The BCL6 proto-oncogene encodes a transcriptional repressor necessary for the development of germinal centers (GCs) and directly implicated in lymphomagenesis. Post-GC development of B cells requires BCL6 downregulation, while its constitutive expression caused by chromosomal translocations leads to diffuse large B cell lymphoma (DLBCL). Herein we identify a signaling pathway that downregulates BCL6 expression in normal GC B cells and is blocked in a subset of DLBCL due to alterations in the BCL6 gene. Activation of the CD40 receptor leads to NF-kappaB-mediated induction of the IRF4 transcription factor, which, in turn, represses BCL6 expression by binding to its promoter region. A subset of DLBCL displays chromosomal translocations or mutations that disrupt the IRF4-responsive region in the BCL6 promoter and block its downregulation by CD40 signaling.

IntroductionBCL6 has emerged as a critical regulator of germinal centers (GCs), the sites where B cells undergo somatic hypermutation (SHM) and class switch recombination of their immunoglobulin genes (Ig) and are then selected on the basis of the production of antibodies with high affinity for the antigen. 1 BCL6 is also a frequently activated oncogene in the pathogenesis of human B-cell lymphomas, most of which derive from the GC B cells. The BCL6 gene encodes a 95-kDA nuclear phosphoprotein belonging to the BTB/POZ zinc-finger (ZF) family of transcription factors. 2-4 BCL6 functions as a transcriptional repressor via its C-terminal zinc-finger domain that binds to specific DNA sequences in the promoter region of target genes and 2 transcriptional repression domains 5 that interact with distinct corepressor complexes during the GC reaction. [6][7][8][9] Within the B-cell lineage, the BCL6 protein is expressed at high levels only in mature B cells within GCs. 10 GC formation and the development of normal T cell-dependent humoral immune responses require expression of BCL6 because BCL6-null mice do not form GCs and are unable to produce high-affinity antibodies. 2,4 BCL6 expression is regulated by several signals that are crucial for GC development. Activation of B-cell receptor (BCR) induces mitogen-activated protein kinase (MAPK)-mediated phosphorylation of the BCL6 protein, which targets BCL6 for rapid degradation by the ubiquitin proteasome pathway. 11 Stimulation of the CD40 receptor by CD40 ligands expressed by T cells leads to transcriptional down-regulation of BCL6 via a signaling pathway that involves nuclear factor (NF)-B-mediated transcriptional activation of interferon regulatory factor 4 (IRF4), which, in turn, directly represses BCL6 transcription. 12,13 BCL6 degradation is induced by DNA damage via a pathway that is distinct from the one induced by BCR, 14 whereas BCL6 function is also inactivated by acetylation, which triggers its dissociation from corepressor complexes. 15 These findings indicate that although BCL6 is required for GC formation, its downregulation may be critical for B cells to exit the GC and differentiate toward memory and plasma cells.A variety of structural alterations of the BCL6 gene are associated with its deregulated expression in B-cell lymphomas. Chromosomal translocations juxtaposing heterologous promoters to the BCL6 coding domain are found in approximately 40% of diffuse large B-cell lymphoma (DLBCL) and in a minority (5%-10%) of follicular lymphoma (FL). [16][17][18] The common denominator of these promoters is their constitutive activity in the B-cell lineage and in particular their persistent activity in post-GC cells such as immunoblasts and plasma cells, in contrast with the GC-specific activity of the BCL6 promoter. 19 In addition, although alterations of the 5Ј noncoding region of BCL6 by SHM is a feature of normal GC B cells, 20,21 specific mutations found only in DLBCL lead to the deregulated expression of BCL6 through disruption of the sequences mediating a...

The electrochemically stable and relatively high conductive room temperature molten salts (RTMS) have been obtained with the use of small ammonium cations such as methoxymethyltrimethylammonium and bis(trifluoromethylsulfonyl)imide. The RTMS showed high conductivity (4.7 mS cm−1 at 25 °C) which is the highest value of all the ammonium based RTMS reported so far.

The full set of microRNAs (miRNAs) in the human genome is not known. Because presently known miRNAs have been identified by virtue of their abundant expression in a few cell types, many tissue-specific miRNAs remain unrevealed. To understand the role of miRNAs in B-cell function and lymphomagenesis, we generated short-RNA libraries from normal human B cells at different stages of development (naïve, germinal-center, memory) and from a Burkitt lymphoma cell-line. A combination of cloning and computational analysis identified 178 miRNAs (miRNome) expressed in normal and/or transformed B-cell libraries. Most notably, the B-cell miRNome included 75 miRNAs which to our knowledge have not been previously reported and of which 66 have been validated by RNA blot and/or RT-PCR analyses. Numerous miRNAs were expressed in a stage- or transformation-specific fashion in B cells, suggesting specific functional or pathologic roles. These results provide a resource for studying the role of miRNAs in B-cell development, immune function, and lymphomagenesis.

Antigen-specific B cells are selected in germinal centers, the structure in which these cells proliferate while accomplishing genome-remodeling processes such as class-switch recombination and somatic hypermutation. These events are associated with considerable genotoxic stress, which cells tolerate through suppression of DNA-damage responses by Bcl-6, a transcription factor required for the formation of germinal centers. Here we show that the expression of Bcl-6 is regulated by DNA damage through a signaling pathway that promotes Bcl-6 degradation. After DNA damage accumulated, the kinase ATM promoted Bcl-6 phosphorylation, leading to its interaction with the isomerase Pin1 and its degradation by the ubiquitin-proteasome system. Because Bcl-6 is required for the maintenance of germinal centers, our findings suggest that the extent of genotoxic stress controls the fate of germinal center B cells by means of Bcl-6.

Interstitial deletions of the chromosome 9p21 segment encoding the p16/CDKN2A tumor suppressor gene (i.e., 9p21 deletions) are frequently observed in a variety of human cancers. A majority of these deletions in lymphoid leukemia have been indicated to be mediated by illegitimate V(D)J recombination. In the present study, to elucidate the molecular processes of 9p21 deletions in nonlymphocytic malignancies, breakpoints for these deletions were analysed in 21 lung cancer cell lines and 32 nonlymphocytic cancer cell lines of nine other histological types. In all, 32 breakpoints in 21 lung cancer cell lines and 56 breakpoints in 32 nonlung cancer cell lines were mapped in a 450-kb segment encompassing the CDKN2A locus with a 10-kb resolution. The largest number of breakpoints (i.e., seven breakpoints in lung cancer and 12 breakpoints in nonlung cancers) was mapped in a 10-kb region containing the CDKN2A gene. More precise mapping of these seven and 12 breakpoints revealed that none of these breakpoints were located within 50-bp intervals to each other in this 10 kb region. Cloning and sequencing of breakpoints in 18 representative cell lines (six lung and 12 nonlung cancers) further revealed that there were no significant homologies among breakpoints in these 18 cell lines. In 11 (61%) cell lines, 1-5-bp nucleotides were overlapped at breakpoint junctions. These results indicate that DNA double-strand breaks triggering 9p21 deletions do not occur at specific DNA sequences, although they preferentially occur in or near the CDKN2A locus. It was also indicated that two broken DNA ends are rejoined by nonhomologous end-joining repair, preferentially utilizing microhomologies of DNA ends, in the occurrence of 9p21 deletions. Oncogene (2003) 22, 3792-3798. doi:10.1038/sj.onc.1206589Keywords: 9p21 deletion; p16/CDKN2A; lung cancer; DNA double-strand break; nonhomologous end joining Chromosome 9p deletions frequently occur in a variety of human cancers including lymphoid leukemia, lung cancer, esophageal cancer, glioma and melanoma, and the 9p21 segment including the p16/CDKN2A, p14/ARF and p15/CDKN2B loci has been defined as a common region for the deletions (Kamb et al., 1994;Nobori et al., 1994;Ohnishi et al., 1995;Tanaka et al., 1997;Drexler, 1998;Hamada et al., 1998Hamada et al., , 2000Park et al., 2002). The CDKN2A tumor suppressor gene and two other related genes, ARF and CDKN2B, encode critical regulators of cell cycle and/or apoptosis (Sherr, 2000). Thus, deletion of the 9p21 segment is a causative generic alteration inactivating the CDKN2A, ARF and/or CDKN2B genes, and is thought to play an important role in the development and/or progression of human cancers. Elucidation of the mechanism(s) for the deletions of the 9p21 segment is, therefore, indispensable in understanding the molecular pathways of multistage human carcinogenesis.Structural analyses of breakpoints for interstitial deletions of the 9p21 segment (i.e., 9p21 deletions) have been performed in lymphoid leukemia to predict the molecular processes of th...

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To understand molecular pathways underlying 9p21 deletions, which lead to inactivation of the p16/CDKN2A, p14/ARF, and/or p15/CDKN2B genes, in lymphoid leukemia, 30 breakpoints were cloned from 15 lymphoid leukemia cell lines. Seventeen (57%) breakpoints were mapped at five breakpoint cluster sites, BCS-LL1 to LL5, each of <15 bp. Two breakpoint cluster sites were located within the ARF and CDKN2B loci, respectively, whereas the remaining three were located >100 kb distal to the CDKN2A, ARF, and CDKN2B loci. The sequences of breakpoint junctions indicated that deletions in the 11 (73%) cell lines were mediated by illegitimate V(D)J recombination targeted at the five BCS-LL and six other sites, which contain sequences similar to recombination signal sequences for V(D)J recombination. An extrachromosomal V(D)J recombination assay indicated that BCS-LL3, at which the largest number of breakpoints (i.e. five breakpoints) was clustered, has a V(D)J recombination potential 150-fold less than the consensus recombination signal sequence. Three other BCS-LLs tested also showed V(D)J recombination potential, although it was lower than that of BCS-LL3. These results indicated that illegitimate V(D)J recombination, which was targeted at several ectopic recombination signal sequences widely distributed in 9p21, caused a large fraction of 9p21 deletions in lymphoid leukemia.V(D)J recombination is a physiological recombination of DNAs, which occurs at the Ig and T cell receptor loci during the differentiation of lymphoid cells (1, 2). These loci have recombination signal sequences (RSSs) 1 consisting of a highly conserved heptamer sequence (consensus 5Ј-CACAGTG) and an AT-rich nonamer sequence (consensus 5Ј-ACAAAAACC). The recombination-activating gene (RAG) complex generates DNA double strand breaks (DSBs) at pairs of RSSs in which one signal has a 12-bp spacer between the heptamer and nonamer (12-signal) and the other signal has a 23-bp spacer (23-signal) (1, 2). This is known as the 12/23 rule. Several nucleotides are inserted as junctional additions at the junctions, and the broken DNA ends are rejoined by the nonhomologous end joining pathway (1-3). The results of V(D)J recombination are that coding junction products and, in some cases such as the Ig locus, signal junction products are left in the human genomic DNA (1). It has been suggested that recurrent chromosomal aberrations in lymphoid malignancies, such as translocations and inversions involving the Ig and T cell receptor loci, are caused by illegitimate actions of the V(D)J recombination machinery at "ectopic" RSSs, the sequences homologous to the consensus RSS at non-Ig, non-T cell receptor loci (4 -9). These chromosomal aberrations cause the activation or fusion of several oncogenes leading to leukemogenesis. In a large portion of these aberrations, insertions of nucleotides were detected between the two joined germ line sequences. In addition, recently, several DNA fragments containing the ectopic RSSs were shown to have potential to undergo V(D)J recombination...