DNA breakage at chromosomal fragile sites (CFS) is believed to promote oncogenic chromosome rearrangement. Notable CFS involvement in leukemia–lymphoma (LL) includes deletions affecting WWOX (located within FRA16D), which is also fused with IGH via t(14;16)(q32;q23) in multiple myeloma, and FHIT at 3p14 (FRA3D) in B-cell lymphoma. Both lesions operate as tumor suppressors and, unlike key LL rearrangements, occur widely in solid tumors. Only 19/89 CFS have been cloned and their coordinates pinpointed.1 Recent mapping data now show that FRA8C, another leading candidate CFS in LL, lies outside the MYC breakpoint cluster at 8q24.2 Hence, there remain tantalizingly few leukemic oncogene chromosome rearrangements definitively mapped within CFS. We describe t(3;7)(q27;q32) in a B-cell lymphoma cell line, which fuses BCL6 to a conserved non-coding region within FRA7H, to serve as a unique model for neoplastic fragile site involvement in LL.
The RC-K8 cell line was established from a 55-year-old man with terminal diffuse large B-cell lymphoma (DLBCL) diagnosed originally as ‘true histiocytic lymphoma’ (www.dsmz.de). G-banding and chromosome painting revealed the following karyotype: 47, XY, der(Y)t(Y;5)(qter;?p15), der(?1)t(1;13)(p32;q11) del(1)(q11), del(2)(p11p13), dup(2)(p11–12p14–15), t(3;7)(q27;q32), der(5)inv(5)(q15q32)add(5)(q32), inv(5)(p?13q?32), der(8)(t(8;8)(p12;q11), del(9)(p24)x2, der(10)t(9;10)(?p24;p11), −13, t(11;14)(q23;q32), der(14)t(X;14)(p11;p11), −15, der(15)(t(X;15)(p11.3;p11), add(17)(p11), der(20)t(13;20)(q11;q13), +2mar. The t(3;7), shown in Figure 1a, is clearly visible in the early passage originators' karyotype where it was then interpreted as t(3;4) indicating stability.
Fluorescence in situ hybridization (FISH) using bacterial artificial chromosome (BAC) and fosmid clones showed that the break at 3q27 lay within BAC RP11-211G11 and fosmid WI2802L21, which both cover the BCL6 major breakpoint region (MBR) (Figure 1b). FISH analysis placed the break at 7q32 within fosmid clone WI2452B9 (Figure 1c) amid the common fragile site FRA7H (Figure 1d) first characterized by Mishmar et al.3. Aphidicolin clastogenicity assays confirmed that RC-K8 cells are prone to breakage within FRA7H (Figure 1e). Interestingly, breakage at FRA7H favored sites in normal chromosomes against translocation derivates, implying hightened DNA stability in the latter.
Long distance inverse (LDI)-PCR was performed over a 35 kbp region 5′ of BCL6 exon 3 including the MBR (Figure 2a and b). Deviant bands were excised and sequenced (Figure 2a) and the respective junction sequences for der(3) and der(7) partners determined (Figure 2c). As shown in Figure 2d, for der(3), the breakpoint on chromosome 3 lies at 188 947 436 bp (March 2006 Assembly) near the start of the promoter region (Figure 2b), 1267 bp upstream of exon 1, outwith both MBR and alternative breakpoint regions (ABR), located respectively in exon 1 (silent) together with part of intron 1 and 245–285 kbp upstream thereof. Hence, LDI–PCR identified a novel BCL6 breakpoint indistinguishable from the MBR by FISH alone. While t(3;7) leaves the promoter region intact, der(3) including the coding region of BCL6 is thereby divested of an upstream binding site for BLIMP1, a major BCL6 repressor.4 Sequencing showed no evidence of somatic hypermutation within the BCL6 promoter.
The breakpoint at 7q32 maps to 130 262 603 bp within FRA7H, confirming the FISH assignment. Although several BCL6 partners map to breakpoints at, or adjacent to, hosting CFS (Supplementary Table 1), none have on cloning been confirmed to lie within CFS until now. The FRA7H breakpoint lies within putative gene transcripts BC039420/3′-FLJ43663, expressed in lymph nodes and B -cells, but silent in myeloid and T cells (http://genome.ucsc.edu). The junction sequence of der(7) differs from that of der(3), with breakpoints at 188 947 802 (chromosome 3) and 130 262 185 bp (chromosome 7) due to deletions of 365 and 416 bp, respectively (Figure 2d). Interestingly, the deletion of chromosome 3 material excises a region highly conserved in mammalian genomes, the breakpoints precisely marking its boundaries, while the breakpoint in chromosome 7 lies within an Alu element.
Shotgun reverse transcription (RT)-PCR analysis performed to detect formation of hybrid mRNA between BCL6 and compatible (minus-strand) mRNA species at 7q32 provided no evidence of chimeric transcription (Table 1, Figure 2d). These findings were supported by the results of 5′-RACE experiments (data not shown). Thus, t(3;7) in RC-K8 cells is probably nonfusogenic, consistent with placement of the BCL6 breakpoint outside the MBR.
Expression of BCL6, measured by real-time quantitative (RQ)-PCR, showed mid-range BCL6 upregulation in RC–K8 cells when compared with other DLBCL cell lines (Figure 3a). Western blot analysis confirmed these findings at the protein level (data not shown). Hence, BCL6 is constitutively, albeit moderately, expressed—as previously reported for comparable non-IGH BCL6 rearrangements.5 To confirm BCL6 signalling, transcriptional profiling was performed on a panel of lymphoma cell lines including RC-K8 (Figure 3b and Supplementary Figure S1). These data show downregulation of major BCL6 program genes, including CITED2, CCND2, ISGF3G, EBI2 and PRDM1, in RC-K8 and other BCL6-rearranged cell lines.
Compared to CFS such as FRA3D and FRA16C, which both exceed 1 Mbp, FRA7H is short (∼161 kbp) lessening the risk of adventitious breakage and bolstering its significance in t(3;7). The 7q32 breakpoint adjoined a mammalian interspersed repeat as implicated in leukemic breakpoints, notably the 8.34 kb MLL breakpoint cluster region, which hosts eight Alu copies.6 To investigate the possible role of DNA sequence instability, stress-induced DNA duplex destabilization (SIDD) analysis of 30 kb surrounding 3q27 and 7q32 breakpoints was performed.7 SIDD data plotted in Supplementary Figure S2 discount clastogenic destabilization, excepting at the der(7) breakpoint on chromosome 3, which crests a minor breakage peak (Supplementary Figure S2A), inviting comparison with another non-coding BCL6 partner breakpoint, which crests a major SIDD peak (Schneider et al., manuscript in preparation). DNA palindromes are implicated in chromosome translocations, including those with CFS involvement. Palindrome analysis of t(3;7) breakpoint regions (http://www.alagu-molbio.net/palin.html) proved negative, however, as did a search for RAG1/2 recombination signal sequences.
Canonical MBR rearrangements deregulate BCL6 by promoter exchange. Both ABR and non-clustered breakpoints observed in several DLBCL-derived cell lines (Schneider et al., in preparation.) may effect juxtaposition of BCL6 with cryptic enhancers. Dysregulation by t(3;7) might follow the removal of the upstream binding site for the BCL6 inhibitory regulator BLIMP1 (Figure 2b) or juxtaposition with a 38 bp tract of abyssally cross-species conserved non-coding DNA at chromosome 7 (130 262 603–130 262 640 bp). This sequence forms a ‘7X Regulatory Region’ (mean value=0.12; http://genome.ucsc.edu/cgi)), warranting its further evaluation. Analogous non-coding regions located ∼1 Mbp downstream of their coordinate target gene, BCL11B, mediate ectopic gene deregulation in T-cell leukemia.8
Although FRA7H lacks RefSeq genes, it bears a micro-RNA gene cluster, located ∼50 kbp centromeric of the breakpoint. One of its two transcripts, miR-29b-1, is downregulated in RC-K8 compared to other DLBCL cell lines lacking 7q32 involvement (Figure 3c) while miR-29a is expressed throughout. Interestingly, miR-29b-1, which regulates target expression in B cells, has recently been highlighted as a target of 7q32 deletions in splenic marginal zone lymphoma.9 Mir genes have attracted attention of late by their placement close to either LL translocation breakpoints or to CFS.10 Uniquely, the miR-29 cluster near the 7q32 breakpoint in RC-K8 cells displays both attributes.
The contribution of CFS to cancer remains poorly understood. The RC-K8 cell line in which BCL6 is fused to a non-coding region within FRA7H thus serves as a unique paradigm and tool for investigating CFS involvement in leukemogenic chromosome rearrangement.
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We thank Professor Batsheva Kerem and the Sanger Institute for supplying clones. This work is partially supported by the Deutsche José Carreras Leukämie-Stiftung e.V.
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Schneider, B., Nagel, S., Kaufmann, M. et al. t(3;7)(q27;q32) fuses BCL6 to a non-coding region at FRA7H near miR-29. Leukemia 22, 1262–1266 (2008) doi:10.1038/sj.leu.2405025
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