scholarly journals Fanca deficiency reduces A/T transitions in somatic hypermutation and alters class switch recombination junctions in mouse B cells

2014 ◽  
Vol 211 (6) ◽  
pp. 1011-1018 ◽  
Author(s):  
Thuy Vy Nguyen ◽  
Lydia Riou ◽  
Saïd Aoufouchi ◽  
Filippo Rosselli
Keyword(s):  
B Cells ◽  
Somatic Hypermutation ◽  
Bone Marrow Failure ◽  
Genetic Disorder ◽  
Point Mutations ◽  
Class Switch Recombination ◽  
Loss Of Function ◽  
Nhej Pathway ◽  
Class Switch ◽  
Increased Risk

Fanconi anemia is a rare genetic disorder that can lead to bone marrow failure, congenital abnormalities, and increased risk for leukemia and cancer. Cells with loss-of-function mutations in the FANC pathway are characterized by chromosome fragility, altered mutability, and abnormal regulation of the nonhomologous end-joining (NHEJ) pathway. Somatic hypermutation (SHM) and immunoglobulin (Ig) class switch recombination (CSR) enable B cells to produce high-affinity antibodies of various isotypes. Both processes are initiated after the generation of dG:dU mismatches by activation-induced cytidine deaminase. Whereas SHM involves an error-prone repair process that introduces novel point mutations into the Ig gene, the mismatches generated during CSR are processed to create double-stranded breaks (DSBs) in DNA, which are then repaired by the NHEJ pathway. As several lines of evidence suggest a possible role for the FANC pathway in SHM and CSR, we analyzed both processes in B cells derived from Fanca−/− mice. Here we show that Fanca is required for the induction of transition mutations at A/T residues during SHM and that despite globally normal CSR function in splenic B cells, Fanca is required during CSR to stabilize duplexes between pairs of short microhomology regions, thereby impeding short-range recombination downstream of DSB formation.

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 AID from bony fish catalyzes class switch recombination

2005 ◽  
Vol 202 (6) ◽  
pp. 733-738 ◽  
Author(s):  
Vasco M. Barreto ◽  
Qiang Pan-Hammarstrom ◽  
Yaofeng Zhao ◽  
Lennart Hammarstrom ◽  
Ziva Misulovin ◽  
...  
Keyword(s):  
B Cells ◽  
Catalytic Domain ◽  
Somatic Hypermutation ◽  
Cytidine Deaminase ◽  
Class Switch Recombination ◽  
Adaptive Immune System ◽  
Bony Fish ◽  
Class Switching ◽  
Class Switch ◽  
Switch Regions

Class switch recombination was the last of the lymphocyte-specific DNA modification reactions to appear in the evolution of the adaptive immune system. It is absent in cartilaginous and bony fish, and it is common to all tetrapods. Class switching is initiated by activation-induced cytidine deaminase (AID), an enzyme expressed in cartilaginous and bony fish that is also required for somatic hypermutation. Fish AID differs from orthologs found in tetrapods in several respects, including its catalytic domain and carboxy-terminal region, both of which are essential for the switching reaction. To determine whether evolution of class switch recombination required alterations in AID, we assayed AID from Japanese puffer and zebra fish for class-switching activity in mouse B cells. We find that fish AID catalyzes class switch recombination in mammalian B cells. Thus, AID had the potential to catalyze this reaction before the teleost and tetrapod lineages diverged, suggesting that the later appearance of a class-switching reaction was dependent on the evolution of switch regions and multiple constant regions in the IgH locus.

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.

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.

Chronic lymphocytic leukemia B cells expressing AID display dissociation between class switch recombination and somatic hypermutation

Blood ◽  
2003 ◽  
Vol 101 (10) ◽  
pp. 4029-4032 ◽  
Author(s):  
Pablo Oppezzo ◽  
Françoise Vuillier ◽  
Yuri Vasconcelos ◽  
Gérard Dumas ◽  
Christian Magnac ◽  
...  
Keyword(s):  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
Gene Product ◽  
Mode Of Action ◽  
Somatic Hypermutation ◽  
Cytidine Deaminase ◽  
Class Switch Recombination ◽  
Lymphocytic Leukemia ◽  
Class Switch ◽  
Activation Induced Cytidine Deaminase

Abstract In B cells, somatic hypermutation (SHM) and class switch recombination (CSR) depend on the activation-induced cytidine deaminase (AID) gene product, although the precise mode of action of AID remains unknown. Because some chronic lymphocytic leukemia (CLL) B cells can undergo CSR without SHM, it constitutes a useful model to dissect AID function. In this work, we have studied AID expression, the presence of mutations in the preswitch μ DNA region, CSR, and the SHM in 65 CLL patients. Our results demonstrate that unmutated CLL B cells can constitutively express AID and that AID expression is associated with the presence of mutations in the preswitch region and in clonally related isotype-switched transcripts. They also demonstrate that in CLL without constitutive AID expression, AID induction on stimulation results in preswitch mutations and the CSR process. Our results show a dissociation between SHM and CSR in CLL and suggest that, in this disease, AID would require additional help for carrying out the SHM process.

A Very Short Mutational Hot-Spot in the IGH 5′Sμ Region Suggests That the Acquisition of a Class Switch Recombination Defect Is an Important Step Toward Lymphomagenesis.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 284-284
Author(s):  
Philippe Ruminy ◽  
Fabrice Jardin ◽  
Dominique Penther ◽  
Françoise Parmentier ◽  
Gérard Buchonnet ◽  
...  
Keyword(s):  
Somatic Hypermutation ◽  
Hot Spot ◽  
Donor Site ◽  
Point Mutations ◽  
Class Switch Recombination ◽  
Lymphoma Cells ◽  
Genetic Event ◽  
Class Switch ◽  
Clustering Effect ◽  
Splicing Site

Abstract The t(14;18), which occurs during VDJ joining, is usually seen as the primary genetic event in follicular lymphoma (FL). Pre-lymphoma cells would then migrate to the secondary lymphoid organs and acquire secondary genetic abnormalities. The mechanisms leading to these secondary lesions are unclear, but both somatic hypermutation (SHM) and class switch recombination (CSR) processes may be involved. Surprisingly, while many FL still express an IgM, aberrant switch junctions were reported on both IGH alleles, indicating dysfunctions of CSR during the onset of the neoplasm. Recently, AID mediated point mutations were shown to accumulate in the switch μ (Sμ) regions of stimulated B cells undergoing CSR. To get insights into a possible CSR deregulation in FL, we sequenced the 5′ end of the Sμ region for 73 t(14;18) positive tumours. 152 point mutations and 5 deletions were identified, for a mutation frequency of 0.31/100 bp. Notably, mutations were found for a majority of cases (49/73, 67.1%) rather than being restricted to few tumours. Significant targeting of RGYW motifs (p<10−4) and bias for transitions over transvertions (p<10−4) both suggested the involvement of AID. More importantly, our data showed a highly significant clustering effect, with 22 mutations (13,9%) found on a 4 bp cluster on the GTAA splicing donor site of the Iμ exon (p<10−6). Those 22 mutations were found in different tumors so that 30.1% of the lymphomas in our series had a mutated splicing site. Analysis of whole blood genomic DNA from 14 patients and 20 healthy donors indicated that these mutations are not polymorphisms but are instead acquired by tumor cells. Importantly, allele specific PCR experiments showed no clustering effect on the translocated alleles, while 15/79 (19%) mutations on the functional alleles were found on the Iμ splicing site (p<10−6). Such mutations were also found, but on the translocated alleles, for two additional lymphomas with complex t(3;14;18)(q27;q32;q21) and t(8;14;18)(q24;q32;q21) translocations, suggesting that they may be linked to downstream rearrangements on the IGH locus. Finally, RT-PCR experiments showed that a mutation of the Iμ splicing site leads to the expression of aberrant germline transcripts. Together, our data thus indicate that mutations leading to an incorrect splicing of the germline Sμ transcripts are probably positively selected during lymphomagenesis, and therefore that an acquired perturbation of the CSR process may have a major impact during the onset of the disease, maybe by preventing pre-lymphoma cells from switching their surface immunoglobulin and/or by favouring subsequent genetic lesions.

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.

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