Regulation Of Class Switch Recombination Research Articles

B cell class switch recombination is regulated by DYRK1A through MSH6 phosphorylation

Protection from viral infections depends on immunoglobulin isotype switching, which endows antibodies with effector functions. Here, we find that the protein kinase DYRK1A is essential for B cell-mediated protection from viral infection and effective vaccination through regulation of class switch recombination (CSR). Dyrk1a-deficient B cells are impaired in CSR activity in vivo and in vitro. Phosphoproteomic screens and kinase-activity assays identify MSH6, a DNA mismatch repair protein, as a direct substrate for DYRK1A, and deletion of a single phosphorylation site impaired CSR. After CSR and germinal center (GC) seeding, DYRK1A is required for attenuation of B cell proliferation. These findings demonstrate DYRK1A-mediated biological mechanisms of B cell immune responses that may be used for therapeutic manipulation in antibody-mediated autoimmunity.

Lung memory B cells promote local IgE production in the respiratory tract

Abstract Allergen-specific IgE mediates allergic asthma. IgE responses including class-switch recombination (CSR) to IgE are tightly regulated. However, the regulation of local CSR to IgE at mucosal sites is poorly understood. This study aims to elucidate cellular sources that promote IgE CSR and mechanisms that regulate IgE production in the respiratory tract. To model allergic airway inflammation, we repeatedly administered intranasal ragweed pollen and OVA to mice. We profiled B cell responses to OVA in the lung, the lung-draining mediastinal lymph nodes (medLN), spleen and bone marrow. We used fluorescent reporter mice to track IgE CSR and identify B cell subsets that promote IgE CSR by flow cytometry and microscopy. We found that intranasal allergen exposure elicited robust local B cell responses with the formation of OVA-specific B cells in the medLN and OVA-specific IgE in the airway. OVA-specific germinal center B cells and memory B cells (MBCs) accumulated in the lungs. Using fluorescent reporter mice, we showed that IgG1+ MBCs are the predominant IgE-switching cells. Importantly, the number of IgE-switching MBCs are the highest in the lungs, and IgG1+ MBCs exhibit an increased frequency of IgE CSR in the lungs compared to lymphoid tissues. These suggest that the lung environment favors IgE production. RNA-seq analyses comparing lung and circulating MBCs revealed elevated expression of Tnfsf4 (OX40L) and Icosl on lung MBCs, suggesting a higher propensity for lung MBCs to engage T cells. Together, these data demonstrate that the allergic lung is a major site of IgE CSR with IgG1+ MBCs promoting local IgE CSR. Our ongoing work focuses on delineating the B cell-intrinsic and extrinsic factors that promote IgE production in the respiratory mucosa. Supported by grants from NIH (1F31 HL156459, 5T32 AI007508, 1R21 AI159456)

MicroRNA-directed pathway discovery elucidates an miR-221/222-mediated regulatory circuit in class switch recombination.

MicroRNAs (miRNAs, miRs) regulate cell fate decisions by post-transcriptionally tuning networks of mRNA targets. We used miRNA-directed pathway discovery to reveal a regulatory circuit that influences Ig class switch recombination (CSR). We developed a system to deplete mature, activated B cells of miRNAs, and performed a rescue screen that identified the miR-221/222 family as a positive regulator of CSR. Endogenous miR-221/222 regulated B cell CSR to IgE and IgG1 in vitro, and miR-221/222-deficient mice exhibited defective IgE production in allergic airway challenge and polyclonal B cell activation models in vivo. We combined comparative Ago2-HITS-CLIP and gene expression analyses to identify mRNAs bound and regulated by miR-221/222 in primary B cells. Interrogation of these putative direct targets uncovered functionally relevant downstream genes. Genetic depletion or pharmacological inhibition of Foxp1 and Arid1a confirmed their roles as key modulators of CSR to IgE and IgG1.

The uncharacterized SANT and BTB domain-containing protein SANBR inhibits class switch recombination

Class switch recombination (CSR) is the process by which B cells switch production from IgM/IgD to other immunoglobulin isotypes, enabling them to mount an effective immune response against pathogens. Timely resolution of CSR prevents damage due to an uncontrolled and prolonged immune response. While many positive regulators of CSR have been described, negative regulators of CSR are relatively unknown. Using an shRNA library screen targeting more than 28,000 genes in a mouse B cell line, we have identified a novel, uncharacterized protein of 82kD (KIAA1841, NM_027860), which we have named SANBR (SANT and BTB domain regulator of CSR), as a negative regulator of CSR. The purified, recombinant BTB domain of SANBR exhibited characteristic properties such as homodimerization and interaction with corepressor proteins, including HDAC and SMRT. Overexpression of SANBR inhibited CSR in primary mouse splenic B cells, and inhibition of CSR is dependent on the BTB domain while the SANT domain is largely dispensable. Thus, we have identified a new member of the BTB family that serves as a negative regulator of CSR. Future investigations to identify transcriptional targets of SANBR in B cells will reveal further insights into the specific mechanisms by which SANBR regulates CSR as well as fundamental gene regulatory activities of this protein.

The role of protein phosphorylation in the regulation of class switch recombination

Antibody is an important part of adaptive immune system and is produced only by B cells. There are five main classes (IgM, IgD, IgG, IgA, IgE) and some subclasses in antibodies. IgM and IgD are produced by mature naïve B ce... | Find, read and cite all the research you need on Tech Science Press

Mechanism and regulation of class switch recombination by IgH transcriptional control elements.

Class switch recombination (CSR) plays an important role in humoral immunity by generating antibodies with different effector functions. CSR to a particular antibody isotype is induced by external stimuli, and occurs between highly repetitive switch (S) sequences. CSR requires transcription across S regions, which generates long non-coding RNAs and secondary structures that promote accessibility of S sequences to activation-induced cytidine deaminase (AID). AID initiates DNA double-strand breaks (DSBs) intermediates that are repaired by general DNA repair pathways. Switch transcription is controlled by various regulatory elements, including enhancers and insulators. The current paradigm posits that transcriptional control of CSR involves long-range chromatin interactions between regulatory elements and chromatin loops-stabilizing factors, which promote alignment of partner S regions in a CSR centre (CSRC) and initiation of CSR. In this review, we focus on the role of IgH transcriptional control elements in CSR and the chromatin-based mechanisms underlying this control.

Bacterial amyloids from biofilms break tolerance in lupus by simultaneous BCR/TLR signaling

Abstract Systemic lupus erythematosus (SLE) is a multifactorial autoimmune disease, in which infections are considered to play a pathogenic role, but the cellular and molecular culprits remain unknown. Bacterial biofilms are multicellular bacterial communities important in the establishment of chronic infection by pathogens. We have previously shown that curli fibers, functional bacterial amyloids present in Salmonella and E. coli biofilms, can complex with DNA. Systemic administration of these curli/DNA complexes accelerated autoimmunity in lupus-prone mice and induced autoantibody production in wild-type mice, suggesting curli/DNA complexes as novel players in SLE pathogenesis. This affect was diminished in TLR2 or TLR9 deficient mice. We present here evidence that curli/DNA complexes polyclonally activate B cells in vivo in wildtype mice, lupus-prone mice and 3H9 mice, the latter expressing an anti-DNA Ig heavy chain and whose B cells are normally tolerized. Interestingly, curli/DNA complexes can activate B cells in the absence of T cell help. Indeed, curli/DNA complexes induce polyclonal proliferation and activation in B cells purified from wild-type and lupus prone mice in vitro. Interestingly, curli/DNA complexes also induce isotype switching and aicda, the master regulator of class switch recombination, in the absence of T cells help in vitro. The induction of non-canonical NFkB in the absence of T cells suggests that the fibrillar structure of curli/DNA complexes can cross-link BCR, some recognizing DNA. Our results suggest that curli/DNA complexes may induce anti-DNA antibody production by simultaneous BCR/TLR signaling, which leads to B cell antibody production in the absence of T cell help.

Regulation of IgA Class Switch Recombination in Immunoglobulin A Nephropathy: Retinoic Acid Signaling and BATF

Background: Immunoglobulin (Ig) A nephropathy (IgAN) is the finding of immune deposits predominantly containing polymeric IgA in the glomerular mesangium on renal biopsy. Increasing evidence suggested that retinoic acid (RA) signaling selectively induces IgA isotype switching and basic leucine zipper transcription factor, ATF-like (BATF) controls the global regulators of class switch recombination (CSR) in lymphocytes. Great effort has been paid to identify whether impaired immune regulation along the ‘mucosa-bone marrow (BM) axis' play an important role in the pathogenesis of IgAN. Methods: The aim of the study was to investigate the expression of all-trans-RA (ATRA) and BATF, and to identify their impact on IgA CSR in IgAN patients and rat animal models. Blood samples and tonsillar tissue specimens were obtained from 22 patients with IgAN and 24 patients with chronic tonsillitis as control. Results: Immunohistochemical, RT-PCR and western blotting examination revealed that RA signaling and BATF productions are activated in IgAN patients compared with controls. Lipopolysaccharide and α-hemolytic streptococcus stimulation upregulated RA receptor (RAR) and BATF expression, promote IgA CSR and ATRA productions in tonsil mononuclear cells. RAR alpha (RARα) or BATF siRNA decreases IgA expression. We also built IgAN rat models and found that RARα, BATF and activation-induced cytidine deaminase were upregulated in the peripheral blood, spleen and BM. With ATRA (500 μg/kg body weight) treatment for 8 weeks, IgA deposition on glomeruli and mesangial cells proliferation increased. It also revealed that ATRA activated BATF and IgA CSR in vivo. Conclusion: These data point toward the role of RA signaling together with BATF in IgA CSR of IgAN, and the data also support the notion that mucosal immunization with neoantigen results in impaired mucosal and systemic IgA responses.

Murine germinal center B cells require functional Fms-like tyrosine kinase 3 signaling for IgG1 class-switch recombination

Switched antibody classes are important for efficient immune responses. Aberrant antibody production to otherwise harmless antigens may result in autoimmunity. The protein kinase fms-like tyrosine kinase 3 receptor (Flt3) has an important role during early B-cell development, but the role of Flt3 in peripheral B cells has not been assessed before. Herein we describe a previously unappreciated role for Flt3 in IgG1 class-switch recombination (CSR) and production. We show that Flt3 is reexpressed on B-cell lymphoma 6(+) germinal center B cells in vivo and following LPS activation of peripheral B cells in vitro. Absence of Flt3 signaling in Flt3 ligand-deficient mice results in impaired IgG1 CSR and accumulation of IgM-secreting plasma cells. On activated B cells, Flt3 is coexpressed and functions in synergy with the common-gamma chain receptor family. B cells from Flt3 ligand-deficient mice have impaired IL-4R signaling, with reduced phosphorylation of signal transducer and activator of transcription (Stat) 6, and demonstrate a failure to initiate CSR to IgG1 with low expression of γ1 germ-line transcripts, resulting in impaired IgG1 production. Thus, functional synergy between Flt3 and IL-4R signaling is critical for Stat-mediated regulation of sterile γ1 germ-line transcripts and CSR to IgG1.

B cells characterization in ADA2 Deficiency patients

ADA2 deficiency, a recently described disease, is characterized by systemic vasculopathy and episodes of strokes. The defect is due to a loss of function mutation of CECR1 gene, codifying for Adenosine Deaminase 2 protein. This protein regulates the catabolism of extracellular adenosine, which we have recently shown is an important regulator of Class Switch Recombination in B lymphocytes. Accordingly DADA2 patients can present hypogammaglobulinemia.

Absence of Activation-induced Cytidine Deaminase, a Regulator of Class Switch Recombination and Hypermutation in B Cells, Suppresses Aorta Allograft Vasculopathy in Mice.

Antibody-mediated rejection is caused in part by increasing circulation/production of donor-specific antibody (DSA). Activation-induced cytidine deaminase (AID) is a key regulator of class switch recombination and somatic hypermutation of immunoglobulin in B cells, yet its role in antibody-mediated transplant rejection remains unclear. We show here that AID deficiency in mice enables suppression of allograft vasculopathy (AV) after aorta transplantation, a DSA-mediated process. Splenocytes from C57BL/6 J (B6) AID(−/−) mice were used for determining in vitro proliferation responses, alloreactivity, cell surface marker expression, and antibody production. BALB/c mouse aortas were transplanted into B6 AID(−/−) mice with or without FK506 treatment. Blood and aorta grafts were harvested on day 30 after transplantation and were subjected to DSA, histological, and immunohistological analyses. The AID(−/−) splenocytes were comparable to wild type splenocytes in proliferation responses, alloreactivity, and expression of cell surface markers in vitro. However, they completely failed to produce immunoglobulin G, although they were not impaired in immunoglobulin M production relative to controls. Furthermore, BALB/c aorta grafts from B6 AID(−/−) recipient mice on day 30 after transplantation showed reduced signs of AV compared to the grafts from B6 wild type recipient mice which had severe vascular intimal hyperplasia, interstitial fibrosis, and inflammation. Treatment with FK506 produced a synergistic effect in the grafts from AID(−/−) recipients with further reduction of intimal hyperplasia and fibrosis scores. The AID deficiency inhibits DSA-mediated AV after aorta transplantation in mice. We propose that AID could be a novel molecular target for controlling antibody-mediated rejection in organ transplantation.

A new 3′RR regulatory role during lymphomagenesis

During B-cell ontogeny and maturation, Ig loci undergo successive transcription coupled remodeling events: V(D)J recombination and class switch recombination (CSR) for the IgH locus, and VJ recombination for IgL loci. Both loci also undergo somatic hypermutation (SHM) to improve antibody affinity. RAG-mediated V(D)J recombination occurs during the early antigen-independent step of B-cell development. AID-mediated CSR and SHM occur later, and require interaction with cognate antigen. These transcription-induced events involve a succession of DNA breaks and repairs, and thus represent potentially oncogenic events, hence implying a strict regulation. Ig cis-regulatory elements and especially transcriptional enhancers are major locus regulators. While 5′ cis-regulatory elements (Eμ and Eκi for IgH and Igκ locus, respectively) control V(D)J recombination, SHM is controlled by the IgH 3′ regulatory region (3′RR)1 (for IgH locus) and the 3′Eκ (for Igκ locus). The 3′RR is also the master regulator of CSR.2 Although less documented, transcriptional enhancers are present in the Igλ locus.3 Chromosomal translocations linking oncogenes to these elements are implicated in various B-cell malignancies.3-5 If the deregulation of the translocated oncogene by enhancer elements located in cis was extensively studied,4,5 little is known about a potential trans effect of the Ig enhancers, for example the role of the IgH 3′RR on lymphomagenesis caused by an oncogene translocation to the IgL locus. Indeed, studies have shown that deletion of the 3′RR modulates the B-cell fate by lowering the BCR signaling,6 which is of key importance in B-cell malignancies. We recently studied the impact of the absence of the 3′RR in mice carrying a transgene mimicking the translocation of c-myc under the transcriptional control of the Igλ enhancers.7 Our results indicate a wide alteration of B-cell lymphoma phenotypes. While wt mice indifferently develop mature or immature lymphomas, 3′RR-deficient mice exhibit a strong preference for immature lymphomas. Furthermore, 3′RR-deficiency leads to a lowered frequency of CD43+ activated mature B-cell lymphomas and to an increased frequency of CD5+ B-cell lymphomas. Transcription level of the c-myc transgene is identical in B-cell lymphomas from wt and 3′RR-deficient mice, suggesting that the 3′RR does not directly regulate the expression of the oncogene bring under the control of the Igλ enhancers.7 Two main models are suggested to explain the heterogeneity of B-cell malignancies: the various tumor subtypes may arise from different mutations within the same target B-cell or from the same mutation occurring during different stages of B-cell differentiation. Although these models are not mutually exclusive, our results reinforce the second hypothesis that the same initial oncogenic event occurring in B-cells with different maturity degree can lead to different B-lymphoma subtypes. Indeed, while wt and 3′RR deficient mice bear the same “mini-locus translocation” (i.e. c-myc under the transcriptional control of Igλ enhancers), they develop different tumor subtypes. The only difference that could explain this variation is the slightly altered B-cell maturation and the abolition of CSR and SHM in 3′RR-deficient mice. B-cells from both genetic backgrounds over express the myc oncogene, but need to accumulate secondary mutations to develop B-cell lymphomas (Fig. 1). Secondary mutations principally occur during the different genetic events that punctuate B-cell ontology. In wt B-cells, these oncogenic events can occur either during early step of maturation (i.e., during V(D)J recombination) or during the later stages (i.e. during CSR or SHM). CSR and SHM are abrogated in 3′RR-deficient mice,1,2 lowering the probability of oncogenic mutations during these later stages of B-cell maturation, hence their increased frequency of immature B-cell lymphomas as compared with wt mice. Figure 1. Schematic hypothesis on the implication of the 3′RR in the development of B-cell lymphomas induced by oncogene translocation in IgL locus. While not directly impacting the expression of the translocated oncogene, the absence of the 3′RR ... Taken together (Fig. 1), our results confirm the importance of considering both the cell of origin and the genetic alterations to determine the B-cell lymphoma phenotype and its behavior. Indeed, B-cells bearing the same initial oncogenic events can lead to different B-cell lymphoma subtypes if their development is impaired, while different mutations occurring in the same cell of origin can induce similar tumors.4 In this context, we also show that the 3′RR can affect the phenotype of B-cell lymphomas due to oncogene translocation in other loci than the IgH one, by acting on B-cell maturation and regulating potentially oncogenic recombination and mutations during late B-cell differentiation.

BANK1 transgenic mice exhibit lupus-associated B cell phenotypes (BA3P.105)

Abstract BANK1 is a B-cell adaptor protein that has been implicated in SLE and systemic sclerosis. Previous studies have interpreted that the overexpression of the full length BANK1 isoform plays a pathogenic and the truncated (∆BANK1) version plays a protective role in lupus. However, there is no mechanistic evidence showing that these 2 isoforms can impact lupus development differentially. We have recently generated 2 transgenic strains on the B6 background, BANK1Tg and ∆BANK1Tg, which will be used to address this knowledge gap. The BANK1Tg mice overexpressing the full length Bank1 under the control of CD19 promoter exhibited a dramatic skewing in the splenic B cell population to MZ and B1a cells with a reduction in FO B cells. These mice also exhibited increased plasma cells along with an increased level of total IgM and anti-dsDNA IgG antibodies. Microarray analysis revealed increased transcript levels of several class switched IgG antibodies with a concomitant increase in the level of activation induced deaminase (AID), a master regulator of class switch recombination (CSR). qPCR confirmed higher level of AID and CSR. Interestingly, these phenotypes are observed only in female Tg mice. In contrast, the ∆BANK1Tg mice don’t exhibit the above phenotypes, supporting the hypothesis that the truncated isoform may play a protective role. These studies indicate that the different isoforms of BANK1 observed in humans contribute differentially to the development of B-cell autoimmunity.

Activation-Induced Cytidine Deaminase in Antibody Diversification and Chromosome Translocation

DNA damage, rearrangement, and mutation of the human genome are the basis of carcinogenesis and thought to be avoided at all costs. An exception is the adaptive immune system where lymphocytes utilize programmed DNA damage to effect antigen receptor diversification. Both B and T lymphocytes diversify their antigen receptors through RAG1/2 mediated recombination, but B cells undergo two additional processes--somatic hypermutation (SHM) and class-switch recombination (CSR), both initiated by activation-induced cytidine deaminase (AID). AID deaminates cytidines in DNA resulting in U:G mismatches that are processed into point mutations in SHM or double-strand breaks in CSR. Although AID activity is focused at Immunoglobulin (Ig) gene loci, it also targets a wide array of non-Ig genes including oncogenes associated with lymphomas. Here, we review the molecular basis of AID regulation, targeting, and initiation of CSR and SHM, as well as AID's role in generating chromosome translocations that contribute to lymphomagenesis.

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.

The Role of Germline Promoters and I Exons in Cytokine-Induced Gene-Specific Class Switch Recombination

Germline transcription precedes class switch recombination (CSR). The promoter regions and I exons of these germline transcripts include binding sites for activation- and cytokine-induced transcription factors, and the promoter regions/I exons are essential for CSR. Therefore, it is a strong hypothesis that the promoter/I exons regions are responsible for much of cytokine-regulated, gene-specific CSR. We tested this hypothesis by swapping the germline promoter and I exons for the murine γ1 and γ2a H chain genes in a transgene of the entire H chain C-region locus. We found that the promoter/I exon for γ1 germline transcripts can direct robust IL-4-induced recombination to the γ2a gene. In contrast, the promoter/I exon for the γ2a germline transcripts works poorly in the context of the γ1 H chain gene, resulting in expression of γ1 H chains that is <1% the wild-type level. Nevertheless, the small amount of recombination to the chimeric γ1 gene is induced by IFN-γ. These results suggest that cytokine regulation of CSR, but not the magnitude of CSR, is regulated by the promoter/I exons.

Regulation of class switch recombination and somatic mutation by AID phosphorylation

Activation-induced cytidine deaminase (AID) is a mutator enzyme that initiates somatic mutation and class switch recombination in B lymphocytes by introducing uracil:guanine mismatches into DNA. Repair pathways process these mismatches to produce point mutations in the Ig variable region or double-stranded DNA breaks in the switch region DNA. However, AID can also produce off-target DNA damage, including mutations in oncogenes. Therefore, stringent regulation of AID is required for maintaining genomic stability during maturation of the antibody response. It has been proposed that AID phosphorylation at serine 38 (S38) regulates its activity, but this has not been tested in vivo. Using a combination of mass spectrometry and immunochemical approaches, we found that in addition to S38, AID is also phosphorylated at position threonine 140 (T140). Mutation of either S38 or T140 to alanine does not impact catalytic activity, but interferes with class switching and somatic hypermutation in vivo. This effect is particularly pronounced in haploinsufficient mice where AID levels are limited. Although S38 is equally important for both processes, T140 phosphorylation preferentially affects somatic mutation, suggesting that posttranslational modification might contribute to the choice between hypermutation and class switching.

APRIL affects antibody responses and early leukocyte infiltration, but not influenza A viral control

A proliferation inducing ligand (APRIL) is implicated in the regulation of class switch recombination to IgA in T-independent B cell responses. Since B cells play an important role in the immunity to influenza A virus and resistance against the virus is partly controlled by T-independent IgA B cell responses, we studied the role of APRIL during an influenza A infection in vivo. APRIL transgenic, wild-type and APRIL deficient mice were intranasally infected with a non-lethal dose of a mouse adapted strain of influenza A. Compared to wild-type mice, APRIL deficient mice showed a twofold reduction in the amount of macrophages in the lungs and a tendency towards decreased granulocyte influx in the early leukocyte recruitment phase. Although the T cell immune response against influenza was unaffected, APRIL Tg mice showed prolonged influenza-specific IgM production and differential class switching. Unexpectedly, the IgA B cell response was completely T helper cell dependent and also not affected by the absence or presence of APRIL. In addition, viral clearance and recovery from the infection was not influenced by APRIL. Combined these results indicate that APRIL affects specific aspects of the anti-influenza response, but plays a limited role in disease recovery.

Mechanism and Regulation of Class Switch Recombination

Antibody class switching occurs in mature B cells in response to antigen stimulation and costimulatory signals. It occurs by a unique type of intrachromosomal deletional recombination within special G-rich tandem repeated DNA sequences [called switch, or S, regions located upstream of each of the heavy chain constant (C(H)) region genes, except Cdelta]. The recombination is initiated by the B cell-specific activation-induced cytidine deaminase (AID), which deaminates cytosines in both the donor and acceptor S regions. AID activity converts several dC bases to dU bases in each S region, and the dU bases are then excised by the uracil DNA glycosylase UNG; the resulting abasic sites are nicked by apurinic/apyrimidinic endonuclease (APE). AID attacks both strands of transcriptionally active S regions, but how transcription promotes AID targeting is not entirely clear. Mismatch repair proteins are then involved in converting the resulting single-strand DNA breaks to double-strand breaks with DNA ends appropriate for end-joining recombination. Proteins required for the subsequent S-S recombination include DNA-PK, ATM, Mre11-Rad50-Nbs1, gammaH2AX, 53BP1, Mdc1, and XRCC4-ligase IV. These proteins are important for faithful joining of S regions, and in their absence aberrant recombination and chromosomal translocations involving S regions occur.

Identification of murine B cell lines that undergo somatic hypermutation focused to A:T and G:C residues

Activation-induced deaminase (AID) is the master regulator of class switch recombination (CSR) and somatic hypermutation (SHM), but the mechanisms regulating AID function are obscure. The differential pattern of switch plasmid activity in three IgM(+)/AID(+) and two IgG(+)/AID(+) B cell lines prompted an analysis of global gene expression to discover the origin of these cells. Gene profiling suggested that the IgG(+)/AID(+) B cell lines derived from germinal center B cells. Analysis of SHM potential demonstrates that the IgVkappa domains are inducibly diversified at high rate during in vitro culture. The mutation spectra focused to A:T base pairs, revealing a component of the hypermutation program that occurs preferentially during phase 2 of SHM. The A:T error spectra were analyzed and were not characteristic of polymerase eta activity. A differential pattern of three consensus motifs used for A:T base substitutions was observed in WT and Poleta-, Msh2- and Msh6-deficient B cells. Strikingly, mutations in our B cell lines recapitulated the mutable motif profile for Poleta and Msh2 deficiency, respectively, and suggest that an additional pathway for the generation of A:T mutations in SHM is conserved in mouse and human.