Oncogene database

604

BCL6

B-cell CLL/lymphoma 6;BCL6;B-cell CLL/lymphoma 6

B-cell lymphoma 5 protein|B-cell lymphoma 6 protein|B-cell lymphoma 6 protein transcript|BCL-5|BCL-6|cys-his2 zinc finger transcription factor|lymphoma-associated zinc finger gene on chromosome 3|protein LAZ-3|zinc finger and BTB domain-containing protein

3q27

protein-coding

One of the most frequent genetic abnormalities associated with non Hodgkin lymphoma is the structural alteration of the 5 non coding/regulatory region of the BCL6 (LAZ3) protooncogene. BCL6 encodes a POZ/Zn finger protein, a structure similar to that of many Drosophila developmental regulators and to another protein involved in a human hematopoietic malignancy, PLZF. BCL6 is a sequence specific transcriptional repressor controlling germinal center formation and T cell dependent immune response. Although the expression of BCL6 negatively correlates with cellular proliferation in different cell types, the influence of BCL6 on cell growth and survival is currently unknown so that the way its deregulation may contribute to cancer remains elusive. To directly address this issue, we used a tetracycline-regulated system in human U2OS osteosarcoma cells and thus found that BCL6 mediates growth suppression associated with impaired S phase progression and apoptosis. Interestingly, overexpressed BCL6 can colocalize with sites of ongoing DNA synthesis, suggesting that it may directly interfere with S phase initiation and/or progression. In contrast, the isolated Zn finger region of BCL6, which binds BCL6 target sequence but lacks transcriptional repression activity, slows, but does not suppress, U2OS cell growth, is less efficient at delaying S phase progression, and does not trigger apoptosis. Thus, for a large part, the effects of BCL6 overexpression on cell growth and survival depend on its ability to engage protein/protein interactions with itself and/or its transcriptional corepressors. That BCL6 restricts cell growth suggests that its deregulation upon structural alterations may alleviate negative controls on the cell cycle and cell survival.

Most acute promyelocytic leukemia (APL) cases are associated with recurrent translocations between the gene of retinoic receptor alpha and that of PML (t(15;17)) or PLZF (t(11;17)). PML localizes onto discrete intranuclear domains, the PML-nuclear bodies, and displays anti-oncogenic and pro-apoptotic properties. PLZF encodes a transcription factor belonging to the POZ/domain and Kruppel zinc finger (POK) family which interacts directly with PML. PLZF is related to another POK protein, LAZ3(BCL6), which is structurally altered, and presumably misexpressed, in many non-Hodgkin lymphoma (NHL) cases. PLZF and LAZ3 share many functional properties: both inhibit cell growth, concentrate into punctated nuclear subdomains and are sequence-specific transcriptional repressors recruiting a histone deacetylase-repressing complex. Given these similarities, we tested whether both proteins could be targeted by each other. Here, LAZ3 and PLZF are shown to colocalize onto nuclear dots. Moreover, truncated derivatives of one protein, which display a diffuse nuclear localization, are recruited onto nuclear dots by the full-length other. The colocalization and the reciprocal rescue is the result of a direct interaction between LAZ3 and PLZF, as indicated by yeast two hybrid assays, in vitro immunoprecipitations, and GST pull down experiments. In contrast to LAZ3 homomerization, LAZ3/PLZF heteromerization in yeast does not solely depend on POZ/POZ contacts but rather also relies on interactions between the two zinc finger regions and cross contacts between the zinc finger region and the POZ domain of each partner. Likewise, LAZ3 shows some colocalization with the PLZF partner PML upon stable overexpression of both proteins in CHO cells and interacts with PML in yeast. Finally, endogenous LAZ3 and PLZF are co-induced and partially colocalized in myeloid MDS cells. These data indicate that a physical interaction between LAZ3 and PLZF underlies their simultaneous recruitment onto multiproteic nuclear complexes, presumably involved in transcriptional silencing and whose integrity (for APL) and/or function (for APL and NHL) may be altered in oncogenesis.

The BCL-6/LAZ3 gene encodes a zinc-finger transcriptional repressor and is located at the breakpoint of the 3q27-associated translocations that occur most frequently in non-Hodgkin s lymphomas (NHLs). A number of chromosomal translocations involving BCL-6 have been analyzed, but the biological functions of this protein remain obscure. To examine cell responses and target genes related to the BCL-6 signaling pathway, we established Ba/F3 pro-B cells carrying a human BCL-6 transgene that is inducible under control of the lactose operon. Using a cDNA array hybridization technique, we found that the induced BCL-6 protein can downregulate the expressions of the genes, cyclin A2, chemokine receptor CXCR4, and insulin-like growth factor binding protein-4 (IGFBP-4) in the Ba/F3 cells. Northern blot analysis established that the expressions of these genes were indeed downregulated by the induced BCL-6 protein but in a somewhat different manner. The induced BCL-6 protein also inhibited cell proliferation of Ba/F3 cells. These findings strongly suggest that three key genes, namely cyclin A2, CXCR4, and IGFBP-4 may play a role in the downstream of the BCL-6 signaling pathway during B-lymphoid differentiation.

The proto-oncogene BCL-6 encodes a transcriptional repressor protein that is expressed at high levels in germinal center B cells and lymphomas with a germinal center B cell phenotype. The BCL-6 gene is a frequent target of chromosomal translocations, micro-deletions, and point mutations in non-Hodgkin s lymphoma. Studies of BCL-6-deficient mice have revealed that BCL-6 is critical for normal lymphocyte differentiation and also that BCL-6 is a negative regulator of inflammation. Recent studies have shed light on how BCL-6 controls these processes by showing that BCL-6 regulates a broad spectrum of target genes. BCL-6 represses transcription of genes involved in lymphocyte activation, differentiation, proliferation, and migration. Although much progress has been made in understanding gene regulation by BCL-6, many important questions are unresolved.

BCL-6 is an important regulator of the immune system. It is required for GC formation and T cell dependent antibody responses. Mice deficient in BCL-6 fail to form GC and mount reduced levels of T cell-dependent antibody responses. BCL-6 (-/-) mice, in addition, develop a massive inflammatory response in many organs characterized by eosinophilic infiltration and hyper-IgE production, a typical Th2 hyperimmune response. This suggests a negative role of BCL-6 in Th2 pathway. The BCL-6 gene encodes a POZ/zinc finger transcription repressor highly expressed in GC B cells, but not in pre-GC B cells or in more differentiated memory or plasma cells. By functioning as a potent transcriptional repressor of various target genes, BCL-6 modulates IL-4, BCR, and CD40L signals for normal B cell development. In B cell lymphomas, structural alterations of the BCL-6 promoter region, including chromosome translocation and somatic hypermutation, represent the most frequent genetic lesions associated with non-Hodgkin lymphoma, especially of diffuse large cell lymphoma, a malignancy often derived from germinal centre (GC) B cells. This suggests that deregulated expression of BCL-6 may contribute to lymphomagenesis.

The BCL6 proto-oncogene encodes a transcriptional repressor whose expression is deregulated by chromosomal translocations in approximately 40% of diffuse large B-cell lymphomas (DLBCLs). The BCL6 regulatory sequences are also targeted by somatic hypermutation in germinal center (GC) B cells and in a fraction of ALL GC-derived lymphomas. However, the functional consequences of these mutations are unknown. Here we report that a subset of mutations specifically associated with DLBCL causes deregulated BCL6 transcription. These mutations affect 2 adjacent BCL6 binding sites located within the first noncoding exon of the gene, and they prevent BCL6 from binding its own promoter, thereby disrupting its negative autoregulatory circuit. These alterations were found in approximately 16% of DLBCLs devoid of chromosomal translocations involving the BCL6 locus, but they were not found in normal GC B cells. This study establishes a novel mechanism for BCL6 deregulation and reveals a broader involvement of this gene in DLBCL pathogenesis.

The multiplicity of transcription factors involved in hematologic malignancies suggests a complicated scenario in which many different molecular mechanisms lead to malignant transformation. We hypothesized that some of these proteins might physically and functionally interact and thus mechanistically link different diseases. The ETO protein of t(8;21) acute myeloid leukemia (AML) is an excellent candidate as a common factor because it is normally expressed in human hematopoietic cells, it binds to histone deacetylases (HDACs), and it interacts with the PLZF protein of t(11;17) acute promyelocytic leukemia. To determine whether ETO functionally links a broader range of disease entities, we asked whether ETO forms a complex with the Bcl-6 oncoprotein of B-cell lymphomas. We found that ETO and Bcl-6 are coexpressed in normal and malignant lymphoid tissue, where they interact and colocalize in nuclear speckles. ETO binds to the fourth zinc finger of Bcl-6, enhances Bcl-6 repression of artificial and endogenous genes in an HDAC-dependent manner, and forms a complex with Bcl-6 on the promoters of its endogenous target genes in B-cell lymphoma cells. Therefore, ETO is a bona fide corepressor that links the transcriptional pathogenesis of acute leukemias and B-cell lymphomas and offers a compelling target for transcriptional therapy of hematologic malignancies.

B-cell lymphoma gene (BCL-6) upregulation contributes to immortalization of mouse embryo fibroblast and primary B cells via upregulation of cyclin D1. As cyclin D1 overexpression is a common phenomenon in different cancers, BCL-6 protein overexpression may not be restricted to lymphomas. In this study, expression of BCL-6 was investigated by immunohistochemistry on paraffin-embedded specimens from 150 breast cancer patients and 10 specimens of normal breast tissue. The results showed BCL-6 overexpression (> or =10% of cells) in 24/150 (16%) breast cancer patients, whereas in normal breast low expression (<1%) of BCL-6 was observed. In linear regression analysis BCL-6 expression was associated with cyclin D1 (r=0.197, P=0.016). Further, in chi2 analyses, BCL-6-positivity was associated with overexpression of p53 (P=0.016), and hypoxia-inducible factor-1alpha (P<0.001). Involvement of BCL-6 in breast carcinogenesis is further underscored by comparative genomic hybridization analysis that showed gains at the BCL-6 locus (3q27) in 14/86 (16%) breast cancer tissues. The cases with amplification in BCL-6 showed an increased (25%) incidence of BCL-6 protein overexpression. Thus, this study is the first to show that BCL-6 oncogene activation plays a role in cancers other than lymphomas.

Primary central nervous system lymphomas (PCNSL) constitute diffuse large B-cell lymphomas arising in and remaining confined to the brain. Little information is available on cytogenetic changes in PCNSL, and recurrent chromosomal translocations have not yet been identified. Fluorescence in situ hybridization (FISH) of a series of 13 PCNSL from immunocompetent patients revealed 3 cases with signal patterns of a BCL6-specific probe suggesting a breakpoint in this oncogene locus in chromosome band 3q27. Here, we describe cloning of the translocation breakpoints by long-distance inverse polymerase chain reaction (LDI-PCR) in 2 of these tumors. Both breakpoints affected the first intron of BCL6. In one PCNSL, the HSPCA (HSP90A) gene in 14q32.31 was identified as BCL6 partner. In the second lymphoma, the gene encoding glyceraldehyde-3-phosphate dehydrogenase (GAPD) on 12p13.31 was detected as a hitherto unknown partner of BCL6. Our results suggest translocation-mediated BCL6 oncogene activation as a so far unknown pathogenetically relevant mechanism in PCNSL.

About 75% of breast tumors are positive for the estrogen receptor (ER) or progesterone receptor (PgR) or both, and estrogen is the main stimulant in the development and growth of these tumors. Tamoxifen, an estrogen receptor antagonist has been endocrine treatment for hormone-sensitive breast cancer for more than 20 years. However, the underlying cause of treatment failure in many breast cancer patients receiving tamoxifen is resistance to tamoxifen. The mechanisms of tamoxifen and the molecular events responsible for resistance to tamoxifen are not fully understood. Two ER subtypes, ERalpha and ERbeta, activate the Activator Protein-1 (AP-1) response elements, and through interactions between ERs and the AP-1 transcription factors c-fos and c-jun, these transcription factors regulate the genes involved in many cellular processes, including proliferation, differentiation, cell motility, and apoptosis. Thus, the interaction between ERs and AP-1 could be important clinically and could have bearing on the response to tamoxifen. Tamoxifen acts as an agonist on genes under the control of an AP-1 response elements when ERalpha or ERbeta is expressed. AP-1 blockade suppresses mitogenic signals from multiple different peptide growth factors as well as estrogen, and inhibits the growth of MCF-7 breast cancer cells both in vitro and in vivo. Tamoxifen actually activate the AP-1 transcription factor. Increased AP-1 activity in breast cancer cells can lead to tamoxifen resistance. The proto-oncogene B-cell lymphoma gene 6 (BCL-6) has been characterized as a regulator of B-lymphocyte growth and development. BCL-6 is also expressed in the mammary epithelium in nonpregnant animals and during early pregnancy and is expressed in 68% of histologically high-grade ductal breast carcinomas, which are clinically the most aggressive. BCL-6 is a potent repressor of transcriptional activity mediated by AP-1 factors. We hypothesize that increased BCL-6 in breast cancer cells might block tamoxifen resistance by repressing AP-1, eventually resulting in apoptosis. We also suggest that BCL-6 expression must be analyzed in ER-positive breast cancer patients and the results must be correlated with predictive and prognostic factors and survival.

The BTB/POZ transcriptional repressor and candidate oncogene BCL6 is frequently misregulated in B-cell lymphomas. The interface through which the BCL6 BTB domain mediates recruitment of the SMRT, NCoR and BCoR corepressors was recently identified. To determine the contribution of this interface to BCL6 transcriptional and biological properties, we generated a peptide that specifically binds BCL6 and blocks corepressor recruitment in vivo. This inhibitor disrupts BCL6-mediated repression and establishment of silenced chromatin, reactivates natural BCL6 target genes, and abrogates BCL6 biological function in B cells. In BCL6-positive lymphoma cells, peptide blockade caused apoptosis and cell cycle arrest. BTB domain peptide inhibitors may constitute a novel therapeutic agent for B-cell lymphomas.

The human proto-oncogene BCL6 encodes a BTB/POZ-zinc-finger transcriptional repressor that is necessary for germinal-centre formation and is implicated in the pathogenesis of B-cell lymphoma. The precise function of BCL6 in germinal-centre development and lymphomagenesis is unclear because very few direct BCL6 target genes have been identified. Here we report that BCL6 suppresses the expression of the p53 (also known as tp53) tumour suppressor gene and modulates DNA damage-induced apoptotic responses in germinal-centre B cells. BCL6 represses p53 transcription by binding two specific DNA sites within the p53 promoter region and, accordingly, p53 expression is absent in germinal-centre B cells where BCL6 is highly expressed. Suppression of BCL6 expression via specific short interfering RNA leads to increased levels of p53 messenger RNA and protein both under basal conditions and in response to DNA damage. Most notably, constitutive expression of BCL6 protects B cell lines from apoptosis induced by DNA damage. These results suggest that an important function of BCL6 is to allow germinal-centre B cells to tolerate the physiological DNA breaks required for immunoglobulin class switch recombination and somatic hypermutation without inducing a p53-dependent apoptotic response. These findings also imply that deregulated BCL6 expression contributes to lymphomagenesis in part by functional inactivation of p53.

Mammalian olfactory sensory neurons harbor a large repertoire of odorant receptors. Yet, each cell chooses a single odorant receptor gene to express, which may then serve as the molecular identification of that cell. This process of differentiation or receptor gene regulation of olfactory sensory neurons has largely been enigmatic. Here, we showed that the important proto-oncogene in B lymphocyte terminal differentiation, B-Cell Leukemia/Lymphoma 6 (BCL-6), which codes for a sequence-specific transcription repressor, is expressed in olfactory sensory neurons in the mouse. We detected BCL-6 mRNA in most, if not all, mature olfactory sensory neurons by in situ hybridization histochemistry. Western blot analysis also revealed the presence of BCL-6 protein in the olfactory epithelium, but immunohistochemical analysis using anti-BCL-6 antibodies showed that only a subset of olfactory sensory neurons highly expressed BCL-6 protein. BCL-6 mRNA was detected as early as embryonic day 14 (E14) in most olfactory sensory neurons as in adults, and at E15, BCL-6 protein was detected in most cells that were likely to be differentiating into mature olfactory sensory neurons. Artificial induction of apoptosis of olfactory sensory neurons resulted in the decrease of BCL-6 mRNA, but during the subsequent phase of regeneration and differentiation, it markedly increased. Taken together, our results suggest the possibility that BCL-6 plays an important role in terminal differentiation not only in B lymphocytes but also in olfactory sensory neurons.

expression profiling of T24 cells revealed that 17 out of 313 human miRNAs were upregulated more than 3-fold by simultaneous treatment with the chromatin-modifying drugs 5-aza-2 -deoxycytidine and 4-phenylbutyric acid. One of these, miR-127, is embedded in a CpG island and is highly induced from its own promoter after treatment. miR-127 is usually expressed as part of a miRNA cluster in normal cells but not in cancer cells, suggesting that it is subject to epigenetic silencing. In addition, the proto-oncogene BCL6, a potential target of miR-127, was translationally downregulated after treatment. These results suggest that DNA demethylation and histone deacetylase inhibition can activate expression of miRNAs that may act as tumor suppressors.

The BCL6 proto-oncogene encodes a nuclear transcriptional repressor, with pivotal roles in germinal center (GC) formation and regulation of lymphocyte function, differentiation, and survival. BCL6 suppresses p53 in GCB-cells and its constitutive expression can protect B-cell lines from apoptosis induced by DNA damage. BCL6-mediated expression may allow GCB-cells to sustain the low levels of physiological DNA breaks related to somatic mutation (SM) and immunoglobulin class switch recombination which physiologically occur in GCB-cells. Three types of genetic events occur in the BCL6 locus and involve invariably the 5 non-coding region and include translocations, deletions and SM actively targeted to the 5 untranslated region. These acquired mutations occur independently of translocations but may be involved in the deregulation of the gene and/or translocation mechanisms. The favorable prognostic value of high levels of BCL6 gene expression in NHL seems well-established. By contrast, the relevance of SM or translocation of the gene remains unclear. However, it is likely that non-Hodgkin s lymphomas (NHL) harboring the most frequent translocation involving BCL6, i.e. t(3;14), are characterized by a common cell of origin and similar oncogenic mechanisms. Several experiments and mouse models mimicking BCL6 translocation occurring in human lymphoma have demonstrated the oncogenic role of BCL6 and constitute a rational to consider BCL6 as a new therapeutic target in NHL. BCL6 blockade can be achieved by different strategies which include siRNA, interference by specific peptides or regulation of BCL6 acetylation by pharmacological agents such as SAHA or niacinamide and would be applicable to most type of B-cell NHL.

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.

The inflammation that occurs during atherosclerosis is characterized by the release of large amounts of group IIA secretory phospholipase A2 (sPLA2-IIA). This study was designed to define the function of the three peroxisome proliferator-activated receptors (PPARs) on sPLA2 expression in vascular smooth muscle cells (VSMCs). We found that PPAR ligands decreased sPLA2-IIA activity and inhibited mRNA accumulation under inflammatory conditions. Furthermore, interleukin-1beta-induced sPLA2-IIA promoter activity was inhibited by the three PPAR ligands and in a similar way when cells were cotransfected with PPARalpha, PPARbeta, or PPARgamma, plus retinoid X receptor alpha (RXRalpha). Our study revealed that the regulation of sPLA2-IIA gene transcription by PPARalpha/RXR and PPARgamma/RXR heterodimers requires an interaction with a PPAR response element (PPRE) of the sPLA2-IIA promoter. In contrast, PPARbeta operates through a PPRE-independent mechanism. In addition, we demonstrated that VSMCs expressed the transcriptional repressor BCL-6. Overexpression of BCL-6 markedly reduced sPLA2-IIA promoter activity in VSMCs, while a dominant negative form of BCL-6 abrogated sPLA2 repression by PPARbeta. The PPARbeta agonist induced a BCL-6 binding to the sPLA2 promoter in VSMCs under inflammatory conditions. The knockdown of BCL-6 by short interfering RNA abolished the inhibitory effect of the PPARbeta ligand on sPLA2 activity and prostaglandin E2 release. Thus, the inhibition of sPLA2-IIA activity by PPARbeta agonists may provide a promising approach to impacting the initiation and progression of atherosclerosis.

OBJECTIVE: To investigate the biological role of BCL-6 oncoprotein in breast cancer disease progression (recurrence and metastasis). METHODS: The series consisted of 93 consecutive female patients with primary breast cancer and median follow-up of 10 years. BCL-6 expression was assessed in vivo by immunohistochemistry and real-time PCR. Breast cancer cell lines and some metastasis-related genes (CXCR4, Itgbeta-3 and FLT-1) were also analysed by molecular techniques. Prognostic evaluation was performed by fitting a multivariate Cox regression model. RESULTS: BCL-6 immunoexpression was positive in 22 (23.7%) tumours and negative in 71 (76.3%). ALL axillary lymph node metastases of 47 node-positive patients were negative, including 12 cases showing BCL-6-positive primary tumours. Likewise, in 9 recurrence cases, BCL-6 expression was similar or decreased compared with primary tumours. No correlation between immunoexpression and gene expression of BCL-6 was observed. BCL-6 was significantly reduced both in derived metastases of a breast cancer cell line (M435) and when the latter was treated with a demethylation agent (5-azacytidine). However, BCL-6-transfected breast cancer cell lines expressed significantly higher levels of CXCR4, Itgbeta-3 and FLT-1. Co-expression of the 4 genes was found in 4 of 17 tumours evaluated, but lacking prognostic significance. BCL-6 oncoprotein revealed no significant influence on outcome. CONCLUSION: The results strongly suggest the loss of BCL-6 expression in breast cancer progression, which might be related with methylation status alterations of still unknown partner gene(s).

For the mammalian olfactory epithelium to continually detect odorant, neuronal survival, apoptosis, and regeneration must be coordinated. Here, we showed that the proto-oncogene BCL6, which encodes a transcriptional repressor required for lymphocyte terminal differentiation, contributes to the survival of olfactory sensory neurons (OSNs). In the olfactory epithelia of the BCL6 null mutant mice, many OSNs were positive for both OMP and GAP43. The epithelium was relatively thinner, showing many apoptotic signals. These characters were phenotypically similar to those of the wild-type mice treated with nasal lectin irrigation, which acutely induces apoptosis of OSNs. Odorant receptors were expressed normally in the epithelia of the mutant mice, and their overall expression profile based on DNA microarray analyses was roughly similar to that of the apoptosis-induced olfactory epithelia of the wild-type mice. Experimental increase of BCL6 together with green fluorescent protein in OSNs using adenovirus-mediated gene transfer made the epifluorescence last longer than the control fluorescence without exogenous BCL6 after the nasal lectin irrigation, indicating that BCL6 made the infected neurons survive longer. We conclude that BCL6 plays an active role in the survival of OSNs as an anti-apoptotic factor and confers immature OSNs enough time to fully differentiate into mature ones.

Constitutive B-cell lymphoma 6 (Bcl-6) expression was undetectable in multiple myeloma (MM) cell lines, except U266 cells. However, it was up-regulated by coculture with bone marrow (BM) stromal cell-culture supernatant (SCCS). Bcl-6 expression in patient MM cells in the BM was positive. Anti-interleukin-6 (IL-6)-neutralizing antibody significantly blocked SCCS-induced Bcl-6 in MM cells. Indeed, IL-6 strongly triggered Bcl-6 expression in MM cells, whereas Janus kinase inhibitor and STAT3 siRNA down-regulated Bcl-6. tumor necrosis factor-alpha (TNF-alpha) also triggered Bcl-6, but independently of STAT3, whereas IkappaB kinasebeta inhibitor down-regulated TNF-alpha-induced Bcl-6, indicating that the canonical nuclear factor-kappaB pathway mediates TNF-alpha-induced Bcl-6 expression. Importantly, down-regulation of Bcl-6 by shRNA significantly inhibited MM cell growth in the presence of SCCS. Our results therefore suggest that Bcl-6 expression in MM cells is modulated, at least in part, via Janus kinase/STAT3 and canonical nuclear factor-kappaB pathways and that targeting Bcl-6, either directly or via these cascades, inhibits MM cell growth in the BM milieu.

BCL6 is a transcriptional repressor which has emerged as a critical regulator of germinal centers (GC), the sites where B cells are selected based on the production of antibodies with high affinity for the antigen. BCL6 is also a frequently activated oncogene in the pathogenesis of human B cell lymphomas, most of which derive from the GC B cells. A thorough understanding of the biological role of BCL6 in normal B cell development and lymphomagenesis depends upon the identification of the full set of genes that are targets of its transcriptional regulatory function. Recently, the identification of BCL6 targets has been implemented with the use of genome-wide chromatin immunoprecipitation and gene expression profiling approaches. A large set of promoters have been shown to be physically bound by BCL6, but only a fraction of them appears to be subjected to transcriptional repression in GC B cells. This set of BCL6 targets points to a number of cellular functions which are likely to be directly controlled by BCL6 during GC development, including activation, survival, DNA-damage response, cell cycle arrest, cytokine-, toll-like receptor-, TGFbeta-, WNT-signaling, and differentiation. Overall, BCL6 is revealing its dual role of "safe-keeper" in preventing centroblasts from responding to signals leading to a premature exit from the GC and of contributor to lymphomagenesis by allowing the instauration of conditions favorable to malignant transformation.

Aberrant DNA methylation commonly occurs in cancer cells where it has been implicated in the epigenetic silencing of tumor suppressor genes. Additional roles for DNA methylation, such as transcriptional activation, have been predicted but have yet to be clearly demonstrated. The BCL6 oncogene is implicated in the pathogenesis of germinal center-derived B cell lymphomas. We demonstrate that the intragenic CpG islands within the first intron of the human BCL6 locus were hypermethylated in lymphoma cells that expressed high amounts of BCL6 messenger RNA (mRNA). Inhibition of DNA methyltransferases decreased BCL6 mRNA abundance, suggesting a role for these methylated CpGs in positively regulating BCL6 transcription. The enhancer-blocking transcription factor CTCF bound to this intronic region in a methylation-sensitive manner. Depletion of CTCF by short hairpin RNA in neoplastic plasma cells that do not express BCL6 resulted in up-regulation of BCL6 transcription. These data indicate that BCL6 expression is maintained during lymphomagenesis in part through DNA methylation that prevents CTCF-mediated silencing.

The PATZ1 gene encoding a POZ/AT-hook/Kruppel zinc finger (PATZ) transcription factor, is considered a cancer-related gene because of its loss or misexpression in human neoplasias. As for other POZ/domain and Kruppel zinc finger (POK) family members, the transcriptional activity of PATZ is due to the POZ-mediated oligomer formation, suggesting that it might be not a typical transactivator but an architectural transcription factor, thus functioning either as activator or as repressor depending on the presence of proteins able to interact with it. Therefore, to better elucidate PATZ function, we searched for its molecular partners. By yeast two-hybrid screenings, we found a specific interaction between PATZ and BCL6, a human oncogene that plays a key role in germinal center (GC) derived neoplasias. We demonstrate that PATZ and BCL6 interact in germinal center-derived B lymphoma cells, through the POZ domain of PATZ. Moreover, we show that PATZ is able to bind the BCL6 regulatory region, where BCL6 itself acts as a negative regulator, and to contribute to negatively modulate its activity. Consistently, disruption of one or both Patz1 alleles in mice causes focal expansion of thymus B cells, in which BCL6 is up-regulated. This phenotype was almost completely rescued by crossing Patz1(+/-) with Bcl6(+/-) mice, indicating a key role for Bcl6 expression in its development. Finally, a significant number of Patz1 knock-out mice (both heterozygous and homozygous) also develop BCL6-expressing lymphomas. Therefore, the disruption of one or both Patz1 alleles may favor lymphomagenesis by activating the BCL6 pathway.

MEF2B encodes a transcriptional activator and is mutated in approximately 11% of diffuse large B cell lymphomas (DLBCLs) and approximately 12% of follicular lymphomas (FLs). Here we found that MEF2B directly activated the transcription of the proto-oncogene BCL6 in normal germinal-center (GC) B cells and was required for DLBCL proliferation. mutation of MEF2B resulted in enhanced transcriptional activity of MEF2B either through disruption of its interaction with the corepressor CABIN1 or by rendering it insensitive to inhibitory signaling events mediated by phosphorylation and sumoylation. Consequently, the transcriptional activity of Bcl-6 was deregulated in DLBCLs with MEF2B mutations. Thus, somatic mutations of MEF2B may contribute to lymphomagenesis by deregulating BCL6 expression, and MEF2B may represent an alternative target for blocking Bcl-6 activity in DLBCLs.

Preeclampsia is a leading cause for maternal and perinatal mortality and morbidity. Microarray-based transcriptional profiling has been widely used for identifying genes responsible for preeclampsia. These studies deliver multiple pictures of gene signatures, implying the complicated pathophysiology. In the present work, we designed our own gene array containing genes involved in various signaling transduction pathways and analyzed placental samples from patients with preeclampsia and controls. We verify that genes associated with angiogenesis and migration pathways are mostly altered in preeclamptic placentas. Interestingly, several genes including B-cell lymphoma 6 have been identified to be linked to preeclampsia. Increased expression of B-cell lymphoma 6 is correlated with enhanced FLT1 and LEPTIN, the hallmarks of preeclampsia. Moreover, the protein level of B-cell lymphoma 6 is elevated in preeclamptic placentas and is predominantly localized in the nucleus of villous cytotrophoblasts lying directly underneath the syncytial layer, suggestive of an involvement in the function of villous trophoblasts. Altered B-cell lymphoma 6, a key oncogene in B-cell lymphomagenesis, may be involved in the pathogenesis of preeclampsia, and further investigations are required to decipher the molecular mechanisms.

Diffuse large B-cell lymphoma (DLBCL) is the most common lymphoma and can be separated into two subtypes based upon molecular features with similarities to germinal centre B-cells (GCB-like) or activated B-cells (ABC-like). Here we identify gain of 3q27.2 as being significantly associated with adverse outcome in DLBCL and linked with the ABC-like subtype. This lesion includes the BCL6 oncogene, but does not alter BCL6 transcript levels or target-gene repression. Separately, we identify expression of BCL6 in a subset of human haematopoietic stem/progenitor cells (HSPCs). We therefore hypothesize that BCL6 may act by hit-and-run oncogenesis. We model this hit-and-run mechanism by transiently expressing Bcl6 within murine HSPCs, and find that it causes mature B-cell lymphomas that lack Bcl6 expression and target-gene repression, are transcriptionally similar to post-GCB cells, and show epigenetic changes that are conserved from HSPCs to mature B-cells. Together, these results suggest that BCL6 may function in a hit-and-run role in lymphomagenesis.

Bcl-6 translocation is a genetic alteration that is commonly detected in Primary Central Nervous System Lymphoma. The role of this protein in cerebral tumors is unclear. In this study we investigated Bcl-6 translocation and its transcriptional and translational levels in formalin-fixed, paraffin-embedded cerebral tissue sections from glioblastoma (GBM), low-grade glioma (Astrocytoma grade II and III), and meningioma patients, and correlated them with apoptotic processes and p53 and caspase-3 expression. The results showed a frequency of 36.6% of Bcl-6 translocation in GBM patients and a decreased expression in low-grade glioma patients, correlated with the severity of the disease. Bcl-6 translocation induced an overexpression of both Bcl-6 protein and messenger in GBM, inhibiting apoptotic processes and caspases 3 expression. On the contrary, in low-grade gliomas and meningiomas Bcl-6 expression was reduced, resulting in an increase of apoptotic processes. Finally, p53 expression levels in brain tumors were comparable to Bcl-6 levels. Overall, these data demonstrate, for the first time, that the Bcl-6 gene translocates in GBM patients and that its translocation and expression are correlated with apoptosis inhibition, indicating a key role for this gene in the control of cellular proliferation. This study offers further insights into glioblastoma biology, and supports Bcl-6 as a new diagnostic marker to evaluate the disease severity.

BACKGROUND: Dysregulation of BCL6 plays critical oncogenic roles and facilitates tumorigenesis in various malignancies. However, whether the aberrant expression of BCL6 in ovarian carcinoma is associated with malignancy, metastasis or prognosis remains unknown. Our study aimed to investigate the expression of BCL6 in ovarian carcinoma and its possible correlation with clinicopathological features as well as patient survival to reveal its biological effects in ovarian tumor progression. METHODS: Immunochemistry analysis was performed in 105 cases of ovarian carcinoma covering the histological types of serous, endometrioid and clear cell. Spearman analysis was used to calculate the correlation between pathological parameters and the expression of BCL6. Kaplan-Meier method and Cox proportional hazards analysis were used to analyze the disease-specific survival (DSS) and disease-free survival (DFS). We also assessed whether overexpression and knockdown of BCL6 influence in vitro cell proliferation, cell cycle progression, as well as tumor cell invasion and migration. RESULTS: The expression of BCL6 was higher in ALL three major kinds of ovarian cancer in comparison with paratumorous epithelium. BCL6 expression was tightly correlated with FIGO staging, lymph node metastasis and recurrence. Higher expression of BCL6 led to a significantly poorer DSS and DFS and multivariate analysis revealed that BCL6 was an independent risk factor of DSS and DFS. Enforced overexpression of BCL6 in ovarian tumor cells stimulated proliferation by inducing G1-S transition, and promoted tumor cell invasion and migration. Conversely, RNA interference-mediated silencing BCL6 expression inhibited proliferation by altered cell cycle progression and reduced the ability of the cells to migrate, and invade the extracellular matrix in culture. CONCLUSIONS: Our study suggests that the inappropriate activation of BCL6 predicts poor prognosis and promotes tumor progression in ovarian carcinoma. Targeting BCL6 could be a novel therapeutic choice for treating ovarian carcinoma patients.

Diffuse large B cell lymphoma (DLBCL) is a complex disease comprising diverse subtypes and genetic profiles. Possibly because of the prevalence of genetic alterations activating canonical NF-kappaB activity, a role for oncogenic lesions that activate the alternative NF-kappaB pathway in DLBCL has remained elusive. Here, we show that deletion/mutation of TRAF3, a negative regulator of the alternative NF-kappaB pathway, occurs in approximately 15% of DLBCLs and that it often coexists with BCL6 translocation, which prevents terminal B cell differentiation. Accordingly, in a mouse model constitutive activation of the alternative NF-kappaB pathway cooperates with BCL6 deregulation in DLBCL development. This work demonstrates a key oncogenic role for the alternative NF-kappaB pathway in DLBCL development.

The LAZ3/BCL6 gene was identified by its disruption in 3q27 translocations associated with diffuse large cell lymphomas. It is predicted to be a transcription factor as it contains six Kruppel-like Zinc finger motifs and a N-terminal BTB/POZ domain, a protein/protein interaction interface that is widely conserved in Metazoans. Using two antisera raised against non overlapping regions of the predicted ORF, we demonstrate that the LAZ3/BCL6 protein appears as a close ca. 79 kDa doublet in B lymphoid cell lines with either a rearranged or a non rearranged LAZ3/BCL6 locus. By immunofluorescence experiments on transiently transfected COS-1 or NIH3T3 cells, we show that the LAZ3/BCL6 protein displays a punctuated nuclear localisation. This appears to rely on LAZ3/BCL6 proper folding and/or activities as it is impaired in a hormone reversible-fashion through fusion of LAZ3/BCL6 to the ligand-binding domain of the oestrogen receptor. Moreover, deletion of its BTB/POZ domain leads to the disappearance of the nuclear dots although the protein remains nuclear. In addition, by using the yeast two-hybrid system, we show that the LAZ3/BCL6 BTB/POZ domain homomerises in vivo. Thus, the LAZ3/BCL6 BTB/POZ domain has the capability to self-interact and target the protein to discrete nuclear substructures.

In up to 45% of reported cases of the non-Hodgkin s lymphoma, diffuse large cell lymphoma, there are translocations of the BCL-6 gene, which are presumed to deregulate its expression. The BCL-6 protein, which is unmutated in these lymphomas, contains six Kruppel-like zinc fingers at its carboxy terminus and a 121 amino acid domain at its amino terminus, termed the POZ domain, which bears homology with amino terminal domains in a subset zinc finger transcription factors. In this study, we tested whether BCL-6 regulates transcription and if the POZ domain has a role in this function. The BCL-6 POZ domain, when fused to the GAL4 DNA binding domain, strongly repressed transcriptional activation initiated from several different promoters including the SV40 enhancer/promoter. Repression was also observed when the fusion protein was bound at a distance of 200 bp 5 of the promoter. When the GAL4/BCL6 POZ domain fusion protein was expressed in yeast, it was able to homodimerize in the nucleus. Nevertheless, in contrast with mammalian cells, the fusion protein did not repress transcription. To test the ability of the full length BC1-6 protein to repress transcription when bound to DNA through its zinc finger DNA binding domain, high affinity BCL-6 binding sites were selected from a pool of random oligonucleotides. Full length BCL-6 was able to strongly repress transcription when bound to its cognate site cloned upstream of the thymidine kinase promoter. This repression was mediated, in large measure, by the POZ domain, although a variant of BCL-6 lacking the POZ domain was able to repress transcription modestly. The ability of BCL-6 to function as a transcriptional repressor may contribute to its ability to transform B lymphocytes in diffuse large cell lymphoma.

Rearrangements and mutations of the LAZ3/BCL6 gene are the most frequent events associated with diffuse large-cell lymphoma, a particular class of non-Hodgkin s lymphomas. This gene encodes a putative regulatory protein with six COOH-terminal Kruppel-like zinc fingers and a NH2-terminal hydrophobic region, the so-called BTB/POZ domain, which mediates homo- as well as heterotypic interactions in other related proteins. Recently, a consensus binding sequence has been defined using the isolated LAZ3/BCL6 zinc finger region produced in bacteria. To understand the normal and oncogenic functions of LAZ3/BCL6, we examined its properties as a transcription factor. We thus demonstrated that its full-length product binds to the same consensus sequence, although the BTB/POZ domain decreases this activity, at least in vitro. In transient transfection experiments, the LAZ3/BCL6 protein exerts a repressive effect, both as a wild-type protein on its own target sequence and as a GAL-4 fusion protein. Furthermore, our results indicate that the BTB/POZ domain plays a prominent role in the mediation of this activity. Indeed, on the LAZ3/BCL6 cognate sequence, deletion of the BTB/POZ domain diminishes the repressive function. Conversely, as a GAL-4 chimera, the isolated LAZ3/BCL6 BTB/POZ domain appears nearly as efficient as the entire protein at inducing transcriptional repression. Taken together, these findings demonstrate that the LAZ3/BCL6 is a sequence-specific transcriptional repressor and point to a novel function for the BTB/POZ region, at least in LAZ3/BCL6, as an autonomous transcriptional inhibitory domain.

Structural alterations of the promoter region of the BCL-6 proto-oncogene represent the most frequent genetic alteration associated with non-Hodgkin lymphoma, a malignancy often deriving from germinal-centre B cells. The BCL-6 gene encodes a zinc-finger transcriptional repressor normally expressed in both B cells and CD4+ T cells within germinal centres, but its precise function is unknown. We show that mice deficient in BCL-6 displayed normal B-cell, T-cell and lymphoid-organ development but have a selective defect in T-cell-dependent antibody responses. This defect included a complete lack of affinity maturation and was due to the inability of follicular B cells to proliferate and form germinal centres. In addition, BCL-6-deficient mice developed an inflammatory response in multiple organs characterized by infiltrations of eosinophils and IgE-bearing B lymphocytes typical of a Th2-mediated hyperimmune response. Thus, BCL-6 functions as a transcriptional switch that controls germinal centre formation and may also modulate specific T-cell-mediated responses. Altered expression of BCL-6 in lymphoma represents a deregulation of the pathway normally leading to B cell proliferation and germinal centre formation.

The LAZ3/BCL6 (lymphoma-associated zinc finger 3/B cell lymphomas 6) gene frequently is altered in non-Hodgkin lymphomas. It encodes a sequence-specific DNA binding transcriptional repressor that contains a conserved N-terminal domain, termed BTB/POZ (bric-a-brac tramtrack broad complex/pox viruses and zinc fingers). Using a yeast two-hybrid screen, we show here that the LAZ3/BCL6 BTB/POZ domain interacts with the SMRT (silencing mediator of retinoid and thyroid receptor) protein. SMRT originally was identified as a corepressor of unliganded retinoic acid and thyroid receptors and forms a repressive complex with a mammalian homolog of the yeast transcriptional repressor SIN3 and the HDAC-1 histone deacetylase. Protein binding assays demonstrate that the LAZ3/BCL6 BTB/POZ domain directly interacts with SMRT in vitro. Furthermore, DNA-bound LAZ3/BCL6 recruits SMRT in vivo, and both overexpressed proteins completely colocalize in nuclear dots. Finally, overexpression of SMRT enhances the LAZ3/BCL6-mediated repression. These results define SMRT as a corepressor of LAZ3/BCL6 and suggest that LAZ3/BCL6 and nuclear hormone receptors repress transcription through shared mechanisms involving SMRT recruitment and histone deacetylation.

Recent works demonstrated that some transcriptional repressors recruit histone deacetylases (HDACs) either through direct interaction, or as a member of a multisubunit repressing complex containing other components referred to as corepressors. For instance, the bHLH-Zip transcriptional repressors MAD/MXI recruit HDACs together with the mSIN3 corepressors, whereas unliganded nuclear receptors contact another corepressor, SMRT (or its relative N-CoR), which, in turn, associates with both mSIN3 and HDACs to form the repressor complex. Recently, we reported that SMRT also directly associates with LAZ3(BCL-6), a POZ/Zn finger transcriptional repressor involvedin the pathogenesis of non-Hodgkin lymphomas. However, whether LAZ3 recruits the HDACs-containing repression complex is currently unknown. We report here that LAZ3 associates with corepressor mSIN3A both in vivo and in vitro , and found that a central region, which harbours autonomous repression activity, is mainly responsible for this interaction. Conversely, the N-terminal half of mSIN3A is both necessary and sufficient to bind LAZ3. Moreover, we show that LAZ3 also interacts with an HDAC (HDAC-1) through its POZ domain in vitro while the immunoprecipitation of LAZ3 results in the coretention of an endogenous HDAC activity in vivo . Finally, inhibitors of HDACs significantly reduce the LAZ3-mediated repression. Taken together, we conclude that LAZ3 recruits a repressing complex containing SMRT, mSIN3A and a HDAC, and that its full repressing potential on transcription requires HDACs activity. Our results identify HDACs as molecular targets of LAZ3 oncogene and further strengthen the connection between aberrant chromatin acetylation and human cancers.

The structural alterations of the LAZ3 (BCL6) gene are one of the most frequent events found in non-Hodgkin lymphoma. LAZ3 encodes a transcriptional repressor with a POZ/zinc finger structure similar to several Drosophila development regulators and to the human promyelocytic leukemia-associated PLZF gene. Consistent with the origin of LAZ3-associated malignancies, LAZ3 is expressed in mature B-cells and required for germinal center formation. However, its ubiquitous expression, with predominant levels in skeletal muscle, suggests that it may act outside the lymphoid system. To study how LAZ3 could be involved in skeletal muscle differentiation, we examined its expression in the C2 muscle cells. We report here that LAZ3 is upregulated at both mRNA and protein levels during the differentiation of proliferating C2 myoblasts into post-mitotic myotubes. This rise in LAZ3 expression is both precocious and sustained, and is not reversed when myotubes are re-exposed to mitogen-rich medium, suggesting that irreversible evens occurring upon myogenic terminal differentiation contribute to lock LAZ3 upregulation. In addition, using two different models, we found that a "simple" growth-arrest upon serum starvation is not sufficient to induce LAZ3 upregulation which rather appears as a feature of myogenic commitment and/or differentiation. Finally, BrdU incorporation assays in C2 cells entering the differentiation pathway indicate that "high" LAZ3 expression strongly correlates with their exit from the cell cycle. Taken as a whole, these findings suggest that LAZ3 could play a role in muscle differentiation. Together with some results reported in other cell types, we propose that LAZ3 may contribute to events common to various differentiation processes, possibly the induction and stabilization of the withdrawal from the cell cycle.

BTB/POZ-domain C2H2 zinc(Zn)-finger proteins are encoded by a subfamily of genes related to the Drosophila gap gene kruppel. To date, two such proteins, PLZF and LAZ-3/BCL-6, have been implicated in oncogenesis. We have now identified a new member of this gene subfamily which encodes a 62 kDa Zn-finger protein, termed LRF, with a BTB/POZ domain highly similar to that of PLZF. Both human and mouse LRF genes, which localized to syntenic chromosomal regions (19p13.3 and 10B5.3, respectively), were widely expressed in adult tissues and cell lines. At approximately 9.5-10.0 days of embryonic development, the mouse LRF gene was expressed in the limb buds, pharyngeal arches, tail bud, placenta and neural tube. The LRF protein associated in vivo with LAZ-3/BCL-6, but not with PLZF to which it was more related. Although the LRF, or LAZ-3/BCL-6, BTB/POZ domain could readily homodimerize, no heterodimerization was detected in vivo between the LRF and LAZ-3/BCL-6 BTB/POZ domains and interaction between full length LRF and LAZ-3/BCL-6 required the presence of both the BTB/POZ domain and Zn-fingers in each partner protein. As expected from the above results, LRF and LAZ-3/BCL-6 also colocalized with each other in the nucleus. Taken together, our findings suggest that BTB/ POZ-domain Zn-finger proteins may function as homo and heterodimeric complexes whose formation, and hence the resultant effect on transcription of their downstream target genes, is determined by the levels and expression domains of a given partner protein.

The Bc16 gene is located at chromosomal band 3q27, a breakpoint for translocation that frequently occurs in B cell lymphomas. Bc16 has been found to be preferentially expressed in germinal center B cells, and expression of this gene has been shown to be essential for germinal center formation in vivo. The physiological function of Bc16 and its role in lymphomagenesis, however, are not yet known. Since significant expression of Bc16 has been demonstrated in skeletal muscle, we have utilized a differentiation-inducible mouse myogenic cell line, C2C12, to elucidate the function of the Bc16 gene product. expression of Bc16 mRNA was very low in growing myocytes, but was increased in differentiating myocytes cultured in serum-starved medium. Incubation of these cells with cytokines or chemicals that are known to block differentiation suppressed this increased Bc16 message abundance, indicating that Bc16 induction is related to the process of terminal differentiation in muscle cells. While a fraction of myocytes is known to undergo apoptosis after serum-starvation to induce differentiation, adenovirus-mediated overexpression of Bc16 enhanced the viability of the differentiating cells by preventing the apoptosis. High levels of Bc16 antisense mRNA expression induced substantial apoptosis during the differentiation of C2C12 cells, but this was effectively prevented by infection with adenovirus that expressed Bc16 sense mRNA. These results indicate that Bc16 acts to prevent apoptotic cell death in differentiating myocytes. The deregulation of expression of this antiapoptotic gene may also contribute to the development of B cell lymphomas.