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 Regulation of class switch recombination and somatic mutation by AID phosphorylation

2008 ◽  
Vol 205 (11) ◽  
pp. 2585-2594 ◽  
Author(s):  
Kevin M. McBride ◽  
Anna Gazumyan ◽  
Eileen M. Woo ◽  
Tanja A. Schwickert ◽  
Brian T. Chait ◽  
...  
Keyword(s):  
Somatic Mutation ◽  
Somatic Hypermutation ◽  
Cytidine Deaminase ◽  
Point Mutations ◽  
Class Switch Recombination ◽  
Variable Region ◽  
Dna Breaks ◽  
Class Switching ◽  
Class Switch

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.

scholarly journals LMP1 signaling can replace CD40 signaling in B cells in vivo and has unique features of inducing class-switch recombination to IgG1

Blood ◽  
2008 ◽  
Vol 111 (3) ◽  
pp. 1448-1455 ◽  
Author(s):  
Julia Rastelli ◽  
Cornelia Hömig-Hölzel ◽  
Jane Seagal ◽  
Werner Müller ◽  
Andrea C. Hermann ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Cell Function ◽  
Somatic Hypermutation ◽  
Class Switch Recombination ◽  
Cd40 Ligand ◽  
Barr Virus ◽  
Class Switch ◽  
Epstein Barr

AbstractThe Epstein-Barr virus (EBV) protein LMP1 is considered to be a functional homologue of the CD40 receptor. However, in contrast to the latter, LMP1 is a constitutively active signaling molecule. To compare B cell–specific LMP1 and CD40 signaling in an unambiguous manner, we generated transgenic mice conditionally expressing a CD40/LMP1 fusion protein, which retained the LMP1 cytoplasmic tail but has lost the constitutive activity of LMP1 and needs to be activated by the CD40 ligand. We show that LMP1 signaling can completely substitute CD40 signaling in B cells, leading to normal B-cell development, activation, and immune responses including class-switch recombination, germinal center formation, and somatic hypermutation. In addition, the LMP1-signaling domain has a unique property in that it can induce class-switch recombination to IgG1 independent of cytokines. Thus, our data indicate that LMP1 has evolved to imitate T-helper cell function allowing activation, proliferation, and differentiation of EBV-infected B cells independent of T cells.

53BP1 Is Targeted to Sites of DNA Breaks within the Immunoglobulin Heavy Chain Locus and Promotes Class Switch Recombination.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2379-2379
Author(s):  
John Manis ◽  
Nicole Walsh ◽  
Phil Carpenter ◽  
Shilpee Dutt
Keyword(s):  
Dna Damage ◽  
B Cells ◽  
Dna Damage Response ◽  
Cellular Response ◽  
Somatic Hypermutation ◽  
Class Switch Recombination ◽  
Dna Breaks ◽  
Class Switch ◽  
Damage Response

Abstract The maintenance of genomic integrity relies on the cellular response to chromosomal damage from both exogenous (e.g. ionizing radiation) and endogenous (e.g. oxidative stress) sources. Various members of the DNA damage-sensing pathway including ATM, H2AX, 53BP1, and MDC1 are necessary to orchestrate the repair of DNA breaks. B cells undergo several programmed DNA alterations during their development: V(D)J recombination, Somatic Hypermutation (SHM), and Class Switch Recombination (CSR). We have previously shown that 53BP1 is relatively dispensable for V(D)J recombination and SHM. In contrast, class switch recombination is largely blocked to all isotypes indicating that regulated DNA breaks in B cells are regarded differentially by the DNA damage response machinery. 53BP1 is thought to promote the joining of DNA ends during CSR thus preventing translocations that could potentially lead to lymphoma. To better understand the damage response to CSR induced DNA breaks, a chromatin immunoprecipitation strategy and a combined immunofluorescence/FISH method was used to examine the components that assemble at IgH switch (S) regions during CSR. H2AX was found at S regions specifically targeted to undergo CSR after in vitro stimulation of B cells, and to a lesser degree, at adjacent S regions that were not activated for a switch event. H2AX was also found at S regions in switch activated 53BP1-deficient B cells. In contrast, 53BP1 was found primarily at S regions specifically targeted for CSR, and not at the adjacent S regions. Moreover, the localization of 53BP1 to S regions appeared to be in part, independent of DNA breaks, and potentially reliant on specialized DNA structures that are generated during CSR. These findings support a differential role for the various components of the DNA damage response program during CSR and have implications for understanding mechanisms of lymphomagenesis.

scholarly journals Human PMS2 deficiency is associated with impaired immunoglobulin class switch recombination

2008 ◽  
Vol 205 (11) ◽  
pp. 2465-2472 ◽  
Author(s):  
Sophie Péron ◽  
Ayse Metin ◽  
Pauline Gardès ◽  
Marie-Alexandra Alyanakian ◽  
Eamonn Sheridan ◽  
...  
Keyword(s):  
Somatic Hypermutation ◽  
Cytidine Deaminase ◽  
Class Switch Recombination ◽  
Dna Breaks ◽  
Gene Encoding ◽  
Class Switch ◽  
Double Strand Dna Breaks ◽  
Switch Regions

Immunoglobulin (Ig) class switch recombination (CSR) deficiencies are rare primary immunodeficiencies characterized by the lack of switched isotype (IgG/IgA/IgE) production. In some cases, CSR deficiencies can be associated with abnormal somatic hypermutation. Analysis of CSR deficiencies has helped reveal the key functions of CSR-triggering molecules, i.e., CD40L, CD40, and effector molecules such as activation-induced cytidine deaminase and uracil N-glycosylase. We report a new form of B cell–intrinsic CSR deficiency found in three patients with deleterious, homozygous mutations in the gene encoding the PMS2 component of the mismatch repair machinery. CSR was found partially defective in vivo and markedly impaired in vitro. It is characterized by the defective occurrence of double-strand DNA breaks (DSBs) in switch regions and abnormal formation of switch junctions. This observation strongly suggests a role for PMS2 in CSR-induced DSB generation.

scholarly journals Disparate roles of ATR and ATM in immunoglobulin class switch recombination and somatic hypermutation

2006 ◽  
Vol 203 (1) ◽  
pp. 99-110 ◽  
Author(s):  
Qiang Pan-Hammarström ◽  
Aleksi Lähdesmäki ◽  
Yaofeng Zhao ◽  
Likun Du ◽  
Zhihui Zhao ◽  
...  
Keyword(s):  
Ataxia Telangiectasia ◽  
Somatic Hypermutation ◽  
Cytidine Deaminase ◽  
Class Switch Recombination ◽  
End Joining ◽  
Class Switch ◽  
Activation Induced Cytidine Deaminase ◽  
Variable Heavy

Class switch recombination (CSR) and somatic hypermutation (SHM) are mechanistically related processes initiated by activation-induced cytidine deaminase. Here, we have studied the role of ataxia telangiectasia and Rad3-related protein (ATR) in CSR by analyzing the recombinational junctions, resulting from in vivo switching, in cells from patients with mutations in the ATR gene. The proportion of cells that have switched to immunoglobulin (Ig)A and IgG in the peripheral blood seems to be normal in ATR-deficient (ATRD) patients and the recombined S regions show a normal “blunt end-joining,” but impaired end joining with partially complementary (1–3 bp) DNA ends. There was also an increased usage of microhomology at the μ-α switch junctions, but only up to 9 bp, suggesting that the end-joining pathway requiring longer microhomologies (≥10 bp) may be ATR dependent. The SHM pattern in the Ig variable heavy chain genes is altered, with fewer mutations occurring at A and more mutations at T residues and thus a loss of strand bias in targeting A/T pairs within certain hotspots. These data suggest that the role of ATR is partially overlapping with that of ataxia telangiectasia–mutated protein, but that the former is also endowed with unique functional properties in the repair processes during CSR and SHM.

scholarly journals Rad52 mediates class-switch DNA recombination to IgD

2021 ◽  
Author(s):  
Yijiang Xu ◽  
Hang Zhou ◽  
Ginell Post ◽  
Hong Zan ◽  
Paolo Casali
Keyword(s):  
B Cells ◽  
Short Range ◽  
Dna Recombination ◽  
Dna Breaks ◽  
Class Switch ◽  
Rad52 Protein ◽  
Class Switch Dna Recombination

While the biology of IgD begins to be better understood, the mechanism of expression of this phylogenetically old and highly conserved Ig class remains unknown. In B cells, IgD is expressed together with IgM as transmembrane receptor for antigen through alternative splicing of long primary VHDJH-Cμ-s-m-Cδ-s-m RNA, which also underpins the secreted form of IgD. IgD is also expressed through class switch DNA recombination (CSR), as initiated by AID-mediated double-strand DNA breaks (DSBs) in Sμ and σδ and resolution of such DSBs by a yet unknown alternative endjoining (A-EJ) mechanism. This synapses Sμ with σδ region DSB resected ends leading to insertion of extensive S-S junction microhomologies, unlike the Ku70/Ku86-dependent NHEJ which resolves DSB blunt ends in CSR to IgG, IgA and IgE with little or no microhomologies. We previously demonstrated a novel role of DNA annealing homologous recombination Rad52 protein in 'short-range' microhomology-mediated synapsis of intra-Sδ region DSBs. This led us to hypothesize that Rad52 is also involved in the short-range microhomology-mediated A-EJ recombination of Sμ with σδ. We found that induction of IgD CSR by T-dependent or T-independent stimuli downregulated Zfp318 (the suppressor of Cδ-s-m transcription termination), promoted Rad52 phosphorylation, recruitment of Rad52 to Sμ and σδ leading to Sμ-σδ recombination with extensive microhomologies, VHDJH-Cδs transcription and sustained IgD secretion. Rad52 ablation in mouse Rad52-/- B cells aborted IgD CSR in vitro and in vivo and dampened the specific IgD antibody response to OVA. Further, Rad52 knockdown in human B cells virtually abrogated IgD CSR. Finally, Rad52 phosphorylation was associated with high levels IgD CSR and anti-nuclear IgD autoantibodies in lupus-prone mice and lupus patients. Thus, Rad52 mediates CSR to IgD by synapsing Sμ-σδ resected DSB ends through microhomology-mediated A-EJ and in concert with Zfp318 modulation. This is a previously unrecognized, critical and dedicated role of Rad52 in mammalian DNA repair.

scholarly journals Epigenetic tethering of AID to the donor switch region during immunoglobulin class switch recombination

2011 ◽  
Vol 208 (8) ◽  
pp. 1649-1660 ◽  
Author(s):  
Beena Patricia Jeevan-Raj ◽  
Isabelle Robert ◽  
Vincent Heyer ◽  
Adeline Page ◽  
Jing H. Wang ◽  
...  
Keyword(s):  
Somatic Hypermutation ◽  
Cytidine Deaminase ◽  
Class Switch Recombination ◽  
Epigenetic Modifications ◽  
Dna Breaks ◽  
Switch Region ◽  
Heterochromatin Protein 1 ◽  
Immunoglobulin Class ◽  
Class Switch

Immunoglobulin class switch recombination (CSR) is initiated by double-stranded DNA breaks (DSBs) in switch regions triggered by activation-induced cytidine deaminase (AID). Although CSR correlates with epigenetic modifications at the IgH locus, the relationship between these modifications and AID remains unknown. In this study, we show that during CSR, AID forms a complex with KAP1 (KRAB domain–associated protein 1) and HP1 (heterochromatin protein 1) that is tethered to the donor switch region (Sμ) bearing H3K9me3 (trimethylated histone H3 at lysine 9) in vivo. Furthermore, in vivo disruption of this complex results in impaired AID recruitment to Sμ, inefficient DSB formation, and a concomitant defect in CSR but not in somatic hypermutation. We propose that KAP1 and HP1 tether AID to H3K9me3 residues at the donor switch region, thus providing a mechanism linking AID to epigenetic modifications during CSR.

scholarly journals Germinal center reutilization by newly activated B cells

2009 ◽  
Vol 206 (13) ◽  
pp. 2907-2914 ◽  
Author(s):  
Tanja A. Schwickert ◽  
Boris Alabyev ◽  
Tim Manser ◽  
Michel C. Nussenzweig
Keyword(s):  
B Cells ◽  
Immune Responses ◽  
Somatic Hypermutation ◽  
Affinity Maturation ◽  
Anatomical Structures ◽  
Class Switch ◽  
Cell Clones ◽  
T Cell Help ◽  
Activated B Cells

Germinal centers (GCs) are specialized structures in which B lymphocytes undergo clonal expansion, class switch recombination, somatic hypermutation, and affinity maturation. Although these structures were previously thought to contain a limited number of isolated B cell clones, recent in vivo imaging studies revealed that they are in fact dynamic and appear to be open to their environment. We demonstrate that B cells can colonize heterologous GCs. Invasion of primary GCs after subsequent immunization is most efficient when T cell help is shared by the two immune responses; however, it also occurs when the immune responses are entirely unrelated. We conclude that GCs are dynamic anatomical structures that can be reutilized by newly activated B cells during immune responses.

scholarly journals Clinical, Immunological, and Functional Characterization of Six Patients with Very High IgM Levels

2020 ◽  
Vol 9 (3) ◽  
pp. 818 ◽  
Author(s):  
Vera Gallo ◽  
Emilia Cirillo ◽  
Rosaria Prencipe ◽  
Alessio Lepore ◽  
Luigi Del Vecchio ◽  
...  
Keyword(s):  
B Cell ◽  
Somatic Hypermutation ◽  
Functional Characterization ◽  
Class Switch Recombination ◽  
Pathogenic Mutation ◽  
Potential Candidate ◽  
Class Switch ◽  
Potential Candidate Gene ◽  
Very High

Very high IgM levels represent the hallmark of hyper IgM (HIGM) syndromes, a group of primary immunodeficiencies (PIDs) characterized by susceptibility to infections and malignancies. Other PIDs not fulfilling the diagnostic criteria for HIGM syndromes can also be characterized by high IgM levels and susceptibility to malignancies. The aim of this study is to characterize clinical phenotype, immune impairment, and pathogenic mechanism in six patients with very high IgM levels in whom classical HIGM syndromes were ruled out. The immunological analysis included extended B-cell immunophenotyping, evaluation of class switch recombination and somatic hypermutation, and next generation sequencing (NGS). Recurrent or severe infections and chronic lung changes at the diagnosis were reported in five out of six and two out of six patients, respectively. Five out of six patients showed signs of lymphoproliferation and four patients developed malignancies. Four patients showed impaired B-cell homeostasis. Class switch recombination was functional in vivo in all patients. NGS revealed, in one case, a pathogenic mutation in PIK3R1. In a second case, the ITPKB gene, implicated in B- and T-cell development, survival, and activity was identified as a potential candidate gene. Independent of the genetic basis, very high IgM levels represent a risk factor for the development of recurrent infections leading to chronic lung changes, lymphoproliferation, and high risk of malignancies.

scholarly journals A nucleotide resolution map of Top2-linked DNA breaks in the yeast and human genome

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
William H. Gittens ◽  
Dominic J. Johnson ◽  
Rachal M. Allison ◽  
Tim J. Cooper ◽  
Holly Thomas ◽  
...  
Keyword(s):  
Dna Cleavage ◽  
Genome Stability ◽  
Dna Double Strand Breaks ◽  
Dna Breaks ◽  
Strand Breaks ◽  
Single Strand Breaks ◽  
Human Genomes ◽  
Nucleotide Resolution

Abstract DNA topoisomerases are required to resolve DNA topological stress. Despite this essential role, abortive topoisomerase activity generates aberrant protein-linked DNA breaks, jeopardising genome stability. Here, to understand the genomic distribution and mechanisms underpinning topoisomerase-induced DNA breaks, we map Top2 DNA cleavage with strand-specific nucleotide resolution across the S. cerevisiae and human genomes—and use the meiotic Spo11 protein to validate the broad applicability of this method to explore the role of diverse topoisomerase family members. Our data characterises Mre11-dependent repair in yeast and defines two strikingly different fractions of Top2 activity in humans: tightly localised CTCF-proximal, and broadly distributed transcription-proximal, the latter correlated with gene length and expression. Moreover, single nucleotide accuracy reveals the influence primary DNA sequence has upon Top2 cleavage—distinguishing sites likely to form canonical DNA double-strand breaks (DSBs) from those predisposed to form strand-biased DNA single-strand breaks (SSBs) induced by etoposide (VP16) in vivo.

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