The gene for interleukin-21 receptor is the partner of BCL6 in t(3;16)(q27;p11), which is recurrently observed in diffuse large B-cell lymphoma

Abstract

BCL6 translocation affecting the chromosomal band 3q27 can involve a number of non-immunoglobulin (non-IG) gene loci as partners. We report here that the gene for interleukin-21 receptor (IL-21R) is the partner of BCL6 in t(3;16)(q27;p11) translocation. The two breakpoints on 16p11 of a lymphoma cell line YM and case no. 1012 with diffuse large B-cell lymphoma, both of which carried t(3;16), were localized within the 27-kb intron 1 of IL-21R. As a result of t(3;16), the promoter region of IL-21R was substituted for the regulatory sequences of BCL6 in the same transcriptional orientation. Reverse transcriptase-mediated polymerase chain reaction revealed chimeric mRNA consisting of two non-coding exons 1a/1b of IL-21R and coding exons of BCL6 in both lymphoma cells. Fluorescence in situ chromosomal hybridization of YM metaphase cells revealed fusion signals that contained both the BCL6 and IL-21R sequences on the der(3)t(3;16) chromosome. IL-21R was actively transcribed in YM cells, while BCL6 that was under the control of the IL-21R promoter was only moderately expressed at the mRNA and protein level. We constructed expression plasmid of BCL6 that followed the promoter sequences of IL-21R. COS-7 cells transiently transfected with the plasmid expressed high level Bcl-6 protein and displayed nuclear staining with a characteristic punctate pattern by immunofluorescence, indicating that expression of BCL6 can be enhanced by t(3;16). This study added to the list of non-IG partners of BCL6 translocations a new class of gene, i.e. cytokine receptor gene, the expression of which is closely associated with lymphoid cells.

Introduction

The BCL6 gene on chromosomal band 3q27 was initially identified at the breakpoints involved in t(3;14)(q27;q32) (Kerckaert et al., 1993; Ye et al., 1993) and t(3;22)(q27;q11) (Miki et al., 1994) translocations, both of which are specifically associated with non-Hodgkin's lymphoma of B-cell type (B-NHL) (Willis and Dyer, 2000). The gene spans 26-kb and contains 9 exons, and encodes the Bcl-6 protein consisting of 706 amino acids (Kerckaert et al., 1993; Miki et al., 1994; Ye et al., 1993). The Bcl-6 protein is a sequence-specific transcriptional repressor that contains two identified functional domains, i.e. the N-terminal POZ domain that plays a role not only in homodimerization but also in heterodimerization, and the C-terminal domain comprising six Cys2–His2 zinc finger motifs (Albagli et al., 1996; Chang et al., 1996). The repressing activity of Bcl-6 has been shown to be exerted by the recruitment of both the SMRT co-repressor and a SMRT/mSin3A/HDAC-containing complex (Dhordain et al., 1998; Wong and Privalsky, 1998). Targeted inactivation of BCL6 in the mouse germline prevents germinal center formation in the lymphoid tissues and alters Th2-mediated immune responses (Fukuda et al., 1997; Ye et al., 1997), indicating that BCL6 is an important regulator of lymphoid development and function.

BCL6 translocation has been identified by cytogenetic analysis and/or Southern blot analysis using a probe for the major translocation cluster (MTC) of BCL6 (Bastard et al., 1994). The range of BCL6 translocation in B-NHL subtypes are 6.4–14.3% in follicular lymphoma, 28.6–35.5% in diffuse large B-cell lymphoma (DLBCL) and its variants, and 20% in acquired immunodeficiency syndrome-associated DLBCL (Willis and Dyer, 2000). The particular feature of BCL6 translocation is that it can involve not only either one of the three immunoglobulin genes (IGs) but also another non-IG partner (Ohno and Fukuhara, 1997). In our series of 58 cases with B-NHL, 30 (52%) cases involved IGs as partners, while 23 (40%) cases affected non-IG partners (Akasaka et al., 2000). Non-IG partner genes that have been identified by the 5′-rapid amplification of cDNA ends strategy or long-distance inverse polymerase chain reaction (LDI–PCR) include: Rho GTP-binding protein, RhoH/TTF (4p13) (Dallery et al., 1995); B cell-specific transcriptional coactivator, BOB1/OBF1 (11q23.1) (Galiegue-Zouitina et al., 1996); H4 histone (6p21.3) (Akasaka et al., 1997); heat shock protein 89α, HSP89α (14q32) (Akasaka et al., 2000; Xu et al., 2000); heat shock protein 90β, HSP90β (6p12) (Akasaka et al., 2000); MHC class II transactivator, CIITA (16p13) (Akasaka et al., 2000; Yoshida et al., 1999); pim-1 proto-oncogene (6p21.2) (Akasaka et al., 2000; Yoshida et al., 1999); eukaryotic initiation factor 4AII, eif4AII (18p11.2) (Yoshida et al., 1999); transferrin receptor, TFRR (3q29) (Akasaka et al., 2000; Yoshida et al., 1999); Ikaros (7p12) (Hosokawa et al., 2000); L-plastin, LCP1 (13q14) (Galiegue-Zouitina et al., 1999); and α-chain of the nascentpolypeptide-associated complex, α-NAC (12q23-q24.1) (Akasaka et al., 2000). These non-IG/BCL6 translocations are recurrent abnormalities that have been observed in 2 cases and/or reported from 2 independent laboratories.

Interleukin-21 (IL-21) and IL-21 receptor (IL-21R) is a recently cloned cytokine–cytokine receptor pair that not only regulates the proliferation of mature B-cells and T-cells in response to activating stimuli but also mediates expansion of NK-cell populations from bone marrow (Ozaki et al., 2000; Parrish-Novak et al., 2000). Structural analysis revealed that IL-21 consists of a 131-residue four-helix-bundle cytokine domain with significant homology to IL-2, IL-4 and IL-15, while IL-21R has highest amino-acid sequence similarity to IL-2Rβ and IL-4Rα. IL-21R is capable of signal transduction through homodimerization or potentially heterodimerization with IL-2Rγ. IL-21R mRNA is restrictively expressed in the thymus and spleen, and is induced by phytohemagglutinin in peripheral blood mononuclear cells. The chromosomal loci of the genes for IL-21/IL-21R has been determined; the IL-21 gene is mapped to 4q26-27 where the IL-2 and IL-15 genes are clustered and the IL-21R gene is close to the locus of the IL-4Rα gene on 16p11. Therefore, these closely related cytokine–cytokine receptor genes may have arisen by gene duplication (Ozaki et al., 2000; Parrish-Novak et al., 2000).

In this study, we found that a recurrent translocation t(3;16)(q27;p11) fused IL-21R on 16p11 to BCL6 on 3q27, resulting in generation of fusion transcripts containing both IL-21R and BCL6 messages. To explore the role of the IL-21R/BCL6 fusion gene in the pathogenesis of DLBCL, we recreated a construct in which the BCL6 was driven by the promoter sequences of IL-21R. Transfection experiments of the plasmid revealed that the IL-21R/BCL6 fusion genes leads to enhanced expression of Bcl-6 in COS-7 cells.

Results

t(3;16)(q27;p11) breakpoints on 16p11 are localized within the intron 1 of IL-21R gene

We previously cloned and sequenced the t(3;16) breakpoint on 16p11 of YM cells (Figure 1a) (Yonetani et al., 1998). The breakpoint of case no. 1012, which was 13.5-kb distant from that of YM, was determined by the LDI–PCR method. A database search revealed that these two breakpoints were localized between the genes for IL-4Rα and the transcription factor TFIIICα subunit, although both genes were substantially distant from the breakpoints (Figure 1b). To identify the gene directly affected by t(3;16), the 50-kb sequences of a bacterial artificial chromosome clone (GenBank accession no. AC002303), which encompassed the t(3;16) breakpoints, were subjected to the GenScan program, leading to prediction of a gene that spans a 20-kb region. We next introduced a DNA fragment that contained three consecutive exons of the virtual gene into an exon trapping vector, pSPL3. Total cellular RNA was recovered from COS-7 cells and RT–PCR was performed using the primers that are complementary to the splice donor and splice acceptor sites of pSPL3. The cDNA amplified was composed of the sequences that were expected from the result of GenScan analysis (Figure 1c,d). Northern blot analysis using the cDNA as a probe demonstrated active transcription in YM cells (data not shown).

Figure 1
figure1

Molecular anatomy of t(3;16)(q27;p11) affecting BCL6 and IL-21R genes. (a) Comparative map encompassing the YM breakpoints (Yonetani et al., 1998). The breakpoints of no. 1012 were determined by long-distance inverse PCR. Boxes are the three most 5′ exons of BCL6. MTC, major translocation cluster. Restriction sites: E, EcoRI; B, BamHI; H, HindIII; X, XbaI; Xh, XhoI. λ-phage clones are aligned accordingly. (b) The two breakpoints on chromosome 16 were localized on two overlapping BAC clones, including IL-4Rα and TFIIICα genes, respectively. Horizontal arrows indicate the transcriptional orientation of the two genes. (c) Genomic organization of the IL-21R gene (Ozaki et al., 2000). The positions of the two breakpoints of t(3;16) are indicated by open triangles. The 3.2-kb fragment (open bar) used for exon trapping was amplified by nested PCR using an outer forward primer 5′-TCATGGCCAAGAACAGAGACCAAGGGCACGAGCAC-3′ and an outer reverse primer 5′-GCTCATTGATTCAGCTAGGGTAGGCAGGGTTATGC-3′, and an inner forward primer 5′-TCTGTCCTCCCCAAACTGGTCTCCTCGGCGGTCAG-3′ and an inner reverse primer 5′-GGTGTGATCATAGCTCACGGCAGCCTCTGAACGAT-3′. The fragment was digested with EcoRI/SacI and cloned into pSPL3. The exon trapping generated 540-bp cDNA composed of exons 6, 7 and 8 (d). (e) Nucleotide sequences of the IL-21R/BCL6 junction of YM. The sequences on der(16) were obtained by PCR using an appropriate primer pair. Vertical lines show nucleotide identity. Twenty-seven nucleotides of IL-21R were deleted at the junction (boldface letters)

A database search revealed that the cDNA we cloned matched the sequences of IL-21R. The IL-21R gene was composed of nine coding exons (2 through 9) that follow two non-coding exons 1a/1b (Figure 1c) (Ozaki et al., 2000). The breakpoint of YM as well as that of no. 1012 were localized within the 27-kb intron 1. Comparison with the der(3) and der(16) sequences of YM revealed that 27 nucleotides of the IL-21R sequences were deleted at the junction (Figure 1e). The breakpoints on 3q27 of both cases were outside the MTC but still close the regulatory sequences of BCL6. Therefore, as the result of translocation, upstream sequences of IL-21R including non-coding exons 1a and 1b were substituted for the promoter area of BCL6 in the same transcriptional orientation.

To confirm the translocation of IL-21R to BCL6 in YM cells, we performed two-color FISH analysis using fluorophore-labeled BAC clones corresponding to each gene. As shown in Figure 2, fusion signals were clearly demonstrated on not only der(3)t(3;16) chromosomes but also interphase nuclei. Judging from the positions of the breakpoints within the two BAC clones (no: AC072022 for BCL6 probe and no. AC004525 in Figure 1b for IL-21R probe, respectively), the fusion gene composed of the IL-21R exons 1a/1b and BCL6 coding regions is localized on the der(3)t(3;16) chromosome.

Figure 2
figure2

Two-color FISH analysis of YM cells showing the IL-21R/BCL6 fusion gene. Two BAC clones corresponding to BCL6 (AC072022) and IL-21R (AC004525) were directly labeled with SpectrumOrange and SpectrumGreen, respectively, and hybridized with metaphase preparations. Fusion signals (yellow) between the two genes are identified on not only a der(3)t(3;16) chromosome but also an interphase nucleus (arrow). Since most of the IL-21R clone was translocated to the der(3) chromosome, no fusion signals were visible on the der(16)

IL-21R/BCL6 fusion mRNA is generated in t(3;16)-carrying cells

To identify fusion mRNA containing both IL-21R and BCL6 messages, we designed a forward primer for IL-21R exon 1a and a reverse primer for BCL6 exon 5 (Figure 3a). Total RNA extracted from YM cells as well as cryopreserved tumor cells of no. 1012 were subjected to RT–PCR analysis. As shown in Figure 3b, two amplified product species were obtained in both cells. Sequencing analysis of the PCR products revealed that the sequences of IL-21R exon 1a/1b or of exon 1a were contiguous with those of BCL6 exon 2 (Figure 3c,d); the latter species of mRNA appeared to be predominant (Figure 3a). In no. 1012, the non-coding exon 1 of BCL6 was not transcribed. RT–PCR analysis using a reverse primer for IL-21R exon 2 (Figure 3b) indicated that transcription initiated from IL-21R exon 1a preferentially splices out exon 1b on not only the IL-21R/BCL6 fusion gene but also on the normal IL-21R gene, suggesting that regulation of the IL-21R promoter is not altered by the translocation.

Figure 3
figure3

RT–PCR analysis of IL-21R/BCL6 fusion mRNA. The IL-21R/BCL6 fusion genes of YM and no. 1012 are schematically presented (a). The forward primer for IL-21R exon 1a was 5′-CATCAGACTGCCCCCAGCACACGGAATGGATTTCT-3′ and the reverse primer for BCL6 exon 5 was 5′-TTATTGAGCCGGCTGGCTTGTGGGATGGTGGAGTC-3′. (b) EtBr-stained gel electrophoresis of RT–PCR products. IL-21R mRNA of YM cells from the normal allele was amplified by a reverse primer for IL-21R exon 2 (5′-ACTATGACAAGCAGGAGGAGAAGCAGCAGGTGAGG-3′). Compositions of the RT–PCR products representing two species of IL-21R/BCL6 fusion mRNA are indicated in (c) and the nucleotide sequences at the junctions are presented in (d)

Expression of IL-21R and BCL6 in lymphoid tumor cells

Figure 4a shows the transcriptional level of IL-21R in YM cells compared with those of a variety of lymphoid tumor cell lines. All cell lines tested except acute T-cell leukemia Jurkat expressed two species of mRNA of 4.7 and 3.2-kb. YM cells expressed more abundant 4.7-kb mRNA than other B-cell lines. Since no BCL6/IL-21R fusion transcripts were detectable by RT–PCR using sets of appropriately primers (data not shown), the IL-21R mRNA in YM cells likely represented transcription from the normal IL-21R allele.

Figure 4
figure4

Expression of IL-21R and BCL6 in lymphoma cells. (a) Northern blotting showing expression levels of IL-21R and BCL6mRNA in lymphoid tumor cells lines. Ten micrograms of total RNA was loaded in each lane. Jurkat, immature T-cell leukemia; ATL2, adult T-cell leukemia; KMH2, Hodgkin's disease; Raji and Ramos, Burkitt's lymphoma; FL-218, follicular lymphoma; KIS-1, diffuse large cell lymphoma with t(9;14); RPMI8226, multiple myeloma; and LCL-OHN, lymphoblastoid B-cell line. (b) Expression levels of Bcl-6 protein were comparable with those of BCL6 mRNA. Cell lysates were prepared from 1×106 cells and an aliquot was loaded. YM showed a lower molecular weight protein in addition to the 79-kDa Bcl-6 protein. (c) Indirect immunofluorescence of follicular lymphoma FL-218 and YM cells using an anti-Bcl-6 polyclonal antibody and a secondary antibody labeled with Alexa 488

Since it is presumed that the BCL6 gene on the translocated allele is under the control of the promoter activity of IL-21R, we investigated whether the expression of BCL6 in YM cells is activated in parallel with that of IL-21R. As shown in Figure 4a,b, YM cells did express a considerable level of BCL6 mRNA as well as Bcl-6 protein; however, the levels were apparently less than those of Burkitt's lymphoma and follicular lymphoma cells. Indirect immunofluorescence of follicular lymphoma FL-218 cells demonstrated characteristic nuclear staining with a granular pattern of signals labeled by an anti-Bcl-6 polyclonal antibody (Cattoretti et al., 1995), while the staining was positive but less intense in YM cells (Figure 4c). This finding is consistent with immunohistochemical observations that lymphoma tissues carrying the BCL6 translocation do not necessarily express high Bcl-6 level (Onizuka et al., 1995).

Bcl-6 protein is a transcriptional repressor, several target genes of which have been identified (Shaffer et al., 2000). To study whether the expression of target genes are in proportion with that of BCL6 in lymphoma cells, we analysed the gene expression profiles of FL-218, Ramos, Raji and YM cell lines. The Atlas Human 1.2 Array used in the present study contained a spot of BCL6 in addition to those of three potential target genes, including MIP-1α, Cyclin D2 and Blimp-1. As summarized in Table 1, the levels of expression of BCL6 in these four cell lines were in accordance with those determined by Northern blot analysis. In contrast, Blimp-1 expression showed a reverse correlation with BCL6, suggesting that transcriptional repression mediated by Bcl-6 may function in lymphoma cells.

Table 1 Expression levels of target genes of Bcl-6 in lymphoma cells analysed by the cDNA array

Promoter activities of IL-21R measured by luciferase assays

We previously demonstrated the common molecular features of non-IG/BCL6 translocations; including the finding that the breakpoint on the partner gene is localized in close proximity to its promoter sequence, and that the complete set of the promoter is fused upstream of the coding region of BCL6 (Akasaka et al., 2000). Thus, it is likely that the regulatory sequences of IL-21R lie upstream of the no. 1012 breakpoint. We, therefore, prepared a 2745-bp fragment including exon 1a (−2550 to +195; the 5′ boundary of exon 1a determined by Ozaki et al. (2000) was +1) and ligated it into the pGL3-Basic reporter plasmid (Figure 5a). Transcriptional activity of the insert was measured as luciferase activity, and compared with that of the co-transfected Renilla plasmid. As shown in Figure 5b, this sequence showed 13-fold higher activity than that obtained by the vector alone. We next constructed a series of deletions linked to the luciferase gene and determined the crucial area for the activity. The results showed that a construct lacking the −60 to +127 region showed reduced luciferase activity to the basal level. Several potential transcription factor-binding sites are identified within this particular area; including AML-1a, MZF1, GATA-1 and NF-E2.

Figure 5
figure5

Comparison of promoter activities determined by luciferase assay between IL-21R and BCL6. (a) Schematic diagram of serial deletion mutants of the promoter regions of IL-21R ((−2550 to +195) and BCL6 (−1353 to +274) that were cloned into the pGL3-Basic luciferase vector. The non-coding exons of the two genes are boxed. (b) Each construct was transiently transfected into COS-7 cells. Luciferase activities were normalized by co-transfected Renilla luciferase activities and were expressed relative to the activity of the pGL3-Basic control. Values are representative of the mean±s.d. of three independent experiments

Recreation of IL-21R/BCL6 fusion DNA and expression of Bcl-6 protein in COS-7 cells

To explore the role that the IL-21R/BCL6 fusion gene may play in the development of DLBCL, we first compared the levels of promoter activity between BCL6 and IL-21R by luciferase assay. The −1353/+274 and −621/+274 fragments of BCL6 were cloned into pGL3; the former fragment was previously reported to exhibit the most potent activity (Ohashi et al., 1995). As indicated in Figure 5a, the luciferase activities driven by the regulatory sequences of BCL6 and IL-21R showed comparable levels in COS-7 cells.

Since there was no significant difference in the promoter activity between IL-21R and BCL6 measured by luciferase assay, we constructed expression plasmids of BCL6 itself that followed the promoter sequences of the two genes, and investigated whether each promoter affected BCL6 expression in a different fashion. To recreate the junctional area as precisely as possible, the plasmids contained BCL6 exons 2 and 3 as well as intronic flanking sequences between the promoter and the BCL6 cDNA (Figure 6a). We transiently introduced the plasmids into COS-7 cells and performed Western blot analysis to confirm that the plasmids expressed Bcl-6 protein in COS-7 cells. As shown in Figure 6b, both transfectants generated the Bcl-6 protein that migrated to an identical position to that of FL-218 cells, and it was apparent that IL-21R/BCL6 transfectant generated more abundant Bcl-6 protein than that driven by the BCL6 promoter. To confirm the Bcl-6 expression, we stained the COS-7 cells by indirect immunofluorescence using a polyclonal antibody against Bcl-6. As shown in Figure 6c, IL-21R/BCL6-transfected cells displayed bright nuclear staining with characteristic punctate pattern. These findings clearly indicated that IL-21R/BCL6 gene fusion leads to enhanced Bcl-6 expression in COS-7 cells.

Figure 6
figure6

Recreation of IL-21R/BCL6 fusion gene and expression of Bcl-6 in COS-7 cells. (a) Schematic presentation of BCL6-expression plasmids that are driven by the SV40, BCL6 and IL-21R promoters (the sequences of the latter two are presented in Figure 5a). Restriction sites: H, HindIII; X, XbaI; G, BglII. The XbaI site within exon 4 is demethylated in DM1 (GIBCO/BRL). MCS, multiple cloning site. (b) Western blot analysis of COS-7 transfectants for Bcl-6 expression. FL-218 was to indicate the position of normal Bcl-6 protein. Cell lysates were prepared from 1×106 cells and an aliquot was loaded. (c) Indirect immunofluorescence of COS-7 cells transfected with BCL6-expression plasmids. The cells were sequentially labeled with a primary rabbit antibody against Bcl-6 and secondary anti-rabbit antibody labeled with Alexa 488. The positions of nuclei, as revealed by DAPI staining, are shown in the bottom

Discussion

We presented here the cloning and sequencing of two breakpoints on 16p11 involved in t(3;16)(q27;p11) translocation. The breakpoints were localized in common within intron 1 of IL-21R and both lymphoma cells transcribed identical species of IL-21R/BCL6 fusion mRNA irrespective of the difference in the position of the breakpoints on the two genes. These findings in addition to the FISH analysis of YM cells clearly indicate that IL-21R is the partner gene of BCL6 in t(3;16) and this molecular lesion can occur recurrently, thereby contributing to the pathogenesis of DLBCL.

The non-IG partner genes identified to date (Akasaka et al., 1997, 2000; Dallery et al., 1995; Galiegue-Zouitina et al., 1996, 1999; Hosokawa et al., 2000; Xu et al., 2000; Yoshida et al., 1999) are too diverse to find common properties that are shared among these genes. However, each non-IG/BCL6 translocation is not random but a recurrent genetic abnormality in B-NHL, indicating that tumor cells carrying a fusion of BCL6 with a specific partner likely gain a growth advantage over normal cells. This study added to the list of non-IG partners a new class of gene, i.e. cytokine receptor gene, whose expression is closely associated with hematopoietic cells. Although regulation of IL-21R expression has not yet been fully clarified, the similarity of the gene product with IL-2Rβ raises the possibility that the regulatory mechanisms of the two genes overlap. Indeed, stimulation of T-cells with phytohemagglutinin can greatly increase the expression of both IL-2Rβ and IL-21R at the mRNA and protein level (Hatakeyama et al., 1989; Ozaki et al., 2000). On the other hand, normal and malignant mature B-cells have been shown to express IL-2Rβ (Zola et al., 1991), although it remains to be determined whether regulatory molecules that were identified to activate IL-2Rβ promoter in Jurkat cells (Lin and Leonard, 1997) are also operating in mature B-cells. We studied here the expression level of IL-21R in a panel of lymphoma cell lines and found that the levels of mRNA were rather low in follicular lymphoma cell lines compared with other types of B-cell lines (Figure 4a, additional data are not shown). This observation may indicate that the basal level of IL-21R expression is unexpectedly low in follicular center B-cells, in which BCL6 translocation is presumed to occur (Kuppers et al., 1999). Thus, t(3;16) may not immediately lead to increased expression of BCL6. Nevertheless, as germinal center B-cells proliferate rapidly in response to a variety of stimuli, it is likely that the BCL6 gene, which is under the control of the IL-21R promoter resulting from t(3;16), is inappropriately expressed during B-cell proliferation.

Subcellular localization of Bcl-6 has been studied. The protein is found in the nucleus in a diffuse microgranular fashion, while overexpression of Bcl-6 by transient transfection to NIH3T3, COS and HeLa cell lines leads to enlarge the granules to produce punctate structures (Bardwell and Treisman, 1994; Dhordain et al., 1995). Although the composition of granules has not been fully understood, the granules include SMRT and N-CoR co-repressors, suggesting that these structures represent multi-subunit repressor complexes that may be associated with chromatin (Dhordain et al., 1997; Huynh and Bardwell, 1998). Since some other POZ domain-containing proteins show identical nuclear localization and the POZ domain-deleted Bcl-6 display a relatively homogeneous nuclear distribution, the POZ domain is essential for generating this large macromolecular complex (Bardwell and Treisman, 1994). In the present study, we performed indirect immunofluorescence of YM and FL-218 lymphoma cell lines as well as COS-7 cells transfected with BCL6-expression vectors. Although it remains to be determined whether the endogeneous Bcl-6 and artificially expressed Bcl-6 in non-lymphoid cell lines can exhibit identical behavior in the nuclei, the sizes and numbers of the granules appeared to be correlated with the amounts of Bcl-6 protein in each cell type. Most importantly, we showed that characteristic punctate structure was reproduced in COS-7 cells by fusion of the BCL6 coding sequences with the IL-21R promoter. Thus, this study provided evidence that expression of Bcl-6 can be quantitatively altered by t(3;16) translocation.

We showed here that the promoter activity measured by luciferase assay does not necessarily predict the expression level of a gene that is physiologically or patho-physiologically linked to the promoter of interest. Kikuchi et al. (2000) identified a negative regulatory region, ES, within exon 1 of BCL6, which had a potential binding site of Bcl-6. They showed that Bcl-6 itself could bind this particular region and exhibited transcriptional repressive activity. However, the constructs used in the present study lacked ES, therefore, this negative auto-regulatory loop cannot account for the low level of Bcl-6 expression in BCL6 promoter-transfectants. Alternatively, the low Bcl-6 expression level, driven by the BCL6 promoter, may have merely reflected the negligible expression of intrinsic Bcl-6 in COS-7 cells. In contrast, high-level expression of Bcl-6 in IL-21R/BCL6-transfectant raises the possibility that Bcl-6 itself and/or its downstream proteins can further activate the IL-21R promoter, leading to the higher level of Bcl-6 expression. We performed analogous transfection experiments by replacing the IL-21R promoter with the promoters of other non-IG partners such as H4 histone and α-NAC. The results showed that COS-7 cells transfected with these constructs generated further higher levels of Bcl-6 protein than that in the IL-21R/BCL6 transfectant (unpublished observation). It is, therefore, suggested that the promoters of non-IG partners may have regulatory elements, whose activity is enhanced by Bcl-6 and/or downstream proteins.

An unresolved issue is whether BCL6 translocation is the primary genetic abnormality or occurs at the time of transformation from low- to high-grade disease, since the translocation sometimes co-exists with other B-NHL-associated translocations (Ohno and Fukuhara, 1997). We previously showed that the majority of lymphoma cells of no. 1012 had t(3;16) as the sole chromosome abnormality, indicating that the translocation was the primary genetic abnormality in this particular case (Ichinohasama et al., 1998). In contrast, YM cells carried a t(2;18)(p11;q21) leading to the fusion between BCL2 and the IG κ-light chain gene (Yonetani et al., 1998), raising the possibility that the t(3;16) was a secondary change that developed in a pre-lymphomatous B-cell with BCL2 translocation and was related to progression to florid lymphoma. However, recombination activation genes (RAG) 1/2 have been shown to be reactivated in the germinal center (Han et al., 1997), therefore, oncogene/IG translocation mediated by V(D)J recombination may not be restricted to B-cell precursors in the bone marrow but can occur in the peripheral lymphoid organs. Thus, the proposed hierarchical order of the translocations, i.e., BCL2 translocation occurs first and BCL6 translocation second, may not be the case. A key experiment to establish the role of t(3;16) in the development of DLBCL would be the creation of a transgenic mouse model, in which the translocation is reproduced by placing the BCL6 under the control of the IL-21R promoter.

Materials and methods

Cells

The YM cell lines was derived from a patient with the immunoblastic variant of DLBCL. Cytogenetic and molecular analysis of YM cells revealed that the cells carried t(3;16)(q27;p11) and t(2;18)(p11;q21) involving BCL6 and BCL2, respectively (Yonetani et al., 1998). Case no. 1012 with DLBCL was previously described in detail; the karyotype was 46, XY, t(3;16)(q27;p11.2) [11 cells]/46, idem, add(18)(q21) [7 cells]/46, XY [2 cells] (Ichinohasama et al., 1998). The cell lines used in the present study were cultured in RPMI 1640 medium supplemented with 10% fetal calf serum (FCS) under the standard culture conditions.

Sequence analysis and gene prediction

Sequence database searches were performed with the sequence comparison algorithms Basic Local Alignment Search Tool at the National Center of Biotechnology Information (http://www.ncbi.nlm.nih.gov/BLAST/). The search of putative coding regions was performed with the GenScan gene-finding program (http://CCR-081.mit.edu/GENSCAN.html).

LDIPCR and reverse transcriptase-mediated PCR (RTPCR)

LDI–PCR to clone non-IG partners of BCL6 translocation was previously described in detail (Akasaka et al., 2000). For RT–PCR, randomly primed cDNA was synthesized from 1 μg of total cellular RNA (First-Strand cDNA Synthesis Kit; Pharmacia, Piscataway, NJ, USA), and PCR assays were performed in 50 μl reaction volumes containing 2 μl of cDNA, reaction buffer, 0.25 mmol each dNTP, 20 pmol of each primer, and 0.5 U Taq polymerase (Takara Shuzo, Kyoto, Japan).

Northern blotting

Total cellular RNA was prepared using an RNeasyTM Total RNA Kit (Qiagen, Hilden, Germany), and electrophoresed on 1.0% agarose gel containing 0.66 M formaldehyde. The 1.6-kb cDNA probe for IL-21R was obtained by RT–PCR using a forward primer for exon 2 (5′-GTGGCTGGGCCGCCCCCTTGCTCCTGCTGCTGCTC-3′) and a reverse primer for exon 9 (5′-TGGGGTCCAGGGCTCGAAAGTGGCGGAGGAATGAC-3′). The M55 probe was a cDNA clone for BCL6 (Kerckaert et al., 1993).

Western blotting

Cells were lysed with 1×loading buffer containing protease inhibitor cocktail. The total cell lysates were loaded onto 10% sodium dodecyl sulfate acrylamide gels and electrotransferred onto Immobilon PVDF transfer members (Millipore, Bedford, MA, USA). The membranes were blocked in phosphate-buffered saline-Tween (PBS-T) buffer containing dried milk, and then incubated with polyclonal rabbit anti-Bcl-6 antiserum against amino acids 687 to 706 of human Bcl-6 (Santa Cruz Biotechnology, Santa Cruz, CA, USA). After extensive washing in PBS-T buffer, the blots were incubated for 1 h with horseradish peroxidase-conjugated secondary antiserum followed by enhanced chemiluminescence reaction (Amersham Pharmacia Biotech, Piscataway, NJ, USA).

Fluorescence in situ hybridization

Bacterial artificial chromosome (BAC) clones containing the DNA sequences of interest were purchased from Research Genetics (Huntsville, AL, USA). The BAC clones were directly labeled with fluorophores (SpectrumOrange-dUTP or SpectrumGreen-dUTP; Vysis, Downers Grove, IL, USA) by nick translation (Vysis). FISH results were analysed with a fluorescence microscope (Olympus, Tokyo, Japan) equipped with DAPI, FITC and tetramethylrhodamine B isothiocyanate (TRITC) as well as a DAPI/FITC/TRITC triple band-pass filter. A charge-coupled device camera (CoolSNAP/OL; Olympus) attached to the fluorescence microscope and Lumina Vision software (Mitani Corporation, Fukui, Japan) were used to capture and process images.

cDNA array

cDNA was prepared and labeled by the Atlas Pure Total RNA Labeling System (Clontech, Palo Alto, CA, USA). Hybridization of the cDNA with Atlas Human 1.2 Array (Clontech) was performed according to the manufacturer's instructions. The membranes were exposed to a BAS2000 imaging analyser (Fuji Photo Film, Tokyo, Japan) and the hybridization signals were quantified by ArrayGauge software (Fuji Photo Film).

Exon trapping

The genomic DNA of interest was cloned into the multiple cloning site of pSPL3 plasmid (GIBCO/BRL, Grand Island, NY, USA). COS-7 cells were grown in 35 mm 6-well plates in Dulbecco's modified Eagle's medium (DMEM) (BioWhittaker, Walkersville, MD, USA) supplemented with 10% FCS and penicillin-streptomycin. Twenty-four hours after passaging at 2×105 cells per well, the cells were transfected with 4 μg of the plasmid by LipofectAMINE 2000 reagents (GIBCO/BRL). Cells were then cultured for 24–48 h. Isolation of RNA and PCR amplification using a set of primers that are complementary to the splice donor and splice acceptor sites of pSPL3 were performed according to the manufacturer.

Luciferase reporter assay

DNA fragments from the promoter region of BCL6 and IL-21R were cloned into a firefly luciferase reporter plasmid, pGL3-Basic Vector (Promega, Madison, WI, USA). The pGL3 plasmid constructs were assayed on COS-7 cells to measure promoter-driven luciferase expression. At 80–90% confluence, cells were co-transfected with 1.0 μg plasmid DNA plus 0.01 μg pRL–TK DNA (Promega) using LipofectAMINE 2000 in serum-free MDEM for 24 h. Cells were lysed and firefly and Renilla luciferase activities were measured using the Dual Luciferase Assay System (Promega) in a luminometer (Lumat LB 9507; Berthold Technologies, Bad Wildbad, Germany).

Construction of BCL6-expression plasmids

To construct BCL6-expression plasmids that mimicked the non-IG/BCL6 fusion gene, we prepared the promoter region of the non-IG partner gene of interest, a genomic DNA fragment which included BCL6 exons 2 through 4 and their flanking sequences, and a partial cDNA of BCL6 composed of exons 4 through 9; the last fragment was obtained from a full-length BCL6 cDNA clone (Miki et al., 1994). These three fragments were sequentially ligated in this order and cloned into pGL3-Enhancer vector (Promega) that deleted the luciferase gene segment. A plasmid in which the SV40 promoter was substituted for the non-IG promoter and that lacked promoter sequences were used as a positive and negative control, respectively. The sequences of each construct were verified by restriction enzyme analysis and sequencing.

Indirect immunofluorescence

COS-7 cells were washed in PBS and fixed for 15 min with 3.0% paraformaldehyde in PBS at room temperature. Cells grown in suspension were washed twice in PBS and then resuspended in 200 μl PBS (1×105 cells/vol). An aliquot of this suspension was spread over poly-L-lysine-coated coverslips (Nacalai Tesque, Kyoto, Japan). The cells were fixed for 15 min with 3.0% paraformaldehyde in PBS at room temperature. All the fixed cells were treated with 0.05% Triton X-100 for 4 min and cold methanol for 5 min. The cells were then permeabilized with 0.05% PBT solution (0.05% Tween 20 in PBS containing 0.1% FCS) for 5 min and blocked with 2 Blocking (DAKO, Carpinteria, CA, USA). After three washes with 0.05% PBT, the specimens were incubated with primary rabbit antibodies against Bcl-6 that was diluted 1 : 100 in 0.05% PBT for 1 h at room temperature. They were washed three times with 0.05% PBT and were incubated with 1 : 50 secondary anti-rabbit antibody conjugated to Alexa 488 (Molecular Probes, Eugene, OR, USA) for 20 min at room temperature. After rinsing in PBS, the DNA was stained with DAPI (Vysis). Preparations were examined and photographed on a fluorescence microscope (Olympus, Tokyo, Japan).

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Acknowledgements

The authors thank Dr I Miura of Akita University School of Medicine for cytogenetic analysis of no. 1012 and Dr T Miki of Tokyo Medical and Dental University for providing the BCL6 cDNA clone. This work was supported by Sankyo Foundation of Life Science and grants-in-aid from the Ministry of Education, Science, Sports and Culture (13470204) of Japan.

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Correspondence to Hitoshi Ohno.

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Ueda, C., Akasaka, T., Kurata, M. et al. The gene for interleukin-21 receptor is the partner of BCL6 in t(3;16)(q27;p11), which is recurrently observed in diffuse large B-cell lymphoma. Oncogene 21, 368–376 (2002). https://doi.org/10.1038/sj.onc.1205099

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Keywords

  • BCL6 gene
  • non-immunoglobulin gene
  • t(3;16)(q27;p11) translocation
  • interleukin-21 receptor gene
  • diffuse large B-cell lymphoma

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