Transcriptional Repression of Stat6-Dependent Interleukin-4-Induced Genes by BCL-6: Specific Regulation of Iε Transcription and Immunoglobulin E Switching

Rearrangement of the BCL-6 proto-oncogene can be detected in 30 to 40% of diffuse large-cell lymphomas (DLCLs) and in 6 to 14% of follicular lymphomas (FLs) (5, 37, 42). In DLCLs and FLs, chromosomal rearrangements affecting the BCL-6 gene are located within a region spanning approximately 4 kb of the promoter and the first exon and result in the juxtaposition of the BCL-6 coding domains downstream of heterologous promoters derived from other chromosomes (53). These alterations lead to the production of chimeric transcripts which encode a wild-type BCL-6 protein, suggesting that the functional consequence of these translocations is the deregulation of BCL-6 expression by promoter substitution (53). The high frequency of dysregulated BCL-6 expression in these tumors suggests that this oncogene plays an important role in the transformation of human B cells. The BCL-6 gene encodes a polypeptide containing six carboxy-terminal zinc-finger motifs homologous to members of the Kruppel subfamily of zinc-finger proteins (30, 38, 54). This domain of BCL-6 has been shown to recognize and bind to specific DNA sequences in vitro (4, 9, 48). The N-terminal portion of BCL-6 contains a ZiN (for zinc-finger N-terminal)/POZ (POX/zinc-finger) domain which is also present in other zinc-finger proteins, including the mammalian transcriptional regulators PLZF, ZF5, and ZID (3, 11, 12, 18, 40, 55). Transfection experiments have demonstrated that BCL-6 can act as a transcriptional repressor, and its ability to mediate repression requires the N-terminal POZ domain (9, 48). These results suggest that BCL-6 modulates transcription not simply through competitive binding, but through a mechanism of active repression. Indeed, the POZ domains of both BCL-6 and PLZF have recently been shown to associate with the SMRT corepressor, and, by extension, the histone deacetylase repression complex (17, 23, 25, 35). BCL-6 is normally expressed in a tissue-specific and developmentally regulated manner. Although many tissues express low levels of BCL-6 mRNA, high levels of the BCL-6 protein have been found only in certain B cells and T cells (6). Within the B-cell lineage, BCL-6 is expressed at high levels in mature, germinal center B cells, but not in other B cells or plasma cells (6, 19, 41). BCL-6 expression in T cells is limited to cortical thymocytes and a population of CD4+ cells within the germinal center and perifollicular zones of the lymph nodes (6). The importance of BCL-6 in normal lymphocyte function has recently been demonstrated in mice in which the gene for BCL-6 has been disrupted by homologous recombination (16, 20, 52). Although these mice contain normal numbers of B and T cells, they fail to form germinal centers or mount T-cell-dependent antibody responses. In addition, many of these mice develop a systemic inflammatory disease characterized by the infiltration of multiple organ systems by eosinophils and immunoglobulin E (IgE)-bearing B cells; these features are indicative of a Th2 polarized inflammatory response, which could potentially result from the inappropriate influence of the Th2 cytokines (interleukin-4 [IL-4], IL-5, and IL-13) on immune function. The striking phenotype of the knockout animal therefore implicates BCL-6 in the normal regulation of the immune system. The evidence suggesting a disruption of cytokine regulation in the BCL-6−/− mice prompted the comparison between the in vitro defined binding site of BCL-6 (B6BS) and the binding sites of STAT proteins, molecules which are important mediators of cytokine signal transduction (reviewed in references 27, 34, and 46). In fact, B6BS shows a marked similarity to STAT recognition sequences, and one study has demonstrated the ability of BCL-6 to bind to the Stat6 site of the IL-4-inducible CD23b promoter (16). Furthermore, transient transfection studies have suggested that BCL-6 may regulate the Stat6-dependent transcription of the CD23b gene (16). However, the regulation of gene expression by BCL-6 under physiological conditions has not yet been tested, and other physiologic targets of BCL-6 repression are so far unknown. In order to identify physiological targets for BCL-6, we have analyzed its ability to bind and regulate Stat6-dependent promoters in vitro and in vivo. Our results demonstrate that although BCL-6 can bind to the Stat6 sites present in several IL-4-responsive promoters in vitro, it can regulate only a subset of Stat6-dependent promoters in vivo; this subset includes the germ line ɛ promoter, but not the CD23b promoter. The germ line ɛ promoter regulates the production of the Ig sterile transcripts necessary for the Ig isotype class switch to IgE (reviewed in reference 13). Consistent with a role for BCL-6 in the regulation of class switching, IgE production is increased in B cells lacking BCL-6 in vitro and in vivo. This dysregulation of IgE production is not observed in B cells lacking Stat6 as well as BCL-6. These results provide evidence for the physiologic regulatory activity of BCL-6 on specific Stat6-dependent IL-4 signaling and identify the regulation of IgE class switching as a target of this activity in vivo.