ABSTRACT
The BAZF (BCL-6b) protein is highly similar to the BCL-6 transcriptional repressor. While BCL-6 has been characterized extensively, relatively little is known about the normal function of BAZF. In order to understand the physiological role of BAZF, we created BAZF-deficient mice. Unlike BCL-6-deficient mice, BAZF-deficient mice are healthy and normal in size. However, BAZF-deficient mice have a hematopoietic progenitor phenotype that is almost identical to that of BCL-6-deficient mice. Compared to wild-type mice, both BAZF-deficient and BCL-6-deficient mice have greatly reduced numbers of cycling hematopoietic progenitor cells (HPC) in the BM and greatly increased numbers of cycling HPC in the spleen. In contrast to HPC from wild-type mice, HPC from BAZF-deficient and BCL-6-deficient mice are resistant to chemokine-induced myelosuppression and do not show a synergistic growth response to granulocyte-macrophage colony-stimulating factor plus stem cell factor. Depletion of CD8 T cells in BAZF-deficient mice reverses several of the hematopoietic defects in these mice. Since both BAZF- and BCL-6-deficient mice have defects in CD8 T-cell differentiation, we hypothesize that both BCL-6 and BAZF regulate HPC homeostasis by an indirect pathway involving CD8 T cells.
Subject(s)
CD8-Positive T-Lymphocytes/physiology , Hematopoiesis , Repressor Proteins/metabolism , Animals , Blood Cell Count , CD8-Positive T-Lymphocytes/drug effects , Chemokines/pharmacology , DNA-Binding Proteins/deficiency , Hematopoiesis/drug effects , Hematopoiesis, Extramedullary/drug effects , Hematopoietic Stem Cells/drug effects , Heterozygote , Mice , Mice, Inbred C57BL , Mutation/genetics , Myeloid Cells/drug effects , Phenotype , Proto-Oncogene Proteins c-bcl-6 , Stem Cell Factor/metabolismABSTRACT
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-gamma 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.
Subject(s)
Gene Expression Regulation/immunology , Interleukin-10/biosynthesis , Interleukin-10/genetics , Proto-Oncogene Proteins c-jun/physiology , Th2 Cells/immunology , Th2 Cells/metabolism , Animals , Base Sequence , Cell Line , Cell Line, Tumor , Cells, Cultured , Chromatin/genetics , Chromatin/metabolism , Clone Cells , Conserved Sequence , Deoxyribonuclease I/genetics , Humans , Interleukin-10/metabolism , Mice , Molecular Sequence Data , NIH 3T3 Cells , Protein Binding , Proto-Oncogene Proteins c-jun/metabolism , Regulatory Sequences, Nucleic Acid , Transcription Factor AP-1/metabolism , Transcription Factor AP-1/physiologyABSTRACT
The B cell lymphoma (BCL)-6 transcriptional repressor protein is an important regulator of Th2 responses. Mice deficient in BCL-6 develop severe Th2-type inflammation that can develop even in the absence of IL-4 signaling. We have investigated the mechanism for how BCL-6 regulates Th2 cell differentiation and have found that IL-6 signaling can promote dramatically increased levels of Th2 differentiation in BCL-6(-/-) CD4 T cells compared with wild-type CD4 T cells. IL-6 can induce a low level of Th2 cytokine expression in BCL-6(-/-)STAT6(-/-) cells but not in STAT6(-/-) cells. Since the promoters for Th2 cytokines such as IL-4, IL-5, IL-10, and IL-13 do not contain consensus BCL-6 DNA binding sites, we investigated whether BCL-6 might regulate the GATA-3 transcription factor that activates the expression of multiple Th2 cytokines. Consistent with the idea that BCL-6 represses GATA-3 expression, we found that GATA-3 levels are up-regulated in BCL-6(-/-)STAT6(-/-) CD4 T cells compared with STAT6(-/-) CD4 T cells. Retrovirus-mediated expression of BCL-6 in BCL-6(-/-)STAT6(-/-) T cells as well as developing wild-type Th2 cells leads to a potent repression of IL-4 and IL-10 secretion. Retrovirus-mediated expression of BCL-6 in both BCL-6(-/-)STAT6(-/-) and wild-type T cells also leads to a significant decrease in GATA-3 protein levels. Surprisingly, BCL-6 does not appear to regulate GATA-3 mRNA levels and thus BCL-6 appears to regulate GATA-3 expression at a posttranscriptional level. Regulation of GATA-3 protein levels is likely a key mechanism for how BCL-6 regulates Th2 cytokine expression and Th2 differentiation independently of STAT6. These data also point to a novel regulatory mechanism for BCL-6 separate from transcriptional repression.
Subject(s)
DNA-Binding Proteins/antagonists & inhibitors , DNA-Binding Proteins/biosynthesis , DNA-Binding Proteins/physiology , Growth Inhibitors/physiology , Proto-Oncogene Proteins/physiology , Th2 Cells/cytology , Th2 Cells/metabolism , Trans-Activators/antagonists & inhibitors , Trans-Activators/biosynthesis , Transcription Factors/physiology , Animals , CD4-Positive T-Lymphocytes/metabolism , CD4-Positive T-Lymphocytes/pathology , Cell Differentiation/genetics , Cell Differentiation/immunology , Cells, Cultured , Cytokines/antagonists & inhibitors , Cytokines/biosynthesis , Cytokines/genetics , DNA-Binding Proteins/deficiency , DNA-Binding Proteins/genetics , GATA3 Transcription Factor , Growth Inhibitors/deficiency , Growth Inhibitors/genetics , Interleukin-6/pharmacology , Mice , Mice, Inbred C57BL , Mice, Knockout , Proto-Oncogene Proteins/deficiency , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins c-bcl-6 , RNA Processing, Post-Transcriptional/immunology , STAT6 Transcription Factor , Signal Transduction/genetics , Signal Transduction/immunology , T-Lymphocyte Subsets/immunology , T-Lymphocyte Subsets/metabolism , Th2 Cells/immunology , Trans-Activators/deficiency , Trans-Activators/genetics , Transcription Factors/deficiency , Transcription Factors/genetics , Up-Regulation/genetics , Up-Regulation/immunologyABSTRACT
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.
Subject(s)
B-Lymphocytes/cytology , DNA-Binding Proteins/genetics , Proto-Oncogene Proteins/genetics , Proto-Oncogenes , Repressor Proteins/genetics , Transcription Factor AP-1/metabolism , Transcription Factors/genetics , B-Lymphocytes/immunology , B-Lymphocytes/metabolism , Base Sequence , Cell Differentiation , Cell Line , Cell Transformation, Neoplastic , DNA/genetics , Humans , Positive Regulatory Domain I-Binding Factor 1 , Proto-Oncogene Proteins c-bcl-6 , Proto-Oncogene Proteins c-jun/genetics , Proto-Oncogene Proteins c-jun/metabolism , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Repressor Proteins/metabolism , Transcription Factors/metabolism , Transcriptional Activation , Two-Hybrid System TechniquesABSTRACT
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.
