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1.
Front Immunol ; 12: 671944, 2021.
Article in English | MEDLINE | ID: mdl-34040612

ABSTRACT

Activation-induced deaminase (AID) is a key enzyme involved in antibody diversification by initiating somatic hypermutation (SHM) and class-switch recombination (CSR) of the Immunoglobulin (Ig) loci. AID preferentially targets WRC (W=A/T, R=A/G) hotspot motifs and avoids SYC (S=C/G, Y=C/T) coldspots. G-quadruplex (G4) structures are four-stranded DNA secondary structures with key functions in transcription, translation and replication. In vitro studies have shown G4s to form and bind AID in Ig switch (S) regions. Alterations in the gene encoding AID can further disrupt AID-G4 binding and reduce CSR in vivo. However, it is still unclear whether G4s form in the variable (V) region, or how they may affect SHM. To assess the possibility of G4 formation in human V regions, we analyzed germline human Ig heavy chain V (IGHV) sequences, using a pre-trained deep learning model that predicts G4 potential. This revealed that many genes from the IGHV3 and IGHV4 families are predicted to have high G4 potential in the top and bottom strand, respectively. Different IGHV alleles also showed variability in G4 potential. Using a high-resolution (G4-seq) dataset of biochemically confirmed potential G4s in IGHV genes, we validated our computational predictions. G4-seq also revealed variation between S and V regions in the distribution of potential G4s, with the V region having overall reduced G4 abundance compared to the S region. The density of AGCT motifs, where two AGC hotspots overlap on both strands, was roughly 2.6-fold greater in the V region than the Constant (C) region, which does not mutate despite having predicted G4s at similar levels. However, AGCT motifs in both V and C regions were less abundant than in S regions. In silico mutagenesis experiments showed that G4 potentials were generally robust to mutation, although large deviations from germline states were found, mostly in framework regions. G4 potential is also associated with higher mutability of certain WRC hotspots on the same strand. In addition, CCC coldspots opposite a predicted G4 were shown to be targeted significantly more for mutation. Our overall assessment reveals plausible evidence of functional G4s forming in the Ig V region.


Subject(s)
G-Quadruplexes , Genes, Immunoglobulin Heavy Chain/physiology , Immunoglobulin Heavy Chains/chemistry , Immunoglobulin Variable Region/chemistry , Deep Learning , Humans , Immunoglobulin Heavy Chains/genetics , Immunoglobulin Variable Region/genetics , Somatic Hypermutation, Immunoglobulin/physiology
2.
Nat Genet ; 53(2): 230-242, 2021 02.
Article in English | MEDLINE | ID: mdl-33526923

ABSTRACT

Noncoding RNAs are exquisitely titrated by the cellular RNA surveillance machinery for regulating diverse biological processes. The RNA exosome, the predominant 3' RNA exoribonuclease in mammalian cells, is composed of nine core and two catalytic subunits. Here, we developed a mouse model with a conditional allele to study the RNA exosome catalytic subunit DIS3. In DIS3-deficient B cells, integrity of the immunoglobulin heavy chain (Igh) locus in its topologically associating domain is affected, with accumulation of DNA-associated RNAs flanking CTCF-binding elements, decreased CTCF binding to CTCF-binding elements and disorganized cohesin localization. DIS3-deficient B cells also accumulate activation-induced cytidine deaminase-mediated asymmetric nicks, altering somatic hypermutation patterns and increasing microhomology-mediated end-joining DNA repair. Altered mutation patterns and Igh architectural defects in DIS3-deficient B cells lead to decreased class-switch recombination but increased chromosomal translocations. Our observations of DIS3-mediated architectural regulation at the Igh locus are reflected genome wide, thus providing evidence that noncoding RNA processing is an important mechanism for controlling genome organization.


Subject(s)
B-Lymphocytes/physiology , Exosome Multienzyme Ribonuclease Complex/genetics , RNA, Untranslated/genetics , Somatic Hypermutation, Immunoglobulin/physiology , Animals , B-Lymphocytes/drug effects , CCCTC-Binding Factor/genetics , CCCTC-Binding Factor/metabolism , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , Chromosomal Proteins, Non-Histone/metabolism , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Embryonic Stem Cells/physiology , Exosome Multienzyme Ribonuclease Complex/metabolism , Exosomes/genetics , Green Fluorescent Proteins/genetics , Mice, Knockout , Mice, Transgenic , Mutation , RNA Processing, Post-Transcriptional , Recombination, Genetic , Tamoxifen/pharmacology , Cohesins
3.
Front Immunol ; 11: 618409, 2020.
Article in English | MEDLINE | ID: mdl-33603748

ABSTRACT

The somatic hypermutation (SHM) of Immunoglobulin (Ig) genes is a key process during antibody affinity maturation in B cells. The mutagenic enzyme activation induced deaminase (AID) is required for SHM and has a preference for WRC hotspots in DNA. Error-prone repair mechanisms acting downstream of AID introduce further mutations, including DNA polymerase eta (Polη), part of the non-canonical mismatch repair pathway (ncMMR), which preferentially generates mutations at WA hotspots. Previously proposed mechanistic models lead to a variety of predictions concerning interactions between hotspots, for example, how mutations in one hotspot will affect another hotspot. Using a large, high-quality, Ig repertoire sequencing dataset, we evaluated pairwise correlations between mutations site-by-site using an unbiased measure similar to mutual information which we termed "mutational association" (MA). Interactions are dominated by relatively strong correlations between nearby sites (short-range MAs), which can be almost entirely explained by interactions between overlapping hotspots for AID and/or Polη. We also found relatively weak dependencies between almost all sites throughout each gene (longer-range MAs), although these arise mostly as a statistical consequence of high pairwise mutation frequencies. The dominant short-range interactions are also highest within the most highly mutating IGHV sub-regions, such as the complementarity determining regions (CDRs), where there is a high hotspot density. Our results suggest that the hotspot preferences for AID and Polη have themselves evolved to allow for greater interactions between AID and/or Polη induced mutations.


Subject(s)
Cytidine Deaminase/metabolism , DNA-Directed DNA Polymerase/metabolism , Genes, Immunoglobulin Heavy Chain/genetics , Immunoglobulin Variable Region/genetics , Somatic Hypermutation, Immunoglobulin/physiology , Cytidine Deaminase/genetics , DNA-Directed DNA Polymerase/genetics , Humans
4.
Eur J Immunol ; 50(3): 380-395, 2020 03.
Article in English | MEDLINE | ID: mdl-31821534

ABSTRACT

Secondary diversification of the Ig repertoire occurs through somatic hypermutation (SHM), gene conversion (GCV), and class switch recombination (CSR)-three processes that are initiated by activation-induced cytidine deaminase (AID). AID targets Ig genes at orders of magnitude higher than the rest of the genome, but the basis for this specificity is poorly understood. We have previously demonstrated that enhancers and enhancer-like sequences from Ig genes are capable of stimulating SHM of neighboring genes in a capacity distinct from their roles in increasing transcription. Here, we use an in vitro proteomics approach to identify E-box, MEF2, Ets, and Ikaros transcription factor family members as potential binders of these enhancers. ChIP assays in the hypermutating Ramos B cell line confirmed that many of these factors bound the endogenous Igλ enhancer and/or the IgH intronic enhancer (Eµ) in vivo. Further investigation using SHM reporter assays identified binding sites for E2A and MEF2B in Eµ and demonstrated an association between loss of factor binding and decreases in the SHM stimulating activity of Eµ mutants. Our results provide novel insights into trans-acting factors that dictate SHM targeting and link their activity to specific DNA binding sites within Ig enhancers.


Subject(s)
Somatic Hypermutation, Immunoglobulin/physiology , Animals , Chickens , Genes, Immunoglobulin , Humans , Transcription Factors/genetics , Transcription Factors/metabolism
5.
Curr Opin Immunol ; 39: 96-102, 2016 Apr.
Article in English | MEDLINE | ID: mdl-26845615

ABSTRACT

Activation induced deaminase is the single B cell specific factor mediating class switch recombination and somatic hypermutation. Numerous studies have shown that AID preferentially targets Ig substrates and also attacks non-Ig substrates to create DNA damage that contributes to lymphomagenesis. AID targeting to Ig loci is linked to transcription but the mechanism governing this process has been obscure. Here we discuss research that illustrates the connection between AID targeting to DNA substrates and transcription processes to reveal rules governing the specificity of AID attack. These observations are woven together to provide a integrated view of AID function and a surprising linkage with global regulation of gene expression.


Subject(s)
B-Lymphocytes/enzymology , Cytidine Deaminase/metabolism , Animals , Cytidine Deaminase/genetics , DNA Methylation , Humans , Immunoglobulin Class Switching/physiology , Somatic Hypermutation, Immunoglobulin/physiology , Transcription, Genetic , Translocation, Genetic
6.
Oncotarget ; 6(15): 13229-40, 2015 May 30.
Article in English | MEDLINE | ID: mdl-25929340

ABSTRACT

Human multiple myeloma (MM) is characterized by accumulation of malignant terminally differentiated plasma cells (PCs) in the bone marrow (BM), raising the question when during maturation neoplastic transformation begins. Immunoglobulin IGHV genes carry imprints of clonal tumor history, delineating somatic hypermutation (SHM) events that generally occur in the germinal center (GC). Here, we examine MM-derived IGHV genes using massive parallel deep sequencing, comparing them with profiles in normal BM PCs. In 4/4 presentation IgG MM, monoclonal tumor-derived IGHV sequences revealed significant evidence for intraclonal variation (ICV) in mutation patterns. IGHV sequences of 2/2 normal PC IgG populations revealed dominant oligoclonal expansions, each expansion also displaying mutational ICV. Clonal expansions in MM and in normal BM PCs reveal common IGHV features. In such MM, the data fit a model of tumor origins in which neoplastic transformation is initiated in a GC B-cell committed to terminal differentiation but still targeted by on-going SHM. Strikingly, the data parallel IGHV clonal sequences in some monoclonal gammopathy of undetermined significance (MGUS) known to display on-going SHM imprints. Since MGUS generally precedes MM, these data suggest origins of MGUS and MM with IGHV gene mutational ICV from the same GC B-cell, arising via a distinctive pathway.


Subject(s)
Genes, Immunoglobulin/genetics , Germinal Center/pathology , Immunoglobulin Heavy Chains/genetics , Immunoglobulin Variable Region/genetics , Multiple Myeloma/genetics , Bone Marrow Cells/pathology , High-Throughput Nucleotide Sequencing , Humans , Monoclonal Gammopathy of Undetermined Significance/pathology , Multiple Myeloma/pathology , Mutation , Plasma Cells/pathology , Somatic Hypermutation, Immunoglobulin/physiology
7.
Int Immunopharmacol ; 23(2): 380-6, 2014 Dec.
Article in English | MEDLINE | ID: mdl-25281392

ABSTRACT

Traditional antibody production technology within non-mammalian cell expression systems has shown many unsatisfactory properties for the development of therapeutic antibodies. Nevertheless, mammalian cell display technology reaps the benefits of producing full-length all human antibodies. Together with the developed cytidine deaminase induced in vitro somatic hypermutation technology, mammalian cell display technology provides the opportunity to produce high affinity antibodies that might be ideal for therapeutic application. This review was concentrated on the development of the mammalian cell display technology as well as the activation-induced cytidine deaminase induced in vitro somatic hypermutation technology and their applications for the production of therapeutic antibodies.


Subject(s)
Antibodies/metabolism , Cell Surface Display Techniques/methods , Cytidine Deaminase/metabolism , Gene Expression Regulation/physiology , Mammals , Somatic Hypermutation, Immunoglobulin/physiology , Animals , Antibodies/therapeutic use , Cell Line , Cytidine Deaminase/genetics
8.
Proc Natl Acad Sci U S A ; 111(25): 9217-22, 2014 Jun 24.
Article in English | MEDLINE | ID: mdl-24927551

ABSTRACT

Somatic hypermutation (SHM) of antibody variable region genes is initiated in germinal center B cells during an immune response by activation-induced cytidine deaminase (AID), which converts cytosines to uracils. During accurate repair in nonmutating cells, uracil is excised by uracil DNA glycosylase (UNG), leaving abasic sites that are incised by AP endonuclease (APE) to create single-strand breaks, and the correct nucleotide is reinserted by DNA polymerase ß. During SHM, for unknown reasons, repair is error prone. There are two APE homologs in mammals and, surprisingly, APE1, in contrast to its high expression in both resting and in vitro-activated splenic B cells, is expressed at very low levels in mouse germinal center B cells where SHM occurs, and APE1 haploinsufficiency has very little effect on SHM. In contrast, the less efficient homolog, APE2, is highly expressed and contributes not only to the frequency of mutations, but also to the generation of mutations at A:T base pair (bp), insertions, and deletions. In the absence of both UNG and APE2, mutations at A:T bp are dramatically reduced. Single-strand breaks generated by APE2 could provide entry points for exonuclease recruited by the mismatch repair proteins Msh2-Msh6, and the known association of APE2 with proliferating cell nuclear antigen could recruit translesion polymerases to create mutations at AID-induced lesions and also at A:T bp. Our data provide new insight into error-prone repair of AID-induced lesions, which we propose is facilitated by down-regulation of APE1 and up-regulation of APE2 expression in germinal center B cells.


Subject(s)
B-Lymphocytes/metabolism , DNA Repair , DNA-(Apurinic or Apyrimidinic Site) Lyase/biosynthesis , Endonucleases/biosynthesis , Gene Expression Regulation, Enzymologic/physiology , Germinal Center/metabolism , Mutation , Somatic Hypermutation, Immunoglobulin/physiology , Animals , B-Lymphocytes/cytology , DNA Glycosylases/genetics , DNA Glycosylases/metabolism , DNA-(Apurinic or Apyrimidinic Site) Lyase/genetics , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Endonucleases/genetics , Germinal Center/cytology , Mice , Mice, Knockout , Multifunctional Enzymes , MutS Homolog 2 Protein/genetics , MutS Homolog 2 Protein/metabolism , Proliferating Cell Nuclear Antigen/genetics , Proliferating Cell Nuclear Antigen/metabolism
9.
Proc Natl Acad Sci U S A ; 110(11): 4261-6, 2013 Mar 12.
Article in English | MEDLINE | ID: mdl-23440204

ABSTRACT

Somatic hypermutation and clonal selection lead to B cells expressing high-affinity antibodies. Here we show that somatic mutations not only play a critical role in antigen binding, they also affect the thermodynamic stability of the antibody molecule. Somatic mutations directly involved in antigen recognition by antibody 93F3, which binds a relatively small hapten, reduce the melting temperature compared with its germ-line precursor by up to 9 °C. The destabilizing effects of these mutations are compensated by additional somatic mutations located on surface loops distal to the antigen binding site. Similarly, somatic mutations enhance both the affinity and thermodynamic stability of antibody OKT3, which binds the large protein antigen CD3. Analysis of the crystal structures of 93F3 and OKT3 indicates that these somatic mutations modulate antibody stability primarily through the interface of the heavy and light chain variable domains. The historical view of antibody maturation has been that somatic hypermutation and subsequent clonal selection increase antigen-antibody specificity and binding energy. Our results suggest that this process also optimizes protein stability, and that many peripheral mutations that were considered to be neutral are required to offset deleterious effects of mutations that increase affinity. Thus, the immunological evolution of antibodies recapitulates on a much shorter timescale the natural evolution of enzymes in which function and thermodynamic stability are simultaneously enhanced through mutation and selection.


Subject(s)
Antibodies, Monoclonal, Murine-Derived/immunology , Antibody Affinity/physiology , Antibody Specificity/immunology , Binding Sites, Antibody/immunology , Immunoglobulin Variable Region/immunology , Somatic Hypermutation, Immunoglobulin/physiology , Animals , Antibodies, Monoclonal, Murine-Derived/genetics , Antibody Specificity/genetics , Binding Sites, Antibody/genetics , HEK293 Cells , Humans , Immunoglobulin Variable Region/genetics , Mice , Mutation , Protein Stability
10.
J Biol Chem ; 287(39): 32415-29, 2012 Sep 21.
Article in English | MEDLINE | ID: mdl-22843687

ABSTRACT

H3K4me3 plays a critical role in the activation-induced cytidine deaminase (AID)-induced DNA cleavage of switch (S) regions in the immunoglobulin heavy chain (IgH) locus during class-switch recombination (CSR). The histone chaperone complex facilitates chromatin transcription (FACT) is responsible for forming H3K4me3 at AID target loci. Here we show that the histone chaperone suppressor of Ty6 (Spt6) also participates in regulating H3K4me3 for CSR and for somatic hypermutation in AID target loci. We found that H3K4me3 loss was correlated with defects in AID-induced DNA breakage and reduced mutation frequencies in IgH loci in both S and variable regions and in non-IgH loci such as metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) and small nucleolar RNA host gene 3 (SNHG3). Global gene expression analysis revealed that Spt6 can act as both a positive and negative transcriptional regulator in B cells, affecting ∼5% of the genes that includes suppressor of Ty4 (Spt4) and AID. Interestingly, Spt6 regulates CSR and AID expression through two distinct histone modification pathways, H3K4me3 and H3K36me3, respectively. Tandem SH2 domain of Spt6 plays a critical role in CSR and H3K4me3 regulation involving Set1 histone methyltransferase. We conclude that Spt6 is a unique histone chaperone capable of regulating the histone epigenetic state of both AID targets and the AID locus.


Subject(s)
Cytidine Deaminase/metabolism , Epigenesis, Genetic/physiology , Histones/metabolism , Molecular Chaperones/metabolism , Transcription Factors/metabolism , Cell Line, Tumor , Cytidine Deaminase/genetics , DNA Breaks , Genetic Loci/physiology , Histone-Lysine N-Methyltransferase/genetics , Histone-Lysine N-Methyltransferase/metabolism , Histones/genetics , Humans , Immunoglobulin Class Switching/physiology , Immunoglobulin Heavy Chains/genetics , Immunoglobulin Heavy Chains/metabolism , Methylation , Molecular Chaperones/genetics , Protein Processing, Post-Translational/physiology , RNA, Long Noncoding/biosynthesis , RNA, Long Noncoding/genetics , Repressor Proteins/genetics , Repressor Proteins/metabolism , Somatic Hypermutation, Immunoglobulin/physiology , Transcription Factors/genetics
12.
Ann Hematol ; 91(7): 981-96, 2012 07.
Article in English | MEDLINE | ID: mdl-22526361

ABSTRACT

Chronic lymphocytic leukemia (CLL) is unique among B cell malignancies in that the malignant clones can be featured either somatically mutated or unmutated IGVH genes. CLL cells that express unmutated immunoglobulin variable domains likely underwent final development prior to their entry into the germinal center, whereas those that express mutated variable domains likely transited through the germinal center and then underwent final development. Regardless, the cellular origin of CLL remains unknown. The aim of this review is to summarize immunological aspects involved in this process and to provide insights about the complex biology and pathogenesis of this disease. We propose a mechanistic hypothesis to explain the origin of B-CLL clones into our current picture of normal B cell development. In particular, we suggest that unmutated CLL arises from normal B cells with self-reactivity for apoptotic bodies that have undergone receptor editing, CD5 expression, and anergic processes in the bone marrow. Similarly, mutated CLL would arise from cells that, while acquiring self-reactivity for autoantigens-including apoptotic bodies-in germinal centers, are also still subject to tolerization mechanisms, including receptor editing and anergy. We believe that CLL is a proliferation of B lymphocytes selected during clonal expansion through multiple encounters with (auto)antigens, despite the fact that they differ in their state of activation and maturation. Autoantigens and microbial pathogens activate BCR signaling and promote tolerogenic mechanisms such as receptor editing/revision, anergy, CD5+ expression, and somatic hypermutation in CLL B cells. The result of these tolerogenic mechanisms is the survival of CLL B cell clones with similar surface markers and homogeneous gene expression signatures. We suggest that both immunophenotypic surface markers and homogenous gene expression might represent the evidence of several attempts to re-educate self-reactive B cells.


Subject(s)
Cell Transformation, Neoplastic/immunology , Leukemia, Lymphocytic, Chronic, B-Cell/immunology , Animals , B-Lymphocytes/immunology , B-Lymphocytes/metabolism , B-Lymphocytes/physiology , Biomarkers, Tumor/analysis , Biomarkers, Tumor/genetics , Biomarkers, Tumor/physiology , Cell Differentiation/genetics , Cell Differentiation/immunology , Cell Transformation, Neoplastic/genetics , Gene Expression Profiling , Humans , Immune Tolerance/genetics , Immune Tolerance/physiology , Leukemia, Lymphocytic, Chronic, B-Cell/diagnosis , Leukemia, Lymphocytic, Chronic, B-Cell/etiology , Models, Biological , Neoplastic Stem Cells/immunology , Neoplastic Stem Cells/physiology , Somatic Hypermutation, Immunoglobulin/genetics , Somatic Hypermutation, Immunoglobulin/physiology
13.
Proc Natl Acad Sci U S A ; 108(48): 19305-10, 2011 Nov 29.
Article in English | MEDLINE | ID: mdl-22080610

ABSTRACT

Somatic hypermutation (SHM) and class-switch recombination (CSR) of the Ig gene require both the transcription of the locus and the expression of activation-induced cytidine deaminase (AID). During CSR, AID decreases the amount of topoisomerase I (Top1); this decrease alters the DNA structure and induces cleavage in the S region. Similarly, Top1 is involved in transcription-associated mutation at dinucleotide repeats in yeast and in triplet-repeat contraction in mammals. Here, we report that the AID-induced decrease in Top1 is critical for SHM. Top1 knockdown or haploinsufficiency enhanced SHM, whereas Top1 overexpression down-regulated it. A specific Top1 inhibitor, camptothecin, suppressed SHM, indicating that Top1's activity is required for DNA cleavage. Nonetheless, suppression of transcription abolished SHM, even in cells with Top1 knockdown, suggesting that transcription is critical. These results are consistent with a model proposed for CSR and triplet instability, in which transcription-induced non-B structure formation is enhanced by Top1 reduction and provides the target for irreversible cleavage by Top1. We speculate that the mechanism for transcription-coupled genome instability was adopted to generate immune diversity when AID evolved.


Subject(s)
Cytidine Deaminase/metabolism , DNA Topoisomerases, Type I/metabolism , Immunoglobulin Class Switching/physiology , Models, Biological , Somatic Hypermutation, Immunoglobulin/physiology , Animals , Blotting, Western , Camptothecin , Cell Line, Tumor , Cloning, Molecular , DNA Topoisomerases, Type I/genetics , Gene Knockdown Techniques , Genetic Vectors/genetics , Green Fluorescent Proteins , Haploinsufficiency , Humans , Mice , Mice, Knockout
14.
Mol Immunol ; 48(15-16): 1993-9, 2011 Sep.
Article in English | MEDLINE | ID: mdl-21724261

ABSTRACT

Ig gene hypermutation is initiated by the activation-induced cytidine deaminase (AID), which converts cytosine to uracil and generates a U:G lesion. One of the unsolved mysteries is how AID-triggered U:G lesions result in efficient induction of mutations at non-damaged A/T bases in the V(H) genes of germinal center (GC) B cells. Genetic and biochemical evidence suggests that components of the mismatch repair pathway and the low fidelity DNA polymerase η are required for the induction of A/T mutations. However, mismatch repair proficient NIH3T3 cells are unable to generate a high frequency of A/T mutations, even after DNA polymerase η overexpression, suggesting that additional mechanisms are involved. Since GC B cells undergo enormous expansion while undergoing hypermutation, we hypothesized that rapid cell division might play a role in the induction of A/T mutations. To test this hypothesis, we utilized an efficient in vitro mutagenesis system, which closely mirrors physiological Ig gene hypermutation, in the human GC-like B cell line Ramos. Ramos cells transduced with AID-IRES-GFP retrovirus were cultured for 10 days in medium supplemented with 20% or 2% fetal bovine serum (FBS) to allow rapid and slow proliferation, respectively. Analysis of the V(H) gene mutations revealed that A/T mutations were significantly reduced in 2% FBS compared with 20% FBS, with transitions more affected than transversions. These results demonstrate that rapid cell division contributes to efficient induction of A/T mutations and suggest that the rate of DNA replication has a profound effect on the processing of AID-triggered U:G lesions.


Subject(s)
B-Lymphocytes/cytology , Cell Division/physiology , DNA Mismatch Repair/physiology , Mutation , Somatic Hypermutation, Immunoglobulin/physiology , Adenine , B-Lymphocytes/immunology , Cell Line , Cytidine Deaminase/genetics , Cytidine Deaminase/immunology , Cytidine Deaminase/metabolism , Germinal Center/cytology , Germinal Center/immunology , Guanine , Humans , Reverse Transcriptase Polymerase Chain Reaction , Thymine , Uracil
15.
J Immunol ; 187(4): 1835-44, 2011 Aug 15.
Article in English | MEDLINE | ID: mdl-21746964

ABSTRACT

B cells are subjected to selection at multiple checkpoints during their development. The selection of Ab H chains is difficult to study because of the large diversity of the CDR3. To study the selection of individual Ab H chain V region genes (V(H)), we performed CDR3 spectratyping of ∼ 75-300 rearrangements per individual V(H) in C57BL6/J mice. We measured the fraction of rearrangements that were in-frame in B cell DNA. We demonstrate that individual V(H)s have different fractions of in-frame rearrangements (IF fractions) ranging from 10 to 90% and that these IF fractions are reproducible in different mice. For most V(H)s, the IF fraction in pro-B cells approximated 33% and then shifted to the nearly final (mature) B cell value by the cycling pre-B cell stage. The frequency of high in-frame (IF) V(H) usage increased in cycling pre-B cells compared with that in pro-B cells, whereas this did not occur for low IF V(H)s. The IF fraction did not shift as much in BCR-expressing B cells and was minimally affected by L chain usage for most V(H). High IF clan II/III V(H)s share more positively charged CDR2 sequences, whereas high IF clan I J558 CDR2 sequences are diverse. These data indicate that individual V(H)s are subjected to differential selection, that V(H) IF fraction is mainly established through pre-BCR-mediated selection, that it may operate differently in clan I versus II/III V(H)s, and that it has a lasting influence on the Ab repertoire.


Subject(s)
Complementarity Determining Regions/metabolism , DNA/metabolism , Precursor Cells, B-Lymphoid/metabolism , Somatic Hypermutation, Immunoglobulin/physiology , Animals , Complementarity Determining Regions/genetics , Complementarity Determining Regions/immunology , DNA/genetics , DNA/immunology , Gene Expression Regulation/physiology , Mice , Precursor Cells, B-Lymphoid/cytology , Precursor Cells, B-Lymphoid/immunology , Proto-Oncogene Proteins c-bcr/biosynthesis , Proto-Oncogene Proteins c-bcr/genetics , Proto-Oncogene Proteins c-bcr/immunology
16.
FASEB J ; 25(9): 2947-55, 2011 Sep.
Article in English | MEDLINE | ID: mdl-21593434

ABSTRACT

Somatic hypermutation diversifies antibody binding sites by introducing point mutations in the variable domains of rearranged immunoglobulin genes. In this study, we analyzed somatic hypermutation in variable heavy-chain (VH) domains of specific IgM antibodies of the urodele amphibian Pleurodeles waltl, immunized either on Earth or onboard the Mir space station. To detect somatic hypermutation, we aligned the variable domains of IgM heavy-chain transcripts with the corresponding VH gene. We also quantified NF-κB and activation-induced cytidine deaminase transcripts. Results were compared with those obtained using control animals immunized on Earth. Our data show that, as in most species of ectotherms, somatic hypermutation in P. waltl exhibits a mutational bias toward G and C bases. Furthermore, we show for the first time that somatic hypermutation occurs in space following immunization but at a lower frequency. This decrease is not due to a decrease in food intake or of the B-cell receptor/antigen interaction or to the absence of the germinal center-associated nuclear protein. It likely results from the combination of several spaceflight-associated changes, such as the severe reduction in T-cell activation, important perturbations of the cytoskeleton, and changes in the distribution of lymphocyte subpopulations and adhesion molecule expression.


Subject(s)
Binding Sites, Antibody/genetics , Immunoglobulin M/genetics , Pleurodeles/immunology , Somatic Hypermutation, Immunoglobulin/genetics , Space Flight , Adaptation, Physiological/immunology , Animals , Gene Expression Regulation , Pleurodeles/genetics , Pleurodeles/metabolism , Somatic Hypermutation, Immunoglobulin/physiology , Time Factors , Weightlessness
17.
FASEB J ; 25(4): 1123-32, 2011 Apr.
Article in English | MEDLINE | ID: mdl-21454370

ABSTRACT

The origin of antibody diversity has intrigued scientists for nearly a century. We now know that the diversity is achieved through a 2-stage process. Gene rearrangement (catalyzed by the RAG1/2 recombinase) allows the production of a primary repertoire of antibodies; targeted deamination of cytosines within these rearranged antibody genes (catalyzed by the DNA deaminase AID) then allows them to be further diversified and matured by somatic hypermutation, gene conversion, and class-switch recombination. Here we review the history of the uncovering of some of these processes, contrasting the relative importance of hypothesis and methodological developments in driving the research at different periods of the work.


Subject(s)
Antibodies/genetics , Antibody Diversity/immunology , Animals , B-Lymphocytes/immunology , Cytidine Deaminase/metabolism , Gene Rearrangement/physiology , Genes, Immunoglobulin , Homeodomain Proteins/physiology , Humans , Immunoglobulins/genetics , Models, Immunological , Somatic Hypermutation, Immunoglobulin/physiology
18.
Mol Biol (Mosk) ; 45(1): 96-107, 2011.
Article in Russian | MEDLINE | ID: mdl-21485501

ABSTRACT

Twenty unique phage antibodies to human tumor necrosis factor alpha were selected from a naive combinatorial library of human single chain fragment variable. Analysis of gene segments encoding selected antibodies shown that repertoire of variable domains of heavy and light chains included variable domains of both naive autoantibodies and antibodies produced as a result of somatic hypermutagenesis.


Subject(s)
Antibodies, Monoclonal/genetics , Autoantibodies/genetics , Bacteriophage M13 , Gene Library , Immunoglobulin Variable Region/genetics , Single-Chain Antibodies/genetics , Tumor Necrosis Factor-alpha , Antibodies, Monoclonal/immunology , Autoantibodies/immunology , Humans , Immunoglobulin Variable Region/immunology , Single-Chain Antibodies/immunology , Somatic Hypermutation, Immunoglobulin/physiology
19.
J Immunol ; 185(12): 7544-52, 2010 Dec 15.
Article in English | MEDLINE | ID: mdl-21076060

ABSTRACT

The mouse Igκ gene locus has three known transcriptional enhancers: an intronic enhancer (Ei), a 3' enhancer (E3'), and a further downstream enhancer (Ed). Previous studies on B lymphocytes derived from mutant embryonic stem cells have shown that deletion of either Ei or E3' significantly reduces Igκ gene rearrangement, whereas the combined deletion of both Ei and E3' eliminates such recombination. Furthermore, deletion of either E3' or Ed significantly reduces rearranged Igκ gene transcription. To determine whether the combined presence of both E3' and Ed are essential for Igκ gene expression, we generated homozygous double knockout (DKO) mice with targeted deletions in both elements. Significantly, homozygous DKO mice were unable to generate κ(+) B cells both in bone marrow and the periphery and exhibited surface expression almost exclusively of Igλ-chains, despite the fact that they possessed potentially functional rearranged Igκ genes. Compared with their single-enhancer-deleted counterparts, Igκ loci in homozygous DKO mice exhibited dramatically reduced germline and rearranged gene transcription, lower levels of gene rearrangement and histone H3 acetylation, and markedly increased DNA methylation. This contributed to a partial developmental block at the pre-B cell stage of development. We conclude that the two downstream enhancers are essential in Igκ gene expression and that Ei in homozygous DKO mice is incapable of triggering Igκ gene transcription. Furthermore, these results reveal unexpected compensatory roles for Ed in E3' knockout mice in triggering germline transcription and Vκ gene rearrangements to both Jκ and RS elements.


Subject(s)
B-Lymphocytes/immunology , Enhancer Elements, Genetic/physiology , Immunoglobulin Variable Region/immunology , Immunoglobulin kappa-Chains/immunology , Somatic Hypermutation, Immunoglobulin/physiology , Transcription, Genetic/physiology , Acetylation , Animals , B-Lymphocytes/metabolism , Base Sequence , DNA Methylation/genetics , DNA Methylation/immunology , Histones/genetics , Histones/immunology , Histones/metabolism , Immunoglobulin Variable Region/biosynthesis , Immunoglobulin Variable Region/genetics , Immunoglobulin kappa-Chains/biosynthesis , Immunoglobulin kappa-Chains/genetics , Mice , Mice, Knockout , Sequence Deletion
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