Signal transducer and activator of transcription 6 (Stat6) and NF-B belong to two distinct families of transcription factors, the STAT family and the NF-B/Rel family, respectively. Members of both families play important roles in immune responses and in cell differentiation induced by cytokines, growth factors, and other cell activators. Stat6, like other STATs, is in the cytoplasm as a latent monomer. Upon cytokine stimulation, mainly by interleukin-4 (IL-4), Stat6 is rapidly phosphorylated by Janus kinases (Jaks) to form homodimers which enter the nucleus where Stat6 binds various target genes containing Stat6 binding sites (3,7,10). Unlike Stat6, NF-B proteins are kept in the cytoplasm by association with IB proteins. When cells are stimulated by CD40L, lipopolysaccharide (LPS), or a variety of other stimuli, NF-B proteins are released from the associated IB proteins and enter the nucleus, where they bind consensus B sites in many genes (1).Some STAT proteins bind other transcription factors or coactivators to activate transcription (2,23,26,40), but Stat6 has not been shown to bind any known transcription factors. NF-B/Rel proteins have also been shown to interact with other transcription factors or proteins of the basal transcription machinery which regulate NF-B/Rel proteins positively or negatively in transcriptional activation (11,13,35,36,39). Recent studies suggest that Stat6 and NF-B, activated by discrete signaling pathways, interact at some unknown point to synergistically activate transcription of certain genes that are induced in response to both signals. Examples of such genes are the germline (GL) immunoglobulin (Ig) Cε and C␥1 genes, the transcription of which is necessary for class switching to IgE and IgG1 (33). Functional studies of the GL Cε and C␥1 promoters indicate that both Stat6 and B sites are required for optimal induction of transcription by IL-4 and/or by CD40L (4, 9, 15, 16, 38), but the molecular mechanism for synergistic activation of promoters by Stat6 and NF-B proteins is not understood. A Stat6 and two or three B sites are closely positioned in the promoters of three relatively well-characterized IL-4-responsive genes, GL Cε, GL C␥1, and FcεRII (CD23) (25), so together with the functional data, this information raises the possibility that Stat6 and NF-B interact directly with each other. In this report, we provide physical evidence that Stat6 and NF-B directly bind each other in vitro and in vivo as well as functional evidence that these two transcription factors cooperatively bind their cognate DNA binding sites and synergistically activate transcription. The significance of their interaction in IL-4 signaling is discussed. MATERIALS AND METHODSCells and cell culture. sIgM ϩ mouse B lymphoma cell line I.29 (34) was grown at 37°C in an atmosphere of 8% CO 2 in RPMI 1640 medium-20% fetal calf serum (FCS) (32). Human embryonic kidney 293 (HEK 293) cells (ATCC CRL-1573) were grown at 37°C in an atmosphere of 5% CO 2 in Dulbecco's modified Eagle's medium-10% FCS. Sf9 insect cells...

Induction of germline (GL) ε transcripts, an essential step preceding Ig isotype switching to IgE, requires activation of transcription factors by IL-4 and a B cell activator, e.g., CD40 ligand or LPS. We demonstrate that AP-1 (Fos and Jun), induced transiently by CD40 ligand or LPS, binds a DNA element in the mouse GL ε promoter. AP-1 synergizes with Stat6 to activate both the intact GL ε promoter and a minimal heterologous promoter driven by the AP-1 and Stat6 sites of the mouse GL ε promoter. By contrast, C/EBPβ, which trans-activates the human GL ε promoter, inhibits IL-4 induction of the mouse promoter, probably by attenuating the synergistic interaction between AP-1 and Stat6. Furthermore, AP-1 does not trans-activate the human GL ε promoter. Thus, induction of GL ε transcripts in mice and humans may be regulated differently. In addition, although mouse GL ε transcripts have a half-life of ∼100 min, the RNA level continues to increase for up to 24 h, and the promoter appears to be active for at least 2 days after B cell activation. Altogether, these data suggest that induction of AP-1 activity, although transient, is required for activation of the mouse GL ε promoter by IL-4-induced Stat6.