Daniel J. Bolland

AID Is Required for the Chromosomal Breaks in c-myc that Lead to c-myc/IgH Translocations

Chromosomal translocation requires formation of paired double-strand DNA breaks (DSBs) on heterologous chromosomes. One of the most well characterized oncogenic translocations juxtaposes c-myc and the immunoglobulin heavy-chain locus (IgH) and is found in Burkitts lymphomas in humans and plasmacytomas in mice. DNA breaks in IgH leading to c-myc/IgH translocations are created by activation-induced cytidine deaminase (AID) during antibody class switch recombination or somatic hypermutation. However, the source of DNA breaks at c-myc is not known. Here, we provide evidence for the c-myc promoter region being required in targeting AID-mediated DNA damage to produce DSBs in c-myc that lead to c-myc/IgH translocations in primary B lymphocytes. Thus, in addition to producing somatic mutations and DNA breaks in antibody genes, AID is also responsible for the DNA lesions in oncogenes that are required for their translocation.

Antisense intergenic transcription in V(D)J recombination.

Antigen receptor genes undergo variable, diversity and joining (V(D)J) recombination, which requires ordered large-scale chromatin remodeling. Here we show that antisense transcription, both genic and intergenic, occurs extensively in the V region of the immunoglobulin heavy chain locus. RNA fluorescence in situ hybridization demonstrates antisense transcription is strictly developmentally regulated and is initiated during the transition from DJH to VDJH recombination and terminates concomitantly with VDJH recombination. Our data show antisense transcription is specific to the V region and suggest transcripts extend across several genes. We propose that antisense transcription remodels the V region to facilitate VH-to-DJH recombination. These findings have wider implications for V(D)J recombination of other antigen receptor loci and developmental regulation of multigene loci.

Complete Sequence Assembly and Characterization of the C57BL/6 Mouse Ig Heavy Chain V Region

The mechanisms that regulate variable (V) gene selection during the development of the mouse IgH repertoire are not fully understood, due in part to the absence of the complete locus sequence. To better understand these processes, we have assembled the entire 2.5-Mb mouse IgH (Igh) V region sequence of the C57BL/6 strain from public sequences and present the first complete annotated map of the region, including V genes, pseudogenes, repeats, and nonrepetitive intergenic sequences. In so doing, we have discovered a new V gene family, VH16. We have identified clusters of conserved region-specific intergenic sequences and have verified our assembly by genic and intergenic Southern blotting. We have observed that V pseudogenes are not evenly spread throughout the V region, but rather cluster together. The largest J558 family, which spans more than half of the locus, has two strikingly different domains, which suggest points of evolutionary divergence or duplication. The 5′ end contains widely spaced J558 genes interspersed with 3609 genes and is pseudogene poor. The 3′ end contains closely spaced J558 genes, no 3609 genes, and is pseudogene rich. Each occupies a different branch of the phylogenetic tree. Detailed analysis of 500-bp upstream of all functional genes has revealed several conserved binding sites, general and B cell-specific, as well as key differences between families. This complete and definitive assembly of the mouse Igh V region will facilitate detailed study of promoter function and large-scale mechanisms associated with V(D)J recombination including locus contraction and antisense intergenic transcription.

The PI3K Isoforms p110alpha and p110delta Are Essential for Pre–B Cell Receptor Signaling and B Cell Development

Different isoforms of phosphatidylinositol 3-kinase have distinct roles in B cell receptor signaling. Relative Importance B cell receptor (BCR) signaling drives the development and survival of B cells and their responses to antigens. Members of the phosphatidylinositol 3-kinase (PI3K) family of lipid kinases mediate BCR signaling. Whereas the p110δ isoform of PI3K is necessary for antigen-dependent BCR signaling, its loss does not affect B cell development in the bone marrow. Ramadani et al. found that, whereas deficiency in individual PI3K isoforms in mice did not prevent early B cell development, deficiency in both p110δ and p110α blocked antigen-independent, so-called tonic, BCR signaling, which was required for B cell development. In contrast, antigen-dependent signaling required p110δ. As Limon and Fruman discuss in the accompanying Perspective, the discovery of this role for p110α suggests that the combined inhibition of p110α and p110δ, rather than of p110δ alone, would be more effective as a therapy to target B cell malignancies that involve chronic BCR signaling. B cell development is controlled by a series of checkpoints that ensure that the immunoglobulin (Ig)–encoding genes produce a functional B cell receptor (BCR) and antibodies. As part of this process, recombination-activating gene (Rag) proteins regulate the in-frame assembly of the Ig-encoding genes. The BCR consists of Ig proteins in complex with the immunoreceptor tyrosine-based activation motif (ITAM)–containing Igα and Igβ chains. Whereas the activation of the tyrosine kinases Src and Syk is essential for BCR signaling, the pathways that act downstream of these kinases are incompletely defined. Previous work has revealed a key role for the p110δ isoform of phosphatidylinositol 3-kinase (PI3K) in agonist-induced BCR signaling; however, early B cell development and mature B cell survival, which depend on agonist-independent or “tonic” BCR signaling, are not substantially affected by a deficiency in p110δ. Here, we show that p110α, but not p110β, compensated in the absence of p110δ to promote early B cell development in the bone marrow and B cell survival in the spleen. In the absence of both p110α and p110δ activities, pre-BCR signaling failed to suppress the production of Rag proteins and to promote developmental progression of B cell progenitors. Unlike p110δ, however, p110α did not contribute to agonist-induced BCR signaling. These studies indicate that either p110α or p110δ can mediate tonic signaling from the BCR, but only p110δ can contribute to antigen-dependent activation of B cells.

Regulation of IgH Gene Assembly: Role of the Intronic Enhancer and 5′DQ52 Region in Targeting DHJH Recombination

The assembly of Ag receptor genes by V(D)J recombination is regulated by transcriptional promoters and enhancers which control chromatin accessibility at Ig and TCR gene segments to the RAG-1/RAG-2 recombinase complex. Paradoxically, germline deletions of the IgH enhancer (Eμ) only modestly reduce DH→JH rearrangements when assessed in peripheral B cells. However, deletion of Eμ severely impairs recombination of VH gene segments, which are located over 100 kb away. We now test two alternative explanations for the minimal effect of Eμ deletions on primary DH→JH rearrangement: 1) Accessibility at the DHJH cluster is controlled by a redundant cis-element in the absence of Eμ. One candidate for this element lies 5′ to DQ52 (PDQ52) and exhibits promoter/enhancer activity in pre-B cells. 2) In contrast to endpoint B cells, DH→JH recombination may be significantly impaired in pro-B cells from enhancer-deficient mice. To elucidate the roles of PDQ52 and Eμ in the regulation of IgH locus accessibility, we generated mice with targeted deletions of these elements. We report that the defined PDQ52 promoter is dispensable for germline transcription and recombination of the DHJH cluster. In contrast, we demonstrate that Eμ directly regulates accessibility of the DHJH region. These findings reveal a significant role for Eμ in the control mechanisms that activate IgH gene assembly and suggest that impaired VH→DHJH rearrangement in enhancer-deficient cells may be a downstream consequence of the primary block in DH→JH recombination.

Antisense Intergenic Transcription Precedes Igh D-to-J Recombination and Is Controlled by the Intronic Enhancer Eμ

ABSTRACT V(D)J recombination is believed to be regulated by alterations in chromatin accessibility to the recombinase machinery, but the mechanisms responsible remain unclear. We previously proposed that antisense intergenic transcription, activated throughout the mouse Igh VH region in pro-B cells, remodels chromatin for VH-to-DJH recombination. Using RNA fluorescence in situ hybridization, we now show that antisense intergenic transcription occurs throughout the Igh DHJH region before D-to-J recombination, indicating that this is a widespread process in V(D)J recombination. Transcription initiates near the Igh intronic enhancer Eμ and is abrogated in mice lacking this enhancer, indicating that Eμ regulates DH antisense transcription. Eμ was recently demonstrated to regulate DH-to-JH recombination of the Igh locus. Together, these data suggest that Eμ controls DH-to-JH recombination by activating this form of germ line Igh transcription, thus providing a long-range, processive mechanism by which Eμ can regulate chromatin accessibility throughout the DH region. In contrast, Eμ deletion has no effect on VH antisense intergenic transcription, which is rarely associated with DH antisense transcription, suggesting differential regulation and separate roles for these processes at sequential stages of V(D)J recombination. These results support a directive role for antisense intergenic transcription in enabling access to the recombination machinery.

RNA-binding proteins ZFP36L1 and ZFP36L2 promote cell quiescence

Reducing the risk of rearrangement As lymphocytes develop, they rearrange their antigen receptor genes and proliferate extensively, potentially putting their genomes at risk. Galloway et al. found that two RNA-binding proteins, ZFP36L1 and ZFP36L2, ensure careful entry and exit into the cell cycle. This helps developing B lymphocytes maintain their genomic integrity. Mice deficient in ZFP36L1 and ZFP36L2 exhibited a profound block in B cell development. ZFP36L1 and ZFP36L2 suppress mRNAs that help B cells progress through the cell cycle, ensuring that cells can enter quiescence and keep their genomes safe when they undergo the risky process of rearranging their antigen receptors. Science, this issue p. 453 RNA-binding proteins enforce quiescence on developing lymphocytes by suppressing cell cycle progression. Progression through the stages of lymphocyte development requires coordination of the cell cycle. Such coordination ensures genomic integrity while cells somatically rearrange their antigen receptor genes [in a process called variable-diversity-joining (VDJ) recombination] and, upon successful rearrangement, expands the pools of progenitor lymphocytes. Here we show that in developing B lymphocytes, the RNA-binding proteins (RBPs) ZFP36L1 and ZFP36L2 are critical for maintaining quiescence before precursor B cell receptor (pre-BCR) expression and for reestablishing quiescence after pre-BCR–induced expansion. These RBPs suppress an evolutionarily conserved posttranscriptional regulon consisting of messenger RNAs whose protein products cooperatively promote transition into the S phase of the cell cycle. This mechanism promotes VDJ recombination and effective selection of cells expressing immunoglobulin-μ at the pre-BCR checkpoint.

ATMIN Is Required for Maintenance of Genomic Stability and Suppression of B Cell Lymphoma

Summary Defective V(D)J rearrangement of immunoglobulin heavy or light chain (IgH or IgL) or class switch recombination (CSR) can initiate chromosomal translocations. The DNA-damage kinase ATM is required for the suppression of chromosomal translocations but ATM regulation is incompletely understood. Here, we show that mice lacking the ATM cofactor ATMIN in B cells (ATMINΔB/ΔB) have impaired ATM signaling and develop B cell lymphomas. Notably, ATMINΔB/ΔB cells exhibited defective peripheral V(D)J rearrangement and CSR, resulting in translocations involving the Igh and Igl loci, indicating that ATMIN is required for efficient repair of DNA breaks generated during somatic recombination. Thus, our results identify a role for ATMIN in regulating the maintenance of genomic stability and tumor suppression in B cells.

Heavy chain-only antibodies are spontaneously produced in light chain-deficient mice

In healthy mammals, maturation of B cells expressing heavy (H) chain immunoglobulin (Ig) without light (L) chain is prevented by chaperone association of the H chain in the endoplasmic reticulum. Camelids are an exception, expressing homodimeric IgGs, an antibody type that to date has not been found in mice or humans. In camelids, immunization with viral epitopes generates high affinity H chain–only antibodies, which, because of their smaller size, recognize clefts and protrusions not readily distinguished by typical antibodies. Developmental processes leading to H chain antibody expression are unknown. We show that L−/− (κ−/−λ−/−-deficient) mice, in which conventional B cell development is blocked at the immature B cell stage, produce diverse H chain–only antibodies in serum. The generation of H chain–only IgG is caused by the loss of constant (C) γ exon 1, which is accomplished by genomic alterations in CH1-circumventing chaperone association. These mutations can be attributed to errors in class switch recombination, which facilitate the generation of H chain–only Ig-secreting plasma cells. Surprisingly, transcripts with a similar deletion can be found in normal mice. Thus, naturally occurring H chain transcripts without CH1 (VHDJH-hinge-CH2-CH3) are selected for and lead to the formation of fully functional and diverse H chain–only antibodies in L−/− animals.

Two Mutually Exclusive Local Chromatin States Drive Efficient V(D)J Recombination.

Summary Variable (V), diversity (D), and joining (J) (V(D)J) recombination is the first determinant of antigen receptor diversity. Understanding how recombination is regulated requires a comprehensive, unbiased readout of V gene usage. We have developed VDJ sequencing (VDJ-seq), a DNA-based next-generation-sequencing technique that quantitatively profiles recombination products. We reveal a 200-fold range of recombination efficiency among recombining V genes in the primary mouse Igh repertoire. We used machine learning to integrate these data with local chromatin profiles to identify combinatorial patterns of epigenetic features that associate with active VH gene recombination. These features localize downstream of VH genes and are excised by recombination, revealing a class of cis-regulatory element that governs recombination, distinct from expression. We detect two mutually exclusive chromatin signatures at these elements, characterized by CTCF/RAD21 and PAX5/IRF4, which segregate with the evolutionary history of associated VH genes. Thus, local chromatin signatures downstream of VH genes provide an essential layer of regulation that determines recombination efficiency.