The B cell lymphoma-6 (BCL-6) transcriptional repressor protein is an important regulator of B cell differentiation and is strongly implicated in the development of B cell lymphoma. Expression of the Blimp-1 transcription factor, which is critical for promoting B cell differentiation into plasma cells, is repressed by BCL-6. We have investigated the mechanism for how BCL-6 represses Blimp-1 transcription, and have found that BCL-6 regulates the Blimp-1 promoter through a novel mechanism involving AP-1 elements. Specifically, BCL-6 is a potent repressor of transcriptional activity mediated by AP-1 factors. We found that the zinc-finger region of BCL-6 interacts with c-Jun, JunB, and JunD proteins but does not bind c-Fos or Fra-2 proteins. An estrogen receptor ligand binding domain fusion with the BCL-6 zinc finger domain can act as a estrogen-inducible dominant negative protein and increase AP-1 activity in BCL-6+ cells but not in BCL-6− cells, indicating that endogenous BCL-6 represses AP-1 activity. Additionally, we have confirmed a specific interaction between c-Jun and the zinc finger domain of BCL-6 in vivo using a mammalian two-hybrid assay. Repression of AP-1 function by BCL-6 may be a key mechanism for how BCL-6 regulates gene expression to control inflammation, lymphocyte differentiation, and lymphomagenesis.

The activation of the AKT/protein kinase B kinases by mutation of the PTEN lipid phosphatase results in enhanced survival of a diversity of tumors. This resistance to apoptosis is partly accomplished by the inhibition of genetic programs induced by a subfamily of forkhead transcription factors including AFX. Here we describe an AFX-regulated pathway that appears to account for at least part of this apoptotic regulatory system. Cells induced to synthesize an active form of AFX die by activating the apoptotic death pathway. An analysis of genes regulated by AFX demonstrated that BCL-6, a transcriptional repressor, is up-regulated ϳ4 -7-fold. An examination of the BCL-6 promoter demonstrated that AFX bound to specific target sites that could activate transcription. BCL-X L , an anti-apoptotic protein, contains potential BCL-6 target sites in its promoter. An analysis of endogenous BCL-X L levels in AFX-expressing cells revealed enhanced down-regulation of the transcript (ϳ1.3-1.7-fold) and protein, and BCL-6 directly binds to and suppresses the BCL-X L promoter. Finally, macrophages isolated from BCL-6؊/؊ mice show enhanced survival in vitro. These results suggest that AFX regulates apoptosis in part by suppressing the levels of anti-apoptotic BCL-XL through the transcriptional repressor BCL-6.The resistance of tumors to diverse apoptotic stimuli is of major clinical importance. A large number of recent studies has highlighted the significance of hyperactivation of the PI3K 1 pathway to the insensitivity of late stage tumors to chemotherapy and radiation treatments (1-6). The induction of this pathway by growth factor or oncogenic stimuli results in the production of three phosphorylated phosphatidylinositol lipids, which act as binding sites for the pleckstrin homology domains of the downstream kinases, PDK-1 and AKT/PKB (7-13). The membrane colocalization of these kinases results in the phosphorylation of a specific activation site in AKT/PKB by PDK-1 (7,8,(13)(14)(15). Importantly, this pathway is reversed by the lipid phosphatase activity of PTEN, a tumor suppressor that is homozygously deleted in a variety of late stage tumors (2-6). Whereas a number of phosphorylation targets has been described for the AKT/PKB kinases, both biochemical and genetic evidence support a subset of forkhead transcription factors including FKHR, FKHR-L1, and AFX as being important substrates for phosphorylation mediated by these kinases (16 -20). Phosphorylation of these forkhead transcription factors by AKT/PKB kinases results in their sequestration to the cytoplasm in which they are unable to activate transcription of their nuclear targets (17)(18)(19)(20). This outcome is an important component of cell survival mediated by the PI3K pathway, because nuclear localization of these forkhead family members results in the induction of transcriptional programs that lead to rapid cell death by apoptosis (17)(18)(19)(20).Whereas it is clear that the inhibition of the apoptotic programs activated by these forkhead transcription factors is ...

The transcriptional repressor protein BCL-6, implicated in the pathogenesis of B cell lymphoma, regulates lymphocyte differentiation and inflammation. We investigated the mechanism for the T helper cell subset 2 (TH2)-type inflammation that occurs in BCL-6-/- mice. Using chimeric mice we found that the TH2-type inflammation is dependent upon nonlymphoid cells. We identified three chemokines, MCP-1, MCP-3 and MRP-1, which are negatively regulated by BCL-6 in macrophages. Promoter analysis revealed that BCL-6 is a potent repressor of MCP-1 transcription. Our results provide a mechanism for the regulation of TH2-type inflammation by BCL-6 and link TH2 differentiation to innate immunity.

IL-10 is a key regulatory cytokine produced by T lymphocytes. Although Th2 cells are a major source of IL-10, little is known about IL-10 gene regulation in Th2 cells. High levels of IL-10 mRNA transcription are induced in the Th2 clone D10 after PMA plus ionomycin (P/I) stimulation; however we found that the IL-10 promoter was not inducible by P/I in D10 cells. We therefore sought regulatory regions in the IL-10 gene that could promote P/I-activated transcription in Th2 cells. Two strong DNase I-hypersensitive sites (DHSSs) were identified in the IL-10 gene in mouse T cells, and conserved noncoding sequences (CNSs) between the mouse and human IL-10 genes were also identified. One IL-10 DHSS maps within or next to a highly conserved CNS region, CNS-3. The CNS-3 region contains an AP-1 site that binds JunB and c-Jun proteins specifically in Th2 cells and not in Th1 cells. The CNS-3 element activates transcription from the IL-10 promoter after P/I stimulation and is responsive to c-Jun and JunB. Retroviral mediated-expression of either c-Jun or JunB in primary T cells led to a large increase in IL-10 expression, and inhibition of AP-1 activity by a dominant negative form of c-Jun in primary T cells strongly repressed IL-10 expression. IFN-γ was relatively unaffected by modulations in AP-1 activity. These data indicate that we have identified a novel regulatory element that can specifically activate transcription of the IL-10 gene in Th2 cells via the AP-1/Jun pathway.