scholarly journals Sγ3 switch sequences function in place of endogenous Sγ1 to mediate antibody class switching

2008 ◽  
Vol 205 (7) ◽  
pp. 1567-1572 ◽  
Author(s):  
Ali A. Zarrin ◽  
Peter H. Goff ◽  
Kate Senger ◽  
Frederick W. Alt
Keyword(s):  
B Cells ◽  
Heavy Chain ◽  
Recombination Event ◽  
Class Switch Recombination ◽  
Constant Region ◽  
Wild Type ◽  
Class Switch ◽  
Specific Factors ◽  
Antibody Class

Immunoglobulin heavy chain (IgH) class switch recombination (CSR) replaces the initially expressed IgH Cμ exons with a set of downstream IgH constant region (CH) exons. Individual sets of CH exons are flanked upstream by long (1–10-kb) repetitive switch (S) regions, with CSR involving a deletional recombination event between the donor Sμ region and a downstream S region. Targeting CSR to specific S regions might be mediated by S region–specific factors. To test the role of endogenous S region sequences in targeting specific CSR events, we generated mutant B cells in which the endogenous 10-kb Sγ1 region was replaced with wild-type (WT) or synthetic 2-kb Sγ3 sequences or a synthetic 2-kb Sγ1 sequence. We found that both the inserted endogenous and synthetic Sγ3 sequences functioned similarly to a size-matched synthetic Sγ1 sequence to mediate substantial CSR to IgG1 in mutant B cells activated under conditions that stimulate IgG1 switching in WT B cells. We conclude that Sγ3 can function similarly to Sγ1 in mediating endogenous CSR to IgG1. The approach that we have developed will facilitate assays for IgH isotype–specific functions of other endogenous S regions.

scholarly journals Immunoglobulin Class Switch Recombination Is Impaired in Atm-deficient Mice

2004 ◽  
Vol 200 (9) ◽  
pp. 1111-1121 ◽  
Author(s):  
Joanne M. Lumsden ◽  
Thomas McCarty ◽  
Lisa K. Petiniot ◽  
Rhuna Shen ◽  
Carrolee Barlow ◽  
...  
Keyword(s):  
B Cells ◽  
Dna Recombination ◽  
Class Switch Recombination ◽  
Intrinsic Defect ◽  
Wild Type ◽  
Immunoglobulin Class ◽  
Class Switch ◽  
Deficient Mice

Immunoglobulin class switch recombination (Ig CSR) involves DNA double strand breaks (DSBs) at recombining switch regions and repair of these breaks by nonhomologous end-joining. Because the protein kinase ataxia telengiectasia (AT) mutated (ATM) plays a critical role in DSB repair and AT patients show abnormalities of Ig isotype expression, we assessed the role of ATM in CSR by examining ATM-deficient mice. In response to T cell–dependent antigen (Ag), Atm−/− mice secreted substantially less Ag-specific IgA, IgG1, IgG2b, and IgG3, and less total IgE than Atm+/+ controls. To determine whether Atm−/− B cells have an intrinsic defect in their ability to undergo CSR, we analyzed in vitro responses of purified B cells. Atm−/− cells secreted substantially less IgA, IgG1, IgG2a, IgG3, and IgE than wild-type (WT) controls in response to stimulation with lipopolysaccharide, CD40 ligand, or anti-IgD plus appropriate cytokines. Molecular analysis of in vitro responses indicated that WT and Atm−/− B cells produced equivalent amounts of germline IgG1 and IgE transcripts, whereas Atm−/− B cells produced markedly reduced productive IgG1 and IgE transcripts. The reduction in isotype switching by Atm−/− B cells occurs at the level of genomic DNA recombination as measured by digestion–circularization PCR. Analysis of sequences at CSR sites indicated that there is greater microhomology at the μ–γ1 switch junctions in ATM B cells than in wild-type B cells, suggesting that ATM function affects the need or preference for sequence homology in the CSR process. These findings suggest a role of ATM in DNA DSB recognition and/or repair during CSR.

scholarly journals Mapping of a Functional Recombination Motif that Defines Isotype Specificity for μ→γ3 Switch Recombination Implicates NF-κB p50 as the Isotype-specific Switching Factor

2004 ◽  
Vol 199 (5) ◽  
pp. 617-627 ◽  
Author(s):  
Amy L. Kenter ◽  
Robert Wuerffel ◽  
Carmen Dominguez ◽  
Ananth Shanmugam ◽  
Hongmei Zhang
Keyword(s):  
B Cells ◽  
Binding Site ◽  
Class Switch Recombination ◽  
Interleukin 4 ◽  
Specific Factor ◽  
Wild Type ◽  
Class Switch ◽  
Activation Induced Deaminase ◽  
Necessary And Sufficient

Ig class switch recombination (CSR) requires expression of activation-induced deaminase (AID) and production of germline transcripts to target S regions for recombination. However, the mechanism of CSR remains unclear. Here we show that an extrachromosomal S plasmid assay is AID dependent and that a single consensus repeat is both necessary and sufficient for isotype-specific CSR. Transfected switch substrates specific for μ→γ3 and μ→γ1 are stimulated to switch with lipopolysaccharide (LPS) alone or LPS and interleukin-4, respectively. An Sγ3/Sγ1 substrate containing only three Sγ3-associated nucleotides reconstituted LPS responsiveness and permitted mapping of a functional recombination motif specific for μ→γ3 CSR. This functional recombination motif colocalized with a binding site for NF-κB p50, and p50 binding to this site was previously established. We show a p50 requirement for plasmid-based μ→γ3 CSR using p50-deficient B cells. Switch junctions from p50-deficient B cells showed decreased lengths of microhomology between Sμ and Sγ3 relative to wild-type cells, indicating a function for p50 in the mechanics of CSR. We note a striking parallel between the affects of p50 and Msh2 deficiency on Sμ/Sγ3 junctions. The data suggest that p50 may be the isotype-specific factor in μ→γ3 CSR and epistatic with Msh2.

scholarly journals How the Signaling Crosstalk of B Cell Receptor (BCR) and Co-Receptors Regulates Antibody Class Switch Recombination: A New Perspective of Checkpoints of BCR Signaling

2021 ◽  
Vol 12 ◽  
Author(s):  
Zhangguo Chen ◽  
Jing H. Wang
Keyword(s):  
B Cells ◽  
Immune Responses ◽  
B Cell ◽  
Dna Recombination ◽  
Class Switch Recombination ◽  
Cell Receptor ◽  
Signaling Crosstalk ◽  
Class Switch ◽  
Antibody Class ◽  
New Perspective

Mature B cells express B cell antigen receptor (BCR), toll-like receptors (TLR) and TNF family receptors including CD40 and B-cell activating factor receptor (BAFFR). These receptors transduce cellular signals to govern the physiological and pathological processes in B cells including B cell development and differentiation, survival, proliferation, and antibody-mediated immune responses as well as autoimmune diseases and B cell lymphomagenesis. Effective antibody-mediated immune responses require class switch recombination (CSR), a somatic DNA recombination event occurring at the immunoglobulin heavy chain (Igh) gene locus. Mature B cells initially express IgM as their BCR, and CSR enables the B cells to switch from expressing IgM to expressing different classes of antibodies including IgG, IgA or IgE that exhibit distinct effector functions. Here, we briefly review recent findings about how the signaling crosstalk of the BCR with TLRs, CD40 and BAFFR regulates CSR, antibody-mediate immune responses, and B cell anergy.

scholarly journals Non-coding Class Switch Recombination-related transcription in human normal and pathological immune responses

2018 ◽  
Author(s):  
Helena Kuri-Magaña ◽  
Leonardo Collado-Torres ◽  
Andrew E. Jaffe ◽  
Humberto Valdovinos-Torres ◽  
Marbella Ovilla-Muñoz ◽  
...  
Keyword(s):  
B Cells ◽  
Immune Responses ◽  
Class Switch Recombination ◽  
Rna Seq ◽  
Ileal Mucosa ◽  
Exon Expression ◽  
Class Switch ◽  
Cytokine Milieu ◽  
Antibody Class ◽  
Cancer Tissues

AbstractBackgroundAntibody class switch recombination (CSR) to IgG, IgA or IgE is a hallmark of adaptive immunity, allowing antibody function diversification beyond IgM. CSR involves a deletion of the IgM/IgD constant region genes placing a new acceptor Constant (CH) gene, downstream of the VDJH exon. CSR depends on non-coding (CSRnc) transcription of donor Iμ and acceptor IH exons, located 5’ upstream of each CH coding gene. Although our knowledge of the role of CSRnc transcription has advanced greatly, its extension and importance in healthy and diseased humans is scarce.MethodsWe analyzed CSRnc transcription in 70,603 publicly available RNA-seq samples, including GTEx, TCGA and the Sequence Read Archive (SRA) using recount2, an online resource consisting of normalized RNA-seq gene and exon counts, as well as coverage BigWig files that can be programmatically accessed through R. CSRnc transcription was validated with a qRT-PCR assay for Iμ, Iγ3 and Iγ1 in humans in response to vaccination.ResultsWe mapped IH transcription for the human IgH locus, including the less understood IGHD gene. CSRnc transcription was restricted to B cells and is widely distributed in normal adult tissues, but predominant in blood, spleen, MALT-containing tissues, visceral adipose tissue and some so-called “immune privileged” tissues. However, significant Iγ4 expression was found even in non-lymphoid fetal tissues. CSRnc expression in cancer tissues mimicked the expression of their normal counterparts, with notable pattern changes in some common cancer subsets. CSRnc transcription in tumors appears to result from tumor infiltration by B cells, since CSRnc transcription was not detected in corresponding tumor-derived immortal cell lines. Additionally, significantly increased I5 transcription in ileal mucosa in Crohn’s disease with ulceration was found.ConclusionsCSRnc transcription occurs in multiple anatomical locations beyond classical secondary lymphoid organs, representing a potentially useful marker of effector B cell responses in normal and pathological immune responses. The pattern of IH exon expression may reveal clues of the local immune response (i.e. cytokine milieu) in health and disease. This is a great example of how the public recount2 data can be used to further our understanding of transcription, including regions outside the known transcriptome.

scholarly journals Mlh1 Can Function in Antibody Class Switch Recombination Independently of Msh2

2003 ◽  
Vol 197 (10) ◽  
pp. 1377-1383 ◽  
Author(s):  
Carol E. Schrader ◽  
Joycelyn Vardo ◽  
Janet Stavnezer
Keyword(s):  
B Cells ◽  
Somatic Hypermutation ◽  
Class Switch Recombination ◽  
Variable Region ◽  
Class Switch ◽  
Switch Regions ◽  
Substitution Mutations ◽  
Antibody Class ◽  
Variable Region Genes ◽  
Mmr Proteins

Mismatch repair proteins participate in antibody class switch recombination, although their roles are unknown. Previous nucleotide sequence analyses of switch recombination junctions indicated that the roles of Msh2 and the MutL homologues, Mlh1 and Pms2, differ. We now asked if Msh2 and Mlh1 function in the same pathway during switch recombination. Splenic B cells from mice deficient in both these proteins were induced to undergo switching in culture. The frequency of switching is reduced, similarly to that of B cells singly deficient in Msh2 or Mlh1. However, the nucleotide sequences of the Sμ-Sγ3 junctions resemble junctions from Mlh1- but not from Msh2-deficient cells, suggesting Mlh1 functions either independently of or before Msh2. The substitution mutations within S regions that are known to accompany switch recombination are increased in Msh2- and Mlh1 single-deficient cells and further increased in the double-deficient cells, again suggesting these proteins function independently in class switch recombination. The finding that MMR functions to reduce mutations in switch regions is unexpected since MMR proteins have been shown to contribute to somatic hypermutation of antibody variable region genes.

The immunoglobulin heavy-chain locus hs3b and hs4 3′ enhancers are dispensable for VDJ assembly and somatic hypermutation

Blood ◽  
2003 ◽  
Vol 102 (4) ◽  
pp. 1421-1427 ◽  
Author(s):  
Caroline Le Morvan ◽  
Eric Pinaud ◽  
Catherine Decourt ◽  
Armelle Cuvillier ◽  
Michel Cogné
Keyword(s):  
B Cells ◽  
B Cell ◽  
Heavy Chain ◽  
Somatic Hypermutation ◽  
Class Switch Recombination ◽  
Immunoglobulin Heavy Chain ◽  
Germinal Centers ◽  
Affinity Maturation ◽  
Class Switch ◽  
Endogenous Locus

Abstract The more distal enhancers of the immunoglobulin heavy-chain 3′ regulatory region, hs3b and hs4, were recently demonstrated as master control elements of germline transcription and class switch recombination to most immunoglobulin constant genes. In addition, they were shown to enhance the accumulation of somatic mutations on linked transgenes. Since somatic hypermutation and class switch recombination are tightly linked processes, their common dependency on the endogenous locus 3′ enhancers could be an attractive hypothesis. VDJ structure and somatic hypermutation were analyzed in B cells from mice carrying either a heterozygous or a homozygous deletion of these enhancers. We find that hs3b and hs4 are dispensable both for VDJ assembly and for the occurrence of mutations at a physiologic frequency in the endogenous locus. In addition, we show that cells functionally expressing the immunoglobulin M (IgM) class B-cell receptor encoded by an hs3b/hs4-deficient locus were fully able to enter germinal centers, undergo affinity maturation, and yield specific antibody responses in homozygous mutant mice, where IgG1 antibodies compensated for the defect in other IgG isotypes. By contrast, analysis of Peyer patches from heterozygous animals showed that peanut agglutinin (PNAhigh) B cells functionally expressing the hs3b/hs4-deficient allele were dramatically outclassed by B cells expressing the wild-type locus and normally switching to IgA. This study thus also highlights the role of germinal centers in the competition between B cells for affinity maturation and suggests that membrane IgA may promote recruitment in an activated B-cell compartment, or proliferation of activated B cells, more efficiently than IgM in Peyer patches.

scholarly journals Kinase-dependent structural role of DNA-PKcs during immunoglobulin class switch recombination

2018 ◽  
Vol 115 (34) ◽  
pp. 8615-8620 ◽  
Author(s):  
Jennifer L. Crowe ◽  
Zhengping Shao ◽  
Xiaobin S. Wang ◽  
Pei-Chi Wei ◽  
Wenxia Jiang ◽  
...  
Keyword(s):  
B Cells ◽  
High Throughput Sequencing ◽  
Class Switch Recombination ◽  
End Joining ◽  
Extensive Resection ◽  
Structural Role ◽  
Class Switch ◽  
Cell Class ◽  
Dependent Protein

The catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) is a classical nonhomologous end-joining (cNHEJ) factor. Loss of DNA-PKcs diminished mature B cell class switch recombination (CSR) to other isotypes, but not IgG1. Here, we show that expression of the kinase-dead DNA-PKcs (DNA-PKcsKD/KD) severely compromises CSR to IgG1. High-throughput sequencing analyses of CSR junctions reveal frequent accumulation of nonproductive interchromosomal translocations, inversions, and extensive end resection in DNA-PKcsKD/KD, but not DNA-PKcs−/−, B cells. Meanwhile, the residual joints from DNA-PKcsKD/KD cells and the efficient Sµ-Sγ1 junctions from DNA-PKcs−/− B cells both display similar preferences for small (2–6 nt) microhomologies (MH). In DNA-PKcs−/− cells, Sµ-Sγ1 joints are more resistant to inversions and extensive resection than Sµ-Sε and Sµ-Sµ joints, providing a mechanism for the isotype-specific CSR defects. Together, our findings identify a kinase-dependent role of DNA-PKcs in suppressing MH-mediated end joining and a structural role of DNA-PKcs protein in the orientation of CSR.

scholarly journals Polζ ablation in B cells impairs the germinal center reaction, class switch recombination, DNA break repair, and genome stability

2009 ◽  
Vol 206 (2) ◽  
pp. 477-490 ◽  
Author(s):  
Dominik Schenten ◽  
Sven Kracker ◽  
Gloria Esposito ◽  
Sonia Franco ◽  
Ulf Klein ◽  
...  
Keyword(s):  
B Cells ◽  
Genome Stability ◽  
Somatic Hypermutation ◽  
Class Switch Recombination ◽  
Dna Breaks ◽  
Dsb Repair ◽  
Direct Role ◽  
Class Switch

Polζ is an error-prone DNA polymerase that is critical for embryonic development and maintenance of genome stability. To analyze its suggested role in somatic hypermutation (SHM) and possible contribution to DNA double-strand break (DSB) repair in class switch recombination (CSR), we ablated Rev3, the catalytic subunit of Polζ, selectively in mature B cells in vivo. The frequency of somatic mutation was reduced in the mutant cells but the pattern of SHM was unaffected. Rev3-deficient B cells also exhibited pronounced chromosomal instability and impaired proliferation capacity. Although the data thus argue against a direct role of Polζ in SHM, Polζ deficiency directly interfered with CSR in that activated Rev3-deficient B cells exhibited a reduced efficiency of CSR and an increased frequency of DNA breaks in the immunoglobulin H locus. Based on our results, we suggest a nonredundant role of Polζ in DNA DSB repair through nonhomologous end joining.

scholarly journals Aberrant recombination and repair during immunoglobulin class switching in BRCA1-deficient human B cells

2015 ◽  
Vol 112 (7) ◽  
pp. 2157-2162 ◽  
Author(s):  
Andrea Björkman ◽  
Per Qvist ◽  
Likun Du ◽  
Margarita Bartish ◽  
Apostolos Zaravinos ◽  
...  
Keyword(s):  
Breast Cancer ◽  
B Cells ◽  
Class Switch Recombination ◽  
Cancer Type ◽  
End Joining ◽  
Immunoglobulin Class ◽  
Class Switch ◽  
Human B Cells ◽  
Breast Cancer Type

Breast cancer type 1 susceptibility protein (BRCA1) has a multitude of functions that contribute to genome integrity and tumor suppression. Its participation in the repair of DNA double-strand breaks (DSBs) during homologous recombination (HR) is well recognized, whereas its involvement in the second major DSB repair pathway, nonhomologous end-joining (NHEJ), remains controversial. Here we have studied the role of BRCA1 in the repair of DSBs in switch (S) regions during immunoglobulin class switch recombination, a physiological, deletion/recombination process that relies on the classical NHEJ machinery. A shift to the use of microhomology-based, alternative end-joining (A-EJ) and increased frequencies of intra-S region deletions as well as insertions of inverted S sequences were observed at the recombination junctions amplified from BRCA1-deficient human B cells. Furthermore, increased use of long microhomologies was found at recombination junctions derived from E3 ubiquitin-protein ligase RNF168-deficient, Fanconi anemia group J protein (FACJ, BRIP1)-deficient, or DNA endonuclease RBBP8 (CtIP)-compromised cells, whereas an increased frequency of S-region inversions was observed in breast cancer type 2 susceptibility protein (BRCA2)-deficient cells. Thus, BRCA1, together with its interaction partners, seems to play an important role in repairing DSBs generated during class switch recombination by promoting the classical NHEJ pathway. This may not only provide a general mechanism underlying BRCA1’s function in maintaining genome stability and tumor suppression but may also point to a previously unrecognized role of BRCA1 in B-cell lymphomagenesis.

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