BCL6 translocations involve not only immunoglobulin(IG) genes but also a number of non-IGloci as partners. Junctional sequences of three IG/BCL6 translocations were readily obtained by long-distance PCR. In cases where partner loci were not determined, we developed a long-distance inverse PCR method, which amplifies unknown fragments flanked by known BCL6sequences. Using these two long-distance PCR-based approaches, we cloned junctional areas of BCL6 translocations from a total of 58 cases of B-cell tumors. Nucleotide sequencing and database searches revealed that 30 cases involved IGs as partners: IG heavy chain gene in 22, IGκ light chain gene in 1, and IG λ light chain gene in 7. In contrast, 23 cases affected non-IG loci,including the H4 histone gene, heat shock protein genes HSP89α and HSP90β, and PIM-1 proto-oncogene. On der(3) chromosomes, complete sets of the promoters of these partner genes replaced that of BCL6 in the same transcriptional orientation. These results suggest that BCL6 gene affected by the translocation is transcriptionally activated by a variety of stimuli,including cell cycle control, changes in the physical environment, and response to cytokines. Break points on BCL6 occurred within the major translocation cluster, and we identified a 120-bp hyper-cluster region a short distance from the 3′ end of exon 1. Gel mobility-shift assay suggested the presence of a protein(s) that bound to this particular region.

Human B-cell tumors are often associated with chromosomal translocations that lead to juxtaposition of cellular oncogenes to the IG4loci (1). The BCL6 gene was first identified at the break points on 3q27 involved in t(3;14)(q27;q32) and t(3;22)(q27;q11) (2, 3, 4). Initial studies suggested that 3q27 translocation and/or rearrangement of the BCL6 gene were specifically associated with the diffuse large cell subtype of B-NHL (4). However, later studies of panels of many types of B-NHL invariably showed that this genetic lesion was found in a significant proportion of follicular lymphomas (5, 6). In contrast to other B-NHL-associated translocations, BCL6translocations are unique in that they can involve not only IGs but also other as yet uncharacterized chromosomal loci as partners. To date, nearly 20 independent chromosomal sites have been reported to be partners (7), and several genes have been identified in the vicinity of break points on partner chromosomes(8, 9, 10). These molecular studies have shown that breakage most frequently occurs within the MTC, encompassing the noncoding exon 1 of BCL6(4, 5). As a result of the translocation, many types of regulatory sequences on each partner chromosomal locus substitute for the 5′ untranslated region of BCL6(11) and the rearranged BCL6gene is presumed to be under the control of the replaced promoter activity. However, the patterns of regulation of each regulatory sequence leading to deregulated BCL6 expression have not yet been completely characterized.

We have developed a LD-PCR method capable of detecting IG/oncogene junctions of up to 30 kb (12, 13). Our previous study showed that LD-PCR successfully detected three IG/BCL6 translocations [i.e.,t(2;3)(p12;q27), t(3;22)(q27;q11), and t(3;14)(q27;q32)], which had been well characterized at the molecular level. For cases in which partner loci were not determined, we have developed a LDI-PCR method,which amplifies unknown fragments flanked by known BCL6sequences. Here, we report the cloning and sequencing of a number of BCL6 translocations from a large series of B-cell tumors using these two LD-PCR-based assays. The purpose of the present study was to explore the diverse molecular anatomy of BCL6translocations and to find structural and functional consequences of the juxtaposition of the BCL6 gene to a wide variety of partner genes.

Tumor Specimens.

Lymphoma tissues or other tumor specimens obtained from patients admitted to Kyoto University Hospital and related hospitals between 1983 and 1997 were analyzed. The lymphomas were originally classified according to the International Working Formulation for Clinical Usage,but their pathological diagnoses were updated by the REAL classification (14). Materials studied were subjected to immunohistochemical analysis and/or surface immunophenotyping, as well as gene rearrangement analysis using probes for the IGHgene, to determine B-cell origin of the lymphoma cells.

Southern Blot Hybridization.

Genomic DNA extracted from lymphoma specimens was digested with appropriate restriction enzymes and electrophoresed through 0.8%agarose gels. DNA was transferred onto nylon membrane filters(GeneScreen Plus; NEN Research products, Boston, MA) and hybridized with probes labeled with 32P using a random labeling system. The F372 probe for the MTC region of the BCL6 gene was a 2.3-kb XhoI/BamHI fragment (5).

LD-PCR.

Designations and sequences of the oligonucleotide primers to detect IG/oncogene fusion genes were listed in our previous studies(12, 13). Each PCR reaction mixture (50 μl) contained 100 ng of genomic DNA, reaction buffer, dNTP mixture, 20 pmol of each primer, and 2.5 units of LA Taq DNA polymerase (Takara Shuzo, Kyoto). PCR cycling variables for long DNA targets were previously described in detail (13). Aliquots of the PCR products were analyzed by agarose gel electrophoresis and visualized under UV illumination after EtBr staining.

LDI-PCR.

High molecular weight genomic DNA was digested with XbaI or BamHI and purified by standard methods. The DNA was diluted to a concentration of 1 μg/ml and incubated at 16°C overnight in the presence of T4 DNA ligase to facilitate intramolecular ligation (15). The self-ligated circular DNA was used as a template for a nested PCR; the circular DNA was not reopened. The position and orientation of the primers are illustrated in Fig. 1 A. The sequences were: 08,5′-CAGCTTGGGACTTTCAGCACCTGGTTTGGGGTCAT-3′; 09,5′-TTCGCCAGGGTTCCAATAACACGGCATCAT-AAAGG-3′; 28,5′-GCCAGTGTTCATTGGAAACCGCTCCCCAGCAG-TCT-3′; 29,5′-GCCAGAATTTGCTCCACAACAGTTCCTCCGTAAAG-3′; 36,5′-CCTGGCAAAGCGGGGGAGTGGGGAGTCGGGTATGG-3′; 37,5′-GGGGCCGTTCCTGGTTTCCACTGGGGCAAAGAGAA-3′; 38,5′-AGGAACGGCCCCTCCCAACCCTCCCGATGTCCACT-3′; 39,5′-AAGACCATACCCGACTCCCCACTCCCCCGCTTTGC-3′; 40,5′-AGCAAAGCGCACTCCCCCTCTTATGTCACCGAATA-3′; 41,5′-AGAATTCCAGAG-GCCGAGCTTTGCTACAGCGAAGG-3′; 42,5′-TGAGGGAGGCCCACA-TAGTGATGCCAACTGGATAC-3′; 45,5′-GGAGAGCATAAGAGAGG-GAGGCAGAGAGGAGAGAA-3′; 46,5′-ATTTGAAGTCAGAGGAAAAA-GCAGATGAGGAGTTT-3′; 47,5′-GCTGAGGTGGGAAGATCACTTG-AGACCAGGGATTC-3′; 48,5′-AGTAAGACCCTGTCTCAAAATAAA-GAAAGAAATAA-3′. The components of LDI-PCR reaction mixture (50 μl) were identical to those of LD-PCR. The first PCR reaction was carried out as described (13),although the annealing and extension time were shortened to 6 min. An aliquot of 1 μl of the first PCR product was subjected to a second PCR using the nested primers.

Molecular Cloning and Nucleotide Sequencing.

The PCR products with “A-overhangs” were ligated into a plasmid vector with 3′ T-overhangs at the cloning site (TA Cloning; Invitrogen,San Diego, CA). Transformation and extraction of DNA were performed by established methods. Nucleotide sequencing of the regions of interest was performed with a BigDye Terminator Cycle Sequencing Kit(Applied Biosystems, Foster City, CA), and the sequencing reactions were resolved on an ABI 310 automated sequencer (Applied Biosystems).

Gel Mobility-Shift Assay.

Nuclear extracts were prepared by the method of Schreiber et al.(16). Binding reactions were performed in a total volume of 10 μl containing 2 μl of extract, 2 μl of 5× binding buffer [20% glycerol, 5 mmMgCl2, 2.5 mm EDTA, 2.5 mm DTT, 250 mm NaCl, 50 mm Tris-HCl (pH 7.5), and 0.25 mg/ml poly(dI-dC)/poly(dI-dC); Promega, Madison, WI], and 1 μl of double-stranded, 32P-end-labeled probes. Incubations were carried out for 20 min at 20°C, and the resulting complexes were resolved on 4% nondenaturing polyacrylamide gels in 0.5× Tris-borate EDTA. For competition experiments, incubations were performed with a 100-fold-excess of unlabeled probe.

BCL6 Translocations in B-Cell Tumors.

This study included a total of 58 patients with B-cell tumors, who showed rearrangement of the BCL6 gene determined by Southern blotting analysis using the MTC probe (5; Fig. 1 A). Fifteen patients had a follicular lymphoma with varying degrees of mixtures of small cleaved and large cells, and 39 patients had diffuse large cell lymphomas. The other histologies, according to the REAL classification (14), included small lymphocytic lymphoma and Burkitt-like lymphoma. The remaining two cases were not classified.

IG/BCL6 Translocations Revealed by LD-PCR.

To detect IG/BCL6 junctions, we performed LD-PCR using primers for BCL6 exon 2 (BCL6 primer),approximately 4 kb upstream of exon 1 (5′-BCL6 primer), and constant region genes of the three IGs (Cprimers; Refs. 12 and 13). Of the 58 cases carrying a BCL6 rearrangement, 18 were positive for LD-PCR amplification (Fig. 2 A). The sizes of these LD-PCR products ranged from 4.3–14 kb and were unique to each tumor specimen. Cλ/BCL6fusions were demonstrated in five cases, and three had a t(3;22)(q27;q11) on cytogenetic analysis. A Cκ/BCL6 fusion was detected in a case carrying both t(14;18)(q32;q21) and t(2;3)(p11;q27) (6). The 5′-BCL6/CH primer pairs amplified fragments,including a junction on der(14)t(3;14) from 12 cases; four had a 5′-BCL6/Cμ fusion, and eight had a 5′-BCL6/Cγ fusion.

The LD-PCR products from the 18 cases were cloned into plasmids. Restriction analysis of the inserts confirmed that relevant regions of BCL6 and IGs were fused in the expected orientation. Nucleotide sequencing of the IGH/BCL6 junctions revealed that Sμor Sγ switch sequences, followed those of 5′-BCL6. The sequences immediately adjacent to the break points sometimes showed considerable complexity. For instance, a 412-bp fragment identified at the 5′-BCL6/Sγ junction of case 726 contained sequences that were identical to those from chromosome band 11q23, in addition to those showing sequence homology to the VH3–11 variable gene (data not shown).

On the other hand, the IGLκ/BCL6 fusion involved the 5′ side of a member of VκIV family (GenBank accession no. HS2 M7VK4) that was associated with the Jκ4 segment (Fig. 3,A). The break points of IGLλ/BCL6fusions occurred upstream of the Vλ2–11/Jλ2 complex (one case), at a point between the Vλ2–1, which is the most 3′ V gene of IGλ and the Jλ1 segment (one case) and between the Jλ and Cλ segments (three cases; Fig. 3, A and B). Thus, the positions of break points on the IGLs were not related to the regions in which V/J recombination normally occurs.

BCL6 Translocations Revealed by LDI-PCR.

Southern blot hybridization of XbaI- or BamHI-digested DNA with the F372 MTC probe generated a 13-kb or 11-kb germ-line band in addition to a rearranged band. Rearranged bands migrating faster than the germ-line bands were targeted by LDI-PCR to avoid preferential amplification of germ-line sequences. As indicated in Fig. 1 A, nested primer pairs in inverse orientation were designed corresponding to sequences 5′ and 3′ of the MTC. The LDI-PCR products, therefore, included regions from unknown sequences involved in each translocation, which were flanked by the known BCL6 sequence. The 3′ primer pairs amplified junctions on the der(3) chromosome, whereas junctional sequences on the partner chromosomes, der(partner), were obtained using the 5′ primer pairs.

We applied LDI-PCR to a total of 40 LD-PCR-negative cases and obtained amplified fragments corresponding to either or both the der(3) and the der(partner). Fig. 2,B shows representative results of EtBr-stained agarose gel electrophoresis of LDI-PCR products. All materials showed a unique PCR product ranging from 1.4–9.4 kb in size,which were the expected sizes from the results of Southern analysis and the distance of the primers used. The LDI-PCR products encompassing junctional points were cloned into plasmids and sequenced with primers from the known BCL6 sequences. The sequences appearing beyond the artificial XbaI or BamHI site represented those from the partners. Homology search of the GenBank database revealed that LDI-PCR products from an additional 12 cases included sequences from IGs; ten cases involved IGH, and IGLλ was the partner in two cases. In the latter fusions, the two translocations involved the 5′ side of the Vλ2–1 gene (Fig. 3 B, 883 and 908).

In contrast, 23 cases involved non-IG loci as partners. We previously reported that a novel H4 histone gene was linked to BCL6 in two B-NHL cases carrying t(3;6)(q27;p21.3)(10). The present LDI-PCR study revealed that an additional three cases involved this particular H4, which has been recently demonstrated to be included in a PAC clone form 6p21.31–6p22.1 (no. HS86C11). Nucleotide sequencing of the break points demonstrated that the translocation was reciprocal at the nucleotide level, and the break points on H4 from the total of five cases were distributed within the single exon to 3′ of the terminal palindrome (Fig. 4,A and Fig. 5 A).

Other previously known genes included two heat shock protein genes, HSP89α and HSP90β, and PIM-1proto-oncogene. The HSP89α gene is composed of 11 exons,and exons 2 through 11 encode the Mr89,000 protein (17). The breakage occurred within intron 1, resulting in exchange of the first noncoding exons of BCL6 and HSP89α (Fig. 4,B). Three heat shock response elements composed of contiguous inverted repeats of a 5-bp sequence, the consensus of which was defined as nGAAn (n is any nucleotide; Ref. 18), were identified in −448 to −439,−429 to −420, and −123 to −114 bp of the break point; all were in the tail-to-tail orientation (Fig. 5,B). On the other hand, HSP90β mapped to 6p12 was composed of 12 exons, and the translation initiation codon lay at the 5′ end of exon 2 (Fig. 4,C; Ref. 19). The break point was close to the 5′ end of exon 3, and three heat shock elements, nGAAnnTTCn, were localized within intron 1 (Fig. 5,C). The PIM-1proto-oncogene consists of 6 exons (Fig. 4,D) and has been mapped to 6p21 (20). The gene has a characteristic G+C-rich housekeeping promoter that lacks TATA or CAAT sequences (Fig. 5 D; Refs. 20 and 21); however,more distant elements have been presumed to regulate tissue-selective expression (21). The breakpoints on der(3) and der(6) were localized within intron 1, both of which were downstream of the translation initiation site.

Other partner sequences registered in the database were TTF(9), transferrin receptor gene (TFRC),α-NAC transcriptional coactivator gene (22),and MHC class II transactivator gene CIITA(23). Two cases had two independent non-IG/BCL6 translocations. The sequences of 11 partners are currently uncharacterized. The remaining five cases did not have a rearrangement with other loci; three had a deletion of 1.0–2.2-kb segments within the MTC, and two had nucleotide substitutions leading to generation of additional XbaI sites(Fig. 1). Table 1 summarizes partner loci identified by the two LD-PCR-based assays. Fifty-two percent of cases involved IG loci as partners, and the major locus was IGH. These frequencies were in agreement with those of 3q27 translocations determined by cytogenetic analysis(24).

Identification of a 120-bp Break Point Hyper-Cluster Region.

We determined the positions of a total of 55 break points in the 53 cases with BCL6 translocations. The results showed that 52 break points fell into the MTC, whereas the remaining three were outside the MTC but still within a region that does not affect the coding exons of BCL6 producing Mr 52,000 Bcl-6 protein. As illustrated in Fig. 1,A, the vast majority of break points were further clustered a short distance downstream of exon 1. Fig. 1 B shows the nucleotide sequences of intron 1 and the positions of break points of 19 cases. Of the 19 break points, 12 involved IG genes as partners, and the remaining break points were associated with non-IG loci. A 120-bp region containing 16 break points was designated as a hyper-cluster region;the region lacked known sequences that have been observed at other IG/oncogene junctions.

Gel Mobility-Shift Assay Suggesting the Presence of a Protein(s)Capable of Binding to the Break Point Hyper-Cluster Region.

We prepared a 30-mer double-stranded oligonucleotide probe (probe 6162;Fig. 1,B) included in the hyper-cluster and studied the presence of a sequence-specific DNA-binding protein(s). Nuclear extracts from hematological cell lines were mixed with the end-labeled probe and subjected to gel mobility-shift assay. Fig. 6 shows representative results for protein/DNA complexes. Sifted bands were prominently observed in a diffuse large cell lymphoma cell line and an EBV-transformed lymphoblastoid cell line, and their specificity was confirmed by competition experiments using excess amounts of unlabeled probe. As indicated in Fig. 1 B, 10 break points were within or close to this probe, suggesting that this particular fragment could include sequences that are related to the underlying mechanism of BCL6 translocation.

IG/BCL6 Translocation Occurs in Mature B-Cells That Have Completed V/(D)/JAssemblies in the Three IGs.

Because translocations involving IGs almost certainly occur as the result of errors of the recombination process (1),the positions of break points on IGs can reflect B-cell stage where the translocation develops. In analogous to t(8;14)(q24;q32) of the sporadic type of Burkitt’s lymphoma, IGH/BCL6 translocation invariably involves switch regions of IGH. Therefore, this translocation is most likely associated with the isotype class switching process following completion of V/D/J assembly and occurs at the mature B-cell stage in the germinal center of lymphoid tissues.

However, this “miss-class switch” mechanism may not be applicable to the variant IGL/BCL6 translocations. In the present study, we cloned a total of eight IGL/BCL6 fusions. Sequencing analysis showed that four break points were 5′ of V genes and two of the four were associated with a J segment, indicating that these translocations involved IGL alleles that had already completed V/J assembly. On the other hand, the remaining four break points were distributed within the IGLλ gene and had no apparent association with Vλ/Jλ joining. Therefore, it is likely that these translocations involved an IGLλ gene with the germ line configuration. Because there is a clear order of events in the formation of an IGL gene (i.e., the IGLκ gene is rearranged first, and the IGLλgene is not affected by the rearrangement process when the IGLκ successfully forms an active gene), our findings led to the conclusion that IGL/BCL6 translocation occurs at the mature B-cell stage when normal V/Jassemblies of the two IGLs have been completed. The mechanisms responsible for the illegitimate recombination between these particular regions of IGL and BCL6 gene remain to be elucidated.

Partner Genes Involved in Non-IG/BCL6 Translocations are Transcriptionally Activated by a Variety of Stimuli.

The common molecular features of non-IG/BCL6translocations include: (a) the gene fusion occurs in the same transcriptional orientation; (b) the break point on the partner gene is localized in close proximity to its promoter sequence;and (c) the complete set of the promoter is fused upstream of the coding region of BCL6 on the der(3) chromosome. It should be noted also that a portion of the 5′ exon(s) of the partner gene, which may or may not include coding regions, was invariably included in the non-IG/BCL6 junction, potentially leading to generation of fusion mRNAs. This may be of functional significance, because it could have substantial impact on the stability and/or translatability of the resulting fusion transcripts. On the other hand, coding exons of the partner gene were interrupted by the upstream sequences of BCL6 in the BCL6/H4, BCL6/HSP90β, and BCL6/PIM-1 fusions, but remained intact in the BCL6/HSP89α fusion. Thus, it seems that partner genes on the translocated allele may not be important although they are fused with the BCL6 promoter.

Expression of the BCL6 gene is tightly regulated during B-cell differentiation; its expression increases in mature B-cells in the germinal center and then decreases during their differentiation into plasma cells (25). Thus, it is conceivable that inappropriate expression of BCL6 may result in transcriptional repression of genes involved in terminal differentiation or in apoptosis, thereby leading to neoplastic cell growth of B cells. In the present study, we identified four unique non-IG/BCL6 fusions in which the pattern of transcriptional regulation of the partner genes has been well studied. The Site II equivalent of the present H4 gene contains consensus binding sites for two transcription factors (i.e.,histone nuclear factor D and IFN regulatory factor 2; Ref.10). histone nuclear factor D is a multicomponent protein containing cyclin A, CDC2, and an RB-related protein, which apparently links H4 gene regulation directly to cell cycle control (26). The regulation of HSP gene expression in eukaryotes is mediated by the conserved HSF(18). HSF acts through the HSEs composed of three contiguous inverted repeats of a 5-bp sequence. On heat stress, HSF binds to the HSEs as a trimer, and all three repeats are required for high-affinity interaction (18).

The PIM-1 proto-oncogene encodes a serine/threonine protein kinase and plays a role in the signal transduction events associated with lymphocyte activation (27). The basal level of PIM-1 expression seems to be regulated in a cell type-specific manner (i.e., highest in myeloid and B cell lines and lowest in T cell lines; Ref. 21). On the other hand, inducible PIM-1 expression has been observed in response to a wide variety of mitogens and cytokines (27). Interleukin-3-induced PIM-1 expression is suppressed by dominant-negative Stat5 mutants, suggesting that PIM-1 is regulated by Jak-Stat pathways (28). Indeed, a canonicalγIFN-activated site-like motif was identified in the promoter region(27).

In summary, our observation suggested that partner genes are transcriptionally activated by a variety of stimuli, including cell cycle control, changes in the physical environment, and response to cytokines. Involvement of the transactivator gene CIITA, the expression of which is induced by IFN-γ (23), is another example of this observation. Thus, it seems that alteration of BCL6 expression resulting from non-IG/BCL6 translocation is much more elegant than simple overexpression or constitutive expression. As germinal center B cells proliferate rapidly in response to antigen stimulation,it is likely that the BCL6 gene affected by the translocation is inappropriately expressed during the B-cell proliferation, although the possible links between the deregulated BCL6 expression and ultimate development of lymphoma remain to be established.

The Intronic Sequence Immediately 3′ of BCL6 Exon 1 Is the Target for Somatic Mutation and Chromosomal Translocation.

Somatic mutations within the 5′ noncoding region of BCL6have been described in B-cell tumors of the germinal center/postgerminal center type (29, 30). The majorities of the mutations are clustering 3′ of exon 1, which has been referred to as the MMC (31). These mutations are often multiple,are frequently biallelic and are independent of BCL6translocation or linkage to IGs (29, 30). On the other hand, somatic mutations within the MMC were recently reported in a large proportion of memory B cells isolated from normal individuals as well as germinal center B cells from a reactive tonsil(32).

The present study clearly showed that the break point hyper-cluster was affected by a variety of BCL6translocations and the MMC overlap. Thus, the two somatic events(i.e., mutations and chromosomal translocation) are apparently related. It is possible that mutations occurring within the MMC might alter the chromatin structure of this particular region,thereby being prone to rearrangement with other independent genes. This may partly account for the lack of known characteristic sequences that potentially mediate chromosome translocations within this region. In this context, we are currently investigating whether binding of the protein(s) identified by the gel mobility-shift assay is enhanced for the hyper-cluster sequence that carries mutations.

Fig. 1.

A, restriction map of the BCL6 gene, and distribution of the break points of BCL6 translocations. The two most 5′ exons of BCL6 are indicated by boxes. The MTC was as determined by Bastard et al.(5). Vertical lines indicate the positions of break points; IG/BCL6 translocations are indicated in red, whereas non-IG/BCL6translocations are shown in blue. Deletions(green bars) and point mutations generating additional XbaI sites (yellow lines) resulted in“false rearrangement” on Southern blotting. Open and closed arrowheads indicate the positions of primers for LD-PCR and LDI-PCR, respectively. Restriction sites: E, EcoRI; B, BamHI; H, HindIII; X, XbaI; S, SacI; Xh, XhoI. B, nucleotide sequences of the break point hyper-cluster region and the positions of break points of IG/BCL6 translocation (red arrowheads) and non-IG/BCL6translocation (blue arrowheads). These break points were determined by sequencing of PCR products either from der(3) or der(partner). The relevant partner gene of each translocation is indicated. The G to A substitution (yellow arrowhead)generated a new XbaI site. Probe 6162 used for gel mobility-shift assay is overlined.

Fig. 1.

A, restriction map of the BCL6 gene, and distribution of the break points of BCL6 translocations. The two most 5′ exons of BCL6 are indicated by boxes. The MTC was as determined by Bastard et al.(5). Vertical lines indicate the positions of break points; IG/BCL6 translocations are indicated in red, whereas non-IG/BCL6translocations are shown in blue. Deletions(green bars) and point mutations generating additional XbaI sites (yellow lines) resulted in“false rearrangement” on Southern blotting. Open and closed arrowheads indicate the positions of primers for LD-PCR and LDI-PCR, respectively. Restriction sites: E, EcoRI; B, BamHI; H, HindIII; X, XbaI; S, SacI; Xh, XhoI. B, nucleotide sequences of the break point hyper-cluster region and the positions of break points of IG/BCL6 translocation (red arrowheads) and non-IG/BCL6translocation (blue arrowheads). These break points were determined by sequencing of PCR products either from der(3) or der(partner). The relevant partner gene of each translocation is indicated. The G to A substitution (yellow arrowhead)generated a new XbaI site. Probe 6162 used for gel mobility-shift assay is overlined.

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Fig. 2.

A, EtBr-stained gel electrophoresis of LD-PCR showing IG/BCL6 fusion genes. Cregion genes involved in each fusion are indicated at the bottom. B, LDI-PCR showing fusions of BCL6 with unknown partners. The LDI-PCR products represent fusions either on der(3) (229, 457a, 816, 124, 943, 908, 276,564) or der(partner) (457b, 764, 458, 201, 190), and partner genes identified by sequencing analysis of the products are indicated at the bottom. Five cases with a deletion or a point mutation refer to Fig. 1. An aliquot of 2–10 μl was loaded in each lane and electrophoresed through a 0.7% agarose gel. HindIII-digested λDNA was used as a molecular weight marker.

Fig. 2.

A, EtBr-stained gel electrophoresis of LD-PCR showing IG/BCL6 fusion genes. Cregion genes involved in each fusion are indicated at the bottom. B, LDI-PCR showing fusions of BCL6 with unknown partners. The LDI-PCR products represent fusions either on der(3) (229, 457a, 816, 124, 943, 908, 276,564) or der(partner) (457b, 764, 458, 201, 190), and partner genes identified by sequencing analysis of the products are indicated at the bottom. Five cases with a deletion or a point mutation refer to Fig. 1. An aliquot of 2–10 μl was loaded in each lane and electrophoresed through a 0.7% agarose gel. HindIII-digested λDNA was used as a molecular weight marker.

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Fig. 3.

A, nucleotide sequences of IGL/BCL6 junctions. The distribution of the break points on IGLλ is illustrated in B. Variable numbers of nucleotides of unknown origin were inserted at the break points (lowercase letters). Nucleotide substitutions deviating from sequences in the available databases are italicized.

Fig. 3.

A, nucleotide sequences of IGL/BCL6 junctions. The distribution of the break points on IGLλ is illustrated in B. Variable numbers of nucleotides of unknown origin were inserted at the break points (lowercase letters). Nucleotide substitutions deviating from sequences in the available databases are italicized.

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Fig. 4.

Schematic representation of non-IG/BCL6 translocations involving H4 histone gene (A), HSP89α heat shock protein gene (B), HSP90β (C), and PIM-1proto-oncogene (D) with reference to that of BCL6(top). Those on the der(partner) are not illustrated. Arrows indicate break points of each translocation. Open (BCL6) and closed(partner genes) boxes indicate the exons, and arrowheads indicate the translation initiation sites of each gene. All of the translocations occurred in the same transcriptional orientation.

Fig. 4.

Schematic representation of non-IG/BCL6 translocations involving H4 histone gene (A), HSP89α heat shock protein gene (B), HSP90β (C), and PIM-1proto-oncogene (D) with reference to that of BCL6(top). Those on the der(partner) are not illustrated. Arrows indicate break points of each translocation. Open (BCL6) and closed(partner genes) boxes indicate the exons, and arrowheads indicate the translation initiation sites of each gene. All of the translocations occurred in the same transcriptional orientation.

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Fig. 5.

Nucleotide sequences of the promoter region of H4 (A; Ref. 10), HSP89α (B; Ref. 17), HSP90β (C; Ref. 19), and PIM-1 (D; Refs. 20 and21). Arrowheads indicate the positions of break points on der(3) and der(partner) of each tumor. The protein coding region of H4, as well as exons of the remaining genes, is represented in boldface letters, and the translation initiation codons of each gene are italicized. Characteristic promoter sequences are underlined: H4, Site II; HSP89α and HSP90β, HSEs; PIM-1, SP1-binding andγIFN-activated site-like motifs (27). The presence of these promoters on the der(3) chromosome from each tumor was confirmed by sequencing of PCR products obtained using appropriate primer combinations.

Fig. 5.

Nucleotide sequences of the promoter region of H4 (A; Ref. 10), HSP89α (B; Ref. 17), HSP90β (C; Ref. 19), and PIM-1 (D; Refs. 20 and21). Arrowheads indicate the positions of break points on der(3) and der(partner) of each tumor. The protein coding region of H4, as well as exons of the remaining genes, is represented in boldface letters, and the translation initiation codons of each gene are italicized. Characteristic promoter sequences are underlined: H4, Site II; HSP89α and HSP90β, HSEs; PIM-1, SP1-binding andγIFN-activated site-like motifs (27). The presence of these promoters on the der(3) chromosome from each tumor was confirmed by sequencing of PCR products obtained using appropriate primer combinations.

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Fig. 6.

Gel mobility-shift assay showing the presence of DNA/protein products. Probe 6162 indicated in Fig. 1 Bwas double-stranded and end-labeled with 32P. Nuclear extracts were obtained from: YM, diffuse large cell lymphoma cell line associated with both BCL2 and BCL6 rearrangements; K562, chronic myelocytic leukemia cell line; HL-60, acute promyelocytic leukemia cell line; LCL-OHN,EBV-transformed lymphoblastoid cell line. Open and closed arrowheads indicate the positions of the probe and DNA/protein products, respectively. Binding was inhibited by the presence of 100-fold excess amounts of unlabeled probe(competitor − or +).

Fig. 6.

Gel mobility-shift assay showing the presence of DNA/protein products. Probe 6162 indicated in Fig. 1 Bwas double-stranded and end-labeled with 32P. Nuclear extracts were obtained from: YM, diffuse large cell lymphoma cell line associated with both BCL2 and BCL6 rearrangements; K562, chronic myelocytic leukemia cell line; HL-60, acute promyelocytic leukemia cell line; LCL-OHN,EBV-transformed lymphoblastoid cell line. Open and closed arrowheads indicate the positions of the probe and DNA/protein products, respectively. Binding was inhibited by the presence of 100-fold excess amounts of unlabeled probe(competitor − or +).

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1

Supported by grants-in-aid from the Ministry of Health and Welfare (7-29 and 9-10) and from the Ministry of Education,Science, Sports and Culture (11670994), Japan.

4

The abbreviations used are: IG,immunoglobulin gene; IGH, IG heavy chain gene; IGL, IG light chain gene; IGLκ, κlight chain gene; IGLλ, λ light chain gene; V, variable; D, diversity; J, joining; S, switch; C,constant; B-NHL, B-cell non-Hodgkin’s lymphoma; MTC, major translocation cluster; LD, long-distance; LDI, LD inverse; EtBr,ethidium bromide; HSF, heat shock transcription factor; HSE, HS response elements; MMC, major mutation cluster.

Table 1

Partner loci of BCL6 tranlocations determined by LD-PCR-based assays

Involved geneChromosomal locusCase number
IG 30 
IGH 14q32 22 
IGLκ 2p12  1 
IGLλ 22q11  7 
Non-IG loci  25 (23)a 
H4 histone 6p21.3  5 
TTF 4p13  2 
TFRC 3q29  2 
HSP89α 14q32  1 
HSP90β 6p12  1 
PIM-1 6p21.2  1 
α-NAC 12q23–q24.1  1 
CIITA 16p13  1 
Currently uncharacterized  11 
Deletion of mutations of BCL6   5 
Total  60 (58)a 
Involved geneChromosomal locusCase number
IG 30 
IGH 14q32 22 
IGLκ 2p12  1 
IGLλ 22q11  7 
Non-IG loci  25 (23)a 
H4 histone 6p21.3  5 
TTF 4p13  2 
TFRC 3q29  2 
HSP89α 14q32  1 
HSP90β 6p12  1 
PIM-1 6p21.2  1 
α-NAC 12q23–q24.1  1 
CIITA 16p13  1 
Currently uncharacterized  11 
Deletion of mutations of BCL6   5 
Total  60 (58)a 
a

Two cases had two independent non-IG/BCL6translocations.

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