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International Immunology Advance Access originally published online on May 15, 2006
International Immunology 2006 18(7):1079-1089; doi:10.1093/intimm/dxl041
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JunD/AP-1 and STAT3 are the major enhancer molecules for high Bcl6 expression in germinal center B cells

Eggi Arguni1, Masafumi Arima1, Nobuhide Tsuruoka1, Akemi Sakamoto1, Masahiko Hatano1,2 and Takeshi Tokuhisa1

1 Department of Developmental Genetics (H2), Graduate School of Medicine, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba 260-8670, Japan
2 Biomedical Research Center, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba 260-8670, Japan

Correspondence to: T. Tokuhisa; E-mail: tokuhisa{at}faculty.chiba-u.jp


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
The Bcl6 proto-oncogene, which encodes a transcriptional repressor, is ubiquitously expressed and predominantly in germinal center (GC) B cells. Although the promoter region of the human Bcl6 gene has been reported, enhancer molecules for its high expression in GC B cells were largely unknown. Here we show that transcriptional start sites of the murine Bcl6 gene were different from the reported human one. DNA sequence around the new promoter region is highly conserved between mice and humans and has no canonical TATA or CCAAT box. Two AP-1-binding elements in the promoter region were the major enhancer elements in GC-derived B lymphoma cells, and JunD/AP-1 was detected in GC B cells. In addition, we identified the silencer region with three Bcl6-binding elements around the start site. Bcl6 bound to the silencer elements and its over-expression repressed the promoter activity through the elements. Activated STAT factors (STATs), especially activated STAT3, also bound to the silencer elements in GC B cells and competed with Bcl6 for the binding, suggesting that JunD/AP-1 and activated STATs drive high Bcl6 expression in GC B cells. Since stimulation of splenic B cells with IL-4 or IL-21 induced high Bcl6 expression with induction of junD and activation of STATs, these cytokines may be inducers for its high expression in GC B cells. However, IL-21 but not IL-4 stimulation activated STAT3 in splenic B cells. Thus, IL-21 may be a major inducer for high Bcl6 expression in GC B cells.

Keywords: cytokines, gene regulation, memory, transcription factors


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 References
 
Chromosomal translocations involving a band 3q27 are often present in diffuse large cell lymphomas (1). The Bcl6 gene has been identified from these translocation break points. The Bcl6 gene encodes a 92- to 98-kDa nuclear phosphoprotein that contains the BTB/POZ domain in the NH2-terminal region and Krüppel-type zinc finger motifs in the COOH-terminal region (2–5). Since the NH2-terminal half of the protein contains repressor domains in vitro (6), Bcl6 can function as a sequence-specific transcriptional repressor. Indeed, the BTB/POZ domain of Bcl6 can bind to silencing mediator of retinoid and thyroid receptors and recruit the histone deacetylase complex to silencer regions of target genes to repress expression of these genes (7). The Bcl6 gene is ubiquitously expressed in human and mouse tissues, and is predominant in germinal center (GC) B cells of human tonsils (8, 9) and mouse lymphoid organs (5). The expression in mouse tissues has been extensively studied. Bcl6 expression is continuously up-regulated in keratinocytes at their terminal stages (10), and was transiently up-regulated in thymocytes and spleen cells as an immediate early gene when these cells were stimulated with phorbol myristate acetate plus ionomycin (5).

To observe the physiological functions of Bcl6, its gene was disrupted in the mouse germ line (11–13). Bcl6-deficient mice displayed Th2-type inflammatory responses in multiple organs, especially in heart and lungs, characterized by infiltration of eosinophils (14). We recently reported that the IL-5 gene (15) and the IL-18 gene (16) are molecular targets of Bcl6. Although all hematopoietic lineages, including mature lymphocytes, can develop in Bcl6-deficient mice, GC formation is impaired after immunization (13). GC is a complex cellular microenvironment that directs generation of high-affinity memory B cells (17). Thus, Bcl6 is an important molecule for maintaining physiological immune responses. Furthermore, the chromosomal translocations in diffuse large cell lymphomas cause ectopic expression of the Bcl6 gene as a result of the juxtaposition of the Bcl6-coding region to heterologous promoters, and its de-regulation may be responsible for lymphomagenesis (18). Its over-expression, however, induced apoptosis not only in B lineage cells but also in CV-1, HeLa, mouse L and NIH3T3 cells (19, 20). Therefore, Bcl6 expression has to be strictly regulated especially in B lymphocytes.

The promoter region of the human Bcl6 (hBcl6) gene has been reported (21). Transcriptional start sites of the hBcl6 gene were identified using mRNA from a human Burkitt's lymphoma cell line, Raji. Nucleotide sequence of the 1.5-kb promoter region contains a TATA box but no CCAAT box and a number of potential regulatory elements including CACCC sequence, E-box, Bcl6-binding sequence and GATA-1-binding sequence. The first 657 nucleotides of the promoter region significantly drove transcription of the reporter gene in Raji cells, and the 657-bp region contains a CACCC sequence, two E-boxes. Furthermore, consensus Bcl6-binding sequences were found in the first exon of hBcl6 gene (22–24), suggesting that Bcl6 expression is negatively regulated by Bcl6 itself. Therefore, there is likely to be mechanisms which inhibit the auto-negative regulation by Bcl6 for its high expression in GC B cells. However, these mechanisms are largely unknown.

Expression profiles of this gene are well conserved between humans and mice (5, 8, 9). However, DNA sequence of the hBcl6 promoter region identified in Raji cells differs from that of homologous region of the murine Bcl6 (mBcl6) gene. Furthermore, the mBcl6 database (Celera, Gene ID: 12053) indicated that a putative transcriptional start site of the mBcl6 gene is further downstream of the reported human one, and genomic DNA sequence of the new murine promoter region is quite similar to that of the homologous region of the hBcl6 gene. Here we show that major transcriptional start sites of the Bcl6 gene in GC B cells and several cell lines including a GC-derived human B lymphoma line, Ramos, were the further downstream of the reported human one as the mBcl6 database pointed. The promoter analysis demonstrated that JunD/AP-1 is the major transcriptional enhancer for Bcl6 expression in human and mouse B lymphoma cell lines. Furthermore, three Bcl6-binding elements were identified in the new promoter region. The exogenous Bcl6 bound to the regions and repressed the promoter activity. Activated STAT factors (STATs) also bound to the silencer regions and competed with Bcl6 for binding to the regions in splenic B cells activated with IFN-{alpha} or LPS. Since STAT3 is the major activated STATs in GC B cells, JunD/AP-1 and STAT3 are important transcriptional factors for high Bcl6 expression in GC B cells. We discuss cytokine signals which induce JunD/AP-1 and activated STAT3 in GC B cells.


   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 References
 
Purification of splenic B and GC B cells
C57BL/6 mice (6–12 weeks), purchased from Japan SLC Co. Ltd (Hamamatsu, Japan), were immunized intra-peritoneally with 50 µg of alum-precipitated (4-hydroxy-3-nitrophenyl) acetyl-chicken {gamma} globulin (NP25-CG) (Biosearch Technologies, Novato, CA, USA). GC B cells were isolated from the spleen of these mice 14 days after immunization. Spleen cells were stained with allophycocyanin–anti-B220 mAb and FITC–peanut agglutinin (PNA, Vector, Burlingame, CA, USA) and GC (B220+PNA+) B cells were sorted by a FACSVantage (Becton Dickinson, San Jose, CA, USA). Purity of FACS-sorted GC B cells exceeded 98%.

Cell culture
Splenic B cells were cultured with LPS (3 µg ml–1; Sigma), IL-2 (50 U ml–1; PeproTech), IL-4 (1000 U ml–1) (25), IL-6 (1000 U ml–1; PeproTech), IL-7 (50 U ml–1; PeproTech) or IL-21 (30 ng ml–1; R&D Systems, Inc.) in RPMI1640 supplemented with 10% FCS, penicillin G (100 U ml–1) and streptomycin (100 µg ml–1) at 37°C in 5% CO2. Ramos cells were cultured with human IFN-{alpha} (1 ng ml–1; PeproTech) for 60 min at 37°C in 5% CO2.

Rapid amplification of 5' cDNA ends analysis
Total RNAs from splenic B and GC B cells were isolated to perform the rapid amplification of 5' cDNA ends (5' RACE) using the 5' RACE system for a RACE version 2.0 kit (Life Technologies). First-strand mBcl6 cDNA was synthesized with the primer m1 (5'-TATGCGAAAAGCTAGATCC-3'), and PCR was done using the primer m2 (5'-CCTACAGTGGGAGAGACGT-3') and the 5' RACE Abridged Anchor Primer followed by nested PCR using the primer m3 (5'-CGAAGATACACATAGGAAACTTGGAGC-3') and the Abridge Universal Amplification Primer. In the case of hBcl6, total RNAs from Ramos (human Burkitt's lymphoma cell line) and Raji cells (human Burkitt's lymphoma cell line ) were used as were the following primers: primer h1 (5'-CAAAACAACACAAGGGAGG-3'), primer h2 (5'-GCAAAAGCCTCCCCAAACC-3') and nested primer h3 (5'-CGACCTATGGTGGGAGAGACGT-3').

Reporter constructs for the mBcl6 promoter region
All genomic DNA fragments were inserted into cloning sites of a pGL3-Basic vector carrying the SV40 enhancer and the firefly Luciferase reporter gene (Promega). The –5200/+5 (BglII/BglII) fragment with an artificial BglII site at +5 was generated by PCR method, using BALB/c genomic DNA as a template. The DNA fragments with deletion at –4200 bp (KpnI), –1300 bp (NheI), –472 bp (XhoI), –367 bp (EcoRI), –227 bp (SmaI), –125 bp (BssHI) or –53 bp (SacI) were inserted into pGL3-Basic vectors. The 3' end of the –472-bp inserted vector (pBcl6 Luc-472) was extended to the position at +114 to generate pBcl6 Luc-472/+114. The mBcl6 expression vector (pcDNA3-Bcl6) was constructed, as described (19).

Mutant Luciferase constructs
Mutant Luciferase constructs were generated by PCR using oligonucleotide primers carrying point mutations (QuikChangeXL site-directed mutagenesis kit, Stratagene). The primers used were mutAP-1(1) (5'-CACGGAGCCCA CGAAAAGGCGGCGGAG-3'), mutAP-1(2) (5'-CGAAGACGCCGACTTTTCGGAGCCC ACGTG-3'), mutNF-{kappa}B (5'-GGCGGCGGAGCTAATGGTTCCATCGGTGGC-3'), mutSp1(1) (5'-CCACGTGACGGCTATTGAGCGGGCGGTTCC-3'), mutSp1(2) (5'-CGCCGGAGCGTATTGTTCCATCGGTGGCCC-3'), mutSTAT(1) (5'-GAGCTCTGTTGATTCGGTTAACTGGGGTTC-3'), mutSTAT(2) (5'-GAACTGGGGTTCGGTTAAGTGGTGATGC-3') and mutBcl6 (5'-GCAAGAAGTTTCGGTTAAAGGCCGGACACC-3').

Transient transfection and Luciferase assay
Transfection of genes was conducted by electroporation using the Bio-Rad Gene PulserII (Bio-Rad, Hercules, CA, USA). Cells (1 x 107) were incubated with DNA (total 20 µg) for 10 min on ice and then electroporated at 960 microfarads with 220 mV for Ramos, Raji and K562 cells (human chronic myelogenous leukemia cell line) or with 260 mV for M12.4.1 (mouse B lymphoma cell line), Jurkat and NIH3T3 cells (Mouse fibroblast cell line). For experiments with trichostatin A (TSA; Biomol), 100 nM TSA was added in the culture immediately after electroporation.

EMSA
Nuclear extracts were prepared from either cell lines or splenic B cells as described (26). The probe was labeled with digoxigenin (Roche Molecular Biochemicals) using digoxigenin oligonucleotide 3'-end labeling kits (Roche Molecular Biochemicals). EMSA was performed as described (15). The following oligonucleotide sequences were used for DNA probes or competitor fragments: Wt AP-1 (5'-ACGCCGACGTCACGGAGCCCACGTGACGGCGGCGG-3'), mutAP-1 (5'-ACGCCGACTTTTCGGAGCCCACGAAAAGGCGGCGG-3'), Wt STAT (5'-TCTGTTGATTCTTAGAACTGGGGTTCTTAGAAGTG-3') and mutSTAT (5'-TCTGTTGATTCGGTTAACTGGGGTTCGGTTAGGTG-3'). For antibody supershift experiments, 1–2 µg of the indicated specific antibodies was added to the binding reaction mixture. Antibodies against c-Fos, FosB, c-Jun, JunB, JunD and IgG were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Measurement of Bcl6 mRNA in activated B cells by northern blot
Northern blot analysis was performed using standard methods with full-length Bcl6 (5), murine c-fos family genes (c-fos, c-jun, junB, junD, fosB) (27) or ß-actin cDNA as a probe.

Western blot analysis
STATs were detected in Ramos and activated B cells using western blot (10). Blots were incubated with rabbit antibodies to phosphorylated (p-)STAT3, p-STAT5, p-STAT6 (Cell Signaling), STAT3, STAT5, STAT6 or actin (Santa Cruz) followed by HRP-conjugated donkey anti-rabbit Igs (Amersham International).

Chromatin immunoprecipitation assay
Chromatin immunoprecipitation (ChIP) assays were performed using the ChIP assay kit (UpState Biotechnology) and were then conducted according to the manufacturer's recommendations. The following primers were used in the ChIP assays: 5'-GAACCACGATCCGCACTATA-3' and 5'-CACATAGGAAACTTGGAGCC-3' for the STAT/Bcl6-binding sites and 5'-AGGAAGAATTAGCCCCAGAC-3' and 5'-TATAGTGCGGATCGTGGTTC-3' for the AP-1-binding sites.

Immunohistochemistry
Tissues were fixed in 10% phosphate-buffered formalin and embedded in paraffin using standard immunohistochemical techniques. Briefly, tissue slides (5 µm thick) were incubated with the primary antibody overnight at 4°C. Anti-p-STAT3, anti-p-STAT5 and anti-p-STAT6 (Cell Signaling) were used at 1:100 dilutions. Antibody binding was detected using a peroxidase-conjugated anti-rabbit IgG and a DAB kit (Nichirei).


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 References
 
Identification of transcriptional start sites and promoter region of the mBcl6 gene
To determine the transcriptional start sites of the mBcl6 gene, we did the 5' RACE analysis with mRNA isolated from FACS-isolated GC B cells or LPS-activated B cells. Since a putative transcriptional start site (+1) in the mBcl6 database is ~500 bp downstream of that of the hBcl6 gene found in Raji cells (21), we planned to make PCR primers specific for the DNA sequences around +280 to +300 bp. We identified major transcriptional start sites of the mBcl6 gene in GC B cells and LPS-activated B cells at +95 and +64, respectively (Fig. 1 and data not shown). An obvious canonical TATA or CCAAT box was not found around these transcriptional start sites. In addition, DNA sequences around the start sites are conserved between mice and humans. We also examined transcriptional start sites of the hBcl6 gene in GC-derived B lymphoma (Ramos) cells, and the major start site was at +38 in the homologous region of the hBcl6 gene.


Figure 1
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  		Fig. 1 Identification of transcription initiation sites of the Bcl6 gene by the 5' RACE method. Nucleotide sequence of the 5'-flanking region of the mBcl6 gene was compared with that of the hBcl6 gene. The transcriptional start site of the mBcl6 gene from the database has been numbered as +1. Arrows indicate transcriptional start sites of mBcl6 mRNA in LPS-activated B cells and GC B cells and that of hBcl6 mRNA in Ramos cells determined by the 5' RACE. Consensus sequences for binding of various potential transcriptional factors are noted and underlined.

 
Although the major transcriptional start site of the mBcl6 gene in GC B cells was +95, there is no known enhancer element but a Bcl6-binding sequence (+6 to +16) between +1 and +95. The Bcl6-binding sequence is conserved in the hBcl6 gene and used as the silencer region in the first exon of hBcl6 gene (22). To identify the promoter region, we made reporter constructs containing DNA fragments between –4200 and +5 bp, in which the Bcl6-binding sequence was excluded. Transcriptional activity of reporter constructs was analyzed in M12.4.1, Ramos and Raji cells. As shown in Fig. 2(A), the deletion from –4200 (pBcl6 Luc-4200) to –472 bp (pBcl6 Luc-472) did not change the promoter activity in these cell lines. However, further truncation of the promoter region to –227 bp (pBcl6 Luc-227) reduced the activity to <10% of the full length. These data suggest that the region between –472 and –227 bp has major promoter activity in GC B cells.


Figure 2
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  		Fig. 2 Identification of the minimal promoter region of the mBcl6 gene in B lineage cells. (A) Serial deletion fragments of pBcl6 Luc-4200 were generated as described in the Methods. Transcriptional activity of these fragments was examined in three B cell lines noted. Data are presented as the mean ± SD from three independent experiments. (B) Basal transcriptional activity of serial deletion fragments of pBcl6 Luc-472 was examined in Ramos cells. Data are presented as the mean ± SD from three independent experiments. (C) Basal transcriptional activity of pBcl6 Luc-472 with mutations was examined in Ramos cells. Activities of those cells transfected with pBcl6 Luc-472 were 100%. Data are presented as the mean ± SD from three independent experiments.

 
Involvement of JunD/AP-1 in promoter activity of the mBcl6 gene in GC B cells
The promoter region between –472 and –227 bp contains a number of consensus DNA-binding sequences for several transcriptional factors detected by a MatInspector TRANSFACTM (GBF-AGBIN, Braunschweig, Germany) database (28). The promoter activity of pBcl6 Luc-472 with deletion or mutations was examined in Ramos cells. The deletion between –367 and –227 bp reduced the activity to ~10% of the pBcl6 Luc-472 (Fig. 2B). The region between –367 and –227 bp contains two AP-1 sites, an NF-{kappa}B site and two Sp1 sites, and we introduced the point mutations into these binding sequences of pBcl6 Luc-472. While a point mutation in the Sp1 sites or the NF-{kappa}B site did not modify the activity, a point mutation in either of the AP-1 sites resulted in about a 50% reduction in the promoter activity (Fig. 2C). Mutations of both AP-1 sites reduced the promoter activity to 25% of the original, suggesting that these AP-1 sites are important for the full activation of the mBcl6 promoter in GC B cells.

We examined the members of the AP-1 family bound to the AP-1-binding sites in Ramos cells and GC B cells using ChIP assays. As shown in Fig. 3(A), the DNA region containing the AP-1 sites was detected in the immune complexes precipitated with anti-c-Fos, anti-c-Jun or anti-JunD, but not in those with anti-JunB or anti-FosB. Since the amount of DNA in the complex precipitated with anti-JunD antibody was the largest among these complexes, the binding of JunD/AP-1 to the DNA region in FACS-isolated GC B cells was examined using ChIP assays. The DNA region was clearly detected in the complexes precipitated with anti-JunD antibody.


Figure 3
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  		Fig. 3 AP-1 as a major transcriptional enhancer factor for Bcl6 expression in Ramos cells. (A) Binding of AP-1 proteins to the AP-1-binding sequences in Ramos cells and FACS-isolated GC B cells was analyzed by ChIP assays. (B) Binding of Ramos nuclear extracts to AP-1-binding sequences of the mBcl6 gene was analyzed by EMSA. Antibody supershift experiments were performed with various anti-AP-1 antibodies. Supershifted AP-1 complexes are indicated by an arrow. Data are presented as a representative of three independent experiments.

 
Next, we confirmed that JunD/AP-1 was the major AP-1 bound to the AP-1 sites in Ramos cells by EMSA with anti-JunD antibody. We detected a retarded band composed of the probe and protein (Fig. 3B). The probe and protein complexes were not detected by the addition of the wild-type cold probe, but were still detected in the presence of the cold probe with the mutated AP-1 sites. In order to confirm the members of AP-1 family in the complexes, we did antibody supershift assays. Although the supershifted band was not detected by the addition of anti-c-Jun, anti-c-Fos, anti-JunB or anti-FosB to nuclear extracts from Ramos cells, the addition of anti-JunD antibody clearly demonstrated the supershifted band (lane 8). These results suggest that JunD/AP-1 is a major enhancer molecule to drive high Bcl6 expression in GC B cells.

A negative feedback regulation of Bcl6 expression
Promoter activity of pBcl6 Luc-4200 was further analyzed in non-B lymphoid cell lines (NIH3T3, Jurkat and K562 cells). Deletion of the DNA fragment between –472 and –227 bp resulted in almost 20% of the full-length promoter activity in NIH3T3 and Jurkat cells (Fig. 4A). Surprisingly, the promoter activity in K562 cells was significantly stronger than that in NIH3T3 and Jurkat cells, and the promoter activity is located in the region between –125 and –53 bp, which contains an NF-{kappa}B site (Fig. 4B). Since K562 cells do not express the endogenous hBcl6 gene (8), the strong promoter activity in K562 cells may be due to the lack of repression by Bcl6 protein which may operate in Ramos cells. The consensus Bcl6-binding sequence contains the STAT-binding sequence (11), and we found two STAT-binding sequences between –39 and –22 bp in the promoter region. We introduced mutations in these two STAT-binding sequences of pBcl6 Luc-472 (mutSTAT), and transiently transfected them into Ramos or K562 cells. The promoter activity of pBcl6 Luc-472 (mutSTAT) increased 3-fold of the original one in Ramos cells but was slightly lower in K562 cells (Fig. 4C). These results suggest that the STAT-binding sequences in the promoter region play a role as silencer elements for Bcl6 expression in GC B cells. Furthermore, mutations in both AP-1 sites in pBcl6 Luc-472 (mutSTAT) reduced the promoter activity to 20% of the original in Ramos cells, suggesting again that AP-1 is a critical factor for Bcl6 expression in GC B cells.


Figure 4
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  		Fig. 4 Negative regulatory elements in the mBcl6 promoter region. (A) Non-B cell lines were transfected with series of deletion fragments of pBcl6 Luc-4200. Data are presented as the mean ± SD from three independent experiments. (B) Basal transcriptional activity of serial deletion fragments of pBcl6 Luc-472 was examined in K562 cells. Data are presented as the mean ± SD from three independent experiments. (C) Basal transcriptional activity of pBcl6 Luc-472 with mutations was examined in K562 and Ramos cells. Activities of these cells transfected with pBcl6 Luc-472 were 100%. Data are presented as the mean ± SD from three independent experiments. (D) Basal transcriptional activity of serial deletion fragments of pBcl6 Luc-472/+114 was examined in K562 and Ramos cells. Data are presented as the mean ± SD from three independent experiments.

 
A major transcriptional start site of the mBcl6 gene in GC B cells is +95, and the region between +1 and +95 contains the Bcl6-binding sequence (+6 to +16) as a silencer element in the hBcl6 gene (22). Thus, we extended the 3' end of pBcl6 Luc-472/+5 to +114 (pBcl6 Luc-472/+114) and introduced the mutation in the Bcl6-binding sequence of pBcl6 Luc-472/+114. These constructs were transfected into Ramos cells. The promoter activity of the pBcl6 Luc-472/+114 (mutBcl6) was ~5-fold stronger than that of the original pBcl6 Luc-472/+114 (Fig. 4D). Although mutations in two STAT-binding sequences increased their promoter activity 15-fold over the original one, mutations in both the STAT-binding sequences and the Bcl6-binding sequence increased ~50-fold of the pBcl6 Luc-472/+114 activity. When these constructs were transfected into K562 cells, the promoter activity was within 3-fold of the original one. Although it remains unclear how the mutations slightly increased the reporter activity in K562 cells, we confirmed Bcl6 binding to the tandem STAT-binding sites (–39 and –22) and to the Bcl6-binding sequence (+6) using EMSA (data not shown). These results suggested that Bcl6 binds to the STAT/Bcl6-binding sequences and reduces Bcl6 expression in GC B cells.

We confirmed the potential of exogenous Bcl6 to repress the promoter activity by co-transfection of pBcl6 Luc-472 and the Bcl6 expression vector (pcDNA3-Bcl6) in K562 cells. Co-transfection of various doses (10–20 µg) of pcDNA3-Bcl6 repressed the promoter activity in K562 cells as a dose-dependent manner (Fig. 5A). Since the repressor activity of Bcl6 can be inhibited by the histone deacetylase inhibitor, TSA, we transfected the optimal dose of pcDNA3-Bcl6 (20 µg) and pBcl6 Luc-472 in Ramos cells in the presence of TSA. Over-expression of Bcl6 reduced the promoter activity, and Bcl6-dependent repression was abolished in the presence of 100 nM of TSA (Fig. 5B). The Bcl6-dependent recruitment of a histone deacetylase complex seemed to be mediated by the STAT-binding sites in the promoter region since co-transfection of pcDNA3-Bcl6 and pBcl6 Luc-472 (mutSTAT) with or without TSA resulted in the similar basal promoter activity.


Figure 5
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  		Fig. 5 Autoregulation of mBcl6 expression by Bcl6. (A) K562 cells were transfected with pBcl6 Luc-472 and various doses (0–20 µg) of pcDNA3-Bcl6 and pcDNA3 (total DNA; 20 µg). Activity of K562 cells transfected with pBcl6 Luc-472 without pcDNA3-Bcl6 was 100%. Data are presented as the mean ± SD from three independent experiments. (B) Ramos cells were transfected with pBcl6 Luc-472 or pBcl6 Luc-472 with mutations and pcDNA3-Bcl6 (20 µg). Transfected cells were cultured with TSA (100 nM). Data are presented as the mean ± SD from three independent experiments.

 
Competition of activated STATs with Bcl6 to bind to the STAT/Bcl6-binding sites in GC B cells
Activated STATs may bind to the STAT/Bcl6-binding sites and inhibit the Bcl6-dependent repression in GC B cells. To prove the possibility, we examined the activation status of STATs in GC cells. Since STAT3 is the major activated STATs in human GC cells by immunohistochemical analysis (29), we repeated the analysis on spleen sections from immunized mice. STAT3 activation was significantly apparent and there were numerous p-STAT3+ cells within and outside the GC (Fig. 6A). However, p-STAT5 and p-STAT6 were present in only a few nuclei of scattered lymphocytes outside of the GC, indicating that STAT3 is also activated in murine GC cells. We confirmed the binding of STAT3 to the negative regulatory elements in GC B cells by ChIP assays. PCR for ChIP assays detected the DNA sequence between –195 and +188 that included three STAT/Bcl6-binding sites. Indeed, STAT3 bound to the STAT/Bcl6-binding sites (Fig. 6B). Although p-STAT5 and p-STAT6 were not clearly detected in GC cells by immunohistochemical analysis, STAT5 and STAT6 also bound to the STAT/Bcl6-binding sites in GC B cells.


Figure 6
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  		Fig. 6 Expression of activated STATs in GC B cells. (A) Expression of p-STAT3, p-STAT5 and p-STAT6 was examined by immunohistochemistry in sections of spleens from immunized mice. Arrows indicate positive staining cells in the sections at low (upper, x100) and high (lower, x400) magnifications. (B) Binding of Bcl6, STAT3, STAT5 and STAT6 proteins in FACS-isolated GC B cells to the STAT/Bcl6-binding sites was analyzed by ChIP assays. Ratios indicate the relative intensity of each sample to that of input as 1. Data are presented as a representative of three independent experiments.

 
Next, we examined the competitive binding of activated STATs and Bcl6 to the STAT/Bcl6-binding sites. Since STAT3 is activated in Ramos cells stimulated with IFN-{alpha} (30), amounts of STATs and Bcl6 bound to the DNA region in Ramos cells stimulated with IFN-{alpha} were analyzed using ChIP assays. First, we analyzed phosphorylation of STATs in Ramos cells after the stimulation by western blots. STAT3, STAT5 and STAT6 were clearly phosphorylated in Ramos cells 20 min after stimulation (Fig. 7A). Then, amounts of the DNA-binding STATs and Bcl6 were analyzed 20 min after stimulation. The amounts of STAT3 and STAT5 but not that of STAT6 bound to the DNA region increased after stimulation (Fig. 7B). On the contrary, the amount of Bcl6 bound to the region decreased to one-third after stimulation.


Figure 7
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  		Fig. 7 Competition of STATs with Bcl6 for binding to the negative regulatory elements. (A) Activated STATs in Ramos cells after IFN-{alpha} stimulation were analyzed by western blot. Actin was used as an internal control for the amount of protein. Data are presented as a representative of three independent experiments. (B) Ramos cells were stimulated with IFN-{alpha}. Binding of Bcl6, STAT3, STAT5 and STAT6 proteins to the STAT/Bcl6-binding sites was analyzed by ChIP assays. Ratios indicate the relative intensity of each sample to that of input as 1. Data are presented as a representative of three independent experiments.

 
High Bcl6 expression with induction of JunD/AP-1 and activation of STATs in splenic B cells stimulated with IL-4 or IL-21
Since stimulation of B cells with IL-4 (31), IL-6 (32) or IL-21 (33) activates STAT3, we examined Bcl6 expression in splenic B cells stimulated with these cytokines using northern blots. Bcl6 expression was strongly induced in splenic B cells stimulated with IL-4 or IL-21 but not with IL-6 (Fig. 8A). However, co-stimulation of B cells with IL-4 and IL-21 did not augment Bcl6 expression (data not shown). Although IL-2 and IL-7 mediate the biological effects through the common cytokine receptor gamma chain, which is shared with receptors for IL-4 and IL-21 (34), these cytokine stimulations did not induce high Bcl6 expression in splenic B cells. Kinetic analysis demonstrated that Bcl6 expression was transiently induced in B cells stimulated with IL-4 or IL-21 30 min after stimulation (Fig. 8B). The expression was further induced in these activated B cells within 6 h after stimulation. IL-4 or IL-21 stimulation also induced expression of junD and fosB, but not that of c-fos, c-jun and junB in splenic B cells. We examined the activation of STATs in splenic B cells stimulated with IL-4 or IL-21 using western blots. IL-4 and IL-21 preferentially activated STAT6 and STAT3 within 20 min after stimulation, respectively (Fig. 8C). STAT5 was also activated in B cells stimulated with IL-4 or IL-21. We confirmed the binding of STATs to the STAT/Bcl6-binding sites in splenic B cells stimulated with IL-4 or IL-21 using ChIP assays. STAT3 bound clearly to the STAT/Bcl6-binding sites after IL-21 stimulation while IL-4 induced modestly STAT6-binding to the sites (Fig. 8D). These results suggest that IL-4 and IL-21, especially IL-21, are crucial cytokines for induction of JunD/AP-1 and activation of STATs to induce high Bcl6 expression in GC B cells.


Figure 8
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  		Fig. 8 Induction of JunD/AP-1 and activation of STAT3 in naive splenic B cells stimulated with IL-21. Splenic B cells were stimulated with various cytokines. (A) Bcl6 expression in activated B cells 6 h after stimulation was analyzed by northern blot. ß-Actin mRNA was used as an internal control for the amount of mRNA. Data are presented as a representative of three independent experiments. (B) Expression of Bcl6 and AP-1 family genes in B cells stimulated with IL-4 or IL-21 for 24 h was analyzed by northern blot. ß-Actin mRNA was used as an internal control for the amount of mRNA. Data are presented as a representative of three independent experiments. (C) Activated STATs in B cells after IL-4 or IL-21 stimulation were analyzed by western blot. Actin was used as an internal control for the amount of protein. Data are presented as a representative of three independent experiments. (D) Binding of Bcl6, STAT3, STAT5 and STAT6 proteins in splenic B cells activated with IL-4 or IL-21 for 6 h to the STAT/Bcl6-binding sites was analyzed by ChIP assays. Data are presented as a representative of two independent experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
References
 
We identified the promoter region of the mBcl6 gene. We found no obvious canonical TATA or CCAAT box around the transcriptional start sites of the mBcl6 gene. The multiple transcription start sites are a common feature of the genes with a TATA-less promoter (35). Transcriptional start sites of the genes lacking the TATA box may rely on the GC-rich region that is bound by transcriptional factors (36), and the start sites of mBcl6 mRNA in GC B cells and LPS-activated B cells are also in the GC-rich region. These start sites are quite different from the transcriptional start site of hBcl6 mRNA in Raji cells reported by Ohashi et al. (21). The 5' RACE analysis of hBcl6 mRNA from Ramos cells revealed a different transcriptional start site from that in Raji cells. Bcl6 expression profiles in various tissues between mice and humans are quite similar, and DNA sequence of the new promoter region of the hBcl6 gene is also similar to that of the homologous region of the mBcl6 gene and does not contain an obvious canonical TATA or CCAAT box (TATA-less promoter). Binding elements for major transcriptional factors including AP-1, NF-AT, NF-{kappa}B and STATs in the mBcl6 promoter region are conserved in the new hBcl6 promoter region. Therefore, the ordinary promoter region of the hBcl6 gene may be similar to that of the mBcl6 gene.

Mutation or deletion of the AP-1-binding sites within the basal mBcl6 promoter mostly reduced the promoter activity in Ramos (Fig. 2) and M12.4.1 cells (data not shown), indicating that AP-1 is the major enhancer molecule for high Bcl6 expression in GC B cells. AP-1-binding sites are also present in the new putative promoter region of the hBcl6 gene, and AP-1 including c-Fos, c-Jun and JunD in Ramos cells bound to these AP-1-binding sites. Among AP-1 family, JunD was the major molecule bound to the AP-1-binding sites in Ramos cells. Jun forms homodimer or heterodimer with Fos, and Fos–Jun heterodimers are more stable than Jun–Jun homodimers and have much stronger DNA-binding activity (37). The AP-1 family confers positive or negative transcriptional regulation depending on its components (38), and composition of AP-1 molecule on AP-1-binding sites may depend on the differentiation state of the cell type or on its affinities to the AP-1 dimers (37). Although we did not examine the presence of Fra-1 and Fra-2, the fosB mRNA was induced in splenic B cells stimulated with IL-4 or IL-21 and c-Fos protein was detected in Ramos cells. Thus, JunD–FosB and/or JunD–c-Fos heterodimers may be the major enhancer molecule for high Bcl6 expression in GC B cells of mice and humans.

We have identified three silencer elements in the putative mBcl6 promoter region, and all contain the STAT-binding (GAS) motif. The silencer elements in the promoter (–36 to –28 and –21 to –13) seem to be STAT3/STAT5-binding elements. GAS motif is very similar to the consensus Bcl6-binding element (11) and Bcl6 binds to STAT6-binding sites from the CD23 promoter (11) and the germ line I{varepsilon} promoter (39). Indeed, Bcl6 also bound to the STAT-binding sites in the mBcl6 promoter. These sequences are conserved in the hBcl6 gene and play a role as a silencer element in human diffuse large B cell lymphomas (23, 24). The other silencer region between +6 and +16 also contains the DNA sequence referring to the consensus Bcl6-binding site (40) and Bcl6 bound to the silencer element detected by EMSA (data not shown). Since major transcriptional start site of mBcl6 mRNA in GC B cells is +95, there are three Bcl6-binding elements within 50 bp of the Bcl6 promoter region. These results indicate the negative feedback regulation of Bcl6 on its own gene expression in GC B cells.

Three STAT/Bcl6-binding sites in the mBcl6 gene are also conserved in the hBcl6 gene. Although these silencer elements were reported to be in the first exon of hBcl6 gene (22–24), the major transcriptional start site of the hBcl6 gene in Ramos cells is around +38 in the homologous region of mBcl6 gene, suggesting that three STAT/Bcl6-binding sites are in the promoter region of hBcl6 gene. Non-Hodgkin's lymphomas and Burkitt's lymphomas carry internal deletions (41) or point mutations (24, 42) within the hBcl6 gene. These alterations are mainly occurring at the promoter region including these silencer elements. The attribution of translocation in tumor cells, therefore, is likely to alter the regulation pattern of Bcl6 expression in part by deleting the silencer elements, and may be a crucial event in the development of B cell lymphomas.

JunD/AP-1 and p-STAT3 are critical molecules for high Bcl6 expression in GC B cells, and stimulation of naive B cells with IL-21 induced high Bcl6 expression with induction of JunD/AP-1 and activation of STAT3 which can bind to the STAT/Bcl6-binding sites. These results suggest that IL-21 is a major inducer for high Bcl6 expression in GC B cells. IL-21 is a recently described type I cytokine produced by Th2 cells (33). Recently, a new major subset of Th2 cells that provide help to B cells was identified in lymphoid tissues through expression of the chemokine receptor CXCR5 (43, 44). These Th2 cells, termed T follicular helper cells, home to the B cell areas of secondary lymphoid tissue and produce large amounts of IL-21 (45). These results strongly suggest that IL-21 derived from T follicular helper cells induces high Bcl6 expression in GC B cells.

High Bcl6 expression in B cells is essential for GC formation (13). However, GC formation was relatively normal in mice lacking IL-21R (34), indicating functional redundancy of IL-21 to induce high Bcl6 expression in GC B cells. Stimulation of splenic B cells with IL-4 induced high Bcl6 expression with activation of STAT5 and STAT6, and binding of STAT5 and STAT6 to the STAT/Bcl6-binding sites was detected in GC B cells, suggesting a physiological role for IL-4 in high Bcl6 expression in GC B cells. These results indicate an important cooperative role for IL-4 and IL-21 in high Bcl6 expression in GC B cells. This cooperation was supported by the evidence that GC formation was normal in IL-4-deficient mice but was disorganized in mice lacking both IL-4 and IL-21R (34). However, co-stimulation of splenic B cells with IL-4 and IL-21 did not augment Bcl6 expression (data not shown) and high Bcl6 expression is continuously detected in GC B cells, suggesting that IL-4 and IL-21 may be required for high Bcl6 expression in GC B cells at different stages.

Another group has recently showed that IL-21 stimulation induces Bcl6 and Blimp1 expression in splenic B cells, and enhanced the important role of IL-21 in plasma cell differentiation (46). However, the Blimp1 gene is not expressed in GC B cells. Since Bcl6 suppresses the differentiation of BCL1 and primary B cells to plasma cells by inhibiting the STAT3-dependent Blimp1 expression (47), Bcl6 can compete with STAT3 to bind to the STAT-binding site in the promoter region of the Blimp1 gene in activated B cells. Furthermore, a recent report demonstrated another Bcl6-binding site in the fifth intron of Blimp1 gene and STAT3 cannot inhibit the Bcl6 binding to the new site (48). Thus, high Bcl6 expression initially induced by IL-4 stimulation may further suppress Blimp1 expression induced by IL-21 stimulation in GC B cells. Since STAT3 is activated in fully developed GC cells and can inhibit the Bcl6-dependent repression of the Bcl6 gene but not that of the Blimp1 gene, IL-21 may be required for the maintenance of high Bcl6 expression in GC B cells. Further study is required to elucidate the functional redundancy of IL-4 and IL-21 in high Bcl6 expression in GC B cells.

In summary, we have identified the transcriptional start sites, the enhancer and silencer elements in the promoter region of the mBcl6 gene. These regulatory elements are conserved in the new promoter region of hBcl6 gene. JunD/AP-1 and STATs, especially STAT3, are crucial molecules to induce high Bcl6 expression in GC B cells. Since IL-21 stimulation induces high Bcl6 expression in splenic B cells with induction of JunD/AP-1 and activation of STAT3, IL-21 may be a crucial inducer for high Bcl6 expression in GC B cells.


   Acknowledgements
 
We thank H. Satake for technical assistance and K. Yagyu for secretarial assistance. This work was supported in part by grants-in-aid from the Ministry of Education, Science, Technology, Sports and Culture of Japan and the Uehara Memorial Foundation.


   Abbreviations
 
ChIP, chromatin immunoprecipitation
GC, germinal center
hBcl6, human Bcl6
mBcl6, murine Bcl6
p-, phosphorylated
PNA, peanut agglutinin
5' RACE, rapid amplification of 5' cDNA ends
STATs, STAT factors
TSA, trichostatin A

   Notes
 
Transmitting editor: H. Karasuyama

Received 16 November 2005, accepted 18 April 2006.


   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

  1. Offit K, Jhanwar S, Ebrahim SA, Filippa D, Clarkson BD, Chaganti RS. (1989) t(3;22)(q27;q11): a novel translocation associated with diffuse non-Hodgkin's lymphoma. Blood 74:1876.[Abstract/Free Full Text]
  2. Ye BH, Lista F, Lo Coco DM, et al. (1993) Alterations of a zinc finger-encoding gene, BCL-6, in diffuse large-cell lymphoma. Science 262:747.[Abstract/Free Full Text]
  3. Kerckaert JP, Deweindt C, Tilly H, Quief S, Lecocq G, Bastard C. (1993) LAZ3, a novel zinc-finger encoding gene, is disrupted by recurring chromosome 3q27 translocations in human lymphomas. Nat. Genet. 5:66.[CrossRef][Web of Science][Medline]
  4. Miki T, Kawamata N, Hirosawa S, Aoki N. (1994) Gene involved in the 3q27 translocation associated with B-cell lymphoma, BCL5, encodes a Kruppel-like zinc-finger protein. Blood 83:26.[Abstract/Free Full Text]
  5. Fukuda T, Miki T, Yoshida T, et al. (1995) The murine Bcl6 gene is induced in activated lymphocytes as an immediate early gene. Oncogene 11:1657.[Web of Science][Medline]
  6. Deweindt C, Albagli O, Bernardin F, et al. (1995) The LAZ3/BCL6 oncogene encodes a sequence-specific transcriptional inhibitor: a novel function for the BTB/POZ domain as an autonomous repressing domain. Cell Growth Differ 6:1495.[Abstract]
  7. Dhordain P, Albagli O, Lin RJ, et al. (1997) Corepressor SMRT binds the BTB/POZ repressing domain of the LAZ3/BCL6 oncoprotein. Proc. Natl Acad. Sci. USA 94:10762.[Abstract/Free Full Text]
  8. Cattoretti G, Chang CC, Cechova K, et al. (1995) BCL-6 protein is expressed in germinal-center B cells. Blood 86:45.[Abstract/Free Full Text]
  9. Onizuka T, Moriyama M, Yamochi T, et al. (1995) BCL-6 gene product, a 92- to 98-kD nuclear phosphoprotein, is highly expressed in germinal center B cells and their neoplastic counterparts. Blood 86:28.[Abstract/Free Full Text]
  10. Yoshida T, Fukuda T, Okabe S, et al. (1996) The BCL6 gene is predominantly expressed in keratinocytes at their terminal differentiation stage. Biochem. Biophys. Res. Commun. 228:216.[CrossRef][Web of Science][Medline]
  11. Dent AL, Shaffer AL, Yu X, Allman D, Staudt LM. (1997) Control of inflammation, cytokine expression, and germinal center formation by BCL-6. Science 276:589.[Abstract/Free Full Text]
  12. Ye BH, Cattoretti G, Shen Q, et al. (1997) The BCL-6 proto-oncogene controls germinal-centre formation and Th2-type inflammation. Nat. Genet. 16:161.[CrossRef][Web of Science][Medline]
  13. Fukuda T, Yoshida T, Okada S, et al. (1997) Disruption of the Bcl6 gene results in an impaired germinal center formation. J. Exp. Med. 186:439.[Abstract/Free Full Text]
  14. Yoshida T, Fukuda T, Hatano M, et al. (1999) A role of Bcl6 in mature cardiac myocytes. Cardiovasc. Res. 42:670.[Abstract/Free Full Text]
  15. Arima M, Toyama H, Ichii H, et al. (2002) A putative silencer element in the IL-5 gene recognized by Bcl6. J. Immunol. 169:829.[Abstract/Free Full Text]
  16. Takeda N, Arima M, Tsuruoka N, et al. (2003) Bcl6 is a transcriptional repressor for the IL-18 gene. J. Immunol. 171:426.[Abstract/Free Full Text]
  17. Toyama H, Okada S, Hatano M, et al. (2002) Memory B cells without somatic hypermutation are generated from Bcl6-deficient B cells. Immunity 17:329.[CrossRef][Web of Science][Medline]
  18. Ye BH, Chaganti S, Chang CC, et al. (1995) Chromosomal translocations cause deregulated BCL6 expression by promoter substitution in B cell lymphoma. EMBO J. 14:6209.[Web of Science][Medline]
  19. Zhang H, Okada S, Hatano M, Okabe S, Tokuhisa T. (2001) A new functional domain of Bcl6 family that recruits histone deacetylases. Biochim. Biophys. Acta 1540:188.[Medline]
  20. Yamochi T, Kaneita Y, Akiyama T, Mori S, Moriyama M. (1999) Adenovirus-mediated high expression of BCL-6 in CV-1 cells induces apoptotic cell death accompanied by down-regulation of BCL-2 and BCL-XL. Oncogene 18:487.[CrossRef][Web of Science][Medline]
  21. Ohashi K, Miki T, Hirosawa S, Aoki N. (1995) Characterization of the promoter region of human BCL-6 gene. Biochem. Biophys. Res. Commun. 214:461.[CrossRef][Web of Science][Medline]
  22. Kikuchi M, Miki T, Kumagai T, et al. (2000) Identification of negative regulatory regions within the first exon and intron of the Bcl6 gene. Oncogene 19:4941.[CrossRef][Web of Science][Medline]
  23. Wang X, Li Z, Naganuma A, Ye BH. (2002) Negative autoregulation of BCL-6 is bypassed by genetic alterations in diffuse large B cell lymphomas. Proc. Natl Acad. Sci. USA 99:15018.[Abstract/Free Full Text]
  24. Pasqualucci L, Migliazza A, Basso K, et al. (2003) Mutations of the BCL6 proto-oncogene disrupt its negative autoregulation in diffuse large B-cell lymphoma. Blood 101:2914.[Abstract/Free Full Text]
  25. Karasuyama H and Melchers F. (1988) Establishment of mouse cell lines which constitutively secrete large quantities of interleukin 2, 3, 4 or 5, using modified cDNA expression vectors. Eur. J. Immunol. 18:97.[Web of Science][Medline]
  26. Schreiber E, Matthias P, Muller MM, Schaffner W. (1989) Rapid detection of octamer binding proteins with ‘mini-extracts’, prepared from a small number of cells. Nucleic Acids Res. 17:6419.[Free Full Text]
  27. Okada S, Obata S, Hatano M, Tokuhisa T. (2003) Dominant-negative effect of the c-fos family gene products on inducible NO synthase expression in macrophages. Int. Immunol. 15:1275.[Abstract/Free Full Text]
  28. Heinemeyer T, Chen X, Karas H, et al. (1999) Expanding the TRANSFAC database towards an expert system of regulatory molecular mechanisms. Nucleic Acids Res. 27:318.[Abstract/Free Full Text]
  29. Skinnider BF, Elia AJ, Gascoyne RD, et al. (2002) Signal transducer and activator of transcription 6 is frequently activated in Hodgkin and Reed-Sternberg cells of Hodgkin lymphoma. Blood 99:618.[Abstract/Free Full Text]
  30. Gupta S, Jiang M, Pernis AB. (1999) IFN-alpha activates Stat6 and leads to the formation of Stat2:Stat6 complexes in B cells. J. Immunol. 163:3834.[Abstract/Free Full Text]
  31. Umeshita-Suyama R, Sugimoto R, Akaiwa M, et al. (2000) Characterization of IL-4 and IL-13 signals dependent on the human IL-13 receptor alpha chain 1: redundancy of requirement of tyrosine residue for STAT3 activation. Int. Immunol. 12:1499.[Abstract/Free Full Text]
  32. Karras JG, Wang Z, Huo L, Howard RG, Frank DA, Rothstein TL. (1997) Signal transducer and activator of transcription-3 (STAT3) is constitutively activated in normal, self-renewing B-1 cells but only inducibly expressed in conventional B lymphocytes. J. Exp. Med. 185:1035.[Abstract/Free Full Text]
  33. Habib T, Nelson A, Kaushansky K. (2003) IL-21: a novel IL-2-family lymphokine that modulates B, T, and natural killer cell responses. J. Allergy Clin. Immunol. 112:1033.[CrossRef][Web of Science][Medline]
  34. Ozaki K, Spolski R, Feng CG, et al. (2002) A critical role for IL-21 in regulating immunoglobulin production. Science 298:1630.[Abstract/Free Full Text]
  35. Smale ST. (1997) Transcription initiation from TATA-less promoters within eukaryotic protein-coding genes. Biochim. Biophys. Acta 1351:73.[Medline]
  36. Philipsen S and Suske G. (1999) A tale of three fingers: the family of mammalian Sp/XKLF transcription factors. Nucleic Acids Res. 27:2991.[Abstract/Free Full Text]
  37. Karin M, Liu Z, Zandi E. (1997) AP-1 function and regulation. Curr. Opin. Cell Biol. 9:240.[CrossRef][Web of Science][Medline]
  38. Kobierski LA, Chu HM, Tan Y, Comb MJ. (1991) cAMP-dependent regulation of proenkephalin by JunD and JunB: positive and negative effects of AP-1 proteins. Proc. Natl Acad. Sci. USA 88:10222.[Abstract/Free Full Text]
  39. Harris MB, Chang CC, Berton MT, et al. (1999) Transcriptional repression of Stat6 dependent interleukin-4-induced genes by BCL-6: specific regulation of iepsilon transcription and immunoglobulin E switching. Mol. Cell. Biol. 19:7264.[Abstract/Free Full Text]
  40. Kawamata N, Miki T, Ohashi K, et al. (1994) Recognition DNA sequence of a novel putative transcription factor, BCL6. Biochem. Biophys. Res. Commun. 204:366.[CrossRef][Web of Science][Medline]
  41. Nakamura Y, Miki T, Miura I, et al. (1996) Internal DNA deletion within the BCL-6 gene on untranslocated chromosome in non-Hodgkin's lymphoma with 3q27 abnormality. Leukemia 10:658.[Web of Science][Medline]
  42. Migliazza A, Martinotti S, Chen W, et al. (1995) Frequent somatic hypermutation of the 5' noncoding region of the BCL6 gene in B-cell lymphoma. Proc. Natl Acad. Sci. USA 92:12520.[Abstract/Free Full Text]
  43. Breitfeld D, Ohl L, Kremmer E, et al. (2000) Follicular B helper T cells express CXC chemokine receptor 5, localize to B cell follicles, and support immunoglobulin production. J. Exp. Med. 192:1545.[Abstract/Free Full Text]
  44. Schaerli P, Willimann K, Lang AB, Lipp M, Loetscher P, Moser B. (2000) CXC chemokine receptor 5 expression defines follicular homing T cells with B cell helper function. J. Exp. Med. 192:1553.[Abstract/Free Full Text]
  45. Chtanova T, Tangye SG, Newton R, et al. (2004) T follicular helper cells express a distinctive transcriptional profile, reflecting their role as non-Th1/Th2 effector cells that provide help for B cells. J. Immunol. 173:68.[Abstract/Free Full Text]
  46. Ozaki K, Spolski R, Ettinger R, et al. (2004) Regulation of B cell differentiation and plasma cell generation by IL-21, a novel inducer of Blimp-1 and Bcl-6. J. Immunol. 173:5361.[Abstract/Free Full Text]
  47. Reljic R, Wagner SD, Peakman LJ, Fearon DT. (2000) Suppression of signal transducer and activator of transcription 3-dependent B lymphocyte terminal differentiation by BCL-6. J. Exp. Med. 192:1841.[Abstract/Free Full Text]
  48. Tunyaplin C, Shaffer AL, Angelin-Duclos CD, Yu X, Staudt LM, Calame KL. (2004) Direct repression of prdm1 by Bcl-6 inhibits plasmacytic differentiation. J. Immunol. 173:1158.[Abstract/Free Full Text]

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