Antibody class switch recombination (CSR) occurs after antigen activation of B cells. CSR is directed to specific heavy chain isotypes by cytokines and B cell activators that induce transcription from the unrearranged, or germline (GL), C(H) region genes. Transforming growth factor (TGF)-beta1 is essential for switch recombination to IgA due to its ability to induce transcription from GL Ig alpha genes. It has been shown that the promoters which regulate transcription of mouse and human GL alpha RNAs contain a TGF-beta1-responsive element that binds Smad and core binding factor (CBFalpha)/AML/PEBPalpha/RUNX: They also contain other elements which bind the transcription factors CREB, BSAP and Ets family proteins. In this manuscript we demonstrate that two tandem Ets sites in the mouse GL alpha promoter bind the transcription factors Elf-1 and PU.1, and that the 3' site is essential for expression of a luciferase reporter gene driven by the GL alpha promoter. Binding of Elf-1 to the GL alpha promoter is inducible by lipopolysaccharide in nuclear extracts from splenic B cells. An NF-kappaB site is identified, although it does not contribute to expression of the promoter in reporter gene assays. Since CSR to IgA is greatly reduced in NF-kappaB/p50-deficient mice, these data support the hypothesis that NF-kappaB has roles in switching in addition to regulation of GL transcription. Finally, we demonstrate that nocodazole, which disrupts microtubules that sequester Smad proteins in the cytoplasm, stimulates transcription from the GL alpha promoter.

Immunoglobulin class switch recombination (SR) occurs by a B cell-specific, intrachromosomal deletional process between switch regions. We have developed a plasmid-based transient transfection assay for SR to test for the presence of transacting switch activities. The plasmids are novel in that they lack a eukaryotic origin of DNA replication. The recombination activity of these switch substrates is restricted to a subset of B cell lines that support isotype switching on their endogenous loci and to mitogen-activated normal splenic B cells. The factors required for extrachromosomal plasmid recombination are constitutively expressed in proliferating splenic B cells and in B cell lines capable of inducibly undergoing immunoglobulin SR on their chromosomal genes. These studies suggest that mitogens that induce switching on the chromosome induce accessibility rather than switch recombinase activity. Finally, we provide evidence for two distinct switching activities which independently mediate mu-->alpha and mu-->gamma3 SR.

Smads are signal transducers for members of the transforming growth factor-beta (TGF-beta) superfamily. Upon ligand stimulation, receptor-regulated Smads (R-Smads) are phosphorylated by serine/threonine kinase receptors, form complexes with common-partner Smad, and translocate into the nucleus, where they regulate the transcription of target genes together with other transcription factors. Polyomavirus enhancer binding protein 2/core binding factor (PEBP2/CBF) is a transcription factor complex composed of alpha and beta subunits. The alpha subunits of PEBP2/CBF, which contain the highly conserved Runt domain, play essential roles in hematopoiesis and osteogenesis. Here we show that three mammalian alpha subunits of PEBP2/CBF form complexes with R-Smads that act in TGF-beta/activin pathways as well as those acting in bone morphogenetic protein (BMP) pathways. Among them, PEBP2alphaC/CBFA3/AML2 forms a complex with Smad3 and stimulates transcription of the germline Ig Calpha promoter in a cooperative manner, for which binding of both factors to their specific binding sites is essential. PEBP2 may thus be a nuclear target of TGF-beta/BMP signaling.

TGF-beta1 directs class switching to IgA by splenic B cells and by the surface IgM+ B cell line, I.29mu, by inducing germline (GL) Ig alpha transcripts. The promoter segment between -130 and +46, relative to the first initiation site for mouse GL alpha transcripts, is sufficient for expression and TGF-beta1 inducibility of a reporter gene in B cell lines. Within this segment resides a TGF-beta1-responsive element (TbetaRE) that is required for induction of the promoter by TGF-beta1 and, when multimerized, is sufficient to transfer TGF-beta1 inducibility to another promoter. In this report we show that a TGF-beta1-inducible complex binds the TbetaRE and contains the transcription factor core-binding factor (CBF; also known as acute myeloid leukemia, AML). Although all three CBF alpha family members activate the GL alpha promoter, only CBF alpha3 (AML-2) is induced by TGF-beta1 in splenic B and I.29mu cells. The TbetaRE contains two CBF binding sites. Mutation of both sites reduces but does not eliminate induction of the GL alpha promoter by TGF-beta1 or by overexpression of CBF, possibly due to the presence of an additional CBF site in the promoter. In addition, the TbetaRE contains two copies of another sequence motif. Mutation of these motifs eliminates TGF-beta1 induction of the GL alpha promoter. Together the data indicate that TGF-beta1 induction of the alpha promoter involves induction of CBF alpha3, which binds to the TbetaRE of the promoter along with one or more proteins.