Immunoglobulin enhancers increase RNA polymerase 2 stalling at somatic hypermutation target sequences

ABSTRACTSomatic hypermutation (SHM) drives the genetic diversity of immunoglobulin (Ig) genes in activated B cells and supports the generation of antibodies with increased affinity for antigen. SHM is targeted to Ig genes by their enhancers (DIVACs; diversification activators), but how the enhancers mediate this activity is unknown. We show using chicken DT40 B cells that highly active DIVACs increase the phosphorylation of RNA polymerase 2 (Pol2) and Pol2 occupancy in the mutating gene with little or no accompanying increase in elongation-competent Pol2 or production of full-length transcripts, indicating accumulation of stalled Pol2. DIVAC has similar effect also in human Ramos Burkitt lymphoma cells. The DIVAC-induced stalling is weakly associated with an increase in the detection of single-stranded DNA bubbles in the mutating target gene. We did not find evidence for antisense transcription, or that DIVAC functions by altering levels of H3K27ac or the histone variant H3.3 in the mutating gene. These findings argue for a connection between Pol2 stalling and cis-acting targeting elements in the context of SHM and thus define a mechanistic basis for locus-specific targeting of SHM in the genome. Our results suggest that DIVAC elements render the target gene a suitable platform for AID-mediated mutation without a requirement for increasing transcriptional output.

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Trade-offs in antibody repertoires to complex antigens

Philosophical Transactions of the Royal Society B Biological Sciences ◽

10.1098/rstb.2014.0245 ◽

2015 ◽

Vol 370 (1676) ◽

pp. 20140245 ◽

Cited By ~ 31

Author(s):

Lauren M. Childs ◽

Edward B. Baskerville ◽

Sarah Cobey

Keyword(s):

B Cells ◽

B Cell ◽

Somatic Hypermutation ◽

Affinity Maturation ◽

Distant Interactions ◽

Trade Offs ◽

Cell Clones ◽

Evolutionary Advantage ◽

Mechanistic Basis ◽

Selection Of

Pathogens vary in their antigenic complexity. While some pathogens such as measles present a few relatively invariant targets to the immune system, others such as malaria display considerable antigenic diversity. How the immune response copes in the presence of multiple antigens, and whether a trade-off exists between the breadth and efficacy of antibody (Ab)-mediated immune responses, are unsolved problems. We present a theoretical model of affinity maturation of B-cell receptors (BCRs) during a primary infection and examine how variation in the number of accessible antigenic sites alters the Ab repertoire. Naive B cells with randomly generated receptor sequences initiate the germinal centre (GC) reaction. The binding affinity of a BCR to an antigen is quantified via a genotype–phenotype map, based on a random energy landscape, that combines local and distant interactions between residues. In the presence of numerous antigens or epitopes, B-cell clones with different specificities compete for stimulation during rounds of mutation within GCs. We find that the availability of many epitopes reduces the affinity and relative breadth of the Ab repertoire. Despite the stochasticity of somatic hypermutation, patterns of immunodominance are strongly shaped by chance selection of naive B cells with specificities for particular epitopes. Our model provides a mechanistic basis for the diversity of Ab repertoires and the evolutionary advantage of antigenically complex pathogens.

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Genomic Uracil and Aberrant Profile of Demethylation Intermediates in Epigenetics and Hematologic Malignancies

International Journal of Molecular Sciences ◽

10.3390/ijms22084212 ◽

2021 ◽

Vol 22 (8) ◽

pp. 4212

Author(s):

Ryszard Olinski ◽

Geir Slupphaug ◽

Marek Foksinski ◽

Hans Einar Krokan

Keyword(s):

B Cells ◽

Adaptive Immunity ◽

Somatic Hypermutation ◽

Hematologic Malignancies ◽

Living Cells ◽

Oxidation Products ◽

Epigenetic Mark ◽

Active Demethylation ◽

Dna Base ◽

Natural Modification

DNA of all living cells undergoes continuous structural and chemical alterations resulting from fundamental cellular metabolic processes and reactivity of normal cellular metabolites and constituents. Examples include enzymatically oxidized bases, aberrantly methylated bases, and deaminated bases, the latter largely uracil from deaminated cytosine. In addition, the non-canonical DNA base uracil may result from misincorporated dUMP. Furthermore, uracil generated by deamination of cytosine in DNA is not always damage as it is also an intermediate in normal somatic hypermutation (SHM) and class shift recombination (CSR) at the Ig locus of B-cells in adaptive immunity. Many of the modifications alter base-pairing properties and may thus cause replicative and transcriptional mutagenesis. The best known and most studied epigenetic mark in DNA is 5-methylcytosine (5mC), generated by a methyltransferase that uses SAM as methyl donor, usually in CpG contexts. Oxidation products of 5mC are now thought to be intermediates in active demethylation as well as epigenetic marks in their own rights. The aim of this review is to describe the endogenous processes that surround the generation and removal of the most common types of DNA nucleobase modifications, namely, uracil and certain epigenetic modifications, together with their role in the development of hematological malignances. We also discuss what dictates whether the presence of an altered nucleobase is defined as damage or a natural modification.

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Modification of hepatitis C virus 1b RNA polymerase to make a highly active JFH1-type polymerase by mutation of the thumb domain

Archives of Virology ◽

10.1007/s00705-009-0366-0 ◽

2009 ◽

Vol 154 (5) ◽

pp. 765-773 ◽

Cited By ~ 15

Author(s):

Leiyun Weng ◽

Jiamu Du ◽

Jingling Zhou ◽

Jianping Ding ◽

Takaji Wakita ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Rna Polymerase ◽

Highly Active ◽

Thumb Domain

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Inactivating SOCS1 mutations are caused by aberrant somatic hypermutation and restricted to a subset of B-cell lymphoma entities

Blood ◽

10.1182/blood-2009-06-225839 ◽

2009 ◽

Vol 114 (20) ◽

pp. 4503-4506 ◽

Cited By ~ 81

Author(s):

Anja Mottok ◽

Christoph Renné ◽

Marc Seifert ◽

Elsie Oppermann ◽

Wolf Bechstein ◽

...

Keyword(s):

B Cells ◽

B Cell ◽

Cell Lymphoma ◽

Somatic Hypermutation ◽

B Cell Lymphoma ◽

Rare Mutations ◽

Large B Cell Lymphoma ◽

Mantle Cell ◽

Follicular Lymphomas ◽

Large B Cell

Abstract STATs are constitutively activated in several malignancies. In primary mediastinal large B-cell lymphoma and Hodgkin lymphoma (HL), inactivating mutations in SOCS1, an inhibitor of JAK/STAT signaling, contribute to deregulated STAT activity. Based on indications that the SOCS1 mutations are caused by the B cell–specific somatic hypermutation (SHM) process, we analyzed B-cell non-HL and normal B cells for mutations in SOCS1. One-fourth of diffuse large B-cell lymphoma and follicular lymphomas carried SOCS1 mutations, which were preferentially targeted to SHM hotspot motifs and frequently obviously inactivating. Rare mutations were observed in Burkitt lymphoma, plasmacytoma, and mantle cell lymphoma but not in tumors of a non–B-cell origin. Mutations in single-sorted germinal center B cells were infrequent relative to other genes mutated as byproducts of normal SHM, indicating that SOCS1 inactivation in primary mediastinal large B-cell lymphoma, HL, diffuse large B-cell lymphoma, and follicular lymphoma is frequently the result of aberrant SHM.

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Constitutive Expression of AID Leads to Tumorigenesis

Journal of Experimental Medicine ◽

10.1084/jem.20030275 ◽

2003 ◽

Vol 197 (9) ◽

pp. 1173-1181 ◽

Cited By ~ 323

Author(s):

Il-mi Okazaki ◽

Hiroshi Hiai ◽

Naoki Kakazu ◽

Shuichi Yamada ◽

Masamichi Muramatsu ◽

...

Keyword(s):

T Cell ◽

B Cells ◽

Genome Stability ◽

Somatic Hypermutation ◽

Cytidine Deaminase ◽

Point Mutations ◽

Constitutive Expression ◽

Cell Receptor ◽

Genetic Alterations ◽

Artificial Substrates

Genome stability is regulated by the balance between efficiencies of the repair machinery and genetic alterations such as mutations and chromosomal rearrangements. It has been postulated that deregulation of class switch recombination (CSR) and somatic hypermutation (SHM), which modify the immunoglobulin (Ig) genes in activated B cells, may be responsible for aberrant chromosomal translocations and mutations of non-Ig genes that lead to lymphocyte malignancy. However, the molecular basis for these genetic instabilities is not clearly understood. Activation-induced cytidine deaminase (AID) is shown to be essential and sufficient to induce both CSR and SHM in artificial substrates in fibroblasts as well as B cells. Here we show that constitutive and ubiquitous expression of AID in transgenic mice caused both T cell lymphomas and dysgenetic lesions of epithelium of respiratory bronchioles (micro-adenomas) in all individual mice. Point mutations, but not translocations, were massively introduced in expressed T cell receptor (TCR) and c-myc genes in T lymphoma cells. The results indicate that AID can mutate non-Ig genes including oncogenes, implying that aberrant AID expression could be a cause of human malignancy.

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Somatic hypermutation in normal and transformed human B cells

Immunological Reviews ◽

10.1111/j.1600-065x.1998.tb01447.x ◽

1998 ◽

Vol 162 (1) ◽

pp. 261-280 ◽

Cited By ~ 239

Author(s):

Ulf Klien ◽

Tina Goasens ◽

Motthias Fischer ◽

Holger Kanzler ◽

Andreas Braeuninger ◽

...

Keyword(s):

B Cells ◽

Somatic Hypermutation ◽

Human B Cells

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Germinal center reutilization by newly activated B cells

Journal of Experimental Medicine ◽

10.1084/jem.20091225 ◽

2009 ◽

Vol 206 (13) ◽

pp. 2907-2914 ◽

Cited By ~ 65

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.

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Single-cell immune repertoire and transcriptome sequencing reveals that clonally expanded and transcriptionally distinct lymphocytes populate the aged central nervous system in mice

Proceedings of The Royal Society B Biological Sciences ◽

10.1098/rspb.2020.2793 ◽

2021 ◽

Vol 288 (1945) ◽

pp. 20202793

Author(s):

Alexander Yermanos ◽

Daniel Neumeier ◽

Ioana Sandu ◽

Mariana Borsa ◽

Ann Cathrin Waindok ◽

...

Keyword(s):

Gene Expression ◽

Central Nervous System ◽

Nervous System ◽

B Cells ◽

Single Cell ◽

Somatic Hypermutation ◽

Expression Profiles ◽

Gene Expression Profiles ◽

Cell Receptor ◽

The Central Nervous System

Neuroinflammation plays a crucial role during ageing and various neurological conditions, including Alzheimer's disease, multiple sclerosis and infection. Technical limitations, however, have prevented an integrative analysis of how lymphocyte immune receptor repertoires and their accompanying transcriptional states change with age in the central nervous system. Here, we leveraged single-cell sequencing to simultaneously profile B cell receptor and T cell receptor repertoires and accompanying gene expression profiles in young and old mouse brains. We observed the presence of clonally expanded B and T cells in the central nervous system of aged male mice. Furthermore, many of these B cells were of the IgM and IgD isotypes, and had low levels of somatic hypermutation. Integrating gene expression information additionally revealed distinct transcriptional profiles of these clonally expanded lymphocytes. Our findings implicate that clonally related T and B cells in the CNS of elderly mice may contribute to neuroinflammation accompanying homeostatic ageing.

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Signatures of B Cell Receptor Repertoire Following Pneumocystis Infection

Frontiers in Microbiology ◽

10.3389/fmicb.2021.636250 ◽

2021 ◽

Vol 12 ◽

Author(s):

Han Sun ◽

Hu-Qin Yang ◽

Kan Zhai ◽

Zhao-Hui Tong

Keyword(s):

B Cells ◽

B Cell ◽

Single Cell ◽

Plasma Cells ◽

Somatic Hypermutation ◽

Cell Receptor ◽

B Cell Receptor ◽

Receptor Repertoire ◽

Elevated Expression ◽

Pneumocystis Infection

B cells play vital roles in host defense against Pneumocystis infection. However, the features of the B cell receptor (BCR) repertoire in disease progression remain unclear. Here, we integrated single-cell RNA sequencing and single-cell BCR sequencing of immune cells from mouse lungs in an uninfected state and 1–4 weeks post-infection in order to illustrate the dynamic nature of B cell responses during Pneumocystis infection. We identified continuously increased plasma cells and an elevated ratio of (IgA + IgG) to (IgD + IgM) after infection. Moreover, Pneumocystis infection was associated with an increasing naïve B subset characterized by elevated expression of the transcription factor ATF3. The proportion of clonal expanded cells progressively increased, while BCR diversity decreased. Plasma cells exhibited higher levels of somatic hypermutation than naïve B cells. Biased usage of V(D)J genes was observed, and the usage frequency of IGHV9-3 rose. Overall, these results present a detailed atlas of B cell transcriptional changes and BCR repertoire features in the context of Pneumocystis infection, which provides valuable information for finding diagnostic biomarkers and developing potential immunotherapeutic targets.

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