Ig Gene Conversion Research Articles

Cell Cycle-Mediated Regulation of Secondary Ig Diversification.

Secondary Ig diversification in B cells requires the deliberate introduction of DNA damage into the Ig genes by the enzyme activation-induced cytidine deaminase (AID) and the error-prone resolution of AID-induced lesions. These processes must be tightly regulated because they may lead to lymphomagenesis if they act on genes other than the Ig genes. Since B cells may limit secondary Ig diversification mechanisms during the cell cycle to minimize genomic instability, we restricted the activity of AID specifically to the G1 or S/G2 phase to investigate the cell cycle contribution to the regulation of somatic hypermutation, class switch recombination, and Ig gene conversion in human, murine, and avian B cells, respectively. The efficient induction of AID in different cell cycle phases allowed us for the first time, to our knowledge, to discriminate G1- from S/G2-specific events of regulation. We show that the processes of Ig gene conversion and C/G mutagenesis during somatic hypermutation can be achieved throughout the cell cycle, whereas A/T mutagenesis and class switch recombination require AID-mediated deamination in G1. Thus, AID activity in G1, but not in S/G2, leads to the efficient accomplishment of all mechanisms of secondary Ig diversification. Our findings refine the current state-of-the-art knowledge in the context of the regulation of secondary Ig diversification.

Development and characterization of a CRISPR/Cas9-mediated RAG1 knockout chicken model lacking mature B and T cells.

Although birds have been used historically as a model animal for immunological research, resulting in remarkable achievements, immune cell development in birds themselves has yet to be fully elucidated. In this study, we firstly generated an immunodeficient chicken model using a CRISPR/Cas9-mediated recombination activating gene 1 (RAG1) knockout, to investigate avian-specific immune cell development. Unlike previously reported immunoglobulin (Ig) heavy chain knockout chickens, the proportion and development of B cells in both RAG1 +/- and RAG1 -/- embryos were significantly impaired during B cell proliferation (embryonic day 16 to 18). Our findings indicate that, this is likely due to disordered B cell receptor (BCR)-mediated signaling and interaction of CXC motif chemokine receptor (CXCR4) with CXCL12, resulting from disrupted Ig V(D)J recombination at the embryonic stage. Histological analysis after hatching showed that, unlike wild-type (WT) and RAG1 +/- chickens, lymphatic organs in 3-week old RAG1 -/- chickens were severely damaged. Furthermore, relative to WT chickens, RAG1+/- and RAG1-/- birds had reduced serum Igs, fewer mature CD4+ and CD8+ T lymphocytes. Furthermore, BCR-mediated B cell activation in RAG1 +/- chickens was insufficient, leading to decreased expression of the activation-induced deaminase (AID) gene, which is important for Ig gene conversion. Overall, this immunodeficient chicken model underlines the pivotal role of RAG1 in immature B cell development, Ig gene conversion during embryonic stages, and demonstrates the dose-dependent regulatory role of RAG1 during immune cell development. This model will provide ongoing insights for understanding chicken immune system development and applied in the fields of immunology and biomedical science.

PDIP38/PolDIP2 controls the DNA damage tolerance pathways by increasing the relative usage of translesion DNA synthesis over template switching.

Replicative DNA polymerases are frequently stalled at damaged template strands. Stalled replication forks are restored by the DNA damage tolerance (DDT) pathways, error-prone translesion DNA synthesis (TLS) to cope with excessive DNA damage, and error-free template switching (TS) by homologous DNA recombination. PDIP38 (Pol-delta interacting protein of 38 kDa), also called Pol δ-interacting protein 2 (PolDIP2), physically associates with TLS DNA polymerases, polymerase η (Polη), Polλ, and PrimPol, and activates them in vitro. It remains unclear whether PDIP38 promotes TLS in vivo, since no method allows for measuring individual TLS events in mammalian cells. We disrupted the PDIP38 gene, generating PDIP38-/- cells from the chicken DT40 and human TK6 B cell lines. These PDIP38-/- cells did not show a significant sensitivity to either UV or H2O2, a phenotype not seen in any TLS-polymerase-deficient DT40 or TK6 mutants. DT40 provides a unique opportunity of examining individual TLS and TS events by the nucleotide sequence analysis of the immunoglobulin variable (Ig V) gene as the cells continuously diversify Ig V by TLS (non-templated Ig V hypermutation) and TS (Ig gene conversion) during in vitro culture. PDIP38-/- cells showed a shift in Ig V diversification from TLS to TS. We measured the relative usage of TLS and TS in TK6 cells at a chemically synthesized UV damage (CPD) integrated into genomic DNA. The loss of PDIP38 also caused an increase in the relative usage of TS. The number of UV-induced sister chromatid exchanges, TS events associated with crossover, was increased a few times in PDIP38-/- human and chicken cells. Collectively, the loss of PDIP38 consistently causes a shift in DDT from TLS to TS without enhancing cellular sensitivity to DNA damage. We propose that PDIP38 controls the relative usage of TLS and TS increasing usage of TLS without changing the overall capability of DDT.

SUMOylation of PCNA by PIAS1 and PIAS4 promotes template switch in the chicken and human B cell lines

DNA damage tolerance (DDT) releases replication blockage caused by damaged nucleotides on template strands employing two alternative pathways, error-prone translesion DNA synthesis (TLS) and error-free template switch (TS). Lys164 of proliferating cell nuclear antigen (PCNA) is SUMOylated during the physiological cell cycle. To explore the role for SUMOylation of PCNA in DDT, we characterized chicken DT40 and human TK6 B cells deficient in the PIAS1 and PIAS4 small ubiquitin-like modifier (SUMO) E3 ligases. DT40 cells have a unique advantage in the phenotypic analysis of DDT as they continuously diversify their immunoglobulin (Ig) variable genes by TLS and TS [Ig gene conversion (GC)], both relieving replication blocks at abasic sites without accompanying by DNA breakage. Remarkably, PIAS1-/-/PIAS4-/- cells displayed a multifold decrease in SUMOylation of PCNA at Lys164 and over a 90% decrease in the rate of TS. Likewise, PIAS1-/-/PIAS4-/- TK6 cells showed a shift of DDT from TS to TLS at a chemosynthetic UV lesion inserted into the genomic DNA. The PCNAK164R/K164R mutation caused a ∼90% decrease in the rate of Ig GC and no additional impact on PIAS1-/-/PIAS4-/- cells. This epistatic relationship between the PCNAK164R/K164R and the PIAS1-/-/PIAS4-/- mutations suggests that PIAS1 and PIAS4 promote TS mainly through SUMOylation of PCNA at Lys164. This idea is further supported by the data that overexpression of a PCNA-SUMO1 chimeric protein restores defects in TS in PIAS1-/-/PIAS4-/- cells. In conclusion, SUMOylation of PCNA at Lys164 promoted by PIAS1 and PIAS4 ensures the error-free release of replication blockage during physiological DNA replication in metazoan cells.

A double-strand break can trigger immunoglobulin gene conversion.

All three B cell-specific activities of the immunoglobulin (Ig) gene re-modeling system—gene conversion, somatic hypermutation and class switch recombination—require activation-induced deaminase (AID). AID-induced DNA lesions must be further processed and dissected into different DNA recombination pathways. In order to characterize potential intermediates for Ig gene conversion, we inserted an I-SceI recognition site into the complementarity determining region 1 (CDR1) of the Ig light chain locus of the AID knockout DT40 cell line, and conditionally expressed I-SceI endonuclease. Here, we show that a double-strand break (DSB) in CDR1 is sufficient to trigger Ig gene conversion in the absence of AID. The pattern and pseudogene usage of DSB-induced gene conversion were comparable to those of AID-induced gene conversion; surprisingly, sometimes a single DSB induced multiple gene conversion events. These constitute direct evidence that a DSB in the V region can be an intermediate for gene conversion. The fate of the DNA lesion downstream of a DSB had more flexibility than that of AID, suggesting two alternative models: (i) DSBs during the physiological gene conversion are in the minority compared to single-strand breaks (SSBs), which are frequently generated following DNA deamination, or (ii) the physiological gene conversion is mediated by a tightly regulated DSB that is locally protected from non-homologous end joining (NHEJ) or other non-homologous DNA recombination machineries.

Checkpoint kinase 2 is required for efficient immunoglobulin diversification

Maintenance of genome integrity relies on multiple DNA repair pathways as well as on checkpoint regulation. Activation of the checkpoint kinases Chk1 and Chk2 by DNA damage triggers cell cycle arrest and improved DNA repair, or apoptosis in case of excessive damage. Chk1 and Chk2 have been reported to act in a complementary or redundant fashion, depending on the physiological context. During secondary immunoglobulin (Ig) diversification in B lymphocytes, DNA damage is abundantly introduced by activation-induced cytidine deaminase (AID) and processed to mutations in a locus-specific manner by several error-prone DNA repair pathways. We have previously shown that Chk1 negatively regulates Ig somatic hypermutation by promoting error-free homologous recombination and Ig gene conversion. We now report that Chk2 shows opposite effects to Chk1 in the regulation of these processes. Chk2 inactivation in B cells leads to decreased Ig hypermutation and Ig class switching, and increased Ig gene conversion activity. This is linked to defects in non-homologous end joining and increased Chk1 activation upon interference with Chk2 function. Intriguingly, in the context of physiological introduction of substantial DNA damage into the genome during Ig diversification, the 2 checkpoint kinases thus function in an opposing manner, rather than redundantly or cooperatively.

Checkpoint kinase 1 negatively regulates somatic hypermutation

Immunoglobulin (Ig) diversification by somatic hypermutation in germinal center B cells is instrumental for maturation of the humoral immune response, but also bears the risk of excessive or aberrant genetic changes. Thus, introduction of DNA damage by activation-induced cytidine deaminase as well as DNA repair by multiple pathways need to be tightly regulated during the germinal center response to prevent lymphomagenesis. In the present study, we show that DNA damage checkpoint signaling via checkpoint kinase 1 (Chk1) negatively regulates somatic hypermutation. Chk1 inhibition in human B cell lymphoma lines as well as inactivation of Chk1 alleles by gene targeting in DT40 B cells leads to increased somatic hypermutation. This is apparently due to changes in DNA repair pathways regulated by Chk1, such as a decreased homologous recombination efficiency that also leads to decreased Ig gene conversion in DT40. Our data show that Chk1 signaling plays a crucial role in regulation of Ig diversification and sheds unexpected light on potential origins of aberrant somatic hypermutation in B cell lymphomagenesis.

A novel activation-induced cytidine deaminase (AID) mutation in Brazilian patients with hyper-IgM type 2 syndrome

Activation-induced cytidine deaminase (AID) is a DNA editing protein that plays an essential role in three major events of immunoglobulin (Ig) diversification: somatic hypermutation, class switch recombination and Ig gene conversion. Mutations in the AID gene (AICDA) have been found in patients with autosomal recessive Hyper-IgM (HIGM) syndrome type 2. Here, two 9- and 14-year-old Brazilian sisters, from a consanguineous family, were diagnosed with HIGM2 syndrome. Sequencing analysis of the exons from AICDA revealed that both patients are homozygous for a single C to G transversion in the third position of codon 15, which replaces a conserved Phenylalanine with a Leucine. To our knowledge, this is a new AICDA mutation found in HIGM2 patients. Functional studies confirm that the homologous murine mutation leads to a dysfunctional protein with diminished intrinsic cytidine deaminase activity and is unable to rescue CSR when introduced in Aicda(-/-)stimulated murine B cells. We briefly discuss the relevance of AICDA mutations found in patients for the biology of this molecule.

MSH6- or PMS2-deficiency causes re-replication in DT40 B cells, but it has little effect on immunoglobulin gene conversion or on repair of AID-generated uracils

The mammalian antibody repertoire is shaped by somatic hypermutation (SHM) and class switch recombination (CSR) of the immunoglobulin (Ig) loci of B lymphocytes. SHM and CSR are triggered by non-canonical, error-prone processing of G/U mismatches generated by activation-induced deaminase (AID). In birds, AID does not trigger SHM, but it triggers Ig gene conversion (GC), a ‘homeologous’ recombination process involving the Ig variable region and proximal pseudogenes. Because recombination fidelity is controlled by the mismatch repair (MMR) system, we investigated whether MMR affects GC in the chicken B cell line DT40. We show here that Msh6−/− and Pms2−/− DT40 cells display cell cycle defects, including genomic re-replication. However, although IgVλ GC tracts in MMR-deficient cells were slightly longer than in normal cells, Ig GC frequency, donor choice or the number of mutations per sequence remained unaltered. The finding that the avian MMR system, unlike that of mammals, does not seem to contribute towards the processing of G/U mismatches in vitro could explain why MMR is unable to initiate Ig GC in this species, despite initiating SHM and CSR in mammalian cells. Moreover, as MMR does not counteract or govern Ig GC, we report a rare example of ‘homeologous’ recombination insensitive to MMR.

The potential mechanism of Bursal-derived BPP-II on the antibody production and avian pre-B cell

The bursa of Fabricius is critical for the normal development of the B lymphocytes responsible for antibody production. However, the mechanism of the bursal-derived bioactive factor on B cell development is little reported. In this paper, chicks were immunized with BPP-II and AIV vaccine or AIV antigen, and antibody and IL-4 production were detected. The results showed that BPP-II played strongly inducing roles on the humoral immune responses. To investigate the gene expression at transcriptional level, avian pre-B lymphocyte DT40 cells were treated with BPP-II, and were analyzed with the gene microarray. The results proved that BPP-II treatment regulated 11 pathways, in which homologous recombination is a vital mechanism which is involved in antibody Ig gene conversion and diversification during B cell development. These results suggested Bursal-derived biological active factor BPP-II might be involved in the antibody production processes and B cell development, which is vital to the humoral central immune organ, the bursa of Fabricius.

Expression of IgA class switching gene in tonsillar mononuclear cells in patients with IgA nephropathy

There are many reports suggesting a relationship between the tonsillar autoimmune response and the pathogenesis of IgA nephropathy (IgAN). Hyperproduction of IgA and IgA1 in tonsils could be caused by activation of the Ig class switching recombination (CSR). αGLT (germline transcripts) plays a critical role in the initiation of switching from Cμ to Cα, resulting in production of IgA. Activation-induced cytidine deaminase (AID) is a molecule essential for CSR and Ig gene conversion. The aim of this study was to investigate IgA and IgA1 levels in the supernatant of tonsillar mononuclear cells (TMCs) and the expression of Iα-Cα germline transcript and AID in TMCs stimulated with lipopolysaccharide or hemolytic streptococcus in IgAN patients and chronic tonsillitis patients. 27 IgAN patients were admitted into our hospital from Jan. 2009 to Feb. 2010. Another 27 patients with chronic tonsillitis but without renal disease were selected as the control group. Tonsillar lymphocytes were isolated by density gradient centrifugation using Lymphocyte Separation Medium. The amount of IgA or IgA1 secreted in the culture supernatants was determined by specific enzyme-linked immunosorbent assay. Expressions of Iα-Cα germline transcript and AID mRNA were examined by reverse transcription real-time PCR. The AID protein was determined by Western blotting. The production of IgA and IgA1 protein, especially the ratio of IgA1/IgA in TMCs stimulated with or without 10μg/ml of lipopolysaccharide or 1×10(8)cfu/ml of hemolytic streptococcus, were significantly increased in the IgAN group compared with that in the non-IgAN group (P<0.05), and the IgA and IgA1 levels in TMCs stimulated with 10μg/ml of lipopolysaccharide or 1×10(8)cfu/ml of hemolytic streptococcus were markedly increased in patients with IgAN compared with the control group (P<0.05).The expressions of Iα-Cα and AID mRNA were significantly upregulated in TMCs stimulated with 10μg/ml of lipopolysaccharide or 1×10(8)cfu/ml of hemolytic streptococcus in patients with IgAN compared with control group (P<0.05). The expression of AID protein in TMCs stimulated with or without 10μg/ml of lipopolysaccharide or 1×10(8)cfu/ml of hemolytic streptococcus was significantly increased in the IgAN group compared with that in the non-IgAN group (P<0.05). The expression of AID protein in TMCs stimulated with 10μg/ml of lipopolysaccharide or 1×10(8)cfu/ml of hemolytic streptococcus was significantly increased in patients with IgAN compared with the control group (P<0.05, P<0.01). Lipopolysaccharide or hemolytic streptococcus can induce the production of IgA and IgA1 and the expression of AID and Iα-Cα in TMCs from patients with IgAN. Our results indicate that the TMCs from patients with IgAN are capable of producing high levels of IgA and IgA1 when stimulated with lipopolysaccharide or hemolytic streptococcus, which may be due to the increased expression of AID and Iα-Cα.

DNA polymerases ν and θ are required for efficient immunoglobulin V gene diversification in chicken

The chicken DT40 B lymphocyte line diversifies its immunoglobulin (Ig) V genes through translesion DNA synthesis-dependent point mutations (Ig hypermutation) and homologous recombination (HR)-dependent Ig gene conversion. The error-prone biochemical characteristic of the A family DNA polymerases Polnu and Pol led us to explore the role of these polymerases in Ig gene diversification in DT40 cells. Disruption of both polymerases causes a significant decrease in Ig gene conversion events, although POLN(-/-)/POLQ(-/-) cells exhibit no prominent defect in HR-mediated DNA repair, as indicated by no increase in sensitivity to camptothecin. Poleta has also been previously implicated in Ig gene conversion. We show that a POLH(-/-)/POLN(-/-)/POLQ(-/-) triple mutant displays no Ig gene conversion and reduced Ig hypermutation. Together, these data define a role for Polnu and Pol in recombination and suggest that the DNA synthesis associated with Ig gene conversion is accounted for by three specialized DNA polymerases.

Transcription factors PU.1 and IRF4 regulate activation induced cytidine deaminase in chicken B cells

The chicken immunoglobulin (Ig) variable region is preceded by multiple pseudo genes. During B cell maturation, the Ig variable region is converted into various pseudo gene sequences, and the diversity of antigen binding site is expanded as a consequence. Ig gene conversion is initiated by a B cell-specific enzyme, activation induced cytidine deaminase (AID). The mechanisms that regulate AID expression and function during Ig gene conversion in chicken B cells are not fully elucidated. In this report, we show that the transcription factor PU.1 and its functional partner, IRF4, are involved in the expression of AID gene in chicken B cells, and the regulation may be mediated in part through a composite PU.1/IRF4 binding site near the AID promoter. Relative to IRF4, PU.1 appears to be more important for AID expression, implying additional IRF4 independent roles for PU.1. Besides regulating AID expression, PU.1 and IRF4 also bind to two composite PU.1/IRF4 binding sites in the Igλ locus, and mutations in these binding sites reduce AID-mediated gene conversion. Moreover, AID-mediated gene conversion is impaired in IRF4 deficient cells, and the defect may be a consequence of both reduced AID expression and lack of IRF4 interaction at the Igλ locus. Based on these observations, we propose that PU.1 and IRF4 regulate AID at two levels in chicken B cells: the expression of AID as well as its gene conversion function at the Igλ locus.

Ubc13 dosage is critical for immunoglobulin gene conversion and gene targeting in vertebrate cells

In contrast to lower eukaryotes, most vertebrate cells are characterized by a moderate efficiency of homologous recombination (HR) and limited feasibility of targeted genetic modifications. As a notable exception, the chicken DT40 B cell line is distinguished by efficient homology-mediated repair of DNA lesions during Ig gene conversion, and also shows exceptionally high gene-targeting efficiencies. The molecular basis of these phenomena is elusive. Here we show that the activity levels of Ubc13, the E2 enzyme responsible for non-canonical K63-linked polyubiquitination, are critical for high efficiency of Ig gene conversion and gene targeting in DT40. Ubc13+/− cells show substantially lower homology-mediated repair, yet do not display changes in somatic hypermutation, overall DNA repair or cell proliferation. Our results suggest that modulation of the activity of K63-linked polyubiquitination may be used to customize HR efficiencies in vertebrate cells.

Temporal Regulation of Ig Gene Diversification Revealed by Single-Cell Imaging

Rearranged Ig V regions undergo activation-induced cytidine deaminase (AID)-initiated diversification in sequence to produce either nontemplated or templated mutations, in the related pathways of somatic hypermutation and gene conversion. In chicken DT40 B cells, gene conversion normally predominates, producing mutations templated by adjacent pseudo-V regions, but impairment of gene conversion switches mutagenesis to a nontemplated pathway. We recently showed that the activator, E2A, functions in cis to promote diversification, and that G(1) phase of cell cycle is the critical window for E2A action. By single-cell imaging of stable AID-yellow fluorescent protein transfectants, we now demonstrate that AID-yellow fluorescent protein can stably localize to the nucleus in G(1) phase, but undergoes ubiquitin-dependent proteolysis later in cell cycle. By imaging of DT40 polymerized lactose operator-lambda(R) cells, in which polymerized lactose operator tags the rearranged lambda(R) gene, we show that both the repair polymerase Poleta and the multifunctional factor MRE11/RAD50/NBS1 localize to lambda(R), and that lambda(R)/Poleta colocalizations occur predominately in G(1) phase, when they reflect repair of AID-initiated damage. We find no evidence of induction of gamma-H2AX, the phosphorylated variant histone that is a marker of double-strand breaks, and Ig gene conversion may therefore proceed by a pathway involving templated repair at DNA nicks rather than double-strand breaks. These results lead to a model in which Ig gene conversion initiates and is completed or nearly completed in G(1) phase. AID deaminates ssDNA, and restriction of mutagenesis to G(1) phase would contribute to protecting the genome from off-target attack by AID when DNA replication occurs in S phase.

Bloom DNA Helicase Facilitates Homologous Recombination between Diverged Homologous Sequences

Bloom syndrome caused by inactivation of the Bloom DNA helicase (Blm) is characterized by increases in the level of sister chromatid exchange, homologous recombination (HR) associated with cross-over. It is therefore believed that Blm works as an anti-recombinase. Meanwhile, in Drosophila, DmBlm is required specifically to promote the synthesis-dependent strand anneal (SDSA), a type of HR not associating with cross-over. However, conservation of Blm function in SDSA through higher eukaryotes has been a matter of debate. Here, we demonstrate the function of Blm in SDSA type HR in chicken DT40 B lymphocyte line, where Ig gene conversion diversifies the immunoglobulin V gene through intragenic HR between diverged homologous segments. This reaction is initiated by the activation-induced cytidine deaminase enzyme-mediated uracil formation at the V gene, which in turn converts into abasic site, presumably leading to a single strand gap. Ig gene conversion frequency was drastically reduced in BLM(-/-) cells. In addition, BLM(-/-) cells used limited donor segments harboring higher identity compared with other segments in Ig gene conversion event, suggesting that Blm can promote HR between diverged sequences. To further understand the role of Blm in HR between diverged homologous sequences, we measured the frequency of gene targeting induced by an I-SceI-endonuclease-mediated double-strand break. BLM(-/-) cells showed a severer defect in the gene targeting frequency as the number of heterologous sequences increased at the double-strand break site. Conversely, the overexpression of Blm, even an ATPase-defective mutant, strongly stimulated gene targeting. In summary, Blm promotes HR between diverged sequences through a novel ATPase-independent mechanism.

Genetic Evidence for Single-Strand Lesions Initiating Nbs1-Dependent Homologous Recombination in Diversification of Ig V in Chicken B Lymphocytes

Homologous recombination (HR) is initiated by DNA double-strand breaks (DSB). However, it remains unclear whether single-strand lesions also initiate HR in genomic DNA. Chicken B lymphocytes diversify their Immunoglobulin (Ig) V genes through HR (Ig gene conversion) and non-templated hypermutation. Both types of Ig V diversification are initiated by AID-dependent abasic-site formation. Abasic sites stall replication, resulting in the formation of single-stranded gaps. These gaps can be filled by error-prone DNA polymerases, resulting in hypermutation. However, it is unclear whether these single-strand gaps can also initiate Ig gene conversion without being first converted to DSBs. The Mre11-Rad50-Nbs1 (MRN) complex, which produces 3′ single-strand overhangs, promotes the initiation of DSB-induced HR in yeast. We show that a DT40 line expressing only a truncated form of Nbs1 (Nbs1p70) exhibits defective HR-dependent DSB repair, and a significant reduction in the rate—though not the fidelity—of Ig gene conversion. Interestingly, this defective gene conversion was restored to wild type levels by overproduction of Escherichia coli SbcB, a 3′ to 5′ single-strand–specific exonuclease, without affecting DSB repair. Conversely, overexpression of chicken Exo1 increased the efficiency of DSB-induced gene-targeting more than 10-fold, with no effect on Ig gene conversion. These results suggest that Ig gene conversion may be initiated by single-strand gaps rather than by DSBs, and, like SbcB, the MRN complex in DT40 may convert AID-induced lesions into single-strand gaps suitable for triggering HR. In summary, Ig gene conversion and hypermutation may share a common substrate—single-stranded gaps. Genetic analysis of the two types of Ig V diversification in DT40 provides a unique opportunity to gain insight into the molecular mechanisms underlying the filling of gaps that arise as a consequence of replication blocks at abasic sites, by HR and error-prone polymerases.

Genetic Variation Stimulated by Epigenetic Modification

Homologous recombination is essential for maintaining genomic integrity. A common repair mechanism, it uses a homologous or homeologous donor as a template for repair of a damaged target gene. Such repair must be regulated, both to identify appropriate donors for repair, and to avoid excess or inappropriate recombination. We show that modifications of donor chromatin structure can promote homology-directed repair. These experiments demonstrate that either the activator VP16 or the histone chaperone, HIRA, accelerated gene conversion approximately 10-fold when tethered within the donor array for Ig gene conversion in the chicken B cell line DT40. VP16 greatly increased levels of acetylated histones H3 and H4, while tethered HIRA did not affect histone acetylation, but caused an increase in local nucleosome density and levels of histone H3.3. Thus, epigenetic modification can stimulate genetic variation. The evidence that distinct activating modifications can promote similar functional outcomes suggests that a variety of chromatin changes may regulate homologous recombination, and that disregulation of epigenetic marks may have deleterious genetic consequences.

Non-conservative homologous recombination in human B lymphocytes is promoted by activation-induced cytidine deaminase and transcription

During secondary immunoglobulin (Ig) diversification in vertebrates, the sequence of the variable region of Ig genes may be altered by templated or non-templated mechanisms. In both cases, cytidine deamination by activation-induced cytidine deaminase (AID) in the transcribed Ig loci leads to DNA lesions, which are repaired by conservative homologous recombination (HR) during Ig gene conversion, or by non-templated mutagenesis during somatic hypermutation. The molecular basis for the differential use of these two pathways in different species is unclear. While experimental ablation of HR in avian cells performing Ig gene conversion may promote a switch to somatic hypermutation, the activity of HR processes in intrinsically hypermutating mammalian cells has not been measured to date. Employing a functional HR assay in human germinal centre like B cell lines, we detect elevated HR activity that can be enhanced by transcription and AID. Products of such recombination events mostly arise through non-conservative HR pathways, while the activity of conservative HR is low to absent. Our results identify non-conservative HR as a novel DNA transaction pathway promoted by AID and suggest that somatic hypermutation in germinal centre B cells may be based on a physiological suppression of conservative HR.

DNA damage response protein ASCIZ links base excision repair with immunoglobulin gene conversion

ASCIZ (ATMIN) was recently identified as a novel DNA damage response protein. Here we report that ASCIZ-deficient chicken DT40 B lymphocyte lines displayed markedly increased Ig gene conversion rates, whereas overexpression of human ASCIZ reduced Ig gene conversion below wild-type levels. However, neither the efficiency of double-strand break repair nor hypermutation was affected by ASCIZ levels, indicating that ASCIZ does not directly control homologous recombination or formation of abasic sites. Loss of ASCIZ led to mild sensitivity to the base damaging agent methylmethane sulfonate (MMS), yet remarkably, suppressed the dramatic MMS hypersensitivity of polβ-deficient cells. These data suggest that ASCIZ may affect the choice between competing base repair pathways in a manner that reduces the amount of substrates available for Ig gene conversion.