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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)-β1 is essential for switch recombination to IgA due to its ability to induce transcription from GL Ig α genes. It has been shown that the promoters which regulate transcription of mouse and human GL α RNAs contain a TGF-β1-responsive element that binds Smad and core binding factor (CBFα)/AML/PEBPα/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 α 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 α promoter. Binding of Elf-1 to the GL α promoter is inducible by lipopolysaccharide in nuclear extracts from splenic B cells. An NF-κB 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-κB/p50-deficient mice, these data support the hypothesis that NF-κB 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 α promoter.

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.

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.

We have compared the synthesis and processing of immunoglobulin alpha chains in two murine cell lines, a B cell lymphoma that expresses membrane-bound IgA and a hybridoma that secretes IgA. Results of biosynthetic labeling experiments demonstrated that membrane-bound and secreted alpha chains have two distinct intracellular precursors, of different molecular weights and isoelectric points. RNAs from both of these cell lines direct the synthesis in vitro of two alpha polypeptides of Mr 59,000 and 62,000, the larger one being the precursor for membrane-bound alpha chain and the smaller one being the precursor for secreted alpha chain. These cell lines each contain three RNAs, 1.7, 2.1, and 3.1 kilobases in length, which hybridize with cDNA for the alpha constant region and which are present in different concentrations. Our results suggest that the smallest RNA encodes the secreted alpha chain and one or both of the larger RNAs encode(s) the membrane-bound alpha chain.

Immunoglobulin heavy-chain switching is effected by recombination events between sites associated with tandemly repeated switch sequences located 5' to immunoglobulin heavy-chain genes. Using the band mobility shift assay, we have identified two distinct sites 5' to the alpha heavy-chain switch sequence with affinity for a single B-cell-specific DNA-binding protein, S alpha-BP. S alpha-BP was present in nuclear extracts from pre-B and B cells but was not detected in extracts from plasmacytomas, B-cell hybridomas, T-cell lymphomas, or a macrophage cell line. It was also not detectable in other nonlymphoid cells tested. Evidence suggests there are S alpha-BP-binding sites near other immunoglobulin switch sequences. As with the S alpha sites, these sites appear to be distinct from the consensus tandem repeats characteristic of immunoglobulin switch sequences. The possible functions of S alpha-BP on contacting its binding sites are discussed in the context of immunoglobulin heavy-chain switch recombination.

Immunoglobulin heavy-chain switching is effected by a DNA recombination event that replaces the C mu gene with one of the other heavy-chain constant-region (CH) genes located 3' to the C mu gene. How the specificity of this event is controlled is unknown. However, it has been shown that IgM+ cells capable of switching to specific isotypes have the corresponding unrearranged CH genes in an accessible or active chromatin state, as demonstrated by the fact that these specific CH genes are hypomethylated and are transcriptionally active. We now report that the RNAs transcribed from specific unrearranged CH genes are induced prior to switching under conditions that promote switching to these specific CH genes. For example, we find that bacterial lipopolysaccharide, which induces the IgM+ cell line I.29 mu to switch to IgA, induces transcripts from the germ-line C alpha gene(s) in I.29 mu cells prior to switch recombination. Two preparations of T-cell lymphokines (recombinant interleukin 4 and supernatant from the T-cell line 2.19, which contains interleukins 4 and 5) that promote switching to specific isotypes by lipopolysaccharide-treated spleen cells induce transcripts from the corresponding germ-line CH genes prior to expression of the new isotypes. For example, interleukin 4, which appears to be necessary for switching to IgE in vitro and in vivo, induces within 2 days large increases in germ-line C epsilon transcripts in lipopolysaccharide-treated spleen cells and in I.29 mu cells. The most straightforward interpretation of our data is that these lymphokines direct switching to specific isotypes by activating specific CH genes, making them accessible to the putative switch recombinase.

Antibody class switching is regulated by transcription of unrearranged C_H genes to produce germline (GL) transcripts which direct the choice of isotype and are required for switching. However, their role is unknown. GL transcripts are initiated at the I exons located upstream of each switch region. Although deletion of the I exon by gene targeting prevents switch recombination to that C_H gene, the I_α exon can be replaced by an entirely different DNA segment, a minigene driven by the phosphoglycerate kinase (PGK) promoter and encoding hypoxanthine phosphoribosyl transferase (HPRT), oriented in the sense direction, without reducing antibody class switching to IgA. To understand why HPRT substitution of the I_α exon does not disrupt switch recombination, we have analyzed the structure of the transcript from the targeted allele in these mice. We identify a spliced transcript in which the HPRT exons are spliced to the C_α gene segments, resulting in a structure similar to normal GL transcripts. The abundance of this transcript is similar to that of the normal α GL RNA. We also demonstrate that switching to the four IgG subclasses in B cells from these mice is reduced in comparison to wild-type mice. We discuss the possibility that the strong PGK promoter inserted at the Ig α locus may interfere with interaction of the promoters for γ GL transcripts with the 3′ IgH enhancer.
Keywords:Ig class switching, gene targeting, germline transcripts

Signal transducer and activator of transcription 6 (Stat6) and NF-kappaB are widely distributed transcription factors which are induced by different stimuli and bind to distinct DNA sequence motifs. Interleukin-4 (IL-4), which activates Stat6, synergizes with activators of NF-kappaB to induce IL-4-responsive genes, but the molecular mechanism of this synergy is poorly understood. Using glutathione S-transferase pulldown assays and coimmunoprecipitation techniques, we find that NF-kappaB and tyrosine-phosphorylated Stat6 can directly bind each other in vitro and in vivo. An IL-4-inducible reporter gene containing both cognate binding sites in the promoter is synergistically activated in the presence of IL-4 when Stat6 and NF-kappaB proteins are coexpressed in human embryonic kidney 293 (HEK 293) cells. The same IL-4-inducible reporter gene is also synergistically activated by the endogenous Stat6 and NF-kappaB proteins in IL-4-stimulated I.29mu B lymphoma cells. Furthermore, Stat6 and NF-kappaB bind cooperatively to a DNA probe containing both sites, and the presence of a complex formed by their cooperative binding correlates with the synergistic activation of the promoter by Stat6 and NF-kappaB. We conclude that the direct interaction between Stat6 and NF-kappaB may provide a basis for synergistic activation of transcription by IL-4 and activators of NF-kappaB.

The B cell lymphoma I.29 consists of a mixture of cells expressing membrane-bound immunoglobulin M (IgM) (lambda) and IgA (lambda) of identical idiotypes. Whereas most of the cells express either IgM or IgA alone, 1 to 5% of the cells in this tumor express IgM and IgA simultaneously within the cytoplasm and on the cell membrane (R. Sitia et al., J. Immunol. 127:1388-1394, 1981; R. Sitia, unpublished data). When IgM+ cells are purified from the lymphoma and passaged in mice or cultured, a portion of the cells convert to IgA+. These properties suggest that some cells of the I.29 lymphoma may undergo immunoglobulin heavy chain switching, although it is also possible that the mixed population was derived by a prior switching event in a clone of cells. We performed Southern blotting experiments on genomic DNAs isolated from populations of I.29 cells containing variable proportions of IgM+ and IgA+ cells and on a number of cell lines derived from the lymphoma. The results were consistent with the deletion model for heavy chain switching, as the IgM+ cells contained rearranged mu genes and alpha genes in the germ line configuration on both the expressed and nonexpressed heavy chain chromosomes, whereas the IgA+ cells had deleted both mu genes and contained one rearranged and one germ line alpha gene. In addition, segments of DNA located within the intervening sequence 5' to the mu gene, near the site of switch recombination, were deleted from both the expressed and the nonexpressed chromosomes. Although mu genes were deleted from both chromosomes in the IgA+ cells, the sites of DNA recombination differed on the two chromosomes. On the expressed chromosome, Smu sequences were recombined with S alpha sequences, whereas on the nonexpressed chromosome, Smu sequences were recombined with S gamma 3 sequences.

The murine B cell lymphoma I.29 contains cells expressing surface IgM or IgA with identical heavy chain variable regions (9, 25, and D. Klein and J. Stavnezer, unpublished data). Purified IgM+ cells from the lymphoma have been adapted to culture and induced to switch to IgA, IgE, or IgG2 by treatment with lipopolysaccharide (LPS) or by treatment with a monoclonal anti-I.29 antiidiotype plus LPS. Clones of IgM+ cells have been obtained and induced to switch. Under optimal conditions, 30% of the cells in the culture expressed IgA 8 d after the inducers were added, and by 15 d 90% of the cells were IgA+. In actively switching cultures, up to 50% of the cells whose cytoplasm stained positively with anti-IgA stained simultaneously with anti-IgM, which indicates that the appearance of IgA+ cells in the cultures was due to isotype switching and not to clonal outgrowth. Examination by Southern blotting experiments of the Ig heavy chain genes in I.29 cells before and after switching revealed that isotype switching was accompanied by DNA recombinations that occurred within or immediately 5' to the tandemly repeated switch sequences. Within 3 d after the addition of inducers of switching, the nonexpressed chromosome underwent a variety of deletions or expansions within the S mu region, and a portion of the S alpha regions had undergone a 0.9-kb deletion. In cultures that contained at least 12% IgA+ cells, rearranged, expressed alpha genes, produced by recombination between the S mu region within the expressed mu gene and the S alpha region, were detected.

Considerable evidence has accumulated suggesting that the antigen receptor(s) on T cells is coded for by genes for the variable (V) region of the immunoglobulin heavy (H) chains. In B cells, a complete gene for the immunoglobulin VH region is formed by somatic recombination of VH and joining region heavy chain (JH) gene segments [through an intermediate diversity(D) region gene segment]. In an attempt to determine whether a complete immunoglobulin VH region is expressed on T cells that bear an antigen receptor, we analyzed the restriction map of the JH-C mu locus in genomic DNA from two cloned murine cytotoxic T-lymphocyte (CTL) lines specific for the x-ray-induced leukemia RL male 1. We found no rearrangement of the JH C mu locus in the CTL lines, indicating that the T-cell antigen receptor(s) in these CTLs is not coded for by a complete immunoglobulin VH gene formed by joining of VH, (DH), and JH genes. In addition, we determined that C mu genes on both chromosomes were present and that there was no rearrangement of the C alpha, C kappa, or lambda chain genes in these CTL cells.

The heavy-chain switch from immunoglobulin M (IgM) expression to IgA expression is mediated by a recombination event between segments of DNA called switch regions. The switch regions lie two to six kilobases upstream of the mu and alpha constant region coding segments. Switch recombination to IgA expression results in a recombinant mu-alpha switch region upstream of the expressed alpha constant region gene. We have characterized the products of switch recombination by a lymphoma cell line, I.29. Two sets of molecular clones represent the expected products of simple mu to alpha switches. Five members of a third set of molecular clones share the same recombination site in both the mu and the alpha switch regions, implying that the five molecular clones were derived from a single switch recombination event. Surprisingly, the five clones fall into two sets of sequences, which differ from each other by several point mutations and small deletions. Duplication of switch region sequences are also found in these five molecular clones. An explanation for these data is that switch recombination involves DNA synthesis, which results in nucleotide substitutions, small deletions, and duplications.