Loop extrusion promotes an alternate pathway for isotype switching.

Class-switch recombination (CSR) involves replacement of the Cµ constant region with another downstream CH region. CSR is initiated by activation-induced cytidine deaminase (AID)-mediated DNA breaks that are targeted to transcriptionally active switch (S) regions. S region promoters (Prs) direct synapsis by associating with the Eµ and 3'Eα enhancers that jointly anchor a chromatin loop. We report that asymmetric loop extrusion allows 3'Eα to track along the locus and form Pr-Pr-E interactions that mediate CSR between downstream S regions, followed by switching to donor Sµ. This alternative pathway bypasses sequential switching and creates immunoglobulin (Ig)E+ B cells in the absence of IgG1 expression. Based on the analysis of diagnostic CSR products in B cell subsets, we identify a BCR-negative cell intermediate that is pivotal to efficient CSR.

ASTE1 promotes shieldin-complex-mediated DNA repair by attenuating end resection.

The shieldin complex functions as the downstream effector of 53BP1-RIF1 to promote DNA double-strand break end-joining by restricting end resection. The SHLD2 subunit binds to single-stranded DNA ends and blocks end resection through OB-fold domains. Besides blocking end resection, it is unclear how the shieldin complex processes SHLD2-bound single-stranded DNA and promotes non-homologous end-joining. Here, we identify a downstream effector of the shieldin complex, ASTE1, as a structure-specific DNA endonuclease that specifically cleaves single-stranded DNA and 3' overhang DNA. ASTE1 localizes to DNA damage sites in a shieldin-dependent manner. Loss of ASTE1 impairs non-homologous end-joining, leads to hyper-resection and causes defective immunoglobulin class switch recombination. ASTE1 deficiency also causes resistance to poly(ADP-ribose) polymerase inhibitors in BRCA1-deficient cells owing to restoration of homologous recombination. These findings suggest that ASTE1-mediated 3' single-stranded DNA end cleavage contributes to the control of DSB repair choice by 53BP1, RIF1 and shieldin.

Sensitive detection of total anti-Spike antibodies and isotype switching in asymptomatic and symptomatic individuals with COVID-19.

Early detection of infection is crucial to limit the spread of coronavirus disease 2019 (COVID-19). Here we develop a flow cytometry-based assay to detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) protein antibodies in individuals with COVID-19. The assay detects specific immunoglobulin M (IgM), IgA, and IgG in individuals with COVID-19 and also acquisition of all IgG subclasses, with IgG1 being the most dominant. The antibody response is significantly higher at a later stage of infection. Furthermore, asymptomatic individuals with COVID-19 also develop specific IgM, IgA, and IgG, with IgG1 being the most dominant subclass. Although the antibody levels are lower in asymptomatic infection, the assay is highly sensitive and detects 97% of asymptomatic infections. These findings demonstrate that the assay can be used for serological analysis of symptomatic and asymptomatic infections, which may otherwise remain undetected.

DNA-PKcs phosphorylation at the T2609 cluster alters the repair pathway choice during immunoglobulin class switch recombination.

The DNA-dependent protein kinase (DNA-PK), which is composed of the KU heterodimer and the large catalytic subunit (DNA-PKcs), is a classical nonhomologous end-joining (cNHEJ) factor. Naïve B cells undergo class switch recombination (CSR) to generate antibodies with different isotypes by joining two DNA double-strand breaks at different switching regions via the cNHEJ pathway. DNA-PK and the cNHEJ pathway play important roles in the DNA repair phase of CSR. To initiate cNHEJ, KU binds to DNA ends and recruits and activates DNA-PK. Activated DNA-PK phosphorylates DNA-PKcs at the S2056 and T2609 clusters. Loss of T2609 cluster phosphorylation increases radiation sensitivity but whether T2609 phosphorylation has a role in physiological DNA repair remains elusive. Using the DNA-PKcs5A mouse model carrying alanine substitutions at the T2609 cluster, here we show that loss of T2609 phosphorylation of DNA-PKcs does not affect the CSR efficiency. Yet, the CSR junctions recovered from DNA-PKcs5A/5A B cells reveal increased chromosomal translocations, extensive use of distal switch regions (consistent with end resection), and preferential usage of microhomology-all signs of the alternative end-joining pathway. Thus, these results uncover a role of DNA-PKcs T2609 phosphorylation in promoting cNHEJ repair pathway choice during CSR.

Parp3 promotes long-range end joining in murine cells.

Chromosomal rearrangements, including translocations, are early and essential events in the formation of many tumors. Previous studies that defined the genetic requirements for rearrangement formation have identified differences between murine and human cells, most notably in the role of classic and alternative nonhomologous end-joining (NHEJ) factors. We reported that poly(ADP)ribose polymerase 3 (PARP3) promotes chromosomal rearrangements induced by endonucleases in multiple human cell types. We show here that in contrast to classic (c-NHEJ) factors, Parp3 also promotes rearrangements in murine cells, including translocations in murine embryonic stem cells (mESCs), class-switch recombination in primary B cells, and inversions in tail fibroblasts that generate Eml4-Alk fusions. In mESCs, Parp3-deficient cells had shorter deletion lengths at translocation junctions. This was corroborated using next-generation sequencing of Eml4-Alk junctions in tail fibroblasts and is consistent with a role for Parp3 in promoting the processing of DNA double-strand breaks. We confirmed a previous report that Parp1 also promotes rearrangement formation. In contrast with Parp3, rearrangement junctions in the absence of Parp1 had longer deletion lengths, suggesting that Parp1 may suppress double-strand break processing. Together, these data indicate that Parp3 and Parp1 promote rearrangements with distinct phenotypes.

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

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.

AID hits the jackpot when missing the target.

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.

Orchestration of B lymphoid cells and their inner myeloid by Bach.

The transcription repressor Bach2 is required for class switch recombination and somatic hypermutation of antibody genes in B cells, and proper development of effector and regulatory T cells. In addition, Bach2 and its related factor Bach1 promote B cell commitment of progenitor cells by repressing myeloid-related genes. Bach2 and the myeloid regulators C/EBPß and C/EBPα mutually repress their expression, forming a gene regulatory network (GRN) that dictates the process of lineage commitment. Bach2 forms another GRN with the plasma cell regulator Blimp-1, in which Bach2 and Blimp-1 mutually repress their expression. Since Bach2 expression is reduced in plasma cells, the repression of myeloid-related genes in B cells may be dissolved upon terminal differentiation of B cells to plasma cells. The Bach2 GRNs support the myeloid-based model of hematopoiesis. Myeloid-like characteristics suppressed or manifested in B cells by modifying differentiation trajectories of B and myeloid cells may be termed as 'inner myeloid' after the concept of 'inner fish'.

The activation of the adaptive immune system: cross-talk between antigen-presenting cells, T cells and B cells.

The adaptive immune system consists of T and B cells that express clonally distributed antigen receptors. To achieve functional adaptive immune responses, antigen-specific T cell populations are stimulated by professional antigen-presenting cells like dendritic cells (DCs), which provide crucial stimulatory signals for efficient expansion and development of effector functions. Antigen-specific B cells receive costimulatory signals from helper T cells to stimulate affinity maturation and isotype switching. Here we elaborate on the interactions between DCs, T cells and B cells, and on the important signals for efficient induction of adaptive immune responses.

Interaction between HIV-1 Tat and DNA-PKcs modulates HIV transcription and class switch recombination.

HIV-1 tat targets a variety of host cell proteins to facilitate viral transcription and disrupts host cellular immunity by inducing lymphocyte apoptosis, but whether it influences humoral immunity remains unclear. Previously, our group demonstrated that tat depresses expression of DNA-PKcs, a critical component of the non-homologous end joining pathway (NHEJ) of DNA double-strand breaks repair, immunoglobulin class switch recombination (CSR) and V(D)J recombination, and sensitizes cells to ionizing radiation. In this study, we demonstrated that HIV-1 Tat down-regulates DNA-PKcs expression by directly binding to the core promoter sequence. In addition, Tat interacts with and activates the kinase activity of DNA-PKcs in a dose-dependent and DNA independent manner. Furthermore, Tat inhibits class switch recombination (CSR) at low concentrations (≤ 4 µg/ml) and stimulates CSR at high concentrations (≥ 8 µg/ml). On the other hand, low protein level and high kinase activity of DNA-PKcs promotes HIV-1 transcription, while high protein level and low kinase activity inhibit HIV-1 transcription. Co-immunoprecipitation results revealed that DNA-PKcs forms a large complex comprised of Cyclin T1, CDK9 and Tat via direct interacting with CDK9 and Tat but not Cyclin T1. Taken together, our results provide new clues that Tat regulates host humoral immunity via both transcriptional depression and kinase activation of DNA-PKcs. We also raise the possibility that inhibitors and interventions directed towards DNA-PKcs may inhibit HIV-1 transcription in AIDS patients.

Generation and repair of AID-initiated DNA lesions in B lymphocytes.

Activation-induced deaminase (AID) initiates the secondary antibody diversification process in B lymphocytes. In mammalian B cells, this process includes somatic hypermutation (SHM) and class switch recombination (CSR), both of which require AID. AID induces U:G mismatch lesions in DNA that are subsequently converted into point mutations or DNA double stranded breaks during SHM/CSR. In a physiological context, AID targets immunoglobulin (Ig) loci to mediate SHM/CSR. However, recent studies reveal genome-wide access of AID to numerous non-Ig loci. Thus, AID poses a threat to the genome of B cells if AID-initiated DNA lesions cannot be properly repaired. In this review, we focus on the molecular mechanisms that regulate the specificity of AID targeting and the repair pathways responsible for processing AID-initiated DNA lesions.

Induction of activation-induced cytidine deaminase-targeting adaptor 14-3-3γ is mediated by NF-κB-dependent recruitment of CFP1 to the 5'-CpG-3'-rich 14-3-3γ promoter and is sustained by E2A.

Class switch DNA recombination (CSR) crucially diversifies Ab biologic effector functions. 14-3-3γ specifically binds to the 5'-AGCT-3' repeats in the IgH locus switch (S) regions. By interacting directly with the C-terminal region of activation-induced cytidine deaminase (AID), 14-3-3γ targets this enzyme to S regions to mediate CSR. In this study, we showed that 14-3-3γ was expressed in germinal center B cells in vivo and induced in B cells by T-dependent and T-independent primary CSR-inducing stimuli in vitro in humans and mice. Induction of 14-3-3γ was rapid, peaking within 3 h of stimulation by LPSs, and sustained over the course of AID and CSR induction. It was dependent on recruitment of NF-κB to the 14-3-3γ gene promoter. The NF-κB recruitment enhanced the occupancy of the CpG island within the 14-3-3γ promoter by CFP1, a component of the COMPASS histone methyltransferase complex, and promoter-specific enrichment of histone 3 lysine 4 trimethylation (H3K4me3), which is indicative of open chromatin state and marks transcription-competent promoters. NF-κB also potentiated the binding of B cell lineage-specific factor E2A to an E-box motif located immediately downstream of the two closely-spaced transcription start sites for sustained 14-3-3γ expression and CSR induction. Thus, 14-3-3γ induction in CSR is enabled by the CFP1-mediated H3K4me3 enrichment in the promoter, dependent on NF-κB and sustained by E2A.

IgG4-related disease: why high IgG4 and fibrosis?

The hallmarks of IgG4-related disease (IgG4-RD) are lymphoplasmacytic tissue infiltration with a predominance of IgG4-positive plasma cells, accompanied by fibrosis, obliterative phlebitis, dacryoadenitis, and elevated levels of IgG4. In a recent issue of Arthritis Research & Therapy, Tsuboi and colleagues demonstrated that regulatory T (Treg) cell-and T helper 2 (Th2) cell-derived cytokines contribute to the pathogenesis of Mikulicz's disease, an activation pathway that appears to be common for IgG4-RD. Additional organ-specific factors may account for the different organ involvement of IgG4-RD.

A combined nuclear and nucleolar localization motif in activation-induced cytidine deaminase (AID) controls immunoglobulin class switching.

Activation-induced cytidine deaminase (AID) is a DNA mutator enzyme essential for adaptive immunity. AID initiates somatic hypermutation and class switch recombination (CSR) by deaminating cytosine to uracil in specific immunoglobulin (Ig) gene regions. However, other loci, including cancer-related genes, are also targeted. Thus, tight regulation of AID is crucial to balance immunity versus disease such as cancer. AID is regulated by several mechanisms including nucleocytoplasmic shuttling. Here we have studied nuclear import kinetics and subnuclear trafficking of AID in live cells and characterized in detail its nuclear localization signal. Importantly, we find that the nuclear localization signal motif also directs AID to nucleoli where it colocalizes with its interaction partner, catenin-ß-like 1 (CTNNBL1), and physically associates with nucleolin and nucleophosmin. Moreover, we demonstrate that release of AID from nucleoli is dependent on its C-terminal motif. Finally, we find that CSR efficiency correlates strongly with the arithmetic product of AID nuclear import rate and DNA deamination activity. Our findings suggest that directional nucleolar transit is important for the physiological function of AID and demonstrate that nuclear/nucleolar import and DNA cytosine deamination together define the biological activity of AID. This is the first study on subnuclear trafficking of AID and demonstrates a new level in its complex regulation. In addition, our results resolve the problem related to dissociation of deamination activity and CSR activity of AID mutants.

Activation-induced deaminase in B lymphocyte maturation and beyond.

Activation-induced deaminase (AID), a member of the AID/apolipoprotein B mRNA-editing enzyme-catalytic (APOBEC) family, deaminates DNA cytidines into uridines and is the major trans-acting player of immunoglobulin (Ig) genes' diversification in mature B lymphocytes. It allows multiple antigen-driven Ig modifications through gene conversion and/or somatic hypermutation of variable region genes and also permits to switch from IgM expression to other antibody classes after class switch recombination, or to stop Ig expression after locus suicide recombination. AID is expressed at high levels into germinal center activated B cells with a very stringent temporal and spatial regulation. Despite multiple levels of regulation, off-target effects of AID are quite frequent in the B cell lineage and can affect a number of non-Ig genes, albeit at lower level than Ig genes. Beyond the immune system, AID also contributes to cytosine demethylation in undifferentiated cells by deaminating methylcytosines into thymines which are further processed by thymidine glycosylase. This contributes to the maintenance of pluripotency and to the limitation of genetic imprinting. Since AID attacks on DNA can induce replication errors, base excision repair, or mismatch repair, they are strongly mutagenic and are also driving forces of tumorigenesis, not only in B cell malignancies but also in some non-lymphoid tumors involving ectopic AID expression.

The histone chaperone Spt6 is required for activation-induced cytidine deaminase target determination through H3K4me3 regulation.

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.

Analysis of IgG4 class switch-related molecules in IgG4-related disease.

INTRODUCTION: Immunoglobulin G4 (IgG4)-related disease (IgG4-RD) is a new disease entity characterized by high serum IgG4 levels, IgG4-positive plasmacytic infiltration, and fibrosis in various organs. The purpose of this study was to determine the mechanism of upregulation of IgG4 class switch recombination in IgG4-RD. METHODS: We extracted RNA from peripheral blood mononuclear cells (PBMCs) of patients with IgG4-RD (n = 6), Sjögren syndrome (SS) (n = 6), and healthy controls (n = 8), from CD3-positive T cells and CD20-positive B cells sorted from PBMCs of patients with IgG4-RD (n = 3), SS (n = 4), and healthy controls (n = 4), as well as from labial salivary glands (LSGs) of patients with IgG4-RD (n = 11), SS (n = 13), and healthy controls (n = 3). The mRNA expression levels of IgG4-specific class switch-related molecules, such as Th2 cytokines (IL-4 and IL-13), Treg cytokines (IL-10 and TGF-ß), and transcriptional factors (GATA3 and Foxp3) were examined with quantitative polymerase chain reaction (PCR). IgG4-nonspecific class switch-related molecules, such as CD40, CD154, BAFF, APRIL, IRF4, and AID, were also examined. RESULTS: The expression levels of Treg cytokines (IL-10 and TGF-ß) and AID were significantly higher in LSGs of IgG4-RD than in SS and the controls (P < 0.05, each). In contrast, those of CD40 and CD154 were significantly lower in PBMCs of IgG4-RD than in SS (P < 0.05, each), whereas CD40 in CD20-positive B cells and CD154 in CD3-positive T cells were comparable in the three groups. CONCLUSION: Overexpression of IL-10, TGF-ß, and AID in LSGs might play important roles in the pathogenesis of IgG4-RD, such as IgG4-specific class-switch recombination and fibrosis. IgG4 class-switch recombination seems to be mainly upregulated in affected organs.

Immunoglobulin class-switch DNA recombination: induction, targeting and beyond.

Class-switch DNA recombination (CSR) of the immunoglobulin heavy chain (IGH) locus is central to the maturation of the antibody response and crucially requires the cytidine deaminase AID. CSR involves changes in the chromatin state and the transcriptional activation of the IGH locus at the upstream and downstream switch (S) regions that are to undergo S-S DNA recombination. In addition, CSR involves the induction of AID expression and the targeting of CSR factors to S regions by 14-3-3 adaptors, and it is facilitated by the transcription machinery and by histone modifications. In this Review, we focus on recent advances regarding the induction and targeting of CSR and outline an integrated model of the assembly of macromolecular complexes that transduce crucial epigenetic information to enzymatic effectors of the CSR machinery.

Iron inhibits activation-induced cytidine deaminase enzymatic activity and modulates immunoglobulin class switch DNA recombination.

Immunoglobulin (Ig) class switch DNA recombination (CSR) and somatic hypermutation (SHM) are critical for the maturation of the antibody response. Activation-induced cytidine deaminase (AID) initiates CSR and SHM by deaminating deoxycytidines (dCs) in switch (S) and V(D)J region DNA, respectively, to generate deoxyuracils (dUs). Processing of dUs by uracil DNA glycosylase (UNG) yields abasic sites, which are excised by apurinic/apyrimidinic endonucleases, eventually generating double strand DNA breaks, the obligatory intermediates of CSR. Here, we found that the bivalent iron ion (Fe(2+), ferrous) suppressed CSR, leading to decreased number of switched B cells, decreased postrecombination Iµ-C(H) transcripts, and reduced titers of secreted class-switched IgG1, IgG3, and IgA antibodies, without alterations in critical CSR factors, such as AID, 14-3-3γ, or PTIP, or in general germline I(H)-S-C(H) transcription. Fe(2+) did not affect B cell proliferation or plasmacytoid differentiation. Rather, it inhibited AID-mediated dC deamination in a dose-dependent fashion. The inhibition of intrinsic AID enzymatic activity by Fe(2+) was specific, as shown by lack of inhibition of AID-mediated dC deamination by other bivalent metal ions, such as Zn(2+), Mn(2+), Mg(2+), or Ni(2+), and the inability of Fe(2+) to inhibit UNG-mediated dU excision. Overall, our findings have outlined a novel role of iron in modulating a B cell differentiation process that is critical to the generation of effective antibody responses to microbial pathogens and tumoral cells. They also suggest a possible role of iron in dampening AID-dependent autoimmunity and neoplastic transformation.