53BP1 links DNA damage-response pathways to immunoglobulin heavy chain class-switch recombination

V(D)J recombination is accomplished by the products of recombination activating genes 1 and 2, which form a complex (RAG) that introduces a DSB between germline antigen receptor gene segments and flanking recombination signal sequences that target RAG activity. Subsequently, the RAG-cleaved gene segments are joined by the enzymes of the ubiquitously expressed NHEJ pathway to generate exons encoding antigen receptor V regions⁶. Productive assembly of immunoglobulin heavy (Igh) and light chain V regions leads to expression of surface IgM on newly generated B cells, which then exit the bone marrow and populate the peripheral lymphoid organs, such as the spleen, lymph node and Peyer's patches. In these settings, further antigen-driven diversification of Igh constant regions (C_H regions) and Igh and Igl V regions occurs through CSR and somatic hypermutation (SHM), respectively. In CSR, the initially expressed Igh constant region exons are exchanged for one of a set of 'downstream' C_H exons ('C_H genes') that specify a new effector function and class (for example IgG, IgA or IgE)⁵. The downstream C_H genes are interspersed over nearly 200 kilobases (kb) of the Igh locus, and all but C are preceded by a 1- to 10-kb repetitive DNA sequence referred to as a switch (S) region. In CSR, a downstream C_H gene is juxtaposed to the expressed V(D)J exon through the fusion of the downstream S region with the S region and the deletion of intervening C_H genes. This recombination event occurs after appropriate stimulation of B cells, usually after encounter with cognate antigen, and is preceded by selective transcriptional activation of the downstream C_H gene targeted for CSR. Thus, productive CSR is limited to activated mature B cells and necessa-rily involves recombination between S regions that are as much as 100 kb apart. The SHM process that also occurs in activated B cells involves the introduction of point mutations into the assembled V(D)J V-region exons at a very high rate, allowing the selection of B cells producing antibodies with higher affinity^{7, 8}.

Both general DSBs and specific DSBs, such as those introduced during V(D)J recombination and probably CSR, activate phosphoinositide-like kinases (PIKs). These include DNA-PKcs (DNA-dependent protein kinase catalytic subunit), ATM (mutated in ataxia-telangiectasia) and ATR (ATM and Rad3-related), which transduce damage signals to various effector molecules^{19, 20}. The PIKs have overlapping substrate specificities and phosphorylate serine and/or threonine residues at motifs found in a variety of DNA damage-response proteins including the histone H2AX, a histone H2A variant, and the p53-binding protein 1 (53BP1). Although 53BP1 was originally discovered as a p53-interacting factor in a yeast two-hybrid screen, it was subsequently found to be rapidly phosphorylated in response to ionizing radiation and to accumulate at presumptive sites of DNA damage^{21, 22, 23, 24}. Accumulation of 53BP1 at sites of irradiation-induced foci is independent of p53 function but is dependent on its interaction with the phosphorylated form of H2AX (-H2AX)²⁴. H2AX represents about 15% of the total H2A cellular pool incorporated into nucleosomes. Once DNA damage occurs, nucleosomal H2AX is rapidly phosphorylated by PIKs to form -H2AX over megabase regions flanking DSBs. The detection of -H2AX precedes the formation of irradiation-induced foci, which include many DNA repair and checkpoint proteins in addition to 53BP1 (refs. 19,25). Evidence at present supports the view that a common biochemical pathway comprising an upstream PIK that activates downstream factors, including H2AX and 53BP1, is central to the global response to DSBs.

To assess B cell development in Trp53bp1^-/- mice, we examined bone marrow from 8- to 12-week-old Trp53bp1^-/- mice and wild-type littermate control mice and found similar numbers of nucleated cells (18.6 10⁶ versus 16.2 10⁶). Likewise, flow cytometry of pro-B (B220⁺CD43⁺) and pre-B (B220⁺CD43^lo) populations from whole bone marrow of three Trp53bp1^-/- mice showed no distinct differences compared with those of wild-type mice (Fig. 1 and data not shown). Thus, whereas two of the Trp53bp1^-/- mice seemed to have slightly reduced pre-B cell numbers, the other had wild-type numbers (Fig. 1 and data not shown). Although a prior study suggested a possible decrease in the percentage of mature B cell populations in Trp53bp1^-/- mice²⁶, we found that the peripheral B cell compartment (spleen and lymph nodes) of Trp53bp1^-/- mice was comparable to that of wild-type in lymphocyte numbers, with no substantial difference in the percentages of mature B cell populations, based on surface expression of IgM and IgD (Fig. 1 and data not shown). Our finding of relatively normal B cell compartments in Trp53bp1^-/- mice indicates that there is no substantial block in V(D)J recombination. Preliminary transient V(D)J recombination substrate assays also did not show any substantial defect in V(D)J recombination (J.C.M. and P.B.C., unpublished data).

Figure 1. Bone marrow and splenic B cell development in Trp53bp1-/- mice. Whole bone marrow (top) or spleen (bottom) was obtained from 7-week-old mice and was stained for flow cytometry. Pro- and pre-B cell compartments were present in 53BP1-deficient mice with no reproducible developmental block. Splenic B220+IgM+ B cells were present in similar numbers and percentages (shown in boxed gates) in five sets of age-matched Trp53bp1-/- and wild-type mice. Full Figure and legend (53K)

Figure 2. Serum Igh isotype expression in 53BP1-deficient mice. Classes of serum Igh isotypes were quantified in serum from five 53BP1-deficient (open circles) and five wild-type (open triangles) mice. Full Figure and legend (13K)

For these experiments, we stimulated cultured splenic B cells derived from Trp53bp1^-/- and Trp53bp1^+/+ mice and assessed surface and secreted immunoglobulin concentrations. We cultured purified B cells in the presence of either lipopolysaccharide (LPS) or antibody to CD40 (anti-CD40) plus interleukin 4 (IL-4) and collected cells on day 4.5 for flow cytometry to assess surface immunoglobulin expression. Such stimulation of normal B cells with LPS induces germline transcription of and CSR to C2b and C3, whereas activation with anti-CD40 plus IL-4 induces germline transcription of and CSR to C1 and C²⁸. After LPS stimulation, 7.4% 3.1% and 20.2% 5.6% of wild-type B cells were IgG3⁺ and IgG2b⁺, respectively, compared with 1.1% 0.8% and 5.1% 2.5 % of Trp53bp1^-/- B cells (Fig. 3a and data not shown). After treatment with anti-CD40 plus IL-4, 32% 6.6% of wild-type B cells were IgG1⁺, in contrast to only 2.1% 2.6% of 53BP1-deficient B cells (Fig. 3a). All stimulated Trp53bp1^-/- B cells retained normal surface expression of IgM (data not shown).

Figure 3. Defective Igh class switching in 53BP1-deficient B cells. (a) Staining for surface IgG2b or IgG1 together with B220 was analyzed by flow cytometry in cultured splenic B cells after 4 d of stimulation with LPS (IgG2b; top) or with anti-CD40 plus IL-4 (IgG1; bottom). Boxes outline the population of switched cells; percentage of total cells in corner. (b) Secreted immunoglobulins were analyzed in supernatants from splenic B cell cultures after 5 d of in vitro stimulation. Open circles, wild-type; open triangles, 53BP1-deficient. (c) CSR to IgG1 was quantified with digestion-circularization PCR for S-S1 recombination products. B cells were stimulated for 4 d with anti-CD40 plus IL-4, and fivefold dilutions (wedges) of genomic DNA obtained from the cells were used in the PCR reaction. Chrnb1 served to normalize for the amount of input DNA. H2O, no input DNA. Full Figure and legend (60K)

The DNA recombination products resulting from CSR can be quantified with a digestion-circularization PCR strategy that amplifies a product only after CSR takes place²⁹. To determine whether the Igh class-switching defect was at the level of CSR, we assayed for S-S1 recombination products in total genomic DNA extracted from B cells stimulated with anti-CD40 plus IL-4 after 4 d of culture. We used the Chrnb1 locus as a control for both total genomic DNA and for efficiency of the endonuclease digestion and recircularization in this assay. The amount of S-S1 recombination products from Trp53bp1^-/- B cells was 4−20% that of wild-type, as determined with samples from two separate experimental cultures (Fig. 3c). These findings demonstrate that the Igh class-switch defect in 53BP1-deficient B cells occurs at the level of DNA recombination.

To further characterize the immunoglobulin switching defect in Trp53bp1^-/- B cells, we first assessed the ability of mutant and control B cells to induce factors required for CSR in vitro. Activation of B cells for CSR leads to the induction of germline transcription of the C_H genes targeted for recombination. Each germline C_H transcriptional unit consists of an activation or cytokine-responsive promoter upstream of a noncoding exon, called an intervening (I) exon, that lies just upstream of the S region³⁰. After appropriate stimulation, transcription is initiated from the I exon promoter, runs through the S region and is ultimately terminated downstream of the corresponding C_H gene. These primary germline C_H transcripts are processed to yield a mature, noncoding transcript in which the I exon is fused to the C_H exons⁵. Germline transcripts of specific C_H genes are detected in B cells 48−72 h after appropriate activation for CSR (discussed above). The presumed function of transcription in CSR is to provide an accessible single-stranded DNA substrate for AID activity^{5, 31}. To determine whether 53BP1 deficiency affected germline C_H transcription, we cultured purified B220⁺CD43^- splenic B cells from wild-type and 53BP1-deficient mice for 48 h with LPS or with anti-CD40 plus IL-4, then isolated total RNA and analyzed it by RT-PCR to quantify the spliced products of I2b-C2b and I1-C1 germline transcripts. We used Cd79a (also known as Mb-1) transcripts to normalize for RNA abundance. Analyses of fivefold dilutions of titrated cDNA indicated that both C2b (after treatment with LPS) and C1 (after treatment with anti-CD40 plus IL-4) germline transcripts were induced similarly in wild-type and 53BP1-deficient B cells (Fig. 4a).

Figure 4. Induction of factors associated with CSR initiation. (a) Germline transcripts from cultured splenic B cells were analyzed after 2 d of activation with LPS (I2b-C2b) or with anti-CD40 plus IL-4 (I1-C1), and are compared with Cd79a transcripts for standardization. Fivefold dilutions of input cDNA (wedges) were used for RT-PCR. (b) Aicda transcription was analyzed in B cells activated with anti-CD40 plus IL-4 from wild-type mice (lane 3) and 53BP1-deficient mice (lane 4) and was compared with that of purified unstimulated splenic B cells (lanes l and 2; wild-type and 53BP1-deficient, respectively). Aicda transcripts were not found in total thymic cDNA or in splenic DNA (lanes 5 and 6, respectively). Gadp transcripts serve to normalize the amount of RNA. Similar results were obtained in an independent experiment. The H2O lanes contain no input cDNA. Full Figure and legend (24K)

Figure 5. Normal growth of activated 53BP1-deficient B cells. (a) Purified splenic B cells were stimulated with LPS or with anti-CD40 plus IL-4 and were cultured at an initial density of 1 106 cells/ml. Live cells were counted on days 2, 3 and 4, as determined by trypan blue exclusion. Data represent the total number of wild-type B cells on day 4, defined as 100%, with the number of Trp53bp1-/- B cells numbers shown as percentage of wild-type. Error bars are shown for a total of five experiments. (b) The number of cell divisions in cultured splenic B cells after stimulation with anti-CD40 plus Il-4 was ascertained by CFSE labeling and quantification by flow cytometry. Each peak represents a cell division, with lower intensity of fluorescence corresponding to a higher number of divisions. Data are representative of three experiments. Full Figure and legend (15K)

Normal S-S1 CSR junctions from 53BP1-deficient B cells

For CSR, AID-initiated DNA modifications are resolved through the coordinated activities of several DNA repair factors; defects in these can affect CSR either quantitatively or qualitatively. The sequences of CSR junctions can offer insights into potential mechanistic defects in the joining process. For example, CSR recombination junctions normally have little junctional sequence homology. However, B cells that are deficient in mismatch repair factors (for example, Mlh1 or Pms2) undergo impaired CSR and form CSR junctions with increased sequence 'microhomology', indicating increased use of an alternative joining pathway in mutant cells. To assay CSR junctions, we cultured Trp53bp1^-/- and control B cells for 4 d in the presence of anti-CD40 plus IL-4 and then assayed total DNA for S1 CSR junctions through PCR amplification of the S-S1 junctions. Although this PCR assay is not fully quantitative, we recovered very few amplified junction sequences from equivalent amounts of DNA from the activated Trp53bp1^-/- cells compared with controls (data not shown), consistent with our digestion-circularization PCR findings indicating that CSR was decreased in these cells. However, sequence analyses of 23 wild-type and 26 Trp53bp1^-/- junctions obtained from two separate experiments showed no substantial qualitative differences (Table 1 and data not shown). Thus, although there seems to be a notable impairment of CSR to S1 in the 53BP1-deficient B cells, the CSR defect is not absolute and the 53BP1-deficient B cells that do undergo CSR to S1 generate junctions qualitatively similar to those found in wild-type B cells.

Table 1. Analysis of S-S1 CSR junctions Full Table

Figure 6. Immunoglobulin gene SHM in Trp53bp1-/- mice. (a) Peyer's patches were isolated for histology and stained for CD45R and Bcl-6. Representative sections of Peyer's patches from wild-type and Trp53bp1-/- mice are stained with anti-Bcl6 (dark brown nuclei), indicating a germinal center B cell. Original magnification, 200. (b) B220+PNAhi B cells were sorted by flow cytometry from wild-type or 53BP1-deficient Peyer's patches, and total DNA was extracted. The sequence immediately downstream of JH4 was amplified by PCR, cloned and sequenced; 51 Trp53bp1+/+ and 55 Trp53bp1-/- clones were analyzed for the number of mutations noted at the perimeters of the plots. The percentage of the total number of clones for each mutation is proportional to the area in each slice. (c) Changes in nucleotide composition detected in the mutations of the JH4 intronic region. Vertical 'axis', germline sequence−determined base; horizontal 'axis', mutated base observed. Full Figure and legend (37K)

To assess and quantify SHM in 53BP1-deficient mice, we purified B220⁺ B cells that also bound the most lectin peanut agglutinin (PNA, a surface marker for germinal centers B cells). This B220⁺PNA^hi population consists mainly of B cells that have undergone SHM. Subsequently, we determined the nucleotide sequence of sets of PCR-amplified V(D)J rearrangements from wild-type and Trp53bp1^-/- germinal center B cells and compared their sequences at the J_H4-distal intronic region, which is a target region for SHM³³. We found a similar overall mutation frequency within the J_H4-intronic region in Trp53BP1^-/- B cells compared with that of wild-type cells (1.24 10² versus 1.5 10²; P = 0.12), as well as a similar percentage of unmutated and more highly mutated sequences among each population (Fig. 6b). Analysis of the substituted nucleotides in the wild-type and 53BP1-deficient sequences showed similar patterns of composition, with most mutations at GC base pairs resulting in transitions. We found a similar 'preference' for mutations at SHM target sequences known to accumulate alterations at a high rate (also known as WRCY 'hotspots') for Trp53bp1^-/- and wild-type B cells⁸ (Fig. 6c and data not shown). Based on this evidence, we conclude that SHM occurs in an apparently normal way in 53BP1-deficient mice.

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PIK family members are major sensors of DNA damage, and one of their key functions is the phosphorylation of a set of downstream factors that includes 53BP1 and H2AX. Both ATM and ATR, and perhaps DNA-PKcs as well, activate 53BP1 (refs. 21,22). ATM is a 'master regulator' of the DSB response and seems to be activated by all types of DSBs throughout the cell cycle²⁰. DNA-PK also seems to sense such DSBs; although it may be more important in DSB repair through its function in NHEJ. In contrast, ATR is key in sensing replication errors that lead to DSBs, but may also partially substitute for some other ATM functions²⁰. Given its phosphorylation by PIK proteins, it seems reasonable to assume that the function of 53BP1 in CSR may involve its activation through this pathway in response to DSBs that occur during CSR. If phosphorylation by PIK members is, indeed, the main route by which 53BP1 acts during CSR, it is notable that the CSR defect in 53BP1 deficiency is much more severe than that seen in ATM deficiency³⁵, suggesting that DNA-PKcs or ATR may also be involved in the DSB response during CSR. Deficiency in DNA-PKcs considerably impairs CSR to all C_H genes except C1, whereas DNA-PKcs kinase domain mutants (the scid mutation) have a much less notable effect on CSR³⁶. Thus, DNA-PKcs might have multiple functions, one involving kinase activity, which we now suggest might target factors such as 53BP1 and H2AX, and another involving its kinase domain−independent ability to create synapses of DNA ends^{36, 37, 38}.

How might known 53BP1 functions in the context of a DSB response be involved in CSR? 53BP1 is involved in several cell cycle checkpoints, potentially by interacting with downstream factors through its BRCA1 C-terminal repeat motifs²¹. Thus, 53BP1 is necessary for effecting a G2-M checkpoint in response to low but not high doses of ionizing radiation^{39, 40, 41}, and it has also been linked to an intra-S-phase checkpoint^{27, 40}. In theory, 53BP1 might be involved in CSR through a checkpoint function by providing cells the opportunity to repair S-region DSBs. This model indicates that CSR would be operative during the cell cycle phases at which 53BP1 checkpoint functions occur. However, if, as proposed, CSR occurs in the G1 phase of the cell cycle⁴², other possibilities must be considered. The -H2AX foci, which are detected early in the DSB response, form at the Igh locus in an AID-dependent way during CSR⁴². Moreover, like 53BP1-deficient mice, H2AX-deficient mice have a substantial CSR block, but no obvious impairment of V(D)J recombination or SHM^{42, 43, 44, 45}. Although colocalization has not been examined for CSR, 53BP1 localizes with -H2AX foci in all other contexts examined^{26, 39, 41, 42}, so it seems reasonable to assume it would be similarly involved in -H2AX foci associated with CSR. Involvement of -H2AX has been suggested in the synapsis of S regions during CSR⁴⁴. Furthermore, it has been proposed that generation of -H2AX foci over megabase regions surrounding DSBs prevents separation and promotes appropriate joining of broken chromosomal DNA ends by serving as a scaffold for recruiting factors, including 53BP1, that interact with H2AX and also with each other or with DNA⁴⁶. Our findings reported here raise the possibility that such a 53BP1 function may be important for the joining phase of CSR.

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Single-cell suspensions of spleen cells were sorted with CD43 magnetic beads (MACS, Miltenyi), and B cells were cultured at a density of 5 10⁵ or 1 10⁶ cells/ml in RPMI medium supplemented with 10% FCS and either 25 ng/ml of LPS or 500 ng/ml of anti-CD40 (HM40-3; Pharmingen) plus 25 ng/ml of IL-4 (R&D) as described³⁶. Cultured cells were maintained daily at a density of 1 10⁶ cells/ml. Cells were collected on various days for flow cytometry and to obtain RNA and DNA, and culture supernatants were obtained for ELISA. Single-cell suspensions from spleens were prepared according to standard methods from mice 6−12 weeks old. Cells from cultures on day 4 were washed twice in PBS plus 2% FCS and were stained with various antibodies conjugated with fluorescein isothiocyanate (IgM-II/41, IgG1-A85-1 and IgG3-R40-82; PharMingen), phycoerythrin (IgM goat anti-mouse; Southern Biotech) or CyChrome (B220-R34-4; PharMingen), or IgG2b−fluorescein isothiocyanate (goat anti-mouse; Southern Biotech). Cells were analyzed with a FACSCalibur and data were interpreted with Cellquest (Becton Dickinson) and Flo-Jo (Tree Star) software. The flow cytometry profiles represented 5,000−10,000 events and were gated for live lymphoid cells, determined by forward scatter versus side scatter. CFSE (5 m; Molecular Probes) was used as described³² except cells were labeled for 10 min at 37 °C in RPMI medium and were washed twice with PBS before in vitro stimulation.

RNA was extracted from cultured cells and was prepared with the TRIPURE reagent (Roche) following the instructions of the manufacturer. Typically, 1 10⁵ to 5 10⁵ cells were collected to generate cDNA with Superscript (Invitrogen), as instructed by the manufacturer. Of the total cDNA obtained, 10% was amplified with primers for germline transcripts, Aicda, Gapd and Cd79a as described^{10, 47}.

For S-S1 junctions, DNA was extracted from cultures of B cells on day 4 that were activated with anti-CD40 plus IL-4. Genomic DNA was prepared with standard methods and PCR was used to amplify the S region junctions as described⁴⁸. ExTaq (Panvera) was used to amplify the junctions, which were subsequently cloned into the Topo-TA vector (Invitrogen) and were sequenced. Sequences were analyzed with DNA-STAR/SeqMan software and were aligned with the corresponding genomic sequences of S and S1. For somatic hypermutation studies, Peyer's patches were dissected from mice and single-cell suspensions were made. Cells were stained with PNA−fluorescein isothiocyanate (Vector), propidium iodide and B220-CyChrome and were sorted for 'live' B220⁺ and PNA^hi surface expression with a FACSVantage (Becton Dickinson). Cells were collected and genomic DNA was prepared for amplification following a published method³³. High-fidelity Pfu (Stratagene) polymerase was used in the PCR to amplify the V_HJ558/FR3 distal sequence and the PCR product was cloned into the Topo-TA vector (Invitrogen) and sequenced. Sequences were compared with Sequencher (Gene Codes) and Seqman (Lasergene) software. Immunohistochemistry was done as described⁴⁹.

MUSIGCD07, S; MUSIGHANB, S1.

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