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Somatic hypermutation (SHM) drives the genetic diversity of Ig genes in activated B cells and supports the generation of Abs with increased affinity for Ag. SHM is targeted to Ig genes by their enhancers (diversification activators [DIVACs]), 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 II (Pol II) and Pol II occupancy in the mutating gene with little or no accompanying increase in elongation-competent Pol II or production of full-length transcripts, indicating accumulation of stalled Pol II. 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 ssDNA 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 Pol II 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., (Copyright © 2021 by The American Association of Immunologists, Inc.)

B lymphopoiesis requires that immunoglobulin genes be accessible to RAG1-RAG2 recombinase. However, the RAG proteins bind widely to open chromatin, which suggests that additional mechanisms must restrict RAG-mediated DNA cleavage. Here we show that developmental downregulation of interleukin 7 (IL-7)-receptor signaling in small pre-B cells induced expression of the bromodomain-family member BRWD1, which was recruited to a specific epigenetic landscape at Igk dictated by pre-B cell receptor (pre-BCR)-dependent Erk activation. BRWD1 enhanced RAG recruitment, increased gene accessibility and positioned nucleosomes 5' to each Jκ recombination signal sequence. BRWD1 thus targets recombination to Igk and places recombination within the context of signaling cascades that control B cell development. Our findings represent a paradigm in which, at any particular antigen-receptor locus, specialized mechanisms enforce lineage- and stage-specific recombination.

Coordinated recombination of homologous antigen receptor loci is thought to be important for allelic exclusion. Here we show that homologous immunoglobulin alleles pair in a stage-specific way that mirrors the recombination patterns of these loci. The frequency of homologous immunoglobulin pairing was much lower in the absence of the RAG-1-RAG-2 recombinase and was restored in Rag1-/- developing B cells with a transgene expressing a RAG-1 active-site mutant that supported DNA binding but not cleavage. The introduction of DNA breaks on one immunoglobulin allele induced ATM-dependent repositioning of the other allele to pericentromeric heterochromatin. ATM activated by the cleaved allele acts in trans on the uncleaved allele to prevent biallelic recombination and chromosome breaks or translocations.

Previously, we described a tetracycline-based autoregulatory system for inducible gene expression in mammalian cells and transgenic mice [Proc. Natl. Acad. Sci. U.S.A. 92 (1995) 6522]. We have tested the ability of this system to drive functional expression in vivo of the V(D)J recombination activating genes, RAG1 and RAG2. In induced transgenic mice, transgenic RAG1 and RAG2 mRNA is observed in thymus and spleen, and expression of both transgenes on the RAG1 or RAG2 knockout backgrounds allows partial, inducible, lymphocyte reconstitution. In thymus and peripheral lymphoid organs of reconstituted animals, cells expressing CD4 and/or CD8 on their surface, also express CD3 and TCR beta chain. In these animals, V(D)J rearrangements are detected in thymus, lymph nodes, and spleen at the TRB locus, and in thymus and lymph nodes at the TRD locus. At the TRA locus, broken ends at V(D)J recombination signals are detected only in thymus, as are reciprocal signal joint products derived from deletional rearrangement. T cell reconstitution occurs in these animals whether they are induced in utero during development, or shortly after birth. A low level of B cell reconstitution is also observed. B220+IgM+ cells are observed in spleen only in induced animals, and rearrangements at IGH and IGK loci are detected in bone marrow and spleen. Broken signal ends at the IGK locus, are not detected in peripheral lymphoid organs. Inducible reconstitution of normal levels of serum immunoglobulin, including heavy chain class switch isotype variants is also observed in these animals. Further, these transgenes do not appear to interfere with lymphocyte development mediated by functionally rearranged TRB chain or IGH chain transgenes in RAG-deficient animals. These mice provide a unique system for the inducible activation of V(D)J recombination and the development of primary lymphocytes.

The activation induced cytosine deaminase (AID) mediates diversification of B cell immunoglobulin genes by the three distinct yet related processes of somatic hypermutation (SHM), class switch recombination (CSR), and gene conversion (GCV). SHM occurs in germinal center B cells, and the transcription factor Bcl6 is a key regulator of the germinal center B cell gene expression program, including expression of AID. To test the hypothesis that Bcl6 function is important for the process of SHM, we compared WT chicken DT40 B cells, which constitutively perform SHM/GCV, to their Bcl6-deficient counterparts. We found that Bcl6-deficient DT40 cells were unable to perform SHM and GCV despite enforced high level expression of AID and substantial levels of AID in the nucleus of the cells. To gain mechanistic insight into the GCV/SHM dependency on Bcl6, transcriptional features of a highly expressed SHM target gene were analyzed in Bcl6-sufficient and -deficient DT40 cells. No defect was observed in the accumulation of single stranded DNA in the target gene as a result of Bcl6 deficiency. In contrast, association of Spt5, an RNA polymerase II (Pol II) and AID binding factor, was strongly reduced at the target gene body relative to the transcription start site in Bcl6-deficient cells as compared to WT cells. However, partial reconstitution of Bcl6 function substantially reconstituted Spt5 association with the target gene body but did not restore detectable SHM. Our observations suggest that in the absence of Bcl6, Spt5 fails to associate efficiently with Pol II at SHM targets, perhaps precluding robust AID action on the SHM target DNA. Our data also suggest, however, that Spt5 binding is not sufficient for SHM of a target gene even in DT40 cells with strong expression of AID. [ABSTRACT FROM AUTHOR]

: Immunoglobulin gene enhancers have a conserved function in targeting somatic hypermutation to immunoglobulin genes, thereby supporting the production of high affinity antibodies. [ABSTRACT FROM AUTHOR]

Genes encoding immunoglobulin heavy chains (Igh) are assembled by rearrangement of variable (VH), diversity (DH) and joining (JH) gene segments. Three critical constraints govern VH recombination. These include timing (VH recombination follows DH recombination), precision (VH gene segments recombine only to DJH junctions) and allele specificity (VH recombination is restricted to DJH-recombined alleles). Here we provide a model for these universal features of VH recombination. Analyses of DJH-recombined alleles showed that DJH junctions were selectively epigenetically marked, became nuclease sensitive and bound RAG recombinase proteins, which thereby permitted DH-associated recombination signal sequences to initiate the second step of Igh gene assembly. We propose that VH recombination is precise, because these changes did not extend to germline DH segments located 5? of the DJH junction. [ABSTRACT FROM AUTHOR]

V(D)J recombination assembles immunoglobulin and T cell receptor genes during lymphocyte development through a series of carefully orchestrated DNA breakage and rejoining events. DNA cleavage requires a series of protein-DNA complexes containing the RAG1 and RAG2 proteins and recombination signals that flank the recombining gene segments. In this review, we discuss recent advances in our understanding of the function and domain organization of the RAG proteins, the composition and structure of RAG-DNA complexes, and the pathways that lead to the formation of these complexes. We also consider the functional significance of RAG-mediated histone recognition and ubiquitin ligase activities, and the role played by RAG in ensuring proper repair of DNA breaks made during V(D)J recombination. Finally, we propose a model for the formation of RAG-DNA complexes that involves anchoring of RAG1 at the recombination signal nonamer and RAG2-dependent surveillance of adjoining DNA for suitable spacer and heptamer sequences. [ABSTRACT FROM AUTHOR]

Somatic hypermutation introduces point mutations into immunoglobulin genes in germinal centre B cells during an immune response. The reaction is initiated by cytosine deamination by the activation-induced deaminase (AID) and completed by error-prone processing of the resulting uracils by mismatch and base excision repair factors. Somatic hypermutation represents a threat to genome integrity and it is not known how the B cell genome is protected from the mutagenic effects of somatic hypermutation nor how often these protective mechanisms fail. Here we show, by extensive sequencing of murine B cell genes, that the genome is protected by two distinct mechanisms: selective targeting of AID and gene-specific, high-fidelity repair of AID-generated uracils. Numerous genes linked to B cell tumorigenesis, including Myc, Pim1, Pax5, Ocab (also called Pou2af1), H2afx, Rhoh and Ebf1, are deaminated by AID but escape acquisition of most mutations through the combined action of mismatch and base excision repair. However, approximately 25% of expressed genes analysed were not fully protected by either mechanism and accumulated mutations in germinal centre B cells. Our results demonstrate that AID acts broadly on the genome, with the ultimate distribution of mutations determined by a balance between high-fidelity and error-prone DNA repair. [ABSTRACT FROM AUTHOR]

Somatic hypermutation (SHM) is a major means by which diversity is achieved in antibody genes, and it is initiated by the deamination of cytosines to uracils in DNA by activation-induced deaminase (AID). However, the process that Leads from these initiating deamination events to mutations at other residues remains poorly understood. We demonstrate that a single cytosine on the top (nontemplate) strand is sufficient to recruit AID and Lead to mutations of upstream and downstream A/T residues. In contrast, the targeting of cytosines on the bottom strand by AID does not lead to substantial mutation of neighboring residues. This strand asymmetry is eliminated in mice deficient in mismatch repair, indicating that the error-prone mismatch repair machinery preferentially targets top-strand uracils in a way that promotes SHM during the antibody response. [ABSTRACT FROM AUTHOR]

After their assembly by V(D)J recombination, immunoglobulin (Ig) genes undergo somatic hypermutation, gene conversion, and class switch recombination to generate additional antibody diversity. The three diversification processes depend on activation-induced cytidine deaminase (AID) and are tightly linked to transcription. The reactions occur primarily on Ig genes and the molecular mechanisms that underlie their targeting to Ig loci have been of intense interest. In this chapter, we discuss the evidence linking transcription and transcriptional control elements to the three diversification pathways, and we consider how various features of chromatin could render parts of the genome permissive for AID-mediated sequence diversification. [ABSTRACT FROM AUTHOR]

Chromosomal translocations involving immunoglobulin heavy chain (Igh) switch regions and an oncogene such as Myc represent initiating events in the development of many B cell malignancies. These translocations are widely thought to result from aberrant class-switch recombination. To test this model, we measured translocations in mice deficient in activation-induced cytidine deaminase (AID) that lack class-switch recombination. We found that AID made no measurable contribution to the generation of initial translocations, indicating that the intrinsic fragility of the switch regions or a pathway unrelated to AID is responsible for these translocations. In contrast, the outgrowth of translocation-positive cells was dependent on AID, raising the possibility that AID is important in tumor progression, perhaps by virtue of its mutagenic properties. [ABSTRACT FROM AUTHOR]

Presents a report on the hypermutation of immunoglobulin variable region genes and on the existence of abundant DNA double-strand breaks (DSBs) in hypermutating sequences. How the hypermutation occurs in B cells during an immune response; Generation of the DSBs and how it is coupled to transcription, enhancer-dependent, and correlates with the appearance of nearby mutations; Proposition of a model for somatic hypermutation in which mutations are introduced into the DNA during repair of DSBs by homologous recombination.

Reports on the transposition mediated by RAG1 and RAG2 proteins and its implications for the evolution of the immune system. How immunglobulin and T-cell-receptor genes are assembled; Suggestion that the results support the theory that RAG1 and RAG2 were once compounds of a transposable element.

Very little is known about how acquired oncogenic mutations arise. In this issue of Cancer Cell, Klemm and colleagues present evidence supporting a role for the antibody diversification enzyme activation-induced deaminase (AID) in the generation of mutations associated with disease progression and drug resistance in chronic myeloid leukemia. [Copyright &y& Elsevier]

A correction to the article "RAG-1 and ATM coordinate monoallelic recombination and nuclear positioning of immunoglobin loci," that was published online on May 17, 2009 is presented.

Discusses the role of activation-induced cytidine deaminase in immunoglobulin class switching and hypermutation. Immunoglobulin gene transcription; R-loop model of class switch recombination; RNA-DNA structure.

Examines the roles of immunoglobulin and antibodies on genetic diversity. Recombination of Ig genes from germline gene; Molecular processes underlying somatic hypermutation; DNA lesions in gene conversion.

A correction to the article "RAG-1 and ATM Coordinate Monoallelic Recombination and Nuclear Positioning of Immunoglobulin Loci" that was published online in the May 17, 2009 issue is presented.

The first step in V(D)J recombination is the formation of specific DNA double-strand breaks (DSBs) by the RAG1 and RAG2 proteins, which form the RAG recombinase. DSBs activate a complex network of proteins termed theDNAdamage response (DDR). A key early event in the DDR is the phosphorylation of histone H2AX around DSBs, which forms a binding site for the tandem BRCA1 C-terminal (tBRCT) domain of MDC1. This event is required for subsequent signal amplification and recruitment of additional DDR proteins to the break site. RAG1 bears a histone H2AX-like motif at its C terminus (R1Ct), making it a putative MDC1-binding protein. In this work we show that the tBRCT domain ofMDC1binds the R1Ct motif of RAG1. Surprisingly, we also observed a second binding interface between the two proteins that involves the Proline-Serine-Threonine rich (PST) repeats of MDC1 and the N-terminal noncore region of RAG1 (R1Nt). The repeats-R1Nt interaction is constitutive, whereas the tBRCT-R1Ct interaction likely requires phosphorylation of the R1Ct motif of RAG1. As the C terminus of RAG1 has been implicated in inhibition of RAG activity, we propose a model in which phosphorylation of the R1Ct motif of RAG1 functions as a self-initiated regulatory signal. [ABSTRACT FROM AUTHOR]

The molecular features that allow activation-induced cytidine deaminase (AID) to target Ig and certain non-Ig genes are not understood, although transcription has been implicated as one important parameter. We explored this issue by testing the mutability of a non-Ig transcription cassette in Ig and non-Ig loci of the chicken B cell line DT40. The cassette did not act as a stable long term mutation target but was able to be mutated in an AID-dependent manner for a limited time post-integration. This indicates that newly integrated DNA has molecular characteristics that render it susceptible to modification by AID, with implications for how targeting and mis-targeting of AID occurs. [ABSTRACT FROM AUTHOR]

Focuses on a study which examined the assembly of immunoglobulin heavy chain (IgH) V gene locus in Pax5-deficient pro-B cells. Expression of IgH V genes; Sterile transcription of IgH D and J gene segments; Recombination of IgH V genes; Histone acetylation of IgH V gene segments.