Molecular Anatomy of BCL6 Translocations Revealed by Long-Distance Polymerase Chain Reaction-Based Assays1

[CANCER RESEARCH 60, 2335–2341, May 1, 2000] Advances in Brief

Molecular Anatomy of BCL6 Translocations Revealed by Long-Distance Polymerase Chain Reaction-based Assays1

Hiroshi Akasaka,2 Takashi Akasaka,2 Masayuki Kurata, Chiyoko Ueda, Akira Shimizu, Takashi Uchiyama, and Hitoshi Ohno3 First Division, Department of Internal Medicine, Faculty of Medicine [H. A., T. A., M. K., C. U., T. U., H. O.], and Center for Molecular Biology and Genetics [T. A., A. S.], Kyoto University, Kyoto 606-8507, Japan

Abstract independent chromosomal sites have been reported to be partners (7), and several genes have been identified in the vicinity of break points BCL6 translocations involve not only immunoglobulin (IG) genes but on partner chromosomes (8–10). These molecular studies have shown also a number of non-IG loci as partners. Junctional sequences of three that breakage most frequently occurs within the MTC, encompassing IG/BCL6 translocations were readily obtained by long-distance PCR. In cases where partner loci were not determined, we developed a long- the noncoding exon 1 of BCL6 (4, 5). As a result of the translocation, distance inverse PCR method, which amplifies unknown fragments many types of regulatory sequences on each partner chromosomal flanked by known BCL6 sequences. Using these two long-distance PCR- locus substitute for the 5Ј untranslated region of BCL6 (11) and the based approaches, we cloned junctional areas of BCL6 translocations from rearranged BCL6 gene is presumed to be under the control of the a total of 58 cases of B-cell tumors. Nucleotide sequencing and database replaced promoter activity. However, the patterns of regulation of searches revealed that 30 cases involved IGs as partners: IG heavy chain each regulatory sequence leading to deregulated BCL6 expression gene in 22, IG ␬ light chain gene in 1, and IG ␭ light chain gene in 7. In have not yet been completely characterized. contrast, 23 cases affected non-IG loci, including the H4 histone gene, heat We have developed a LD-PCR method capable of detecting IG/ ␣ ␤ shock protein genes HSP89 and HSP90 , and PIM-1 proto-oncogene. On oncogene junctions of up to 30 kb (12, 13). Our previous study der(3) chromosomes, complete sets of the promoters of these partner genes showed that LD-PCR successfully detected three IG/BCL6 transloca- replaced that of BCL6 in the same transcriptional orientation. These results suggest that BCL6 gene affected by the translocation is transcrip- tions [i.e., t(2;3)(p12;q27), t(3;22)(q27;q11), and t(3;14)(q27;q32)], tionally activated by a variety of stimuli, including cell cycle control, which had been well characterized at the molecular level. For cases in changes in the physical environment, and response to cytokines. Break which partner loci were not determined, we have developed a LDI- points on BCL6 occurred within the major translocation cluster, and we PCR method, which amplifies unknown fragments flanked by known identified a 120-bp hyper-cluster region a short distance from the 3؅ end BCL6 sequences. Here, we report the cloning and sequencing of a of exon 1. Gel mobility-shift assay suggested the presence of a protein(s) number of BCL6 translocations from a large series of B-cell tumors that bound to this particular region. using these two LD-PCR-based assays. The purpose of the present study was to explore the diverse molecular anatomy of BCL6 trans- Introduction locations and to find structural and functional consequences of the Human B-cell tumors are often associated with chromosomal trans- juxtaposition of the BCL6 gene to a wide variety of partner genes. locations that lead to juxtaposition of cellular oncogenes to the IG4 loci (1). The BCL6 gene was first identified at the break points on Materials and Methods 3q27 involved in t(3;14)(q27;q32) and t(3;22)(q27;q11) (2–4). Initial Tumor Specimens. Lymphoma tissues or other tumor specimens obtained studies suggested that 3q27 translocation and/or rearrangement of the from patients admitted to Kyoto University Hospital and related hospitals BCL6 gene were specifically associated with the diffuse large cell between 1983 and 1997 were analyzed. The lymphomas were originally subtype of B-NHL (4). However, later studies of panels of many types classified according to the International Working Formulation for Clinical of B-NHL invariably showed that this genetic lesion was found in a Usage, but their pathological diagnoses were updated by the REAL classifi- significant proportion of follicular lymphomas (5, 6). In contrast to cation (14). Materials studied were subjected to immunohistochemical analysis other B-NHL-associated translocations, BCL6 translocations are and/or surface immunophenotyping, as well as gene rearrangement analysis unique in that they can involve not only IGs but also other as yet using probes for the IGH gene, to determine B-cell origin of the lymphoma uncharacterized chromosomal loci as partners. To date, nearly 20 cells. Southern Blot Hybridization. Genomic DNA extracted from lymphoma specimens was digested with appropriate restriction enzymes and electrophore- Received 8/30/99; accepted 3/17/00. sed through 0.8% agarose gels. DNA was transferred onto nylon membrane The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with filters (GeneScreen Plus; NEN Research products, Boston, MA) and hybrid- 18 U.S.C. Section 1734 solely to indicate this fact. ized with probes labeled with 32P using a random labeling system. The F372 1 Supported by grants-in-aid from the Ministry of Health and Welfare (7-29 and 9-10) probe for the MTC region of the BCL6 gene was a 2.3-kb XhoI/BamHI and from the Ministry of Education, Science, Sports and Culture (11670994), Japan. fragment (5). 2 These two authors equally contributed to this work. T. A. is a fellow in Cancer Research of the Japan Society for the Promotion of Science and a recipient of the Award LD-PCR. Designations and sequences of the oligonucleotide primers to for Young Investigators from the American Association for Cancer Research. detect IG/oncogene fusion genes were listed in our previous studies (12, 13). 3 To whom requests for reprints should be addressed, at First Division, Department of Each PCR reaction mixture (50 ␮l) contained 100 ng of genomic DNA, Internal Medicine, Faculty of Medicine, Kyoto University, 54-Shogoin-Kawaramachi, reaction buffer, dNTP mixture, 20 pmol of each primer, and 2.5 units of LA Sakyo-ku, Kyoto 606-8507, Japan. Phone and fax: 81-75-751-3155; E-mail: hohno@ kuhp.kyoto-u.ac.jp. Taq DNA polymerase (Takara Shuzo, Kyoto). PCR cycling variables for long 4 The abbreviations used are: IG, immunoglobulin gene; IGH, IG heavy chain gene; DNA targets were previously described in detail (13). Aliquots of the PCR IGL, IG light chain gene; IGL␬, ␬ light chain gene; IGL␭, ␭ light chain gene; V, variable; products were analyzed by agarose gel electrophoresis and visualized under D, diversity; J, joining; S, switch; C, constant; B-NHL, B-cell non-Hodgkin’s lymphoma; UV illumination after EtBr staining. MTC, major translocation cluster; LD, long-distance; LDI, LD inverse; EtBr, ethidium bromide; HSF, heat shock transcription factor; HSE, HS response elements; MMC, major LDI-PCR. High molecular weight genomic DNA was digested with XbaI mutation cluster. or BamHI and purified by standard methods. The DNA was diluted to a 2335

Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 2000 American Association for Cancer Research. ANATOMY OF BCL6 TRANSLOCATIONS concentration of 1 ␮g/ml and incubated at 16°C overnight in the presence of 20 min at 20°C, and the resulting complexes were resolved on 4% nondena- T4 DNA ligase to facilitate intramolecular ligation (15). The self-ligated turing polyacrylamide gels in 0.5ϫ Tris-borate EDTA. For competition ex- circular DNA was used as a template for a nested PCR; the circular DNA was periments, incubations were performed with a 100-fold-excess of unlabeled not reopened. The position and orientation of the primers are illustrated in Fig. probe. 1A. The sequences were: 08, 5Ј-CAGCTTGGGACTTTCAGCACCTGGTTT- GGGGTCAT-3Ј; 09, 5Ј-TTCGCCAGGGTTCCAATAACACGGCATCAT- Results AAAGG-3Ј; 28, 5Ј-GCCAGTGTTCATTGGAAACCGCTCCCCAGCAG- TCT-3Ј; 29, 5Ј-GCCAGAATTTGCTCCACAACAGTTCCTCCGTAAAG- BCL6 Translocations in B-Cell Tumors. This study included a 3Ј; 36, 5Ј-CCTGGCAAAGCGGGGGAGTGGGGAGTCGGGTATGG-3Ј; 37, total of 58 patients with B-cell tumors, who showed rearrangement of 5Ј-GGGGCCGTTCCTGGTTTCCACTGGGGCAAAGAGAA-3Ј; 38, 5Ј-AG- the BCL6 gene determined by Southern blotting analysis using the Ј Ј GAACGGCCCCTCCCAACCCTCCCGATGTCCACT-3 ; 39, 5 -AAGAC- MTC probe (5; Fig. 1A). Fifteen patients had a follicular lymphoma CATACCCGACTCCCCACTCCCCCGCTTTGC-3Ј; 40, 5Ј-AGCAAAGCG- with varying degrees of mixtures of small cleaved and large cells, and CACTCCCCCTCTTATGTCACCGAATA-3Ј; 41, 5Ј-AGAATTCCAGAG- GCCGAGCTTTGCTACAGCGAAGG-3Ј; 42, 5Ј-TGAGGGAGGCCCACA- 39 patients had diffuse large cell lymphomas. The other histologies, TAGTGATGCCAACTGGATAC-3Ј; 45, 5Ј-GGAGAGCATAAGAGAGG- according to the REAL classification (14), included small lympho- GAGGCAGAGAGGAGAGAA-3Ј; 46, 5Ј-ATTTGAAGTCAGAGGAAAAA- cytic lymphoma and Burkitt-like lymphoma. The remaining two cases GCAGATGAGGAGTTT-3Ј; 47, 5Ј-GCTGAGGTGGGAAGATCACTTG- were not classified. AGACCAGGGATTC-3Ј; 48, 5Ј-AGTAAGACCCTGTCTCAAAATAAA- IG/BCL6 Translocations Revealed by LD-PCR. To detect IG/ GAAAGAAATAA-3Ј. The components of LDI-PCR reaction mixture (50 ␮l) BCL6 junctions, we performed LD-PCR using primers for BCL6 exon were identical to those of LD-PCR. The first PCR reaction was carried out as 2(BCL6 primer), approximately 4 kb upstream of exon 1 (5Ј-BCL6 described (13), although the annealing and extension time were shortened to 6 primer), and constant region genes of the three IGs(C primers; Refs. min. An aliquot of 1 ␮l of the first PCR product was subjected to a second PCR 12 and 13). Of the 58 cases carrying a BCL6 rearrangement, 18 were using the nested primers. Molecular Cloning and Nucleotide Sequencing. The PCR products with positive for LD-PCR amplification (Fig. 2A). The sizes of these “A-overhangs” were ligated into a plasmid vector with 3Ј T-overhangs at the LD-PCR products ranged from 4.3–14 kb and were unique to each cloning site (TA Cloning; Invitrogen, San Diego, CA). Transformation and tumor specimen. C␭/BCL6 fusions were demonstrated in five cases, extraction of DNA were performed by established methods. Nucleotide se- and three had a t(3;22)(q27;q11) on cytogenetic analysis. A C␬/BCL6 quencing of the regions of interest was performed with a BigDye Terminator fusion was detected in a case carrying both t(14;18)(q32;q21) and Cycle Sequencing Kit (Applied Biosystems, Foster City, CA), and the se- t(2;3)(p11;q27) (6). The 5Ј-BCL6/CH primer pairs amplified frag- quencing reactions were resolved on an ABI 310 automated sequencer (Ap- ments, including a junction on der(14)t(3;14) from 12 cases; four had plied Biosystems). a5Ј-BCL6/C␮ fusion, and eight had a 5Ј-BCL6/C␥ fusion. Gel Mobility-Shift Assay. Nuclear extracts were prepared by the method The LD-PCR products from the 18 cases were cloned into plasmids. of Schreiber et al. (16). Binding reactions were performed in a total volume of 10 ␮l containing 2 ␮l of extract, 2 ␮lof5ϫ binding buffer [20% glycerol, 5 Restriction analysis of the inserts confirmed that relevant regions of BCL6 and IGs were fused in the expected orientation. Nucleotide mM MgCl2, 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 sequencing of the IGH/BCL6 junctions revealed that S␮ or S␥ switch ␮l of double-stranded, 32P-end-labeled probes. Incubations were carried out for sequences, followed those of 5Ј-BCL6. The sequences immediately

Fig. 1. A, restriction map of the BCL6 gene, and distribution of the break points of BCL6 translocations. The two most 5Ј 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/BCL6 translocations 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/BCL6 translocation (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. 2336

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 IGL␬/BCL6 fusion involved the 5Ј side of a member of V␬IV family (GenBank accession no. HS2 M7VK4) that was associated with the J␬4 segment (Fig. 3A). The break points of IGL␭/BCL6 fusions occurred upstream of the V␭2–11/J␭2 complex (one case), at a point between the V␭2–1, which is the most 3Ј V gene of IG␭ and the J␭1 segment (one case) and between the J␭ and C␭ 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. 1A, nested primer pairs in inverse orientation were designed corresponding to sequences 5Ј and 3Ј 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 3Ј primer pairs amplified junctions on the der(3) chromosome, whereas junctional sequences on the partner chromosomes, der(partner), were obtained using the 5Ј 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. 2B 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 Fig. 2. A, EtBr-stained gel electrophoresis of LD-PCR showing IG/BCL6 fusion genes. analysis and the distance of the primers used. The LDI-PCR products C region 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 encompassing junctional points were cloned into plasmids and se- on der(3) (229, 457a, 816, 124, 943, 908, 276, 564) or der(partner) (457b, 764, 458, 201, quenced with primers from the known BCL6 sequences. The se- 190), and partner genes identified by sequencing analysis of the products are indicated at quences appearing beyond the artificial XbaIorBamHI site repre- the bottom. Five cases with a deletion or a point mutation refer to Fig. 1. An aliquot of 2–10 ␮l was loaded in each lane and electrophoresed through a 0.7% agarose gel. sented those from the partners. Homology search of the GenBank HindIII-digested ␭DNA was used as a molecular weight marker. database revealed that LDI-PCR products from an additional 12 cases included sequences from IGs; ten cases involved IGH, and IGL␭ was the partner in two cases. In the latter fusions, the two translocations adjacent to the break points sometimes showed considerable complex- involved the 5Ј side of the V␭2–1 gene (Fig. 3B, 883 and 908). ity. For instance, a 412-bp fragment identified at the 5Ј-BCL6/S␥ In contrast, 23 cases involved non-IG loci as partners. We previ- junction of case 726 contained sequences that were identical to those ously reported that a novel H4 histone gene was linked to BCL6 in two

Fig. 3. A, nucleotide sequences of IGL/BCL6 junctions. The distribution of the break points on IGL␭ 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. 2337

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), ␣-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. 1A, the vast majority of break points were further clustered a short distance downstream of exon 1. Fig. 1B 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. Fig. 4. Schematic representation of non-IG/BCL6 translocations involving H4 histone Gel Mobility-Shift Assay Suggesting the Presence of a Pro- gene (A), HSP89␣ heat shock protein gene (B), HSP90␤ (C), and PIM-1 proto-oncogene tein(s) Capable of Binding to the Break Point Hyper-Cluster (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 Region. We prepared a 30-mer double-stranded oligonucleotide genes) boxes indicate the exons, and arrowheads indicate the translation initiation sites of probe (probe 6162; Fig. 1B) included in the hyper-cluster and studied each gene. All of the translocations occurred in the same transcriptional orientation. 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 B-NHL cases carrying t(3;6)(q27;p21.3) (10). The present LDI-PCR representative results for protein/DNA complexes. Sifted bands were study revealed that an additional three cases involved this particular prominently observed in a diffuse large cell lymphoma cell line and an H4, which has been recently demonstrated to be included in a PAC EBV-transformed lymphoblastoid cell line, and their specificity was clone form 6p21.31–6p22.1 (no. HS86C11). Nucleotide sequencing confirmed by competition experiments using excess amounts of un- of the break points demonstrated that the translocation was reciprocal labeled probe. As indicated in Fig. 1B, 10 break points were within or at the nucleotide level, and the break points on H4 from the total of close to this probe, suggesting that this particular fragment could five cases were distributed within the single exon to 3Ј of the terminal include sequences that are related to the underlying mechanism of palindrome (Fig. 4A and 5A). BCL6 translocation. Other previously known genes included two heat shock protein ␣ ␤ genes, HSP89 and HSP90 , and PIM-1 proto-oncogene. The Discussion HSP89␣ gene is composed of 11 exons, and exons 2 through 11 encode the Mr 89,000 protein (17). The breakage occurred within IG/BCL6 Translocation Occurs in Mature B-Cells That Have Com- intron 1, resulting in exchange of the first noncoding exons of BCL6 pleted V/(D)/J Assemblies in the Three IGs. and HSP89␣ (Fig. 4B). Three heat shock response elements composed Because translocations involving IGs almost certainly occur as the of contiguous inverted repeats of a 5-bp sequence, the consensus of result of errors of the recombination process (1), the positions of break which was defined as nGAAn (n is any nucleotide; Ref. 18), were points on IGs can reflect B-cell stage where the translocation devel- identified in Ϫ448 to Ϫ439, Ϫ429 to Ϫ420, and Ϫ123 to Ϫ114 bp of ops. In analogous to t(8;14)(q24;q32) of the sporadic type of Burkitt’s the break point; all were in the tail-to-tail orientation (Fig. 5B). On the lymphoma, IGH/BCL6 translocation invariably involves switch re- other hand, HSP90␤ mapped to 6p12 was composed of 12 exons, and gions of IGH. Therefore, this translocation is most likely associated the translation initiation codon lay at the 5Ј end of exon 2 (Fig. 4C; with the isotype class switching process following completion of Ref. 19). The break point was close to the 5Ј end of exon 3, and three V/D/J assembly and occurs at the mature B-cell stage in the germinal heat shock elements, nGAAnnTTCn, were localized within intron 1 center of lymphoid tissues. (Fig. 5C). The PIM-1 proto-oncogene consists of 6 exons (Fig. 4D) However, this “miss-class switch” mechanism may not be applica- and has been mapped to 6p21 (20). The gene has a characteristic ble to the variant IGL/BCL6 translocations. In the present study, we GϩC-rich housekeeping promoter that lacks TATA or CAAT se- cloned a total of eight IGL/BCL6 fusions. Sequencing analysis showed quences (Fig. 5D; Refs. 20 and 21); however, more distant elements that four break points were 5Ј of V genes and two of the four were 2338

Fig. 5. Nucleotide sequences of the promoter region of H4 (A; Ref. 10), HSP89␣ (B; Ref. 17), HSP90␤ (C; Ref. 19), and PIM-1 (D; Refs. 20 and 21). 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; HSP89␣ and HSP90␤, HSEs; PIM-1, SP1-binding and ␥IFN-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. associated with a J segment, indicating that these translocations in- of BCL6 may result in transcriptional repression of genes involved in volved IGL alleles that had already completed V/J assembly. On the terminal differentiation or in apoptosis, thereby leading to neoplastic other hand, the remaining four break points were distributed within cell growth of B cells. In the present study, we identified four unique the IGL␭ gene and had no apparent association with V␭/J␭ joining. non-IG/BCL6 fusions in which the pattern of transcriptional regula- Therefore, it is likely that these translocations involved an IGL␭ gene tion of the partner genes has been well studied. The Site II equivalent with the germ line configuration. Because there is a clear order of of the present H4 gene contains consensus binding sites for two events in the formation of an IGL gene (i.e., the IGL␬ gene is transcription factors (i.e., histone nuclear factor D and IFN regulatory rearranged first, and the IGL␭ gene is not affected by the rearrange- factor 2; Ref. 10). histone nuclear factor D is a multicomponent ment process when the IGL␬ successfully forms an active gene), our protein containing cyclin A, CDC2, and an RB-related protein, which findings led to the conclusion that IGL/BCL6 translocation occurs at apparently links H4 gene regulation directly to cell cycle control (26). the mature B-cell stage when normal V/J assemblies of the two IGLs The regulation of HSP gene expression in eukaryotes is mediated by have been completed. The mechanisms responsible for the illegitimate the conserved HSF (18). HSF acts through the HSEs composed of recombination between these particular regions of IGL and BCL6 three contiguous inverted repeats of a 5-bp sequence. On heat stress, gene remain to be elucidated. HSF binds to the HSEs as a trimer, and all three repeats are required Partner Genes Involved in Non-IG/BCL6 Translocations are for high-affinity interaction (18). Transcriptionally Activated by a Variety of Stimuli. The common The PIM-1 proto-oncogene encodes a serine/threonine protein ki- molecular features of non-IG/BCL6 translocations include: (a) the nase and plays a role in the signal transduction events associated with gene fusion occurs in the same transcriptional orientation; (b) the lymphocyte activation (27). The basal level of PIM-1 expression break point on the partner gene is localized in close proximity to its seems to be regulated in a cell type-specific manner (i.e., highest in 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 5Ј exon(s) of the partner Table 1 Partner loci of BCL6 tranlocations determined by LD-PCR-based assays gene, which may or may not include coding regions, was invariably Involved gene Chromosomal locus Case number included in the non-IG/BCL6 junction, potentially leading to genera- IGs30 tion of fusion mRNAs. This may be of functional significance, be- IGH 14q32 22 IGL␬ 2p12 1 cause it could have substantial impact on the stability and/or translat- IGL␭ 22q11 7 ability of the resulting fusion transcripts. On the other hand, coding Non-IG loci 25 (23)a exons of the partner gene were interrupted by the upstream sequences H4 histone 6p21.3 5 TTF 4p13 2 of BCL6 in the BCL6/H4, BCL6/HSP90␤, and BCL6/PIM-1 fusions, TFRC 3q29 2 but remained intact in the BCL6/HSP89␣ fusion. Thus, it seems that HSP89␣ 14q32 1 HSP90␤ 6p12 1 partner genes on the translocated allele may not be important although PIM-1 6p21.2 1 they are fused with the BCL6 promoter. ␣-NAC 12q23–q24.1 1 Expression of the BCL6 gene is tightly regulated during B-cell CIITA 16p13 1 Currently uncharacterized 11 differentiation; its expression increases in mature B-cells in the ger- Deletion of mutations of BCL6 5 minal center and then decreases during their differentiation into Total 60 (58)a plasma cells (25). Thus, it is conceivable that inappropriate expression a Two cases had two independent non-IG/BCL6 translocations. 2339

1, which has been referred to as the MMC (31). These mutations are often multiple, are frequently biallelic and are independent of BCL6 translocation 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 BCL6 translocations 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.

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Hiroshi Akasaka, Takashi Akasaka, Masayuki Kurata, et al.

Cancer Res 2000;60:2335-2341.

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