Illegitimate recombination generates a class switch from C mu to C delta in an IgD-secreting plasmacytoma.

We present here the sequence characterization of a C mu----C delta immunoglobulin (Ig) heavy chain class switch. In the murine IgD-secreting plasmacytoma TEPC 1017, deletion of most of the mu switch recombination region (S mu) and the entire C mu gene occurred in the absence of switch region sequences 5' to C delta. This unique rearrangement resulted from an illegitimate recombination of sequences with only patchy homology to each other. The infrequent and variable nature of illegitimate recombination may explain the low frequency of IgD-secreting plasma cells in normal mouse tissues.     

Negative regulation of activation-induced cytidine deaminase in B cells

Both class switch recombination (CSR) and somatic hypermutation (SHM) of the Ig genes require the activity of activation-induced cytidine deaminase (AID). Expression of AID is restricted to B cells in the germinal centers of the lymphoid organs, where activated B cells undergo CSR and SHM. We previously showed that constitutive and systemic expression of AID leads to tumorigenesis in T cells and lung epithelium, but not in B cells. This finding led us to suspect that transgenic AID may be inactivated at least in part in B cells. To address this issue, we generated conditional AID-transgenic mice that constitutively express AID only in B cells. Studies on the cross between the AID-transgenic and AID-deficient mice showed that abundant AID protein accumulated by constitutive expression is inactivated in B cells, possibly providing an explanation for the absence of deregulation of CSR and SHM in AID-transgenic B cells.     

Class switch recombination is IgG1 specific on active and inactive IgH loci of IgG1-secreting B-cell blasts

Mouse B lymphocytes can be activated polyclonally by bacterial lipopolysaccharide (LPS) to secrete Ig and perform Ig class switch. In the presence of the T-cell lymphokine B-cell differentiation factor, the frequency of IgG1-secreting cells is drastically enhanced. We show here that IgG1-secreting B cells isolated from such cultures have undergone a similar DNA rearrangement of the switch regions (S mu, S gamma 1) of the Ig heavy chain constant region genes C mu and C gamma 1 on both active and inactive IgH loci. This result argues against a stochastic model of class switch recombination and suggests programmed class-specific switch recombination in the case of the switch to IgG1. In accord with this notion, cells expressing IgM but not IgG on the surface have not deleted or rearranged C mu or S gamma 1 on either chromosome.     

PKA-mediated phosphorylation regulates the function of activation-induced deaminase (AID) in B cells

During humoral immune responses, two distinct genetic modification events diversify the Ig genes in germinal center (GC) B cells: somatic hypermutation and class switch recombination (CSR). Both processes require the activity of activation-induced cytidine deaminase (AID), an enzyme expressed specifically in GC B cells. However, the mechanisms that regulate AID activity are largely unknown. Here we report that protein kinase A (PKA) phosphorylates AID and regulates its activity in GC B cells. AID physically interacts with the PKA holoenzyme in the cytoplasm and is phosphorylated by the PKA catalytic subunit at specific residues. AID phosphorylation is required for CSR, because substitution of the two phosphorylation targets impairs its ability to rescue CSR in AID-deficient B cells. Pharmacologic inhibition of PKA prevents isotype class switching in a murine B-cell lymphoma cell line; conversely, B cells from mice where PKA activity is made constitutive by conditional deletion of the PKA regulatory subunit gene display enhanced CSR. These findings implicate PKA in the regulation of AID function and suggest that the control of T cell-dependent immune responses may be modulated, via AID, by signals that activate PKA.     

Expression of activation-induced cytidine deaminase is regulated by cell division, providing a mechanistic basis for division-linked class switch recombination

Class switch recombination (CSR) is the process by which B cells alter the effector function properties of their Ig molecules. The decision to switch to a particular Ig isotype is determined primarily by the mode of B cell activation and cytokine exposure. More recent work indicates that the likelihood or probability of switching increases with successive cell divisions and is largely independent of time. We have analyzed different molecular features of CSR using cell division as a reference point in an attempt to gain insight into the mechanism of division-linked switching. Our results indicated that the accessibility of Ig heavy chain constant regions targeted for CSR was established after the cells had undergone a single cell division and did not vary significantly with subsequent cell divisions. In contrast, expression of activation-induced cytidine deaminase (AID) mRNA was found to increase with successive divisions, exhibiting a striking correlation with the frequency of CSR. Levels of AID in a given division remained constant at different time points, strongly suggesting that the regulation of AID expression was division-linked and independent of time. In addition, constitutive AID expression from a transgene accelerated division-linked CSR. Thus, we propose that the division-linked increase in AID expression provides an underlying molecular explanation for division-linked CSR.     

M17, a gene specific for germinal center (GC) B cells and a prognostic marker for GC B-cell lymphomas, is dispensable for the GC reaction in mice

In T-cell-dependent antibody responses, antigen-specific B cells undergo a phase of secondary antibody diversification in germinal centers (GCs). Somatic hypermutation (SHM) introduces mutations into the rearranged immunoglobulin (Ig) variable (V) region genes, and class-switch recombination (CSR) alters the Ig heavy (H) chain constant region. Aberrant SHM or CSR is thought to contribute to the development of GC-derived B-cell malignancies. Diffuse large B-cell lymphomas (DLBCLs) are a heterogeneous group of such GC-derived tumors. Based on their gene expression profile, DLBCLs can be divided into activated B-cell-like and GC-like subgroups. The human gene HGAL is predominantly expressed in GCs. It is also part of the gene expression signature of GC-like DLBCL, and its high expression in DLBCL has been associated with a better clinical prognosis. We have generated mice deficient of the HGAL homologue M17 in order to investigate its functional significance. The mutant animals form normal GCs, undergo efficient CSR and SHM, and mount T-cell-dependent antibody responses similar to wild-type controls. Thus, M17 is dispensable for the GC reaction, and its potential function in the pathogenesis of DLBCL remains elusive.     

Histone chaperone Spt6 is required for class switch recombination but not somatic hypermutation

Activation-induced cytidine deaminase (AID) is shown to be essential and sufficient to induce two genetic alterations in the Ig loci: class switch recombination (CSR) and somatic hypermutation (SHM). However, it is still unknown how a single-molecule AID differentially regulates CSR and SHM. Here we identified Spt6 as an AID-interacting protein by yeast two-hybrid screening and immunoprecipitation followed by mass spectrometry. Knockdown of Spt6 resulted in severe reduction of CSR in both the endogenous Ig locus in B cells and an artificial substrate in fibroblast cells. Conversely, knockdown of Spt6 did not reduce but slightly enhanced SHM in an artificial substrate in B cells, indicating that Spt6 is required for AID to induce CSR but not SHM. These results suggest that Spt6 is involved in differential regulation of CSR and SHM by AID.     

Transcriptional regulatory elements stimulate recombination in extrachromosomal substrates carrying immunoglobulin switch-region sequences.

We have developed a sensitive genetic assay to analyze DNA sequences and regulatory elements required for immunoglobulin heavy chain isotype switch recombination. Recombination substrates containing mu and gamma 3 chain switch (S)-region sequences, S mu and S gamma 3, are transiently introduced into primary murine B cells cultured with lipopolysaccharide to induce isotype switching. Recombination involving S-region sequences deletes a conditionally lethal marker, the leftward promoter of phage lambda (lambda PL), enabling recovered plasmids to transform Escherichia coli. In substrates carrying S mu-lambda PL-S gamma 3, about 2% of replicated molecules undergo deletion of lambda PL during transfection; insertion of either the immunoglobulin heavy chain promoter and enhancer sequences or cytomegalovirus IE1 promoter region upstream of S mu increases recombination 10-fold or more to 25% of replicated molecules. Guanosine-rich S-region sequences are essential for efficient recombination of these substrates.     

AID-induced decrease in topoisomerase 1 induces DNA structural alteration and DNA cleavage for class switch recombination

To initiate class switch recombination (CSR) activation-induced cytidine deaminase (AID) induces staggered nick cleavage in the S region, which lies 5′ to each Ig constant region gene and is rich in palindromic sequences. Topoisomerase 1 (Top1) controls the supercoiling of DNA by nicking, rotating, and religating one strand of DNA. Curiously, Top1 reduction or AID overexpression causes the genomic instability. Here, we report that the inactivation of Top1 by its specific inhibitor camptothecin drastically blocked both the S region cleavage and CSR, indicating that Top1 is responsible for the S region cleavage in CSR. Surprisingly, AID expression suppressed Top1 mRNA translation and reduced its protein level. In addition, the decrease in the Top1 protein by RNA-mediated knockdown augmented the AID-dependent S region cleavage, as well as CSR. Furthermore, Top1 reduction altered DNA structure of the Sμ region. Taken together, AID-induced Top1 reduction alters S region DNA structure probably to non-B form, on which Top1 can introduce nicks but cannot religate, resulting in S region cleavage.     

Artemis-independent functions of DNA-dependent protein kinase in Ig heavy chain class switch recombination and development

Assembly of Ig genes in B lineage cells involves two distinct DNA rearrangements. In early B cell development, site-specific double strand breaks (DSBs) at germ-line V, D, and J gene segments are joined via nonhomologous end-joining (NHEJ) to form variable region exons. Activated mature B cells can change expressed Ig heavy chain constant region exons by class switch recombination (CSR), which also involves DSB intermediates. Absence of any known NHEJ factor severely impairs joining of cleaved V, D, and J segments. In NHEJ, DNA-dependent protein kinase (DNA-PK), which is comprised of the Ku70/80 end-binding heterodimer and the catalytic subunit (DNA-PKcs), activates Artemis to generate a nuclease that processes DSBs before ligation. Because inactivation of DNA-PKcs components also severely affects CSR, we tested whether DNA-PK also functions in CSR via activation of Artemis. To obviate the requirement for V(D)J recombination, we generated DNA-PKcs- and Artemis-deficient B cells that harbored preassembled Ig heavy chain and kappa-light chain "knock-in" (HL) alleles. We found that Artemis-deficient HL B cells undergo robust CSR, indicating that DNA-PKcs functions in CSR via an Artemis-independent mechanism. To further elucidate potential Artemis-independent functions of DNA-PKcs, we asked whether the embryonic lethality associated with double-deficiency for DNA-PKcs and the related ataxia-telangiectasia-mutated (ATM) kinase was also observed in mice doubly deficient for ATM and Artemis. We found that ATM/Artemis double-deficient mice were viable and born in normal Mendelian numbers. Therefore, we conclude that DNA-PKcs has Artemis-independent functions in CSR and normal development.     

Impaired induction of DNA lesions during immunoglobulin class-switch recombination in humans influences end-joining repair

Ig class-switch recombination (CSR) is a region-specific process that exchanges the constant Ig heavy-chain region and thus modifies an antibody's effector function. DNA lesions in switch (S) regions are induced by activation-induced cytidine deaminase (AID) and uracil-DNA glycosylase 2 (UNG2), subsequently processed to DNA breaks, and resolved by either the classical nonhomologous end-joining pathway or the alternative end-joining pathway (XRCC4/DNA ligase 4- and/or Ku70/Ku80-independent and prone to increased microhomology usage). We examined whether the induction of DNA lesions influences DNA end-joining during CSR by analyzing Sμ-Sα recombination junctions in various human Ig CSR defects of DNA lesion induction. We observed a progressive trend toward the usage of microhomology in Sμ-Sα recombination junctions from AID-heterozygous to AID-autosomal dominant to UNG2-deficient B lymphocytes. We thus hypothesize that impaired induction of DNA lesions in S regions during CSR leads to unusual end-processing of the DNA breaks, resulting in microhomology-mediated end-joining, which could be an indication for preferential processing by alternative end-joining rather than by classical nonhomologous end-joining.     

Overlapping activation-induced cytidine deaminase hotspot motifs in Ig class-switch recombination

Ig class-switch recombination (CSR) is directed by the long and repetitive switch regions and requires activation-induced cytidine deaminase (AID). One of the conserved switch-region sequence motifs (AGCT) is a preferred site for AID-mediated DNA-cytosine deamination. By using somatic gene targeting and recombinase-mediated cassette exchange, we established a cell line-based CSR assay that allows manipulation of switch sequences at the endogenous locus. We show that AGCT is only one of a family of four WGCW motifs in the switch region that can facilitate CSR. We go on to show that it is the overlap of AID hotspots at WGCW sites on the top and bottom strands that is critical. This finding leads to a much clearer model for the difference between CSR and somatic hypermutation.     

Somatic rearrangements forming active immunoglobulin mu genes in B and T lymphoid cell lines.

We have cloned an active gene for an immunoglobulin mu heavy (H) chain, bearing the variable (VH), joining (JH), and constant (C mu) sequences expressed in the IgM-secreting mouse plasmacytoma HPC-76. The mu gene was formed by somatic recombination between a VH gene and one of several JH genes, which are located about 7.7 kilobase pairs from the C mu gene in embryo DNA. The JH-C mu intervening sequence has suffered a deletion of about 2.7 kilobase pairs in HPC-76. Because the delection encompasses sequences required to switch an expressed VH-JH gene from C mu to another CH gene, it may represent a mechanism for "freezing" a lymphocyte clone at the stage of IgM expression. For the second (inactive) C mu allele in HPC-76, the entire joining and switch regions have been deleted; functional inactivation of one allele may thus represent one mechanism by which a lymphocyte clone restricts expression to a single allele (allelic exclusion). Probes generated from the cloned mu gene allowed examination of the JH locus in B, Abelson "pre-B," and T lymphoma cell lines and a myeloid line, all of which cotain RNA species bearing C mu sequences. The B and pre-B lines exhibited recombination within both alleles of the JH locus, suggesting that both alleles may be expressed in some cells. The absence of the JH gene 5' to the recombination sites favors a deletion mechanism for VH-JH joining. Recombination within the JH locus was also detected in two out of four T lymphoma lines, but not in the myeloid line. This indicates that the mechanism by which B cells generate immunoglobulin diversity is operational in some T cells. Lines that synthesize mu RNA without JH rearrangement may have activated the C mu gene directly or have undergone recombination at a more distant locus.