpoints was searched for
the 5'-CAC trinucleotide in the forward direction and the 3'-GTG
trinucleotide in the reverse direction. This was due to the fact that
the CAC trinucleotide in the heptamer-RSSs is the only part of the RSSs
indispensable for V(D)J recombination (26, 27) and nucleotide loss as a
result of V(D)J recombination does not extend over 20 bp (28). Either
the CAC or GTG trinucleotide was detected in total in 12 (67%) of the
18 sites (Fig. 1). Notably, the trinucleotide was detected at all of
the BCS-LL sites (Fig. 2B).
In 11 (73%) cell lines consisting of seven T- and four B-lineage
lines, the CAC trinucleotide flanked both the distal and proximal
breakpoints (Fig. 3A). In
addition, all of these cell lines had insertions of 1-10-bp
nucleotides at the breakpoint junctions (Fig. 3A). Thus,
they had features of V(D)J recombination (1, 2). In six of the cell
lines, both the proximal and distal breakpoints were located at BCS-LLs
(cases in red, Fig. 3A), whereas, in four others,
either a proximal or distal breakpoint was located at BCS-LLs (cases in
blue, Fig. 3A). Neither of the breakpoints were
located at BCS-LLs in the remaining one cell line, KOPN-30.
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Fig. 3.
DNA sequences of breakpoint junctions for
9p21 deletions. Alignment of the germ line sequences and the
deletion junction sequences are shown. Heptamer-RSS-like sequences
flanking breakpoints are boxed. A, junctions with
evidence of V(D)J recombination. Six cell lines, in which both the
proximal and distal breakpoints were located at BCS-LLs, are in
red, whereas four cell lines, in which either a proximal or
distal breakpoint was located at BCS-LLs, are in blue
(see "Results"). N-nucleotide-like sequences are indicated
in lowercase type, whereas P-nucleotide-like
sequences are underlined. B, junctions without
evidence of V(D)J recombination. Nucleotides overlapped at breakpoint
junctions are boxed.
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In four (27%) cell lines consisting of two T- and two B-lineage lines,
evidence for V(D)J recombination was not observed at the breakpoint
junctions. In two cell lines, P12/Ichikawa and KAWAI, the GTG
trinucleotide flanked the proximal breakpoints, whereas the CAC
trinucleotide did not flank the distal breakpoints (Fig.
3B). In one of them, P12/Ichikawa, the proximal breakpoint was located at BCS-LL2. In the remaining two cell lines, HPB-NULL and
THP-7, neither the CAC nor GTG trinucleotide flanked the breakpoints. No nucleotides were inserted at the junctions of these four cell lines.
Overlaps of 1-bp nucleotide were observed at the junctions of HPB-NULL
and THP-7.
V(D)J Recombination Potential of BCS-LL3
Fragment--
Detection of the CAC trinucleotide in the vicinity of
breakpoints, especially of those at BCS-LLs, suggested that
illegitimate V(D)J recombination is a major cause of 9p21 deletions.
However, a variety of tetranucleotides followed CAC (Figs.
2B and 3A), and there were no obvious
nonamer-RSS-like sequences 12 or 23 bp downstream of the
heptamer-RSS-like sequences, CACNNNN. Therefore, it was not clear that
these breakpoint junctions were generated by the V(D)J recombination
machinery. Thus, we went on to do functional assessment of V(D)J
recombination potential of DNA fragments containing breakpoints. For
this purpose, we carried out the extrachromosomal V(D)J recombination
assay using the pGG49 plasmid (24, 29).
pGG49 has 12- and 23-signals, which contains the consensus heptamer-
and nonamer-RSSs with 12- and 23-bp intervals, in its SalI
and BamHI sites, respectively (Table
I). Since V(D)J recombination gives rise
to rearranged plasmids with the signal joint configuration in which the
12- and 23-signals are joined with or without insertions of several
nucleotides (i.e. N-nucleotides), the resultant recombinants are resistant to chloramphenicol due to the lack of the transcription terminator sequence of 247 bp (Fig. 4).
The plasmid was introduced into a human pre-B ALL cell line, Reh, which
has been shown to have V(D)J recombination activity (29), was harvested
after 48 h incubation, and was introduced into E. coli
for genetic discrimination of recombinants. The recombination frequency
of pGG49 was 2.2 × 10
1 (Table
II), similar to the results of previous
studies (24, 29). The signal joint configuration in the recombined
plasmids was examined by PCR amplification of DNA fragments
encompassing the 12- and 23-signals and subsequent sequencing of them.
All of the 20 recombinants randomly picked and sequenced were signal joint products, since the consensus 12- and 23-signal sequences were
joined with or without nucleotide insertions (Table
III). pGG49 was also introduced into HEL,
a leukemia cell line of the myeloid lineage, which has been shown to
have no V(D)J recombination activity (29). As expected, pGG49 did not
yield recombinant products, and its recombination frequency was
estimated as being <5.1 × 105 (data not
shown).
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Fig. 4.
V(D)J recombination substrates and predicted
V(D)J recombinants in an extrachromosomal recombination assay.
Signal joint products with or without N-nucleotide insertion are shown.
Plac, lac promoter driving cat
expression. STOP, prokaryotic transcription terminator of
247 bp.
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Next, we assessed the V(D)J recombination potential of the DNA
fragments containing breakpoints by examining the frequency and
fidelity of V(D)J recombination of modified pGG49 plasmids, in which
the 12- and 23-signals were replaced with 33- and 43-bp sequences
containing putative RSS sequences at the breakpoints, respectively
(Table I, Fig. 4). First, we tested the BCS-LL3 fragment, in which the
largest portion of breakpoints was clustered (Fig. 2A). We
constructed two plasmids, pLL3-RSS and pRSS-LL3, in which the 12- and
23-signals of pGG49 were replaced with 33- and 43-bp sequences of
BCS-LL3, respectively (Table I, Fig. 4). The pLL3-RSS plasmid yielded
recombinants at a frequency of 1.5 × 103 in Reh
cells, whereas the pRSS-LL3 did not yield recombinants; thus, its
recombination frequency was estimated as being at least 1000-fold lower
than that of pLL3-RSS (Table II). In all of the 20 recombinants from
pLL3-RSS, which were randomly picked and sequenced, the BCS-LL3 and
consensus 23-signal sequences were joined with or without nucleotide
insertions and thus were considered to be signal joint products (Table
III). This result indicated that BCS-LL3 has a specific recombination
potential as a 12-signal, and its potential is 150-fold lower than that
of the consensus 12-signal (Table II).
The pLL3-RSS plasmid recovered from HEL cells did not yield recombinant
colonies, and its recombination frequency in HEL was estimated as being
<1.6 × 106 (data not shown). Thus, the
recombination frequency of the pLL3-RSS plasmid in Reh cells was
obviously higher than that in HEL cells, indicating that the
rearrangements of the pLL3-RSS plasmid in Reh cells were carried out by
V(D)J recombination. To further confirm that the BCS-LL3 fragment
functions as an RSS, we assessed the recombination frequency of the
pLL3M-RSS plasmid in Reh cells. pLL3M-RSS had a mutant BCS-LL3 fragment
positioned as a 12-signal, in which the first position of the CAC
trinucleotide in a putative heptamer-RSS was substituted by a guanine
(Table I). pLL3M-RSS showed a recombination frequency 30-fold lower
than that of pLL3-RSS (Table II). In addition, none of the 20 recombinants were considered to be true signal joint products, since
none of them retained the signal joint configuration. Instead, they had
mutations or interstitial deletions in the transcription terminator or
suffered gross rearrangements involving mutated BCS-LL3 and/or
23-signal sequences. Therefore, the mutation introduced in the BCS-LL3
fragment significantly inhibited the production of recombinants with
signal joint configuration. This result was consistent with a previous finding that the CAC trinucleotide in the heptamer-RSS is crucial for
V(D)J recombination (25, 26). Thus, it was further confirmed that the
BCS-LL3 fragment functioned as an RSS in vivo.
V(D)J Recombination Potential of BCS-LL1, -2, and -5 Fragments--
Next, we assessed the recombination potentials of three
other BCS-LL fragments, BCS-LL1, BCS-LL2, and BCS-LL5. The pRSS-LL1 plasmid carrying the BCS-LL1 fragment as a 23-signal yielded
recombinants at a frequency of 2.4 × 104, whereas
the pLL1-RSS plasmid carrying the BCS-LL1 fragment as a 12-signal did
not yield recombinants, and its frequency was estimated as <9.8 × 106. The pLL5-RSS plasmid carrying the BCS-LL5
fragment as a 12-signal yielded recombinants at a frequency of 1.8 × 104, whereas the pRSS-LL5 plasmid carrying the BCS LL5
fragment as a 23-signal did not yield recombinants, and its frequency
was estimated as <5.4 × 106. Twenty recombinants
from pRSS-LL1 and pLL5-RSS plasmids were picked and sequenced,
respectively. All of them were considered to be signal joint products
(Table III). Thus, it was indicated that the BCS-LL1 and BCS-LL5
fragments have specific recombination potential as a 23- and as a
12-signal, respectively. Their efficiencies were estimated as 900- and
1200-fold lower than that of the consensus 23- and 12-signals,
respectively. The pRSS-LL1 and pLL5-RSS plasmids recovered from HEL
cells did not yield recombinant colonies, and their recombination
frequencies in HEL were estimated as being <1.3 × 106 and <4.6 × 106, respectively
(data not shown).
In contrast to the results above, both the pLL2-RSS and pRSS-LL2
plasmids, in which the BCS-LL2 fragment was positioned as a 12- and as
a 23-signal, respectively, yielded recombinant products at similar
frequencies (3.5 × 105 and 2.9 × 105, respectively) (Table II). All of the 18 recombinants
obtained from the two experiments using the pLL2-RSS plasmid were
sequenced. Sixteen of them (89%) were signal joint products (Tables II
and III). However, the remaining 2 were not considered as recombinants by V(D)J recombination using the BCS-LL2 sequence as a RSS. One recombinant did not have the deletion of the transcription terminator but had two G to A substitutions in the transcription terminator sequence. The other recombinant apparently retained a wild type sequence of the terminator, suggesting that unknown genetic alterations on the substrate caused the transcriptional activation of the cat gene. Therefore, we estimated the V(D)J recombination
frequency of pLL2-RSS as 3.1 × 105 (equal to
3.5 × 105 × 16/18) (Table II). pLL2-RSS recovered
from HEL cells did not yield recombinant colonies, and their
recombination frequencies in HEL were estimated as being <7.0 × 106 (data not shown). Thus, the V(D)J recombination
frequency of BCS-LL2 as a 12-signal was higher in Reh than in HEL,
supporting the fact that the fragment functioned as a 12-signal
in vivo. Twenty recombinants from pRSS-LL2 were also
randomly picked and sequenced. Four of them (20%) were signal joint
products (Tables II and III), and they consisted of one identical
recombinant with the same 5-bp insertion. The remaining 16 were not
considered as V(D)J recombinants using the BCS-LL2 sequence as a RSS.
Fifteen of them consisted of six types of recombinants in which the
transcription terminator was deleted along with the BCS-LL2 or the
12-signal sequence. The remaining one allowed amplification of multiple DNA fragments, suggesting that gross rearrangement occurred in this
clone. Thus, we estimated the V(D)J recombination frequency of pRSS-LL2
as 5.9 × 106 (equal to 2.9 × 105 × 4/20) (Table II). pRSS-LL2 plasmids recovered from
HEL cells did not yield recombinant colonies, and the recombination
frequency was <2.9 × 105 (data not shown). Thus,
it was inferred that the V(D)J recombination potential of BCS-LL2 as a
23-signal was present but at a low level.
V(D)J Recombination by Pairing BCS-LL3 and BCS-LL1--
An
additional plasmid, pLL3-LL1, in which BCS-LL3 and BCS-LL1 were
inserted in the place of the 12- and the 23-signals, respectively, was
tested to examine whether or not V(D)J recombination by pairing the
BCS-LL3 and BCS-LL1 fragments occurs. If we assume that the BCS-LL3 and
BCS-LL1 fragments undertake recombination at efficiencies 1/150
and 1/900 less than consensus RSSs, respectively (Table II), the
recombination frequency of pLL3-LL1 could be calculated as being
1.6 × 106 (equal to 2.2 × 101 × 1/150 × 1/900). The pLL3-LL1 plasmid yielded two recombinant clones in the six experiments (Table II). However, sequence analysis of
these two recombinants indicated that they were not the signal joint
products generated by pairing BCS-LL3 and BCS-LL1 but rather recombinants with different interstitial deletions within the transcription terminator. Hence, the actual recombination frequency was
<0.9 × 106; thus, the use of two inefficient RSSs
did not give rise to V(D)J recombination with enough efficiency to be
detected by the assay used.
| |
DISCUSSION |
In the present study, the distribution of 33 breakpoints for 9p21
deletions in 21 lymphoid leukemia were examined in the 240-kb segment
covering the 40-kb region containing the CDKN2A,
ARF, and CDKN2B loci, the region where the
breakpoint distribution was previously examined in T-ALL (17). This
breakpoint mapping and the subsequent breakpoint cloning enabled us to
identify three breakpoint cluster sites, BCS-LL1 to LL3. These three
sites contained four or five breakpoints; thus, they were considered as
major breakpoint cluster sites (Fig. 2, A and B).
BCS-LL2 was the same as MTS1bcr in the ARF locus, where
the significant portion (>60%) of the breakpoints in T-ALL had been
previously mapped; therefore, the result of Cayuela et al.
(17) was confirmed. In the present study, an additional major
breakpoint cluster site, BCS-LL3, was identified in the
CDKN2B locus, and another site, BCS-LL1, was identified in
the MTAP locus of >100 kb distal to the CDKN2A, ARF, and CDKN2B loci (Fig. 2A).
Furthermore, two additional breakpoint cluster sites, BCS-LL4 and -LL5,
were also identified at loci >100 kb distal to these loci (Fig.
2A). Since two or three breakpoints among those identified
by us or Cayuela et al. (17) were located at these sites,
they were considered as being minor breakpoint cluster sites. Cayuela
et al. (17) also identified two minor breakpoint cluster
sites, MTS1bcr and MTS2bcr1, in the vicinity of the
CDKN2A and CDKN2B loci, respectively (Fig.
2A). However, none of the breakpoints in this study were
mapped at these two sites. Therefore, they are likely to be minor
cluster sites as previously reported (17). Thus, the present result
further supported the previous finding that breakpoints for 9p21
deletions in lymphoid leukemia are clustered. In addition, it was found
that both the breakpoints of T- and B-lineage cell lines were located
at the BCS-LL sites. Thus, the present result also indicates that the breakpoint cluster sites are susceptible to DNA breaks during lymphoid
leukemogenesis irrespective of cell lineages.
All BCS-LLs contained the CAC trinucleotide, which is identical to the
first three nucleotides of heptamer-RSSs indispensable for V(D)J
recombination. In addition, 7 of the 13 sites, where a single
breakpoint was mapped in this study, also contained the CAC
trinucleotide within 23 bp of each breakpoint. Therefore, in total, 12 (67%) of the 18 sites contained the CAC trinucleotide (Fig. 1). Since
the probability of finding CAC in the genome is 1 in 64 bp (1/4 × 1/4 × 1/4 = 1/64), 18 sites of 23-bp, each of
which contains 21 trinucleotides, could contain six copies of CAC
(18 × 21 × 1/64 = 5.9). Therefore, the CAC
trinucleotide was overrepresented at the breakpoints. Breakpoint
junctions in 11 (73.3%) cell lines consisting of seven T- and four
B-lineage lines showed typical evidence of V(D)J recombination (Fig.
3A). The CAC trinucleotide, which flanked both the distal
and proximal breakpoints, was deleted with the segment encoding the
CDKN2A, ARF, and CDKN2B genes, and nucleotides of 1-10 bp were inserted at the breakpoint junctions. The
deletions in these cases were typical of coding junctions of V(D)J
recombination. DSBs generated by the RAG complex are processed to the
P-nucleotide and/or N-nucleotide additions in the case of coding
junctions (1). Three of the 22 breakpoints in these 11 cases, distal
ones of L-SMY and CCRF-CEM and a proximal one of KOPT-K1,
were located next to the CAC trinucleotide, respectively, and one or
two nucleotides complementary to the germ line sequences upstream of
the CAC trinucleotide were inserted at the junctions (underlined in
Fig. 3A). These nucleotides are likely to be
P-nucleotides. The remaining 19 breakpoints were located 1-15 bp
away from the heptamer-RSS-like sequences; therefore, inserted
N-nucleotides (rather than P-nucleotides) probably account for
nucleotide sequences beside those breakpoints. Thus, overrepresentation
of CAC in the vicinity of breakpoints as well as the sequences of the
breakpoint junctions indicated that the majority (>70%) of 9p21
deletions in lymphoid leukemia of both T- and B-lineages are caused by
illegitimate V(D)J recombination.
In the remaining four (27%) cell lines, V(D)J recombination was not
evident at the breakpoint junctions (Fig. 3B). In two T-lineage cell lines, P12/Ichikawa and KAWAI, only proximal breakpoints were flanked by the CAC trinucleotide, and nucleotides were not inserted at the junctions. In two B-lineage cell lines, HPB-NULL and
THP-7, breakpoints were not flanked by the CAC trinucleotide. Therefore, it was likely that deletions in these cell lines were generated by mechanisms other than illegitimate V(D)J recombination. Joining of nonhomologous DNA without evidence of V(D)J recombination has been detected at the breakpoint junctions for the interstitial deletions in nonhematological malignancies, including 9p21 deletions in
two cases of glioma (30-33). Therefore, as hypothesized in
nonhematological malignancies (3, 34, 35), 9p21 deletions in a subset
of lymphoid leukemias, such as these four cell lines, may have also been generated by nonhomologous end joining of DSBs caused by unknown
mechanisms. Meanwhile, the proximal breakpoint of P12/Ichikawa was
located at BCS-LL2. Therefore, it might be that the DSB at the proximal
breakpoint in this cell line was generated by the illegitimate action
of the RAG complex. The broken DNA ends generated by the RAG complex
may have rejoined with other broken ends generated by other mechanisms.
Notably, in 10 of the 11 cell lines whose 9p21 deletions were possibly
caused by illegitimate V(D)J recombination, one or both of the proximal
and distal breakpoints were located at BCS-LLs. However, the RSS-like
sequences, even those at BCS-LLs, showed only a partial homology to the
consensus RSSs (Fig. 2B, Table I). Thus, we assessed the
V(D)J recombination potential of DNA fragments containing the RSS-like
sequences. We subjected four BCS-LL fragments, BCS-LL1, -LL2, -LL3, and
-LL5, to the extrachromosomal V(D)J recombination assay in Reh cells
with V(D)J recombination activity (29). The BCS-LL3 fragment positioned
as a 12-signal (i.e. pLL3-RSS) showed the highest
recombination frequency (Table II), indicating that this fragment had
the highest recombination potential among the four fragments tested.
This result was consistent with the fact that the largest portions
(i.e. five breakpoints) of the breakpoints were clustered at
BCS-LL3 (Fig. 2A). Other cluster sites, BCS-LL1 and BCS-LL5,
that were located >100-kb apart from the CDKN2A,
ARF, and CDKN2B loci, were also shown to undergo
V(D)J recombination as RSSs, respectively. It was inferred that BCS-LL2
has a V(D)J recombination potential both as 12- and 23-signals, but
its potential, especially as a 23-signal, was considerably
lower than other BCS-LL fragments. This result was contrary to the fact
that a considerable number of breakpoints were clustered at the BCS-LL2
site in this (Fig. 2B) and a previous studies (17). BCS-LL2
is located in a CA/GT tract, and this tract was not cloned into the
pLL2-RSS and pRSS-LL2 plasmids (Table I). CA/GT tracts are speculated
to adopt the Z-DNA structure (17), which might enhance V(D)J
recombination; thus, the recombination potential of BCS-LL2 could be
enhanced by the CA/GT tract.
In the present study, all four BCS-LL fragments tested showed potential
as RSSs, although the efficiency and specificity as RSS were different
among the fragments. Especially, the estimated V(D)J recombination
potential of BCS-LL1 and -LL3, two major cluster sites, were similar to
that of other ectopic RSSs, which have been predicted to pair with
physiological RSSs to give rise to recurrent translocations in lymphoid
leukemia (11). In the 9p21 deletions analyzed in this study, the
BCS-LL3 site was joined with the BCS-LL1 site, but not with the BCS-LL5
site, to give rise to deletions (Fig. 3A). This is
consistent with the fact that the BCS-LL3 and BCS-LL5 sites
preferentially acted as 12-signals, whereas the BCS-LL1 site acted as a
23-signal. The BCS-LL5 site with a 12-signal potential was joined with
the BCS-LL2 site, for which a weak potential as a 23-signal was
suggested (Fig. 3A). Thus, it is likely that the occurrence
of 9p21 deletions conformed to the 12/23 rule (1, 2). V(D)J
recombination by pairing of two ectopic RSSs, BCS-LL1 and BCS-LL3, was
not recapitulated in the extrachromosomal recombination assay; however,
this was conceivable, since its predicted recombination frequency was
too low to be detected by this system (11). Thus, the present results strongly support the fact that illegitimate V(D)J recombination, targeted at ectopic RSSs in 9p21, causes a large fraction of 9p21 deletions in lymphoid leukemia.
The majority of the breakpoints were clustered at BCS-LLs despite the
fact that the 9p21 segment analyzed in this study contained thousands
of CAC trinucleotides. Thus, it is possible that BCS-LLs have specific
structures enhancing V(D)J recombination other than the CAC
trinucleotide. It is noted that BCS-LL1, -LL3, and -LL5 specifically
functioned as a 12- or 23-signal contrary to the fact that they have no
obvious nonamer-RSS-like sequences (Table I). In addition, the
recombination potential of BCS-LLs was not correlated to the degrees of
their homologies with the consensus RSSs. These results indicate that
BCS-LL fragments can take specific configurations as RSSs, irrespective
of their homologies with the consensus RSSs. In addition, it is also
noted that all of the five BCS-LLs were located in, or upstream of, the
genes in this region (Fig. 2A). The MTAP and
CDKN2A genes are expressed ubiquitously in a variety of
human tissues, including hematopoietic progenitor cells (20, 36, 37).
Recent studies have indicated that the open chromatin structure
associated with gene expression is a critical factor determining the
efficiency of V(D)J recombination, probably changing the accessibility
of the V(D)J recombination machinery (38, 39). Therefore, the local
chromatin structure of BCS-LLs might further enhance the susceptibility
to illegitimate V(D)J recombination. Illegitimate V(D)J recombination
has been indicated as being involved in a variety of chromosomal
aberrations in lymphoid leukemias (8, 9). However, only a few candidate ectopic RSSs have been functionally assessed for their potential as
RSSs (10-12). Thus, genomic structures underlying illegitimate V(D)J
recombination should be further investigated to define the mechanisms
underlying 9p21 deletions and other recurrent chromosomal aberrations
that occur in lymphoid leukemogenesis.
§,
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ABSTRACT
INTRODUCTION
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