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The Journal of Immunology, 2005, 175: 5615-5618.
Copyright © 2005 by The American Association of Immunologists

CUTTING EDGE

Cutting Edge: Double-Stranded DNA Breaks in the IgV Region Gene Were Detected at Lower Frequency in Affinity-Maturation Impeded GANP–/– Mice1

Yousuke Kawatani*,{ddagger}, Hideya Igarashi*,{ddagger}, Takeshi Matsui*,{ddagger}, Kazuhiko Kuwahara*,{ddagger}, Satoru Fujimura*,{ddagger}, Nobukazu Okamoto*,{ddagger}, Katsumasa Takagi{dagger} and Nobuo Sakaguchi2,*,{ddagger}

* Department of Immunology and {dagger} Department of Orthopedics, Graduate School of Medical Sciences, Kumamoto University, Kumamoto, Japan; and {ddagger} Core Research for Evolutional Science and Technology, Japan Science and Technology Agency, Kawaguchi, Japan


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Double-stranded DNA breaks (DSBs) at the IgV region (IgV) genes might be involved in somatic hypermutation and affinity-maturation of the B cell receptor in response to T cell-dependent Ag. By ligation-mediated PCR, we studied IgV DSBs that occurred in mature germinal center B cells in response to nitrophenyl-chicken {gamma}-globulin in a RAG1-independent, Ag-dependent, and IgV-selective manner. We quantified their levels in GANP-deficient B cells that have impaired generation of high-affinity Ab. GANP–/– B cells showed a decreased level of DSBs with blunt ends than control B cells and, on the contrary, the ganp gene transgenic (GANPTg) B cells showed an increased level. These results suggested that the level of IgV DSBs in germinal center B cells is associated with GANP expression, which is presumably required for B cell receptor affinity maturation.


   Introduction
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 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Double-stranded DNA breaks (DSBs)3 occur in the IgV gene during B cell maturation in peripheral lymphoid organs following immunization with T cell-dependent Ag (TD-Ag) (1, 2, 3). This type of break is probably not dependent on recombination signal sequence or S sequences, but is frequently observed during the maturation of B cells in germinal center (GC) regions (1, 2, 3). DSBs target the RGY(W) hot spot (1, 2) and their generation is coupled to enhancer-dependent IgV expression of rearranged V(D)J genes (1), suggesting their association with the somatic hypermutation (SHM) at the IgV gene during proliferation and maturation of GC-B cells after TD-Ag immunization. For induction of SHM, AID is required as a primary molecule that attacks the cytidine nucleotide either in the mRNA (4) or in the DNA (5, 6, 7, 8, 9, 10, 11, 12, 13). However, the frequency of IgV DSBs in AID knockout B cells is the same as in wild-type B cells (14, 15). Therefore, the role of DSBs has remained unclear with respect to generation of IgV SHM and the affinity maturation of B cell receptors.

In this study, we characterize the B cell subpopulation that undergoes DSB processes in the spleen in detail by cell sorting after immunization with the TD-Ag nitrophenyl chicken {gamma}-globulin (NP-CG). Next, the level of IgV DSBs was examined in a mutant (B-GANP–/–) mouse with impaired generation of high-affinity Abs against TD-Ag. The B-GANP–/– mouse was conditionally targeted at the ganp gene loci in B cells using the CD19-Cre knock-in mouse. Lack of GANP caused a decrease of Ab affinity in vivo, which was accompanied by the decreased SHM frequency of the VH-186.2, especially of the high-affinity type W33L mutation (16, 17, 18). The results presented here suggest that GANP had effects on the level of IgV DSBs, which might be involved in generation of high-affinity Ab in vivo.


   Materials and Methods
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 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Cell surface staining, flow cytometric analysis, and cell sorting

The rag1/gfp knock-in mouse is as described previously (19). The B-GANP–/– mouse conditionally targeted at the ganp gene in B cells was reported previously (18), which is now given a correct formal nomenclature of Mcm3aptm1Imku (MGI:3583894). GANPTg mouse and the immunization protocols were as described elsewhere (20). The purified splenic B cells were stained with PE-anti-CD45R/B220 mAb (RA3-6B2) and FITC-anti-mouse T and B cell activation Ag (GL7). Cells were stained with PE-anti-CD95/Fas mAb (Jo2) and biotinylated anti-rat IgM (G53-238) with streptavidin-CyChrome (BD Pharmingen) and were sorted by FACSVantage (BD Immunocytometry Systems) or JSAN (Bay Bioscience). Apoptotic cells were studied as described elsewhere with 7-aminoactinomycin D (7AAD; BD Pharmingen).

DSB analysis by ligation-mediated PCR (LM-PCR)

The DNA from 5 x 104 cells (equivalent) was ligated with the BW linker (0.2 µM) in a 50-µl volume with 250 U of T4 DNA ligase (Nippon gene) at 16°C for 16 h and the diluted samples were PCR amplified by two PCR rounds: 10 cycles, 50°C annealing step, followed by 28 cycles, 64°C annealing step. For the second reaction, an aliquot (1 µl) of the mixture was added in a 25-µl reaction with sets of linker-specific and gene-specific nested primers using hotstart AmpliTaq Gold (Applied Biosystems), and the products were separated, blotted, and hybridized with radioactive oligonucleotide probes (14, 21). The Cµ region reaction was used for the controls.

Real-time quantitative RT-PCR

PCR was by TaqMan Universal PCR Master Mix in the ABI PRISM 7700 Sequence Detection System using Sequence Detector version 1.6.3 software (Applied Biosystems).


   Results
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 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Selective induction of IgV DSBs in mature GL7+Fas+ GC B cells

To address the question whether RAG is responsible for IgV DSBs, we used rag1/gfp knock-in mice to follow RAG1 reinduction with GFP tagging. Splenic B220+ B cells were sorted by GL7 and RAG signals to the GL7+GFP– and GL7+GFP+ fractions from NP-CG-immunized rag1/gfp knock-in mice. The Fas expression further differentiates the GL7+ B cells into immature transitional B cells and mature GC B cells (22). The GFP– fraction expresses Fas to a greater extent than the GFP+ fraction (data not shown). From sorted cells, DSB levels were examined using the LM-PCR method (21). Fig. 1A shows that GL7+GFP+ cells had the lower level of DSBs but GL7+GFP– cells had the higher level, which confirmed that DSBs in the IgV genes occurred independently of RAG expression in GC B cells. The DSBs appeared preferentially in IgV, as shown by measuring the Cµ in comparison to the loading controls.



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  		FIGURE 1. Detection of IgV DSBs in GC B cells. B220+GL7+ spleen cells of rag1-gfp knock-in mice at day 16 after immunization with NP-CG were fractionated by GFP. A, DSBs at VH-186.2 and Cµ by LM-PCR are shown in GFP– and GFP+ cells. Splenic B220+GL7+ cells, obtained from C57BL/6J mice at day 10 after immunization with NP-CG, were purified by Fas expression. B, DSBs at VH-186.2 and Cµ were measured in Fas+ and Fas– subpopulations. C, C57BL/6J mice were immunized i.p. with NP-CG or adjuvant alone. B220+GL7+ spleen cells were obtained at day 9.

 
We next characterized the B cell populations manifesting these IgV DSBs by separating the B220+GL7+ cells into Fas+ and Fas– cells (Fig. 1B). IgV DSBs were found predominantly in the GL7+Fas+ cells, indicating that IgV DSBs occurred exclusively in mature GC B cells. We compared the DSBs in GC-like B cells in NP-CG/alum-immunized and alum-only mice. Ag immunization induced an increase of DSBs in GL7+ GC B cells, implying that DSBs at the VH-186.2 occurred in B cells stimulated with NP-CG (Fig. 1C). Adjuvant alone induced few DSBs in an Ag-independent manner, but the results suggested that far more IgV DSBs were induced in Ag-driven B cells.

IgV DSBs in B cells of B-GANP–/– mice

We examined their generation in B-GANP–/– mice, which fail to produce high-affinity Abs against TD-Ag. The mutant mice were immunized with NP-CG and the generation of IgV DSBs was examined in comparison to control B-GANP+/– mice. GC B cells were sorted into a B220+GL7+Fas+ subpopulation at day 9. In comparison to the controls, B-GANP–/– mice did not show any alteration of VH-186.2 DSBs in the GC B cell (B220+GL7+) population (data not shown), but showed a significant decrease in the mature GC B cell subpopulation (B220+GL7+Fas+) (Fig. 2A). We attempted to prove the difference more carefully by measuring the relative intensities of DSB bands based on the control Cµ signals. DSBs at IgV in GANP–/– B cells occurred at a higher level than in control B cells (Fig. 2A, right panel). This was also observed after a secondary challenge (data not shown). These results suggested that GANP is involved in generation or regulation of Ag-induced DNA breakage and/or in the postcleavage processes.



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  		FIGURE 2. IgV DSBs in mature GC B cells of GANP mutant mice. A, B-GANP–/– and heterozygous control B-GANP+/– mice were immunized with NP-CG. IgV DSBs in mature GC B cells were measured at day 9. The relative values with that of Cµ were measured by the intensities of bands corresponding to DSBs and are shown as the percentage compared with those of B-GANP+/– mice. B, Detection of apoptotic cells by staining with 7AAD. C and D, Rad51, Ku80, and AID, relative to the control mRNA, in B220+GL7+Fas+ and B220+GL7+Fas– subpopulations were assessed by the TaqMan system in triplicate and are shown as the mean ± SEM with p values by Student’s t test: *, p < 0.01; **, p < 0.05. E, The frequencies of DSBs in wild-type (WT) and GANPTg mice after immunization of NP-CG were evaluated as in A: *, p < 0.05.

 
Apoptotic cells increased in the spleen of B-GANP–/– mice (18). The reduction of DSBs found in mature GC B cells in these mice might have been caused by the elimination of apoptotic cells undergoing IgV DSBs. We asked whether the apoptotic cells were eliminated from the GL7+Fas+ GC B cell subpopulation in B-GANP–/– mice (Fig. 2B). The mature GC B cell subpopulation contained 7AAD+ cells (Fig. 2Bc; 2.68%) in B-GANP–/– mice in comparison to no dead cells (Fig. 2Ba) in the control mice. The GL7+Fas– GC B cells also showed the increase of 7AAD+ cells (Fig. 2Bd; 6.83%) in B-GANP–/– mice. These results suggested that the decrease of IgV DSBs in mature GC B cells of B-GANP–/– mice did not simply represent the elimination of apoptotic cells undergoing IgV DSBs. The increase of apoptotic DNA-damaged cells did not have an effect on the level of IgV DSBs in the GL7+Fas– GC B cells (data not shown).

Rad51, Ku80, and AID transcripts in mature GC B cells

We compared the transcripts of the DNA repair molecules as the markers to confirm whether IgV DSBs indeed decreased in GANP–/– GC B cells. Rad51 and Ku80 were measured by real-time RT-PCR with purified, mature GL7+Fas+ GC B cells. Rad51, which is crucial in DNA homologous recombination (HR), increased 3-fold in GANP–/– mature GC B cells in comparison to the controls (Fig. 2C) (23). This was also the case in Ku80, which is pivotal for nonhomologous end-joining repair (NHEJ) (24). This observation supported the observation that Ag-driven GC B cells bear the lower frequency of IgV DSBs in B-GANP–/– mice after TD-Ag immunization. Not only the DNA editing but also the modification of DSB ends might be affected by AID in GC B cells (5). We examined whether AID transcription occurred in the same GC B cell population as that in which IgV DSBs appeared. AID transcription was found to be restricted to GL7+Fas+ GC B cells (Fig. 2D), indicating that AID is selectively expressed in the same mature GC B cells. B-GANP–/– mice showed the higher level of AID transcripts in mature GC B cells. These results indicated that the level of IgV DSB might be dependent on GANP expression rather than on AID expression in GC B cells. We demonstrated that a loss of GANP caused the reduction of blunt-end IgV DSBs generated selectively in Ag-driven GL7+Fas+ GC B cells (Fig. 2A).

IgV DSBs in GANPTg B cells

In contrast, B cells of GANPTg mice showed the increased frequency of DSBs in B220+GL7+Fas+ GC B cells after immunization with NP-CG (Fig. 2E). The GANPTg mice displayed the increase of high-affinity Abs after immunization with TD-Ag (20). The overexpression of GANP induced the increase of IgV DSBs, further supporting the notion that GANP plays a significant role in the level of IgV DSBs.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
DSBs within IgV genes occurred selectively in Ag-driven GC B cells. The induction of IgV DSBs was RAG1 independent, Ag dependent, and IgV selective.

The generation of IgV DSBs has been proposed to be a requisite event for SHM (1, 2, 3), but the generation of DSBs does not necessarily seem to be associated with SHM because the absence of AID did not alter the frequency of DSBs (14, 15). DSBs might not be a major type of DNA damage directly associated with the facilitation of SHM. Nevertheless, Wu et al. (3) proposed an integrated model for SHM with the involvement of AID and IgV DSBs (3). According to their model, IgV SHM would begin with the introduction of DSBs that would then be subject to repair by the NHEJ or HR mechanism. B cells undergoing SHM at the IgV gene would be further affected by AID or the AID-associated attack. Thereafter, error-prone DNA polymerases might introduce mismatches during the filling or extending of the DNA strand in the HR process.

Several possibilities are considered as the role of IgV DSBs in the Ab affinity maturation associated with GANP expression. Lack of GANP may cause the early cell elimination of the apoptotic Ag-driven B cells undergoing IgV DSBs. Alternatively, GANP might be involved in the generation of DNA injury or in the post-DSB processes in Ag-driven B cells. Loss of GANP might have caused the increase of the molecules required for the repair of both NHEJ and HR, which reduced the remaining IgV DSBs with blunt ends. GANP has two functional domains of RNA primase and MCM3-binding/acetylating activities (16, 17), which may be involved in transcriptional regulation by the interaction with the transcriptional regulatory molecules. It remains to be studied how GANP plays a molecular function in regulation of GC B cells harboring IgV DSBs.

IgV DSBs decreased in GANP–/– B cells but, conversely, increased in GANPTg B cells, supporting the observation that IgV DSBs have a significant role in the affinity maturation of B cell receptors associated with SHM.


   Acknowledgments
 
We acknowledge the technical assistance of H. Niizato and Y Kawasho.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by Special Coordination Funds for Promoting Science and Technology from the Ministry of Education, Culture, Sports, Science, and Technology of Japan, by grants from Pharmaceuticals and Medical Devices Agency, and by grants from Core Research for Evolutional Science and Technology of the Japan Science and Technology Agency. Back

2 Address correspondence and reprint requests to Dr. Nobuo Sakaguchi, Department of Immunology, Graduate School of Medical Sciences, Kumamoto University, 1-1-1, Honjo, Kumamoto, 860-8556, Japan. E-mail address: nobusaka{at}kaiju.medic.kumamoto-u.ac.jp Back

3 Abbreviations used in this paper: DSB, dsDNA break; SHM, somatic hypermutation; NP-CG, nitrophenyl-chicken {gamma}-globulin; TD, T cell-dependent; GC, germinal center; 7AAD, 7-aminoactinomycin D; LM, ligation mediated; HR, homologous recombination; NHEJ, nonhomologous end-joining repair. Back

Received for publication March 30, 2005. Accepted for publication August 23, 2005.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Papavasiliou, F. N., D. G. Schatz. 2000. Cell-cycle-regulated DNA double-stranded breaks in somatic hypermutation of immunoglobulin genes. Nature 408:216.-221. [Medline]
  2. Bross, L., Y. Fukita, F. McBlane, C. Demolliere, K. Rajewsky, H. Jacobs. 2000. DNA double-strand breaks in immunoglobulin genes undergoing somatic hypermutation. Immunity 13:589.-597. [Medline]
  3. Wu, X., J. Feng, A. Komori, E. C. Kim, H. Zan, P. Casali. 2003. Immunoglobulin somatic hypermutation: double-strand DNA breaks, AID and error-prone DNA repair. J. Clin. Immunol. 23:235.-246. [Medline]
  4. Doi, T., K. Kinoshita, M. Ikegawa, M. Muramatsu, T. Honjo. 2003. De novo protein synthesis is required for the activation-induced cytidine deaminase function in class-switch recombination. Proc. Natl. Acad. Sci. USA 100:2634.-2638. [Abstract/Free Full Text]
  5. Martin, A., P. D. Bardwell, C. J. Woo, M. Fan, M. J. Shulman, M. D. Scharff. 2002. Activation-induced cytidine deaminase turns on somatic hypermutation in hybridomas. Nature 415:802.-806. [Medline]
  6. Okazaki, I. M., K. Kinoshita, M. Muramatsu, K. Yoshikawa, T. Honjo. 2002. The AID enzyme induces class switch recombination in fibroblasts. Nature 416:340.-345. [Medline]
  7. Yoshikawa, K., I. M. Okazaki, T. Eto, K. Kinoshita, M. Muramatsu, H. Nagaoka, T. Honjo. 2002. AID enzyme induces class switch recombination in fibroblasts. Science 296:2033.-2036. [Abstract/Free Full Text]
  8. Pham, P., R. Bransteitter, J. Petruska, M. F. Goodman. 2003. Processive AID-catalysed cytosine deamination on single-stranded DNA simulates somatic hypermutation. Nature 424:103. 107–107. [Medline]
  9. Bransteitter, R., P. Pham, M. D. Scharff, M. F. Goodman. 2003. Activation-induced cytidine deaminase deaminates deoxycytidine on single-stranded DNA but requires the action of RNase. Proc. Natl. Acad. Sci. USA 100:4102.-4107. [Abstract/Free Full Text]
  10. Chaudhuri, J., M. Tian, C. Khuong, K. Chua, E. Pinaud, F. W. Alt. 2003. Transcription-targeted DNA deamination by the AID antibody diversification enzyme. Nature 422:726.-730. [Medline]
  11. Dickerson, S. K., E. Market, E. Besmer, F. N. Papavasiliou. 2003. AID mediates hypermutation by deaminating single stranded DNA. J. Exp. Med. 197:1291.-1296. [Abstract/Free Full Text]
  12. Sohail, A., J. Klapacz, M. Samaranayake, A. Ullah, A. S. Bhagwat. 2003. Human activation-induced cytidine deaminase causes transcription-dependent, strand-biased C to U deaminations. Nucleic Acids Res. 31:2990.-2994. [Abstract/Free Full Text]
  13. Yu, K., F. T. Huang, M. R. Lieber. 2004. DNA substrate length and surrounding sequence affect the activation-induced deaminase activity at cytidine. J. Biol. Chem. 279:6496.-6500. [Abstract/Free Full Text]
  14. Papavasiliou, F. N., D. G. Schatz. 2002. The activation-induced deaminase functions in a postcleavage step of the somatic hypermutation process. J. Exp. Med. 195:1193.-1198. [Abstract/Free Full Text]
  15. Bross, L., M. Muramatsu, K. Kinoshita, T. Honjo, H. Jacobs. 2002. DNA double-strand breaks: prior to but not sufficient in targeting hypermutation. J. Exp. Med. 195:1187.-1192. [Abstract/Free Full Text]
  16. Kuwahara, K., M. Yoshida, E. Kondo, A. Sakata, Y. Watanabe, E. Abe, Y. Kouno, S. Tomiyasu, S. Fujimura, T. Tokuhisa, H. Kimura, T. Ezaki, N. Sakaguchi. 2000. A novel nuclear phosphoprotein, GANP, is up-regulated in centrocytes of the germinal center and associated with MCM3, a protein essential for DNA replication. Blood 95:2321.-2328. [Abstract/Free Full Text]
  17. Kuwahara, K., S. Tomiyasu, S. Fujimura, K. Nomura, Y. Xing, N. Nishiyama, M. Ogawa, S. Imajoh-Ohmi, S. Izuta, N. Sakaguchi. 2001. Germinal center-associated nuclear protein (GANP) has a phosphorylation-dependent DNA-primase activity that is up-regulated in germinal center regions. Proc. Natl. Acad. Sci. USA 98:10279.-10283. [Abstract/Free Full Text]
  18. Kuwahara, K., S. Fujimura, Y. Takahashi, N. Nakagata, T. Takemori, S. Aizawa, N. Sakaguchi. 2004. Germinal center-associated nuclear protein contributes to affinity maturation of B cell antigen receptor in T cell-dependent responses. Proc. Natl. Acad. Sci. USA 101:1010.-1015. [Abstract/Free Full Text]
  19. Kuwata, N., H. Igarashi, T. Ohmura, S. Aizawa, N. Sakaguchi. 1999. Cutting edge: absence of expression of RAG1 in peritoneal B-1 cells detected by knocking into RAG1 locus with green fluorescent protein gene. J. Immunol. 163:6355.-6359. [Abstract/Free Full Text]
  20. Sakaguchi, N., T. Kimura, S. Matsushita, S. Fujimura, J. Shibata, M. Araki, T. Sakamoto, C. Minoda, K. Kuwahara. 2005. Generation of high-affinity antibody against T cell-dependent antigen in the Ganp gene-transgenic mouse. J. Immunol. 174:4485.-4494. [Abstract/Free Full Text]
  21. Schlissel, M., A. Constantinescu, T. Morrow, M. Baxter, A. Peng. 1993. Double-strand signal sequence breaks in V(D)J recombination are blunt, 5'-phosphorylated, RAG-dependent, and cell cycle regulated. Genes Dev. 7:2520.-2532. [Abstract/Free Full Text]
  22. Nagaoka, H., G. Gonzalez-Aseguinolaza, M. Tsuji, M. C. Nussenzweig. 2000. Immunization and infection change the number of recombination activating gene (RAG)-expressing B cells in the periphery by altering immature lymphocyte production. J. Exp. Med. 191:2113.-2120. [Abstract/Free Full Text]
  23. Daboussi, F., A. Dumay, F. Delacote, B. S. Lopez. 2002. DNA double-strand break repair signalling: the case of RAD51 post-translational regulation. Cell. Signal. 14:969.-975. [Medline]
  24. Jeggo, P. A.. 1998. Identification of genes involved in repair of DNA double-strand breaks in mammalian cells. Radiat. Res. 150:S80.-S91. [Medline]



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