RAG-mediated Recombination Research Articles

Regulation of VH Replacement by B Cell Receptor–Mediated Signaling in Human Immature B Cells

VH replacement provides a unique RAG-mediated recombination mechanism to edit nonfunctional IgH genes or IgH genes encoding self-reactive BCRs and contributes to the diversification of Ab repertoire in the mouse and human. Currently, it is not clear how VH replacement is regulated during early B lineage cell development. In this article, we show that cross-linking BCRs induces VH replacement in human EU12 μHC(+) cells and in the newly emigrated immature B cells purified from peripheral blood of healthy donors or tonsillar samples. BCR signaling-induced VH replacement is dependent on the activation of Syk and Src kinases but is inhibited by CD19 costimulation, presumably through activation of the PI3K pathway. These results show that VH replacement is regulated by BCR-mediated signaling in human immature B cells, which can be modulated by physiological and pharmacological treatments.

The role of germinal centers in T cell receptor revision (P1421)

Abstract Vβ5 transgenic CD4 T cells are tolerized to the endogenous superantigen Mtv-8 by either deletion or TCR revision. Revision occurs when a peripheral CD4 T cell downregulates Vβ5 and undergoes RAG-mediated recombination and surface expression of an endogenous TCR. Given that revision occurs in germinal centers, and that follicular helper T cells, or Tfh, in Vβ5 Tg mice increase in an Mtv-8-dependent manner, we are studying whether revising cells have a Tfh phenotype and whether germinal center interactions are required for revision. Revising cells have a transcription factor and surface phenotype resembling that of Tfh, with low Blimp-1 and elevated Bcl-6, CXCR5, PD-1, IL-21 receptor, and OX-40 expression. The frequency of post-revision T cells increases in mice heterozygous for Blimp-1, a transcriptional repressor of Tfh formation, but revision intermediates are unaffected. The adaptor molecule SAP is required for prolonged B and T cell interactions in the germinal center, and SAP null mice have reduced numbers of post-revision T cells, but no reduction in revision intermediates. These results suggest that revising T cells share some traits with Tfh and that Tfh formation and prolonged B-T cell interactions are required for the completion, but not initiation, of TCR revision. Ongoing studies will analyze revision in the absence of Bcl-6, a transcription factor required for Tfh formation, and TNFR1, a receptor required for the formation of germinal center structures.

Contribution of VH Replacement Products in Mouse Antibody Repertoire

VH replacement occurs through RAG-mediated recombination between the cryptic recombination signal sequence (cRSS) near the 3′ end of a rearranged VH gene and the 23-bp RSS from an upstream unrearranged VH gene. Due to the location of the cRSS, VH replacement leaves a short stretch of nucleotides from the previously rearranged VH gene at the newly formed V-D junction, which can be used as a marker to identify VH replacement products. To determine the contribution of VH replacement products to mouse antibody repertoire, we developed a Java-based VH Replacement Footprint Analyzer (VHRFA) program and analyzed 17,179 mouse IgH gene sequences from the NCBI database to identify VH replacement products. The overall frequency of VH replacement products in these IgH genes is 5.29% based on the identification of pentameric VH replacement footprints at their V-D junctions. The identified VH replacement products are distributed similarly in IgH genes using most families of VH genes, although different families of VH genes are used differentially. The frequencies of VH replacement products are significantly elevated in IgH genes derived from several strains of autoimmune prone mice and in IgH genes encoding autoantibodies. Moreover, the identified VH replacement footprints in IgH genes from autoimmune prone mice or IgH genes encoding autoantibodies preferentially encode positively charged amino acids. These results revealed a significant contribution of VH replacement products to the diversification of antibody repertoire and potentially, to the generation of autoantibodies in mice.

Novel Genomic Structural Variations in Relapsed Childhood ETV6/RUNX1 (TEL/AML1) Acute Lymphoblastic Leukemias Identified by Next Generation Sequencing

AbstractAbstract 518 IntroductionThe chimeric fusion gene ETV6/RUNX1 generated by the interchromosomal translocation t(12;21) presents the most frequent chromosomal aberration in childhood acute lymphoblastic leukemia (ALL), occurring in approximately 25% of all patients. This ALL subtype is associated with an overall favorable prognosis, nevertheless 10–20% of children will suffer from relapse. ETV6/RUNX1-positive preleukemic clones arise already in utero, but require additional cooperating oncogenic lesions for the development of overt leukemia. The nature of the assisting genetic alterations and the mechanisms driving the development of leukemia and recurrent disease are still not well understood. MethodsWe applied state-of-the-art whole genome and whole exome next-generation-sequencing to comprehensively analyze the assisting oncogenic alterations in pediatric patients with initial and/or recurrent ETV6/RUNX1+ ALL (primary disease n=11, recurrent disease n=7). Matched sample sets taken at initial diagnosis, remission and relapse were compared. Mate pair and/or paired end sequencing was carried out for whole genome analysis with inserts spanning 2 kb or 500 kb, respectively. Constructed libraries were sequenced from both ends with 36- or 50-bp reads on a Genome Analyzer IIx or a HiSeq 2000 (Illumina/Solexa), respectively. Reads were aligned against the human reference genome (GRCh37) using BWA. Duplicate reads were removed. Unique reads with high mapping quality (q>35) served as input for GASV which detected translocations and inversions based on the mapping coordinates, insert sizes and read orientation. Variations covered by at least three reads in the tumor sample and not detected in the remission sample or the Database of Genomic Variants were reported. For detection of copy number variations, the program FREEC carried out coverage normalization, computation of copy number ratios between paired leukemia and remission samples (with up to 10 kb resolution) and subsequent segmentation. A subset of six selected patients was further investigated by targeted enrichment of whole exomic regions employing SeqCap EZ libraries (Roche) and 100 bp single read next-generation-sequencing on a HighSeq 2000. Mutations were called using GATK and further processed by an in-house bioinformatic pipeline. Putative somatically acquired mutations were validated by PCR, Sanger sequencing and FISH analysis. ResultsGenomes were sequenced to at least 13× physical coverage (mate pair) and 6.7× sequence coverage (paired-end). Exome sequencing achieved a minimum of 25× sequence coverage. In silico we detected 155 tumor-specific intragenic translocations. On average each tumor harbored 9 acquired translocations. With the exception of one case (13 translocations at diagnosis, 9 at relapse), the number of translocations was higher in relapse than in the matched diagnosis sample (additional 3 translocations on average). Ongoing validation studies confirmed the defining ETV6/RUNX1 translocation t(12;21) and identified 13 novel translocations. The genes affected are involved in essential signaling pathways, such as cytokine signaling (LIFR), calcium signaling (RCAN2), insulin and anti-apoptotic signaling (PHIP). Interestingly, also a factor essential for pre-mRNA splicing (IBP160) and genes encoding regulatory RNAs (miRNAs, lincRNAs and RNAs involved in splicing) were rearranged. A validated intragenic deletion of 836 bp leading to a frameshift and premature stop affected a calcium ion sensor of the ferlin protein family. Recurrent deletions in 9 of 11 cases (82%) ranging from 5 to 200 kb were detected in the immunoglobulin lambda variable gene cluster (IGLV) on chromosome 22q11. Some of the deletions were extending into the pre-B lymphocyte 1 gene (VPREB1) locus. In silico the probabilty of illegitimate RAG-mediated recombination at the breakpoint sites was determined by evaluation of RIC scores. RIC scores indicated that aberrant V(D)J rearrangements involving cryptic recombination sequence signals had caused the deletions on chromosome 22q11. ConclusionWe present somatic mutations that are promising novel candicate genes (e.g. LIFR, RCAN2, PHIP, IBP160) for cooperating secondary mutations in ETV6/RUNX1+ ALL and discuss their impact on the molecular pathology of primary and recurrent disease. Disclosures:No relevant conflicts of interest to declare.

Contribution of VH replacement products to the generation of anti-HIV antibodies

V(H) replacement occurs through RAG-mediated secondary recombination to change unwanted IgH genes and diversify antibody repertoire. The biological significance of V(H) replacement remains to be explored. Here, we show that V(H) replacement products are highly enriched in IgH genes encoding anti-HIV antibodies, including anti-gp41, anti-V3 loop, anti-gp120, CD4i, and PGT antibodies. In particular, 73% of the CD4i antibodies and 100% of the PGT antibodies are encoded by potential VH replacement products. Such frequencies are significantly higher than those in IgH genes derived from HIV infected individuals or autoimmune patients. The identified V(H) replacement products encoding anti-HIV antibodies are highly mutated; the V(H) replacement "footprints" within CD4i antibodies preferentially encode negatively charged amino acids within the IgH CDR3; many IgH encoding PGT antibodies are likely generated from multiple rounds of V(H) replacement. Taken together, these findings uncovered a potentially significant contribution of V(H) replacement products to the generation of anti-HIV antibodies.

A NUP98-HOXD13 leukemic fusion gene leads to impaired class switch recombination and antibody production

Myelodysplastic syndrome is a clonal process characterized by ineffective hematopoiesis and progression to acute leukemia. Although many myelodysplastic syndrome and leukemic patients have compromised immunity, the role of underlying mutations in regulating immune function is poorly understood. Recent studies show that NUP98-HOXD13 (NHD13) fusion gene results in myelodysplastic syndrome and impairs lymphocyte differentiation in transgenic mice. In our studies, we sought to elucidate the mechanism by which NHD13 affects B-lymphocyte development and function. Based on our preliminary findings that transgenic mice had increased levels of IgM and reduced IgG1 and IgE, we hypothesized that the fusion gene might impair class switch recombination (CSR). Mice were immunologically challenged with dinitrophenol. NHD13 mice showed a marked reduction in B-lymphocyte differentiation in their bone marrow and spleen following dinitrophenol stimulation and had reduced production of dinitrophenol-specific antibodies. Spleen follicles from these mice were small and hypocellular, indicating failure of clonal expansion. When isolated NHD13 B lymphocytes were stimulated in vitro using Escherichia coli lipopolysaccharide or lipopolysaccharide + interleukin-4, they failed to undergo sufficient CSR and proliferation. Taken together, our findings show that expression of NUP98-HOXD13 impairs CSR and reduces the antibody-mediated immune response, in addition to its role in leukemia. Further delineation of the NUP98-HOXD13 transgene may reveal novel pathways involved in CSR.

Phylogenetic conservation of the 3′ cryptic recombination signal sequence (3′cRSS) in the VH genes of jawed vertebrates

The VH replacement process is a RAG-mediated secondary recombination in which the variable region of a rearranged VHDJH is replaced by a different germline VH gene. In almost all human and mouse VH genes, two sequence features appear to be crucial for VH replacement. First, an embedded heptamer, which is located near the 3′ end of the rearranged VH gene, serves as a cryptic recombination signal sequence (3′cRSS) for the VH replacement process. Second, a short stretch of nucleotides located downstream of the 3′cRSS serve as a footprint of the original VH region, frequently encoding charged amino acids. In this review, we show that both of these two features are conserved in the VH genes of all jawed vertebrates, which suggests that the VH replacement process may be a conserved mechanism.

Identification and molecular characterization of recurrent genomic deletions on 7p12 in the IKZF1 gene in a large cohort of BCR-ABL1–positive acute lymphoblastic leukemia patients: on behalf of Gruppo Italiano Malattie Ematologiche dell'Adulto Acute Leukemia Working Party (GIMEMA AL WP)

AbstractThe BCR-ABL1 fusion gene defines the subgroup of acute lymphoblastic leukemia (ALL) with the worst clinical prognosis. To identify oncogenic lesions that combine with BCR-ABL1 to cause ALL, we used Affymetrix Genome-Wide Human SNP arrays (250K NspI and SNP 6.0), fluorescence in situ hybridization, and genomic polymerase chain reaction to study 106 cases of adult BCR-ABL1–positive ALL. The most frequent somatic copy number alteration was a focal deletion on 7p12 of IKZF1, which encodes the transcription factor Ikaros and was identified in 80 (75%) of 106 patients. Different patterns of deletions occurred, but the most frequent were those characterized by a loss of exons 4 through 7 (Δ4-7) and by removal of exons 2 through 7 (Δ2-7). A variable number of nucleotides (patient specific) were inserted at the conjunction and maintained with fidelity at the time of relapse. The extent of the Δ4-7 deletion correlated with the expression of a dominant-negative isoform with cytoplasmic localization and oncogenic activity, whereas the Δ2-7 deletion resulted in a transcript lacking the translation start site. The IKZF1 deletion also was identified in the progression of chronic myeloid leukemia to lymphoid blast crisis (66%) but never in myeloid blast crisis or chronic-phase chronic myeloid leukemia or in patients with acute myeloid leukemia. Known DNA sequences and structural features were mapped along the breakpoint cluster regions, including heptamer recombination signal sequences recognized by RAG enzymes during V(D)J recombination, suggesting that IKZF1 deletions could arise from aberrant RAG-mediated recombination.

BCR–ABL1 lymphoblastic leukaemia is characterized by the deletion of Ikaros

The Philadelphia chromosome, a chromosomal abnormality that encodes BCR-ABL1, is the defining lesion of chronic myelogenous leukaemia (CML) and a subset of acute lymphoblastic leukaemia (ALL). To define oncogenic lesions that cooperate with BCR-ABL1 to induce ALL, we performed a genome-wide analysis of diagnostic leukaemia samples from 304 individuals with ALL, including 43 BCR-ABL1 B-progenitor ALLs and 23 CML cases. IKZF1 (encoding the transcription factor Ikaros) was deleted in 83.7% of BCR-ABL1 ALL, but not in chronic-phase CML. Deletion of IKZF1 was also identified as an acquired lesion at the time of transformation of CML to ALL (lymphoid blast crisis). The IKZF1 deletions resulted in haploinsufficiency, expression of a dominant-negative Ikaros isoform, or the complete loss of Ikaros expression. Sequencing of IKZF1 deletion breakpoints suggested that aberrant RAG-mediated recombination is responsible for the deletions. These findings suggest that genetic lesions resulting in the loss of Ikaros function are an important event in the development of BCR-ABL1 ALL.

Editing IgH Genes Encoding Anti‐SM Antibodies by VH Replacement

VH replacement occurs through RAG‐mediated recombination between a cryptic RSS within a rearranged IgH gene and an upstream VH gene. Our previous studies showed that VH replacement occurs spontaneously in human EU12 cells and generates new IgH genes even after formation of functional IgH genes. Recently, we found that one of the dominantly expressed IgH genes in the EU12 muHC+ cells, A19H, directly encodes anti‐SM antigen antibodies when paired with two endogenous IgLambda chains. SM antigen can be detected from EU12 culture supernatant, presumably released from dead cells in the culture, suggesting that SM antigens might be the initial stimuli of ongoing VH replacement in the EU12 cell system. Indeed, treatment of EU12 muHC+ cells with purified bovine SM antigen strongly induces VH replacement. Moreover, when the A19H gene was artificially paired with a panel of 14 randomly selected light chains to express recombinant antibodies, 10 out of the 14 antibodies still react with SM antigens; 5 of them even have higher affinities against SM antigens. Our results suggest that the SM reactivity contributed by the A19H IgH gene can not be efficiently neutralized by light chain editing and VH replacement might offer a unique mechanism to delete IgH genes encoding anti‐SM antibodies.

Early TCR Expression and Aberrant T Cell Development in Mice with Endogenous Prerearranged T Cell Receptor Genes

The factors that regulate the rate of production of T cells by the thymus remain incompletely defined. To test whether generation of functional T cell receptors limits the rate of thymic T cell export, we made use of a line of mice, LN3alphabeta, that have endogenously prerearranged TCR genes. The prerearranged TCR genes were expressed abnormally early in hemopoietic development, indicating that RAG-mediated recombination, rather than transcription factor expression, is the key determinant of the initiation of robust TCR transcription. Thymic T cell export rates were similar between wild-type (wt) and LN3alphabeta mice, indicating that T cell maturation rates in these mice are determined by factors other than TCR gene rearrangement. In competitive bone marrow chimeras, however, LN3alphabeta thymocytes were out-competed by wt cells and failed to develop beyond the double-negative 4 stage. Furthermore, wt progenitors transplanted intrathymically into LN3alphabeta mice proliferated excessively, suggesting that increased proliferative signals in the LN3alphabeta thymus compensate for faulty T cell development driven by early TCR expression.

Gene conversion in human rearranged immunoglobulin genes

Over the past 20 years, many DNA sequences have been published suggesting that all or part of the V(H) segment of a rearranged immunoglobulin gene may be replaced in vivo. Two different mechanisms appear to be operating. One of these is very similar to primary V(D)J recombination, involving the RAG proteins acting upon recombination signal sequences, and this has recently been proven to occur. Other sequences, many of which show partial V(H) replacements with no addition of untemplated nucleotides at the V(H)-V(H) joint, have been proposed to occur by an unusual RAG-mediated recombination with the formation of hybrid (coding-to-signal) joints. These appear to occur in cells already undergoing somatic hypermutation in which, some authors are convinced, RAG genes are silenced. We recently proposed that the latter type of V(H) replacement might occur by homologous recombination initiated by the activity of AID (activation-induced cytidine deaminase), which is essential for somatic hypermutation and gene conversion. The latter has been observed in other species, but not in human Ig genes, so far. In this paper, we present a new analysis of sequences published as examples of the second type of rearrangement. This not only shows that AID recognition motifs occur in recombination regions but also that some sequences show replacement of central sections by a sequence from another gene, similar to gene conversion in the immunoglobulin genes of other species. These observations support the proposal that this type of rearrangement is likely to be AID-mediated rather than RAG-mediated and is consistent with gene conversion.

Potential Contribution of VH Gene Replacement in Immunity and Disease

VH replacement occurs through RAG-mediated recombination between a cryptic recombination signal sequence (cRSS) presented in a rearranged VH gene and a 23-bp RSS from an upstream VH gene. VH replacement renews the entire VH coding region and extends the immunoglobulin heavy-chain (IgH) CDR3 regions preferentially with charged amino acids. VH replacement occurs in bone marrow-immature B cells and contributes significantly to the primary B-cell repertoire in humans. However, the biological significance of VH replacement is not clear. Our recent studies revealed elevated frequencies of VH replacement products in different autoimmune diseases, including rheumatoid arthritis, systemic lupus erythematosus, and Sjögren's syndrome. Moreover, elevated frequencies of VH replacement products were also found in patients with human immunodeficiency virus or hepatitis C virus infections. These results provide the first clue that VH replacement contributes to autoimmune disease and antiviral immunity, and they also suggest a potential link between viral infection and autoimmune disease.

Homozygous deletions of CDKN2A caused by alternative mechanisms in various human cancer cell lines

The CDKN2A tumor-suppressor locus on chromosome band 9p21, which encodes p16(INK4A), a negative regulator of cyclin-dependent kinases, and p14(ARF1), an activator of TP53, is inactivated in many human cancers by point mutation, promoter hypermethylation, and, often, deletion. Homozygous deletions are unusually prevalent at this locus in very different human cancers. In the present study, we compared deletions in squamous cell carcinoma of the head and neck (SCCHN) cell lines to those in T-cell acute lymphatic leukemia (T-ALL), glioma, and bladder carcinoma (TCC) cell lines. Of 14 SCCHN lines, 10 showed homozygous deletions of CDKN2A, one displayed promoter hypermethylation with gene silencing, and one had a frameshift deletion in exon 2. Many deletion ends were in or proximal to the repetitive sequence clusters flanking the locus. Breakpoint junctions displayed variable microhomologies or insertions characteristic of DNA repair by nonhomologous end-joining. In general, deletions were much smaller in SCCHN than in TCC and glioma. In T-ALL, breakpoints were near consensus sites for recombination mediated by RAG (recombination activating genes) enzymes, and the structure of the junctions was consistent with this mechanism. We suggest that different mechanisms of CDKN2A deletion prevail in different human cancers. Aberrant RAG-mediated recombination may be responsible in T-ALL, and exuberant DNA repair by nonhomologous end-joining is the likely prevailing mechanism in SCCHN, but a distinct mechanism in TCC and glioma remains to be elucidated.

Bone Marrow Microenvironmental Changes Underlie Reduced RAG-mediated Recombination and B Cell Generation in Aged Mice

During aging, adaptive immunity is severely compromised, due in part to decreased production of B lymphocytes and loss of immunoglobulin (Ig) diversity. However, the molecular mechanisms that underlie age-associated diminished B cell production remain unclear. Using in vivo labeling, we find that this reduction in marrow pre–B cells reflects increased attrition during passage from the pro–B to pre–B cell pool. Analyses of reciprocal bone marrow chimeras reveal that the magnitude and production rates of pre–B cells are controlled primarily by microenvironmental factors, rather than intrinsic events. To understand changes in pro–B cells that could diminish production of pre–B cells, we evaluated rag2 expression and V(D)J recombinase activity in pro–B cells at the single cell level. The percentage of pro–B cells that express rag2 is reduced in aged mice and is correlated with both a loss of V(D)J recombinase activity in pro–B cells and reduced numbers of pre–B cells. Reciprocal bone marrow chimeras revealed that the aged microenvironment also determines rag2 expression and recombinase activity in pro–B cells. Together, these observations suggest that extrinsic factors in the bone marrow that decline with age are largely responsible for less efficient V(D)J recombination in pro–B cells and diminished progression to the pre–B cell stage.

Mechanisms of antigen receptor evolution

The adaptive immune system, which utilizes RAG-mediated recombination to diversify immune receptors, arose in ancestors of the jawed vertebrates approximately 500 million years ago. Homologs of immunoglobulins (Igs), T cell antigen receptors (TCRs), major histocompatibility complex (MHC) I and II, and the recombination activating genes (RAGs) have been identified in all extant classes of jawed vertebrates; however, no definitive ortholog of any of these genes has been identified in jawless vertebrates or invertebrates. Although the identity of the “primoridal” receptor that likely was interrupted by the recombination mechanism in the common ancestor of jawed vertebrates may never be established, many different families of genes that exhibit predicted characteristics of such a receptor have been described both within and outside the jawed vertebrates. Various model systems point toward a range of immune receptor diversity, encompassing many different families of recognition molecules, including non-diversified and diversified Ig-type variable (V) regions, as well as diversified VJ domains, whose functions are integrated in an organism's response to pathogenic invasion. The transition from the primordial antigen receptor to the monomeric Ig-/TCR-like domain and subsequent antigen-specific heterodimer likely involved progressive refinement of unique intermolecular associations in parallel with the acquisition of combinatorial diversity and antigen-specific recognition through somatic modification of the V region. RAG-mediated recombination and associated junctional diversification of both Ig and TCR genes occurs in all jawed vertebrates. In the case of Igs, somatic variation is expanded further through class switching, gene conversion, and somatic hypermutation. Various approaches, including both genomic and protein functional analyses, currently are being applied in jawless vertebrates, protochordates and other invertebrate deuterostome model systems in order to examine both RAG-mediated and alternative forms of antigen receptor diversification. Such studies have uncovered previously unknown mechanisms of generating receptor diversity.

The phylogenetic origins of the antigen-binding receptors and somatic diversification mechanisms.

The adaptive immune system arose in ancestors of the jawed vertebrates approximately 500 million years ago. Homologs of immunoglobulins (Igs), T-cell antigen receptors (TCRs), major histocompatibility complex I (MHC I) and MHC II, and the recombination-activating genes (RAGs) have been identified in all extant classes of jawed vertebrates; however, no definitive homolog of any of these genes has been identified in jawless vertebrates or invertebrates. RAG-mediated recombination and associated junctional diversification of both Ig and TCR genes occurs in all jawed vertebrates. In the case of Igs, somatic variation is expanded further through class switching, gene conversion, and somatic hypermutation. Although the identity of the 'primordial' receptor that was interrupted by the recombination mechanism in jawed vertebrates may never be established, many different families of genes that exhibit predicted characteristics of such a receptor have been described both within and outside the jawed vertebrates. Recent data from various model systems point toward a continuum of immune receptor diversity, encompassing many different families of recognition molecules whose functions are integrated in an organism's response to pathogenic invasion. Various approaches, including both genomic and protein-functional analyses, currently are being applied in jawless vertebrates, protochordates, and other invertebrate deuterostome systems and may yield definitive evidence regarding the presence or absence of adaptive immune homologs in species lacking adaptive immune systems. Such studies have the potential for uncovering previously unknown mechanisms of generating receptor diversity.

Molecular mechanism of serial VH gene replacement.

The molecular mechanism of serial VH replacement was analyzed using a human B cell line, EU12, that undergoes continuous spontaneous differentiation from pro-B to pre-B and then to B cell stage. In earlier studies, we found that this cell line undergoes intraclonal V(D)J diversification. Analysis of the IgH gene sequences in EU12 cells predicted the occurrence of serial VH replacement involving the cryptic recombination signal sequences (cRSS) embedded within framework 3 regions and concurrent extension of the CDR3 region. Detection of double-stranded DNA breaks at the cRSS site and different VH replacement excision circles confirm the ongoing nature of this diversification process in the EU12 cells. In vitro binding and cleavage assays using recombinant RAG-1 and RAG-2 proteins further validated the cRSS participation in this RAG-mediated recombination process. Serial VH replacements may represent an additional mechanism for diversification of the primary B cell repertoire.

Chromatin remodeling directly activates V(D)J recombination.

V(D)J recombination substrate choice is regulated to ensure that the appropriate gene segments are rearranged during lymphocyte development. It has been proposed that regulation of substrate usage is determined by changes in accessibility of the DNA targets. We show that Rag-mediated recombination of an episomal substrate in cells is affected by its packaging into chromatin. Chromatinized substrates were inefficiently rearranged, and methylation further reduced recombination. Disruption of nucleosomes by using butyrate on methylated substrates was sufficient to activate recombination, and dexamethasone could activate recombination in the absence of detectable transcription. Therefore, chromatin structure, and its manipulation by altering nucleosome positioning, can directly affect recombination efficiencies.