Immunoglobulin Gene Segments Research Articles

A case of convergence: why did a simple alternative to canonical antibodies arise in sharks and camels?

The success of our adaptive humoral immune response, mediated by B cells, relies on its capacity to produce multiple antigen-recognition specificities against any possible invader. This antigen-recognition specificity is attributed to antibodies, which display remarkable flexibility and diversity. That diversity is generated by cutting and pasting of immunoglobulin (Ig) gene segments during B-cell development to generate functional variable (V) genes, which are selected from two separate regions of the genome to yield so-called “heavy” (H) and “light” (L) chains (Figure 1; see Section 1 in Text S1). Ig H and L chain V genes have been expanded in various vertebrate taxa to yield multiple families and subfamilies [1]. Given the obvious advantage of the diversity afforded by such multichain antibodies, it was extremely surprising to discover that some species—notably cartilaginous fish (holocephalins, e.g., chimeras, and elasmobranchs, e.g., sharks, skates, and rays) and camelids (e.g., camels and llamas)—developed functional H-chain-only antibodies (HCAbs) (Figures 1 and ​and2)2) [2],[3]. Loss of the combinatorial H-L diversification of heterotetrameric (H2L2) antibodies should result in a handicapped, less diversified antigen-binding repertoire in HCAbs; yet, contrary to expectations, high-affinity, antigen-reactive HCAbs can be elicited to all studied antigens [4],[5]. Even more peculiar, the HCAbs function alongside conventional H2L2 antibodies. What explains this remarkable evolutionary convergence of functional HCAbs in sharks and camels? What forces drove such distantly related species to produce antibodies with this simple structure? And, if they are so effective why is their prevalence not more widespread in evolution? Figure 1 Schematic of classical H2L2 and H2 homodimeric HCAbs. Figure 2 Simplified vertebrate phylogenetic tree emphasizing those taxa possessing single-domain Igs or TCRs. Occurrence of Functional Heavy Chain-Only Antibodies in Camelids and Shark The antigen-binding fragment of camelid HCAbs consists of a single domain known as a variable domain of the heavy chain of HCAbs (VHH) (Figure 1) [2]. It was demonstrated that HCAbs from dromedaries (one-humped camels) infected with trypanosomes are capable of associating tightly and specifically with these parasite antigens [2]. Subsequently, HCAbs were elicited to many different foreign antigens. HCAbs are present in all living species of the mammalian family Camelidae, but not in other ungulates (hoofed mammals). Approximately equal levels of HCAbs and H2L2 antibodies are present in camel blood [6]. The origin of camelid Ig genes encoding HCAbs is unambiguous. Phylogenetic analyses have demonstrated that the HCAb-dedicated γ genes were derived from γ genes coding for conventional antibodies. The genes emerged and diverged ∼25 million years ago [6],[7], after Tylopoda split from other mammals (60–80 million years ago) and before the camel and llama speciation (∼11 million years ago) (Figure 2) [6]. Also, the VHH germline genes descended from the classical variable region of a heavy-chain (VH) genes, and the diversity (D) and JH genes are even shared in the formation of the VH or VHH domains. Thus, the emergence of HCAbs was a relatively recent event in these species. In the nurse shark (Ginglymostoma cirratum), a novel secreted antigen receptor was reported and named the new antigen receptor (NAR) [3]. It too is composed of two covalently associated H-chains with no associated L-chains, but having six domains (Figure 1). Since the molecule shares several functional features with Ig isotypes, it was renamed IgNAR [8], and its N-terminal V domain is known as V-NAR. Later studies showed IgNAR to be induced by immunization with protein antigens with similar kinetics as in a typical IgM response [9]. Although the origin of shark IgNAR is poorly understood, IgNAR is found in all elasmobranchs and thus emerged at least 220 million years ago (Figure 2). However, the single-domain V is also found as a T-cell receptor (TCR) in all living cartilaginous fish (so-called NAR-TCR) [10],[11], showing that this immune system feature appeared at least 350 million years ago (Figure 2). In addition, V-NAR gene clusters actually are present in the TCR-δ loci of all cartilaginous fish and thus it remains uncertain whether they originated as a TCR, an Ig, or perhaps a common antigen receptor ancestor [10].

Construction and molecular characterization of mouse single-chain variable fragment antibodies against Burkholderia mallei and Burkholderia pseudomallei

We have selected two lipopolysaccharide (LPS) specific Burkholderia mallei mouse monoclonal antibodies (mAbs) and four anti-capsular B. pseudomallei-specific mAbs to generate mouse single-chain variable fragment (scFv) antibodies. This selection was made through extensive in vitro and in vivo assay from our library of mAbs against B. mallei and B. pseudomallei. We initially generated the mouse immunoglobulin variable heavy chain (VH) and light chain (VL) regions from each of these six selected mAbs using a phage display scFv technology. We determined the coding sequences of the VH and VL regions and successfully constructed two B. mallei-specific scFv phage antibodies consisting of two different VH (VH1 and VH2) and one Vλ1 families. Four scFvs constructed against B. pseudomallei had two VH (VH1 and VH6) and two VL (Vκ4/5 and Vκ21) families. All of six scFv antibodies constructed demonstrated good binding activity without any rounds of biopanning against B. mallei (M5D and M18F were 0.425 and 0.480 at OD405nm) and B. pseudomallei (P1E7, P2I67, P7C6, and P7F4 were 0.523, 0.859, 0.775, and 0.449 at OD405nm) by ELISA, respectively. A comparison of the immunoglobulin gene segments revealed that the gene sequences in complementarity-determining regions (CDRs) of three out of four B. pseudomallei-specific scFvs are highly conserved. We determined that the two B. mallei-specific scFvs have different CDRs in the VH, but the amino acid sequences of CDRs in the VL are conserved. This high sequence homology found in CDRs of VH or VL of these mAbs contributes to our better understanding and determination of binding to the specific antigenic epitope(s). The scFv phage display technology may be a valuable tool to develop and engineer mAbs with improved antigen-binding affinity.

Analysis of human immunoglobulin gene repertoire in NOD/scid IL2Rγ null mice engrafted with human cord blood derived hematopoietic stem cells (36.14)

Abstract The humanized NOD/scid IL2rg-/- (hNSG) and similar mouse models hold promise as testing platforms for preclinical studies. Here, we analyzed the human immunoglobulin (IG) variable heavy and light chain gene repertoire from sorted single B-cells of hNSG mice engrafted with cord blood derived hematopoietic stem cells. Unique IGHV (339) and IGLV (364, kappa and lambda) gene segments were derived from major B cell subsets identified in a largely normal B cell development pathway. However, the hNSG mice expressed elevated CD5+ mature B-1-like cells in the periphery. In mice and humans, B-1 cells are the major source of autoantibodies. Although the hNSG mice expressed a highly diverse repertoire comparable to humans, detailed genetic analysis of IG gene segments from the mature B cell subset revealed the presence of long IGH-CDR3 regions with positive charges—a hallmark for auto/polyreactivity. For functional tests, several in vitro expressed scFv-Fc IG candidates, derived from cloned heavy and light chain cognate pairs of single B cells, tested positive in an anti-nuclear antibody assay for auto/polyreactivity and/or bound to HIVgp120 in ELISA assays. The natural induction during HIV-1 infection of broadly neutralizing antibodies (BnAbs) which display autoantibody-like characteristics may be curtailed by the checkpoint control mechanisms during B cell ontogeny. We propose the hNSG mouse can be a model platform to test this hypothesis and interrogate how BnAbs are formed.

Violations of the 12/23 rule at the mouse immunoglobulin kappa locus, including Vκ-Vκ rearrangement

Classically, recombination between immunoglobulin gene segments uses a pair of recombination signal sequences (RSSs) with dissimilar spacers (the “12/23 rule”). Using a series of different genotyping assays, four different kinds of atypical rearrangements were identified at the murine kappa locus: (1) Vκ to Vκ, (2) Jκ to Jκ, (3) Vκ to iRS, a heptameric sequence found in the JκCκ intron, and (4) a possible by-product of a rearrangement between a Vκ and the hypothetical 12-RSS side of a pre-existing signal joint. The novel Vκ-Vκ structure prompted further characterization. Sequence analysis of 14 different Vκ-Vκ rearrangements cloned from murine splenocytes and hybridomas revealed a Vκ4 family member as one participant in 13 rearrangements, but no rearrangements contained two Vκ4 genes. The Vκ4 partner in the Vκ-Vκ rearrangement exhibited more trimming of nucleotides at the Vκ-Vκ junction. A signal joint derived from the inversional rearrangement of two neighboring Vκs was also recovered. These data suggest that the Vκ-Vκ structures arise via RAG-mediated, intrachromosomal recombination.

Use of V H, D and J H immunoglobulin gene segments in Brazilian patients with chronic lymphocytic leukaemia (CLL)

Chronic lymphocytic leukaemia (CLL) is a haematological malignancy for which reliable prognostic markers are needed in view of its clinical heterogeneity. In approximately 50% of CLL patients, immunoglobulin (Ig) rearrangements are modified by somatic hypermutation (SHM), a process that represents a reliable prognostic indicator of favourable progression. In this study, we investigated SHM in 37 Brazilian CLL patients and identified the preferential involvement of specific immunoglobulin gene families and segments through PCR-amplified fragments or subcloned fragments. Forty-one rearrangements were observed and 37 of them were functional. A 98% homology cut-off with germinal sequences showed 18 patients (48.7%) with SHM. Unmutated cases showed a poorer clinical outcome. VH3 was the most frequent VH family, followed by VH4. The VH4-39 segment was the most frequently used, mainly in unmutated cases, while the VH3 family was predominant in mutated cases. The D3 and JH4/JH6 families were the most frequently observed.

A Plant Homeodomain in Rag-2 that Binds Hypermethylated Lysine 4 of Histone H3 Is Necessary for Efficient Antigen-Receptor-Gene Rearrangement

V(D)J recombination is initiated by the recombination activating gene (RAG) proteins RAG-1 and RAG-2. The ability of antigen-receptor-gene segments to undergo V(D)J recombination is correlated with spatially- and temporally-restricted chromatin modifications. We have found that RAG-2 bound specifically to histone H3 and that this binding was absolutely dependent on dimethylation or trimethylation at lysine 4 (H3K4me2 or H3K4me3). The interaction required a noncanonical plant homeodomain (PHD) that had previously been described within the noncore region of RAG-2. Binding of the RAG-2 PHD finger to chromatin across the IgH D-J(H)-C locus showed a strong correlation with the distribution of trimethylated histone H3 K4. Mutation of a conserved tryptophan residue in the RAG-2 PHD finger abolished binding to H3K4me3 and greatly impaired recombination of extrachromosomal and endogenous immunoglobulin gene segments. Together, these findings are consistent with the interpretation that recognition of hypermethylated histone H3 K4 promotes efficient V(D)J recombination in vivo.

Evidence for Non-Random Immunoglobulin VH Gene and Light Chain Isotype Usage in Follicular Non-Hodgkin's Lymphoma.

Background: Antibody diversity is generated by recombination of individual immunoglobulin (Ig) gene segments and subsequent somatic diversification driven by antigen recognition. In the repertoire of expressed B cell receptors (BCR) among normal peripheral B cells, variable heavy (VH) gene segments are not equally represented. The ratio of kappa to lambda light chain usage is also skewed; the normal κ/λ is 1.5. Investigation of the BCR repertoire may provide clues to the genesis of B cell malignancies, as suggested in chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL). BCR V gene identification during the production of recombinant antibodies used in our ongoing PhII and PhIII FavId® (idiotype/KLH) immunotherapy studies has enabled us to analyze V gene usage from 475 B cell follicular lymphoma (FL) tissue samples. This study reports the results of VH gene and κ/λ gene expression in this FL sample collection.Methods: Ig heavy chain (HC) and light chain (LC) isotypes from B cell FL samples were identified by flow cytometry. VH and VL regions were sequenced from gene specific cDNA libraries prepared from these samples. VH gene usage and κ/λ ratios were compared to frequencies determined for normal peripheral B cells isolated from six healthy volunteers as well as published reports for normal peripheral B cells and other B cell malignancies.Results: Compared to VH gene family usage determined for normal B cells, VH3 usage is higher (68% vs. 42%), VH1 usage is lower (7.8% vs. 22%) and VH4 usage is equivalent (22% vs. 26%) in our cohort of FL patients while VH2, 5, 6 and 7 are infrequently used in both populations. Usage of the VH3 genes within FL derived sequences also depends upon isotype, in that this gene family is preferentially associated with the IgM HC isotype relative to IgG (76% and 57% respectively). Additionally, the combined usage of the specific genes VH3-23 and VH3-48 in our patient collection accounts for over 29% of all VH genes - compared to 9% among normal B cells. These VH gene usages also differ from reports of VH gene expression among CLL and MCL patients. With respect to LC usage, VH3 isolates are associated with a normal κ/λ ratio of 1.6 while VH4 gene isolates are preferentially associated with λ light chains with a κ/λ ratio of 0.9. Finally, FL B cells expressing the IgM HC isotype preferentially co-express κ light chains (κ/λ ratio of 2.4) while IgG expressing cells preferentially utilize λ chains (κ/λ ratio of 0.6).Conclusions: Non-random V gene and LC expression among patients with FL is noted. These distortions in Ig gene expression suggest that lymphomagenesis in FL may be associated with B cell stimulation by common antigens. A program to investigate the epitopes recognized by FL derived BCRs via binding of recombinant FL derived antibodies to protein arrays containing common auto-antigens is currently underway.

Antibody repertoire development in teleosts—a review with emphasis on salmonids and Gadus morhua L

The group of teleosts is highly diverse, comprising more than 23000 extant species. Studies of the teleost antibody repertoire have been conducted in many different species within different orders, though some species and families have been better characterised than others. The Atlantic cod ( Gadus morhua L.) and several species within the Salmoninae (e.g. Salmo salar and Oncorynchus mykiss) are among the best-studied teleosts in terms of the antibody repertoire. The estimated size of the repertoire, the organisation of immunoglobulin (IG) gene segments, the expressed IG repertoire, the IgM serum concentration, and the serum antibody responses reveal some fundamental differences between these species. The serum IgM concentration of G. morhua is some ten times higher than that of S. salar, though G. morhua is characterised as a ‘low’ (or ‘non’) responder in terms of specific antibody production. In contrast, an antibody response is readily induced in S. salar, although the response is strongly regulated by antigen induced suppression. The IGHD gene of G. morhua has a unique structure, while the IGHM and IGHD genes of S. salar have a characteristic genomic organisation in two parallel loci. In addition, salmonids, express a broad repertoire of IGH and IGI V-region gene segments, while a single V gene family dominates the expressed heavy and light chain repertoire of G. morhua. Little is known about the developing antibody repertoire during ontogeny, in different stages of B-cell maturation, or in separate B-cell subsets. Information on the establishment of the preimmune repertoire, and the possible role of environmental antigens is also sparse.

Immunoglobulin gene segment usage, location and immunogenicity in mutated and unmutated chronic lymphocytic leukaemia

The mutational status of the variable region of the immunoglobulin heavy chain gene (IgV(H)) is an important prognostic marker in B-cell chronic lymphocytic leukaemia (B-CLL), with mutated patients having improved outcome. To examine the impact of mutational status on V(H), D(H), and J(H) gene segment location and immunogenicity, we analysed 375 IgH sequences from 356 patients with B-CLL. Although V(H) and D(H) gene usage was different in mutated compared to unmutated patients, there was no impact of gene location on frequency of use or clinical outcome. Surprisingly, somatic mutations did not increase the immunogenicity of the Ig, as assessed by predicted binding affinity of Ig-derived peptides to major histocompatibility Class I and Class II molecules. Even excluding patients using V(H)1-69, cases using the V(H)1 gene family had a poor outcome. Both mutated and unmutated CLL patients demonstrated evidence of antigen selection. The worst outcome was seen in the subset of 14 unmutated patients with similar HCDR3 amino acid sequence using V(H)1-69, D(H)3-3 and J(H)6, suggesting an antigen-driven process modulating the clinical course.

Directed molecular evolution by somatic hypermutation

After rearrangement of immunoglobulin gene segments, the immune system evolves the antibody repertoire by mutating the immunoglobulin variable region at a high rate. While this somatic hypermutation was thought to occur only at the variable region, recent studies suggest that hypermutation can occur at locations throughout the genome. Building upon this notion, we sought to exploit this mechanism as a mutagenesis tool. We created a substrate based on GFP that could be screened using flow cytometry and showed that retroviral infection can deliver the transgene to genomic locations that support hypermutation. Infected cells generated various GFP mutants with increased fluorescence intensity and analysis revealed mutations not only at the chromophore, but also an unexpected mutation at a distant residue. Our results demonstrate in principle that immunoglobulin somatic hypermutation can be a potent means of mutagenesis. With appropriate selection conditions it may be utilized to evolve gene products with desired properties.

Somatic diversification of variable lymphocyte receptors in the agnathan sea lamprey.

Although jawless vertebrates are apparently capable of adaptive immune responses, they have not been found to possess the recombinatorial antigen receptors shared by all jawed vertebrates. Our search for the phylogenetic roots of adaptive immunity in the lamprey has instead identified a new type of variable lymphocyte receptors (VLRs) composed of highly diverse leucine-rich repeats (LRR) sandwiched between amino- and carboxy-terminal LRRs. An invariant stalk region tethers the VLRs to the cell surface by means of a glycosyl-phosphatidyl-inositol anchor. To generate rearranged VLR genes of the diversity necessary for an anticipatory immune system, the single lamprey VLR locus contains a large bank of diverse LRR cassettes, available for insertion into an incomplete germline VLR gene. Individual lymphocytes express a uniquely rearranged VLR gene in monoallelic fashion. Different evolutionary strategies were thus used to generate highly diverse lymphocyte receptors through rearrangement of LRR modules in agnathans (jawless fish) and of immunoglobulin gene segments in gnathostomes (jawed vertebrates).

Regulating antigen-receptor gene assembly.

The genes encoding antigen receptors are unique because of their high diversity and their assembly in developing lymphocytes from gene segments through a series of site-specific DNA recombination reactions known as V(D)J rearrangement. This review focuses on our understanding of how recombination of immunoglobulin and T-cell receptor gene segments is tightly regulated despite being catalysed by a common lymphoid recombinase, which recognizes a widely distributed conserved recombination signal sequence. Probable mechanisms involve precise expression of the lymphoid-restricted recombination-activating genes RAG1 and RAG2, and developmentally regulated epigenetic alterations in template accessibility, which are targeted by transcriptional regulatory elements and involve chromatin-modifying enzymes.

Somatic Hypermutation Patterns in Germinal Center B Cell Malignancies

(2003). Somatic Hypermutation Patterns in Germinal Center B Cell Malignancies. Hematology: Vol. 8, No. 5, pp. 319-328.

Receptor revision of immunoglobulin heavy chain genes in human MALT lymphomas

Background/Aims: Rearrangement of immunoglobulin gene segments, leading to B cells with functional receptors, is thought to be largely restricted to developing immature B cells in bone marrow. However, accumulating evidence...

The pathogenetic role of oncogenes deregulated by chromosomal translocation in B-cell malignancies.

Chromosomal translocations involving the immunoglobulin (IG) loci play a pivotal role in the pathogenesis of many subtypes of mature B-cell malignancy. Although all the common IG translocations have been cloned, cloning of rare but nonetheless recurrent translocations continues to allow identification of genes of importance to the development of both normal and malignant B-cells. Clustering of breakpoints within the IG gene segments has allowed development of polymerase chain reaction methods that facilitate cloning. IG translocations result in overexpression of a wide variety of genes ranging from cell surface receptors to transcriptional repressors. Genes recently shown to be involved in such translocations include BCL11A and MALT1. As with the acute leukemias, different translocations in B-cell lymphomas may target different proteins that interact directly. A common endpoint for several translocations is activation of the nuclear factor kappaB pathway. Analysis of the mechanisms of transformation may define new therapeutic strategies.

ATP-dependent DNA ligases.

By catalyzing the joining of breaks in the phosphodiester backbone of duplex DNA, DNA ligases play a vital role in the diverse processes of DNA replication, recombination and repair. Three related classes of ATP-dependent DNA ligase are readily apparent in eukaryotic cells. Enzymes of each class comprise catalytic and non-catalytic domains together with additional domains of varying function. DNA ligase I is required for the ligation of Okazaki fragments during lagging-strand DNA synthesis, as well as for several DNA-repair pathways; these functions are mediated, at least in part, by interactions between DNA ligase I and the sliding-clamp protein PCNA. DNA ligase III, which is unique to vertebrates, functions both in the nucleus and in mitochondria. Two distinct isoforms of this enzyme, differing in their carboxy-terminal sequences, are produced by alternative splicing: DNA ligase IIIalpha has a carboxy-terminal BRCT domain that interacts with the mammalian DNA-repair factor XrccI, but both alpha and beta isoforms have an amino-terminal zinc-finger motif that appears to play a role in the recognition of DNA secondary structures that resemble intermediates in DNA metabolism. DNA ligase IV is required for DNA non-homologous end joining pathways, including recombination of the V(D)J immunoglobulin gene segments in cells of the mammalian immune system. DNA ligase IV forms a tight complex with Xrcc4 through an interaction motif located between a pair of carboxy-terminal BRCT domains in the ligase. Recent structural studies have shed light on the catalytic function of DNA ligases, as well as illuminating protein-protein interactions involving DNA ligases IIIalpha and IV.

Recombinant monoclonal antibody technology

With the development of murine hybridoma technology over a quarter century ago, the ability to produce large quantities of well-characterized monoclonal antibody preparations revolutionized diagnostic and therapeutic medicine. For many applications in transfusion medicine, however, the production of serological reagents in mice has certain biological limitations relating to the difficulty in obtaining murine monoclonal antibodies specific for many human blood group antigens. Furthermore, for therapeutic purposes, the efficacy of murine-derived immunoglobulin preparations is limited by the induction of anti-mouse immune responses. Technical difficulties inherent in human hybridoma formation have led to novel molecular approaches that facilitate the isolation and production of human antibodies without the need for B-cell transformation, tissue culture, or even immunized individuals. These technologies, referred to as ‘repertoire cloning’ or ‘Fab/phage display’, involve the rapid cloning of immunoglobulin gene segments to create immune libraries from which antibodies with desired specificities can be selected. The use of such recombinant methods in transfusion medicine is anticipated to play an important role in the development and production of renewable supplies of low-cost reagents for diagnostic and therapeutic applications.

Pro-B-cell to pre-B-cell development in B-lineage acute lymphoblastic leukemia expressing the MLL/AF4 fusion protein

AbstractThe most common chromosomal abnormality of infant acute lymphoblastic leukemia (ALL) is the t(4;11)(q21;q23) that gives rise to the MLL/AF4 fusion gene. Leukemic blasts expressing MLL/AF4 are arrested at an early progenitor stage with lymphoid or monocytoid characteristics. A novel B-lineage ALL cell line termedB-lineage–3 (BLIN-3) requiring human bone marrow (BM) stromal cell contact and interleukin-7 (IL-7) for optimal proliferation has been established. BLIN-3 cells have a CD19+/CD10− phenotype typical of infant ALL, and they harbor the t(4;11)(q21;q23) chromosomal translocation. Reverse transcription–polymerase chain reaction and Western blot analysis confirmed the presence of the MLL/AF4 fusion mRNA and protein in BLIN-3. Initial BLIN-3 cultures had a pro-B cell phenotype and did not express cytoplasmic or surface μ heavy chain. After approximately 5 months in culture on BM stromal cells plus IL-7, BLIN-3 sublines emerged expressing μ heavy chain and VpreB on the cell surfaces (ie, pre-B-cell receptor [BCR]+). BLIN-3 cells expressing pre-BCR had the t(4;11)(q21;q23) translocation and expressed the MLL/AF4 fusion protein. Cross-linking the BLIN-3 pre-BCR led to enhanced cell proliferation, demonstrating that BLIN-3 expressed a functional pre-BCR. Increased acquisition of surface pre-BCR in BLIN-3 sublines was associated with loss of DJ rearrangements and the appearance of VDJ rearrangements. These results indicate that expression of the MLL/AF4 fusion protein is compatible with BM stromal cell and cytokine dependency, functional immunoglobulin gene segment rearrangement, and subsequent expression of a potentially diverse antigen receptor repertoire. Thus, the expression of MLL/AF4 is compatible with the normal developmental program of human B-lineage cells.

Keeping alleles quiet

With rare exceptions, each lymphocyte is destined to express only one antigen-receptor specificity for life, but the mechanisms by which this is enforced have not been fully resolved. During B-cell development, immunoglobulin gene segments ? variable (V), diversity (D) and joining (J) ? recombine to form functional immunoglobulin genes, generating a diverse B-cell repertoire. Regulated expression of the recombination activating genes (RAG1 and RAG2) during B-cell development helps to ensure that each B cell expresses a single heavy (IgH) chain and a single light-chain (Ig or Ig) allele, a phenomenon known as allelic exclusion. Now, reporting in Nature Immunology, Skok et al. provide the first evidence that epigenetic mechanisms could reinforce monoallelic expression of immunoglobulin genes at later stages.

Localized gene-specific induction of accessibility to V(D)J recombination induced by E2A and early B cell factor in nonlymphoid cells.

Accessibility of immunoglobulin (Ig) gene segments to V(D)J recombination is highly regulated and is normally only achieved in B cell precursors. We previously showed that ectopic expression of E2A or early B cell factor (EBF) with recombination activating gene (RAG) induces rearrangement of IgH and IgL genes in nonlymphoid cells. VkappaI genes throughout the locus were induced to rearrange after transfection with E2A, suggesting that the entire Vkappa locus was accessible. However, here we show that Ig loci are not opened globally but that recombination is localized. Gene families are interspersed in the D(H), Vkappa, and Vlambda loci, and we show that certain families and individual genes undergo high levels of recombination after ectopic expression of E2A or EBF, while other families within the same locus are not induced to rearrange. Furthermore, in some families, induction of germline transcription correlates with the level of induced recombination, while in others there is no correlation, suggesting that recombination is not simply initiated by induction of germline transcription. The induced repertoire seen at 24 hours does not change significantly over time indicating the absence of many secondary rearrangements and also suggesting a direct targeting mechanism. We propose that accessibility occurs in a local manner, and that binding sites for factors facilitating accessibility are therefore likely to be associated with individual gene segments.