Pathogenetic and clinical implications of non-immunoglobulin ; BCL6 translocations in B-cell non-Hodgkin's lymphoma.

Abstract

Chromosomal translocations affecting band 3q27, where BCL6 gene is located, are among the most common genetic abnormalities in non-Hodgkin's lymphoma of B-cell type (B-NHL). The BCL6 gene encodes a BTB/POZ zinc finger transcription factor, which exerts repressive activity by recruiting corepressor molecules. The 3q27/BCL6 translocation is unique in that it can involve not only immunoglobulin (Ig) genes but also non-Ig chromosomal loci as a partner. To date, around 20 non-Ig partner genes have been identified. As a result of non-Ig ; BCL6 translocations, many types of regulatory sequences of each partner gene substitute for the 5' untranslated region of BCL6, and the rearranged BCL6 comes under the control of the replaced promoter. The introduction of non-Ig ; BCL6 constructs into transformed cells led to high-level Bcl-6 protein expression in the nucleus, while BCL6 mRNA levels in clinical materials of diffuse large B-cell lymphoma (DLBCL) with non-Ig ; BCL6 translocations were unexpectedly low. A comparative study suggested that non-Ig ; BCL6 translocation and a low level of BCL6 mRNA expression are concordant indicators of a poor clinical outcome in cases of DLBCL. The coexistence of a non-Ig ; BCL6 translocation with t(14 ; 18)(q32 ; q21) in a single clone did not significantly affect the clinical features of follicular lymphoma. The pathogenetic and clinical implications of non-Ig ; BCL6 translocations in B-NHL subtypes may not be identical to those of Ig ; BCL6.

Full text

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Review Article
Pathogenetic and Clinical Implications of
Non-Immunoglobulin ; BCL6 Translocations in B-Cell
Non-Hodgkin’s Lymphoma
Hitoshi Ohno
Chromosomal translocations affecting band 3q27, where BCL6 gene is located, are among the most common genetic
abnormalities in non-Hodgkin’s lymphoma of B-cell type (B-NHL). The BCL6 gene encodes a BTB/ POZ zinc finger transcrip-
tion factor, which exerts repressive activity by recruiting corepressor molecules. The 3q27/BCL6 translocation is unique in that
it can involve not only immunoglobulin (Ig) genes but also non-Ig chromosomal loci as a partner. To date, around 20 non-Ig
partner genes have been identified. As a result of non-Ig ; BCL6 translocations, many types of regulatory sequences of each
partner gene substitute for the 5′ untranslated region of BCL6, and the rearranged BCL6 comes under the control of the replaced
promoter. The introduction of non-Ig ; BCL6 constructs into transformed cells led to high-level Bcl-6 protein expression in the
nucleus, while BCL6 mRNA levels in clinical materials of diffuse large B-cell lymphoma (DLBCL) with non-Ig ; BCL6
translocations were unexpectedly low. A comparative study suggested that non-Ig ; BCL6 translocation and a low level of
BCL6 mRNA expression are concordant indicators of a poor clinical outcome in cases of DLBCL. The coexistence of a non-Ig ;
BCL6 translocation with t(14 ; 18)(q32 ; q21) in a single clone did not significantly affect the clinical features of follicular
lymphoma. The pathogenetic and clinical implications of non-Ig ; BCL6 translocations in B-NHL subtypes may not be
identical to those of Ig ; BCL6. 〔J Clin Exp Hematopathol 46(2) : 43-53, 2006〕
Keywords: non-Hodgkin’s lymphoma of B-cell type, BCL6 gene, non-immunoglobulin gene, prognostic marker, concurrent
translocations
INTRODUCTION
Chromosomal translocations and rearrangements of on-
cogenes located at breakpoints are observed in a large propor-
tion of patients with non-Hodgkin’s lymphoma of B-cell type
(B-NHL). These genetic aberrations, in general, involve im-
munoglobulin (Ig) gene loci as a partner1. As a result of the
translocation, the oncogene is transcriptionally deregulated
under the influence of the juxtaposed Ig regulatory sequences.
t(3 ; 14)(q27 ; q32) and its variants t(2 ; 3)(p11 ; q27) and t
(3 ; 22)(q27 ; q11), involving Ig heavy chain (IgH), k light
chain (IgLk) and l light chain (IgLl) genes, respectively, were
first reported to be detected in 6.3% of cytogenetically ana-
lyzed B-NHLs2. By analogy with previously well-
characterized translocations, an oncogene was expected to be
located at 3q27, and three groups independently cloned BCL6
(B-cell CLL/ lymphoma 6) gene on this particular chromoso-
mal band3-5. A group at Columbia University headed by Dr.
Dalla-Favera very recently engineered transgenic mouse
strains harboring a BCL6 gene driven by the IgH Im pro-
moter, mimicking t(3 ; 14)(q27 ; q32), using the knock-in
strategy6. The mice at between 15 and 20 months of age
developed B-cell tumors showing features of diffuse large B-
cell lymphoma (DLBCL) at a frequency of 36% to 62%,
confirming clearly the oncogenetic role of Ig ; BCL6 translo-
cations in the pathogenesis of B-NHL.
In contrast to other B-NHL-specific translocations, 3q27/
BCL6 translocation is unique in that it can involve not only Ig
gene loci but also non-immunoglobulin (non-Ig) chromoso-
mal loci as a partner7. Molecular cloning of these transloca-
tions revealed that a gene is located at each partner locus and
rearranged with BCL6 so that the normal regulatory sequences
of BCL6 are replaced with those of the partner gene7, 8. Some
of these translocations are not random but recurrent, indicat-
ing that non-Ig ; BCL6 translocations as well as Ig ; BCL6
play a pathogenetic role in B-NHL. In this review, I first
describe the molecular anatomy of non-Ig ; BCL6 transloca-
tions. Second, I present our experiments to show to what
43
J Clin Exp Hematopathol
Vol. 46, No. 2, Nov 2006
Received : Nov 15, 2005
Accepted : Nov 17, 2005
Department of Internal Medicine, Takeda General Hospital, Kyoto, Japan.
Address correspondence and reprint request to Hitoshi Ohno, Department of Internal
Medicine, Takeda General Hospital, 28-1 Ishida-Moriminami-cho, Fushimi-ku, Kyoto
601-1495, Japan.

Page 2

extent non-Ig ; BCL6 translocations affect the level of Bcl-6
protein expression. Finally, I discuss the clinical implications
of this unique type of translocation in subtypes of B-NHL.
THE BCL6 GENE AND ITS PRODUCT
The BCL6 gene spans 24, 310 bases and contains 11
exons, generating 4 types of splicing patterns (http :
//www.genecards.org/cgi-bin/carddisp?BCL6). The ATG sig-
nal for the initiation of protein synthesis is within exon 3 and
is followed by an open reading frame. The 3q27/BCL6 trans-
location occurs within the major translocation cluster (MTC)
spanning the non-coding exon 1 and intron 1 ; in a majority
of cases, breakpoints are located immediately 3′ of exon 13,5,9.
The translocation, therefore, does not interrupt the protein-
coding region of BCL6.
The Bcl-6 protein, consisting of 706 amino acids with a
calculated molecular weight of 78,846 Da, is a sequence-
specific transcription factor that can repress transcription from
promoters containing its DNA-binding site (Fig. 1)10. The C-
terminal region of Bcl-6 contains six C2-H2 type zinc fingers,
each separated by a conserved stretch of seven amino acids.
The two cysteine and two histidine residues coordinate a zinc
ion and fold the domain into a finger-like projection that can
interact with DNA.
The BTB/POZ domain (broad-complex, tramtrack, and
bric-a-brac/pox virus and zinc finger) at the N-terminus is a
conserved 120-amino acid motif, which is found in 5 to 10%
of zinc finger proteins11. The primary function of the BTB/
POZ domain appears to be the mediation of protein-protein
interactions. It has been shown that the repressive effect of
Bcl-6 on the target gene is exerted via the recruitment of
SMRT (silencing mediator of retinoid and thyroid receptor),
NCoR (nuclear receptor corepressor) and BCoR (BCL6
corepressor) corepressors12-15. Crystallographic analysis of
the BTB/POZ domain revealed that it forms a butterfly-
shaped homodimer to generate a ‘lateral grove’ motif that
interfaces with a 17-residue sequence (BBD motif, Bcl-6
binding domain) of SMRT12,15. A fusion protein that contains
the HIV-TAT protein transduction domain and the BBD se-
quence can penetrate the cell membrane and bind to Bcl-6,
thereby blocking the BTB/POZ-mediated recruitment of
SMRT16.
The middle portion of Bcl-6 contains a second domain
required for the repressive transcriptional activity (Fig. 1).
Bereschchenko et al.17showed that the KKYK motif within
the PEST sequence is targeted by p300-mediated acetylation.
This post-translational modification disrupts the ability of
Bcl-6 to recruit histone deacetylase (HDAC), thereby hinder-
ing its capacity to repress transcription.
Targeted inactivation of BCL6 in the mouse germline
leads to impaired production of secondary IgG antibody
against T-cell-dependent antigens and the spleen of the
BCL6−/ −mouse lacks germinal center (GC) formation18-20. A
cDNA array analysis along with loss-of-function experiments
revealed a set of genes that are negatively regulated by Bcl-
621. The BLIMP1 (B-lymphocyte-induced maturation
protein 1) gene, which is a representative target gene, plays a
key role in the differentiation of B-cells into plasma cells by
turning off the entire mature B-cell gene expression
program22,23. It is presumed that BCL6 is the master gene for
the generation by B-cells of a GC, acting to modulate the
transcription of genes involved in cell proliferation, differen-
tiation and apoptosis.
MOLECULAR ANATOMY OF NON-IG ; BCL6
TRANSLOCATIONS
To determine the partner of the BCL6 translocation, two
polymerase chain reaction (PCR)-based approaches have been
applied, i.e. 5′-rapid amplification of cDNA ends (5′-RACE)
and long-distance inverse (LDI-) PCR (Fig. 2)9,24,25. Both
methods isolate sequences adjacent to the junction, a database
search of which leads to identification of the partner gene.
Table 1 lists non-Ig partners that have been identified to
date9, 24-35. These include the genes for a transcription factor,
serine/threonine-protein kinase, cytokine receptor, Ras small
Ohno H
44
Fig. 1.
Functional domains of the Bcl-6 protein
10. The domains
are involved in protein-protein interaction, transcriptional repres-
sion of target genes, post-translational modification and DNA
binding.
*ETO/ MTG8 binds to the fourth zinc finger
61.
**
MTA3/ NuRD interacts with the region spanning aa 141 to 507
PEST, where P＝proline, E＝glutamic acid, S＝serine and T＝
threonine ; KKYK, where K＝lyine and Y＝tyrosine.
62.
?Homodimerization
?Transcriptional repression
?Interaction with:
??? Corepressors
??? ?SMRT/NcoR
?????BcoR
?????HDAC1
??? Related proteins
?????BAZF
?????PLZF
?????LRF
??? Other zinc fingers
?????Evi-9
?DNA binding
??? TTCCT(A/C)GAA
?Transcriptional repression
?Interaction with
??? Related proteins
?????PLZF
?????LRF
??? Class II HDACs
??? Jun
??? ETO/MTG8*
BTB/POZ domain
1 130 191 386 520 681
zinc-finger motif
KKYK
Bcl-6
706
300
417
?Transcriptional repression
?Interaction with:
??? Corepressors
?????MTA3/NuRD**
?????mSIN3A
?????HDACs
?PEST sequence
???Phosphorylation by
??MAPK → Degradation through
??the ubiquitin/proteasome
??pathway
?KKYK motif
???Inactivation by aoetylation

Page 3

GTPase, heat shock proteins and so on. In spite of this
marked diversity of protein products, there are common fea-
tures in the molecular anatomy of non-Ig ; BCL6 transloca-
tions. First, the gene fusion occurs in the same transcriptional
orientation ; second, the breakpoint on the partner gene is
located in close proximity to the promoter sequence ; and
third, the complete sequence of the promoter is fused up-
stream of the coding region of BCL6 on the der(3) chromo-
some (Fig. 3). As the result of translocation, many types of
regulatory sequences of each partner gene substitute for the 5′
untranslated region of BCL6, and the rearranged BCL6 comes
under the control of the replaced promoter (promoter
substitution36). The translocated allele generates chimeric
transcripts composed of 5′ sequences of the partner followed
by the open reading frame of BCL6, which is readily detect-
able by reverse transcriptase-mediated PCR33,37.
Although the transcriptional control of these non-Ig
genes has not been fully determined, it should be noted that
some genes are transcriptionally activated by a variety of
stimuli, including cell cycle control (H4), changes in the
physical environment (HSP89a and HAP90b)38, and response
to cytokines (PIM1 and CIITA). Considering that GC B-
cells, in which the BCL6 translocation is presumed to occur,
proliferate rapidly in response to antigen, it is likely that a
BCL6 gene affected by the translocation is inappropriately
expressed during B-cell proliferation.
There is evidence to suggest that the somatic hypermuta-
Non-Ig ; BCL6 translocation in B-NHL
45
Fig. 2.
Schematic diagram of the long-distance inverse PCR
assay used to detect non-Ig ; BCL6 translocations
9. PCR pri-
mers (open arrows) were designed for the BCL6 sequences in the
divergent orientation. Genomic DNA from tumor tissues was
digested with XbaI, and self-ligated at a low DNA concentration.
These circular DNAs are targeted by nested PCR amplification.
The PCR product contains a fragment of an unknown partner that
is flanked by the known BCL6 sequences. The fragment beyond
the artificial XbaI site is the sequence of the partner.
BCL6
MTC
2kb
5'
123
3'
HSP89α
5'3'
HSP89α
BCL6
23
HSP90β
5'
3'
HSP90β
BCL6
23
Breakpoint of the translocation
Translation initiation site
Heat shock element :
nGAAnnTTCn (n = any nucleotide)
Fig. 3.
Molecular anatomy of BCL6 translocations in-
volving two heat shock protein (HSP) genes. The break-
points on the BCL6 gene were within the MTC region,
while those on the HSP genes were either 5′ or 3′ of the
translation initiation sites. Transcriptional control of HSP
genes is mediated by three tandem copies of heat shock
element (HSE). In response to heat shock, the heat shock
factors as a trimer bind to these HSEs
38. As the result of
translocation, the complete set of the HSEs is fused up-
stream of BCL6. Therefore, the expression of BCL6 ap-
parently comes under the control of the heat shock factors
that bind to the HSEs
1） ???? digestion
der(3)
partner
Junctions
exon 2
der
(partner)
BCL6
exon 1
partner
2） Self-ligation
der(3)
exon 1
der(partner)
3） Nested PCR
partner
exon 1
partner
Xba Ⅰ
Xba Ⅰ
Xba Ⅰ
Xba Ⅰ
Xba Ⅰ
Xba Ⅰ
Xba Ⅰ
Xba Ⅰ
Xba Ⅰ
BCL6

Page 4

tion (SHM) machinery of the Ig gene is involved in the
development of non-Ig ; BCL6 translocations. SHM of
BCL6 occurs in a large proportion of memory B-cells isolated
from normal individuals, in GC-B-cells from reactive tonsils,
and across the spectrum of GC/post-GC type B-cell
tumors39-41. The mutations are clustered within the first exon-
intron boundary of the gene, which overlaps with the MTC42,
suggesting that SHM and translocations involving BCL6 are
mediated by common molecular mechanisms. On the other
hand, PIM1 and RHOH, both of which are non-Ig partners
(Table 1), are mutated in DLBCL and the regions involved in
the mutation match those in the translocation43. These obser-
vations suggest that the SHM machinery, which generates
double-stranded breaks of DNA, targets both the BCL6 and
non-Ig partners of GC-B-cells, thereby predisposing these
genes to exchanges of genetic material.
DEREGULATED EXPRESSION OF BCL-6 PRO-
TEIN BY HISTONE H4; BCL6 TRANSLOCATION
We showed that t(3 ; 6)(q27 ; p21) results in the fusion
of BCL6 with a particular histone H4 gene (HUGO nomencla-
ture : HIST1H4I) on 6p2127. The H4 gene is composed of a
single exon followed by a terminal palindrome.
Transcriptional control of the histone H4 gene is mediated by
two multipartite proximal promoter elements (Sites I and II),
the activity of which is augmented by two distal domains
(Sites III and IV)44. The Site II-equivalent of the H4 gene
contains consensus binding sites for the transcription factors
HiNF-M, HiNF-D and HiNF-P. These Site II-binding pro-
teins, in addition to other co-regulatory molecules, contribute
to enhancing the H4 gene transcription at the G1/ S phase
transition44. On the other hand, the terminal palindrome se-
quence initially contributes to cleavage of the primary H4
gene transcript and mediates mRNA destabilization at the end
of S phase45. The sequence approximately 16 bp downstream
of the palindrome is also essential and through base-pairing
interactions mediates the U7 snRNP-dependent processing of
the mRNA 3′ end45.
We cloned 5 H4 ; BCL6 fusion genes and found that the
breakpoints on H4 were distributed within the 3′ half of the
H4 protein-coding region or in the vicinity of the
palindrome37. Therefore, the mechanism of 3′ end formation
of H4 is perturbed and the resulting fusion mRNAs with
BCL6 are predicted to be processed like normal polyadeny-
lated mRNAs. It appears then that the deregulation of Bcl-6
protein expression is facilitated by ‘capturing’ sequences that
support cell cycle control of H4 gene transcription during the
Ohno H
46
Table 1.
Non-Ig partner genes of BCL6 translocation
Gene symbol : HUGO (Alias)Gene product
Chromosomal locus
1
References
MBNL1 (KIAA0428)Muscleblind-like protein (Triplet-expansion RNA-binding protein)3q25/ 3q25.134
TFRC
Transferrin receptor (p90, CD71)3q26.2-qter/ 3q299, 28
ST6GAL1 (CD75) Sialyltransferase 1 (beta-galactoside alpha-2, 6-sialytransferase)3q27-q28/ 3q27.334
EIF4A2
Eukaryotic translation initiation factor 4A, isoform 2 3q28/ 3q27.328
RHOH (RhoH, TTF)Rho-related GTP-binding protein RhoH (GTP-binding protein TTF)4p13/ 4p1424, 43
HSPCB (HSP90b)Heat shock 90kDa protein 1, beta6p12/ 6p21.19
PIM1
Pim-1 oncogene product6p21.29, 28, 43
SFRS3 (SRp20)Splicing factor, arginine/ serine-rich 3 (Pre-mRNA splicing factor
SRP20)
6p21/ 6p21.3132
HIST1H4I (H4/ m) H4 histone family, member M6p21.33 27, 37
U50HG
2
Small nucleolar RNA6q1530
ZNFN1A1 (IKAROS)Ikaros (zinc finger protein)7p13-p11.19, 29
GRHPR (GLXR)Glyoxylate reductase/ hydroxypyruvate reductase9q12/ 9p13.2 34
POU2AF1 (BOB1, OBF-1)POU domain class 2, associating factor 1 (B-cell-specific coactivator
OBF-1) (OCT binding factor 1) (BOB-1) (OCA-B)
11q23.125
LRMP (JAW1)Lymphoid-restricted membrane protein 12p12.134
GAPDH
Glyceraldehyde-3-phosphate dehydrogenase 12p13.3135
NACA
Nascent-polypeptide-associated complex alpha polypeptide12q23-q24.1/ 12q13.3 9
LCP1
L-plastin (Lymphocyte cytosolic protein 1) (LCP-1) (LC64P) 13q14.3/ 13q14.13 26
HSPCA (HSP90a)Heat shock 90kDa protein 1, alpha14q32.33/ 14q32.319, 31
IL21R
Interleukin-21 receptor16p11/ 16p12.133
CIITA
MHC class II transactivator 16p13/ 16p13.139, 28
1LocusLink and/ or Ensembl cytogenetic band
2not included in the HUGO gene nomenclature database

Page 5

cell cycle, while simultaneously inactivating the regulatory
sequences required for post-transcriptional control of H4 gene
expression.
To determine the level of Bcl-6 protein expression di-
rected by the H4 ; BCL6 fusion gene as compared with the
germ-line BCL6, we constructed expression plasmids that
mimicked the structure of t(3 ; 6)(q27 ; p21) (Fig. 4A)37.
Transient transfection of the plasmids into COS-7 cells re-
sulted in transcription of H4 ; BCL6 fusion mRNA that had
the same structure as mRNA from clinical materials with t(3 ;
6)(q27 ; p21). Comparison of the levels of Bcl-6 expression
revealed that H4 ; BCL6-transfected cells produced mark-
edly more Bcl-6 than cells transfected with a plasmid carry-
ing BCL6 driven by its normal promoter (Fig. 4B). We next
subjected the COS-7 cells to indirect immunofluorescence
microscopy using a polyclonal antibody against Bcl-6 and
found that H4 ; BCL6-transfected cells displayed bright nu-
clear staining with a characteristic granular pattern (Fig. 4C) ;
the granules have been shown to contain SMRT and N-CoR
co-repressors13. The introduction of a series of deletion mu-
tants that lacked the Site II sequences led to a reduction in the
expression of Bcl-6 to the basal level (Fig. 4C). These find-
ings indicate that H4 ; BCL6 gene fusion leads to enhanced
Bcl-6 protein expression, which is promoted by the H4 regu-
Non-Ig ; BCL6 translocation in B-NHL
47
Fig. 4.
tion into COS-7 cells37. (A) Diagram of the Bcl-6-expression plasmids driven by the SV40 promoter,
Construction of H4 ; BCL6 fusion gene mimicking t(3 ; 6)(q27 ; p21) and the effect of transfec-
normal BCL6 promoter, and H4 ; BCL6 fusion gene of case no. 457. Transcription initiated from the H4 ;
BCL6 fusion gene was contiguous with the BCL6 exon2 at the cryptic 5' splice-donor site ( ▲ ). A series of
deletion mutants of the last fragments was generated by digestion with exonuclease III and mung bean
nuclease. IRF2, interferon regulatory factor 2 ; B, BamHI ; X, XbaI ; H, HindIII ; G, BglII ; and S, SacI.
(B) Western blot analysis of COS-7 transfectants for Bcl-6 expression. FL-218 is a follicular lymphoma
cell line. (C) Effect of the promoters of H4 upon the level of Bcl-6 expression. The COS-7 transfectants
were subjected to Western blot analysis and indirect immunofluorescence microscopy. (D) Scatter plot
analysis comparing the expression profile of H4 ; BCL6-transfected cells (Y axis) to that of normal BCL6
promoter-transfected cells (X axis). Alteration of the levels of expression was determined by the Atlas
Human Array (Clontech) and ArrayGauge software (Fuji Photo Film). Up- or down-regulation by >
1. 5-fold, indicated by two diagonal lines, was considered significant.
A
No promoter
B
Genome DNA
XX
cDNA
SV40
enhancer
23 4~9
poly A
SV40 promoter
BCL6
exon 1
#457
H4, BCL6
H
IRF2 Site II
H4 exon
H4 exon
H4 exon
H4 exon
GS
IRF2Site II
Site II
ΔH(?669) ～H(?224)
ΔH(?669)～H(?202)
ΔH(?669) ～H(?10)
ΔH(?669) ～B(?1978)
B
79kDa
FL-218
No
WB :
Bcl-6
promoter
SV40
promoter
BCL6
?621＋273
#457
H4 ; BCL6
C
79kDa
#457
H4 ; BCL6
ΔH(?669)
～H(?202)
ΔH(?669)
～H(?224)
ΔH(?669)
～H(?10)
ΔH(?669)
～B(?1978)
WB :
Bcl-6
IF :
Bcl-6
DAPI
D
H4 ; BCL6
#457
Normal BCL6 promoter
10
BCL6
RBBP7
PFDN4
ATF3
BLIMP1
CCND2
10 10 10
10
10
10
103
2
1
0
0123
?????-621～＋273
BCL6