Mutations occur in the Ig S region but rarely in S regions prior to class switch recombination

Class switch recombination (CSR) allows B cells to make effective protective antibodies. CSR involves the replacement of the constant region with one of the downstream constant regions by recombination between the donor and recipient switch (S) regions. Although histone H3 hyperacetylation in recipient S regions was recently reported to coincide with CSR, the relative histone H3 and H4 acetylation status of the donor and recipient S regions and the relationship between the generation of mutations and histone hyperacetylation in S regions have not been addressed. Here we report that histone H3 and H4 were constitutively hyperacetylated in the donor S region before and after different mitogen and cytokine treatments. We observed an increased frequency of mutations in hyperacetylated S␥ DNA segments immunoprecipitated with anti-acetyl histone antibodies. Furthermore, time course experiments revealed that the pattern of association of RNA polymerase II with S regions was much like that of H3 hyperacetylation but not always like that of H4 hyperacetylation. Collectively, our data suggest that H3 and H4 histone hyperacetylation in different S regions is regulated differently, that RNA polymerase II distribution and H3 hyperacetylation reflect the transcriptional activity of a given S region, and that transcription, hyperacetylation, and mutation are not sufficient to guarantee CSR. These findings support the notion that there are additional modifications and͞or factors involved in the complex process of CSR.H igh-affinity IgG, IgA, and IgE antibodies protect higher organisms from infection by pathogenic organisms and other environmental threats. The generation of effective protective antibodies requires B cells to carry out somatic hypermutation (SHM) and class switch recombination (CSR), two related but quite different DNA transactions (1). SHM introduces many point mutations in the variable (V) regions of the Ig heavy and light chain genes that encode the antigen-binding site of the antibody molecule (2). In contrast to SHM, CSR is a region-specific recombinationdeletion process that requires the generation of double-stranded DNA breaks (DSB) (3). These DSB are generated in the donor switch (S) region that is just upstream of the constant (C) region gene and in a downstream recipient ␥, , or ␣ S region (4, 5). Recombination then brings one of those downstream C regions into proximity to the V region. This allows the mutated heavy chain V region to be expressed with one of the C regions so that each antigen-binding site will be able to mediate different effector functions and be distributed throughout the body.Despite the different outcomes of SHM and CSR, activationinduced cytidine deaminase (AID) is required for both processes (6) presumably because of its ability to deaminate deoxycytidine to deoxyuridine on single-stranded DNA (ssDNA) (7-11). Both SHM and CSR require transcription (12), suggesting that the ssDNA substrate for AID is created by the generation of transcription bubbles (7) and͞or perhaps triplex RNA...

Section: Resultssupporting

confidence: 83%

Section: Resultssupporting

confidence: 77%

Differential regulation of histone acetylation and generation of mutations in switch regions is associated with Ig class switching

Luo

Scharff

2004

“…Large ISDs typically are assayed by activating B cells in culture to switch to a particular C H and then generating hybridomas and assaying for ISD in IgM-producing hybridomas that have not undergone bona fide CSR (11,12). Most such studies have found that large ISDs occur far more frequently in Sμ than in downstream target S regions (6,12,13). The almost complete absence of detectable ISDs or SHMs in downstream S regions in prior studies (6,12) led to the proposal that AID targeting in Sμ is required to promote AID targeting of downstream S regions (6).…”

mentioning

confidence: 99%

“…Most such studies have found that large ISDs occur far more frequently in Sμ than in downstream target S regions (6,12,13). The almost complete absence of detectable ISDs or SHMs in downstream S regions in prior studies (6,12) led to the proposal that AID targeting in Sμ is required to promote AID targeting of downstream S regions (6). However, it is possible that absence of downstream ISD or SHM in those studies might reflect, in part, IgM + hybridomas arising from B cells not fully activated for CSR (2,12).…”

mentioning

confidence: 99%

Alternative end-joining catalyzes robust IgH locus deletions and translocations in the combined absence of ligase 4 and Ku70

Boboilă

Janković

Yan

et al. 2010

167

170

Class switch recombination (CSR) in B lymphocytes is initiated by introduction of multiple DNA double-strand breaks (DSBs) into switch (S) regions that flank immunoglobulin heavy chain ( IgH ) constant region exons. CSR is completed by joining a DSB in the donor Sμ to a DSB in a downstream acceptor S region (e.g., Sγ1) by end-joining. In normal cells, many CSR junctions are mediated by classical nonhomologous end-joining (C-NHEJ), which employs the Ku70/80 complex for DSB recognition and XRCC4/DNA ligase 4 for ligation. Alternative end-joining (A-EJ) mediates CSR, at reduced levels, in the absence of C-NHEJ, even in combined absence of Ku70 and ligase 4, demonstrating an A-EJ pathway totally distinct from C-NHEJ. Multiple DSBs are introduced into Sμ during CSR, with some being rejoined or joined to each other to generate internal switch deletions (ISDs). In addition, S-region DSBs can be joined to other chromosomes to generate translocations, the level of which is increased by absence of a single C-NHEJ component (e.g., XRCC4). We asked whether ISD and S-region translocations occur in the complete absence of C-NHEJ (e.g., in Ku70/ligase 4 double-deficient B cells). We found, unexpectedly, that B-cell activation for CSR generates substantial ISD in both Sμ and Sγ1 and that ISD in both is greatly increased by the absence of C-NHEJ. IgH chromosomal translocations to the c-myc oncogene also are augmented in the combined absence of Ku70 and ligase 4. We discuss the implications of these findings for A-EJ in normal and abnormal DSB repair.

“…Point mutations occurring close to the recombination junction are supposedly generated during the resolving step of CSR, given that they are generally more frequent and mostly affect GC residues, in comparison with point mutations in the germline Sμ region, which exhibit a pattern similar to SHM in V regions and thus likely involve a different mechanism (28,29). A lower number of mutations around the Sμ-Sα junctions (±15 bp) has been reported in patients with Artemis deficiency and Ataxia-telangiectasia compared with controls, suggesting that DNA ends are not normally processed in these individuals (27).…”

Section: Position Of Sμ and Sα Breakpoints In Switchmentioning

confidence: 99%

Impaired induction of DNA lesions during immunoglobulin class-switch recombination in humans influences end-joining repair

Kracker

Imai

Gardès

et al. 2010

Ig class-switch recombination (CSR) is a region-specific process that exchanges the constant Ig heavy-chain region and thus modifies an antibody's effector function. DNA lesions in switch (S) regions are induced by activation-induced cytidine deaminase (AID) and uracil-DNA glycosylase 2 (UNG2), subsequently processed to DNA breaks, and resolved by either the classical nonhomologous endjoining pathway or the alternative end-joining pathway (XRCC4/ DNA ligase 4-and/or Ku70/Ku80-independent and prone to increased microhomology usage). We examined whether the induction of DNA lesions influences DNA end-joining during CSR by analyzing Sμ-Sα recombination junctions in various human Ig CSR defects of DNA lesion induction. We observed a progressive trend toward the usage of microhomology in Sμ-Sα recombination junctions from AID-heterozygous to AID-autosomal dominant to UNG2-deficient B lymphocytes. We thus hypothesize that impaired induction of DNA lesions in S regions during CSR leads to unusual end-processing of the DNA breaks, resulting in microhomology-mediated end-joining, which could be an indication for preferential processing by alternative end-joining rather than by classical nonhomologous end-joining.antibody maturation | switch junction | DNA repair D uring immune responses, mature B cells diversify their Ig genes through class-switch recombination (CSR) and somatic hypermutation (SHM). The latter mechanism introduces nontemplated point mutations in the variable region of Ig genes and thereby enables the selection of antibodies with increased affinity for the antigen. CSR modulates antibody effector function by replacing one constant region with another in a deletionmediated recombination process, while retaining the binding specificity (variable region) of the B-cell receptor. Both processes are initiated by the enzyme activation-induced cytidine deaminase (AID), which deaminates cytosine to produce U:G mismatches in target DNA (1-3). GC-rich regions, so-called "switch" (S) regions, present in front of each constant region and of varying lengths, are the target DNAs for AID during CSR. The main route for processing AID-induced lesions involves uracil excision through several pathways, primarily the pathway involving uracil-DNA glycosylase (UNG2) (4, 5). Abasic sites are further processed to generate DNA double-strand breaks (DSBs), which are obligate intermediates in CSR (6, 7). The DSBs activate damage response proteins, such as PI3-like protein kinase ataxia-telangiectasia mutated, the phosphorylated histone variant H2AX, the MRN complex (MRE11, RAD50, and NBS1), MDC1, and 53BP1, all of which are known to play roles in CSR in promoting appropriate repair and efficient long-range, region-specific recombination (8)(9)(10)(11)(12)(13)(14). In CSR, the resolution step is normally mediated by the classical nonhomologous end-joining pathway (c-NHEJ); however, recent reports strongly suggest that resolution also can be mediated (albeit at a lower frequency) by Ku 70-, Ku 80-, and/or XRCC4/DNA ligase 4-independen...