The DNA Glycosylases Ogg1 and Nth1 Do Not Contribute to Ig Class Switching in Activated Mouse Splenic B Cells

During activation of B cells to undergo class switching, B cell metabolism is increased, and levels of reactive oxygen species (ROS) are increased. ROS can oxidize DNA bases resulting in substrates for the DNA glycosylases Ogg1 and Nth1. Ogg1 and Nth1 excise oxidized bases, and nick the resulting abasic sites, forming single-strand DNA breaks (SSBs) as intermediates during the repair process. In this study, we asked whether splenic B cells from mice deficient in these two enzymes would show altered class switching and decreased DNA breaks in comparison with wild-type mice. As the c-myc gene frequently recombines with the IgH S region in B cells induced to undergo class switching, we also analyzed the effect of deletion of these two glycosylases on DSBs in the c-myc gene. We did not detect a reduction in S region or c-myc DSBs or in class switching in splenic B cells from Ogg1- or Nth1-deficient mice or from mice deficient in both enzymes.


Introduction
Immunoglobulin (Ig) class switching occurs in activated mature B cells after immunization or infection in vivo, and causes an exchange of the IgM isotype for IgG, IgE or IgA isotypes, resulting in increased effectiveness of the humoral immune response. Class switching occurs by a DNA recombination event, termed class switch recombination (CSR), which can be induced in cultured mouse splenic B cells by treatment with bacterial lipopolysaccharide (LPS). CSR occurs via a mechanism that involves induction of double-strand DNA breaks (DSBs) in special switch (S) region sequences located upstream of each isotype gene, followed by nonhomologous end joining between donor and acceptor S regions. Activation-induced cytidine deaminase (AID) initiates DSB formation in S regions by deamination of cytosines, converting them to uracils. Then DSBs are created by the action of both the base excision repair pathway and by mismatch repair [1]. Although AID primarily targets Ig genes, it also deaminates other sites in the genome, and this can lead to mutations, DNA breaks, and to translocations [2,3,4,5,6]. In fact, translocations between the c-myc gene and the IgH locus are a relatively frequent event in mouse B cells induced with LPS, and also occur in vivo and lead to lymphomagenesis.
During B cell activation in culture by LPS, large amounts of reactive oxygen species (ROS) are generated [7,8,9]. High levels of ROS can result in oxidized DNA bases, which like AID-induced dUs, are also repaired by base excision repair (BER), a pathway that results in single-strand DNA breaks (SSBs) as intermediates. Ogg1 and Nth1 are two BER glycosylases that perform both the initial excision of the oxidized base and the incision of the DNA backbone during the process of removing oxidized guanine (8-oxo-G) and oxidized pyrimidines (cytosine glycol and thymine glycol), respectively [10,11]. We hypothesized that if the increased ROS produced during LPS-activation of B cells were to result in oxidized dG and pyrimidines, then Ogg1 and Nth1 might contribute to formation of DNA breaks in S regions or in the cmyc locus in these cells. To test this hypothesis, we determined whether deficiencies in Ogg1 and/or Nth1 would lead to decreased DNA DSBs in Sm or c-myc. We also examined the efficiency of CSR, because if these Sm DSBs were important for CSR, then CSR might be decreased in B cells deficient in these enzymes.

Results
Ogg1 and Nth1 are ubiquitously expressed, and Ogg1 mRNA has been shown to be expressed in both germinal center and nongerminal center splenic B cells [12]. We performed RT-PCR and confirmed that both Ogg1 and Nth1 mRNAs are expressed in splenic B cells induced to undergo CSR to IgG3, IgG2a, or IgA in culture (Fig. 1).
To examine whether Ogg1 or Nth1 affect IgH S region DSBs or c-myc DSBs in B cells induced to undergo CSR, we used ligationmediated PCR (LM-PCR) to assay DSBs in the IgH Sm region, the donor S region which is involved in initial CSR events, and also in the c-myc locus. The breaks in Sm are greatly reduced in aid 2/2 B cells, consistent with previous data indicating the dependence of Sm DSBs upon AID, uracil DNA glycosylase, AP endonuclease 1 and 2, and mismatch repair [13,14,15,16,17,18]. However, we did not observe any consistent change in DSB frequencies in the Sm region or c-myc locus in ogg1 2/2 , nth1 2/2 or double-knock out (DKO) cells relative to wild-type (WT) splenic B cells (Fig. 2). We conclude that these glycosylases do not make a substantial contribution to the induction of DSBs in the IgH Sm region or in the c-myc locus. Although c-myc mutations and c-myc-IgH translocations have been shown to be dependent upon AID [2], [4], we could not detect a consistent reduction in c-myc DSBs in aid 2/2 cells by LM-PCR. This could be explained by the fact that AID-induced mutations in c-myc occur at very low levels, except in cells deficient in the DNA repair proteins UNG and Msh2, indicating that most AID-induced lesions in the c-myc gene are correctly repaired in cells with intact repair systems [2].
As another approach to determine whether Nth1 and/or Ogg1 generate DNA breaks that contribute to CSR, we assayed CSR in ogg1 2/2 , nth1 2/2 or DKO splenic B cells activated to switch in culture. As CSR is dependent on cell proliferation, we first tested whether splenic B cells from mice deficient in Ogg1 or Nth1 or both (DKO) proliferated as well as wild-type (WT) littermate B cells when induced to undergo CSR in culture with LPS and other activators that induce isotype specific CSR (see Methods). As shown in Fig. 3A, proliferation as assayed by dilution of CFSE dye ,44 hrs after initiation of the B cell cultures, was equivalent between the WT and mutant B cells. At this same timepoint, we assayed CSR to several different isotypes by staining of surface Ig expression, but found no consistent differences in IgG1, IgG3 or IgG2a CSR, although there was a slight increase in IgA CSR in nth1 2/2 and DKO cells (Fig. 3B, C). Fig. 3C summarizes the CSR results in the mutant B cells as a percent of WT CSR.

Discussion
As ROS are induced in splenic B cells induced to undergo CSR in culture by LPS-treatment, we hypothesized that oxidized DNA bases would be generated during CSR, and the glycosylases Ogg1 and Nth1 would be important for their repair. Further, since repair of oxidized bases by these base excision repair enzymes involves the formation of single-strand break intermediates, we hypothesized that B cells deficient in these enzymes would show decreased DNA DSBs, and perhaps reduced CSR. However, deletion of the genes for Ogg1 and Nth1 has no consistent effect on the frequency of DSBs in the Ig Sm region or in the c-myc gene or on CSR efficiency in these cells. Our results are consistent with a previous report that Ogg1 does not contribute to somatic hypermutation of antibody variable regions, another process initiated by AID, but one that does not involve formation of DNA DSBs [12]. Furthermore, another protein involved in repair of oxidized dAs, alkyladenine DNA glycosylase, has also been shown to not be involved in CSR [19]. However, as glycosylases have partially redundant functions, for example [20], it is possible that if the mice were deficient in other combinations of glycosylases, a CSR phenotype might be observed.
Previously, we found that p53-deficient LPS-activated splenic B cells have increased levels of ROS relative to WT cells, increased CSR to IgG2a, and also increased mutations at recombination junctions between Sm and Sc3 in IgG3-expressing cells [9]. One possible interpretation of these data was that ROS-instigated DNA breaks might contribute to CSR and S region mutations and DSBs. As no CSR is observed in the absence of AID and the deamination reaction performed by AID is an oxidation reaction, we prefer the interpretation that ROS actually stimulated the deamination activity of AID itself, thereby increasing mutations and DNA breaks. However, due to the redundancy among enzymes that repair oxidized bases, our current results do not rule out the possibility that ROS contribute to DNA breaks in Sm and c-myc.

Mice
Both the ogg1 2/2 [10] and nth1 2/2 [11] mice were back-crossed for 8 generations to C57BL/6 mice prior to use in these experiments. Mice were housed in the Institutional Animal Care and Use Committee-approved specific pathogen-free facility at the University of Massachusetts Medical School. The mice were bred and used according to the guidelines from the University of Massachusetts Animal Care and Use Committee, Assurance number A-3306-01. Experiments are performed using spleens taken from mice sacrificed by CO2 asphyxiation followed by  bilateral pneumothorax. Nth1 mice were typed using the published primers [11]. Ogg1 mice were typed using the following primers: CH597, 59 primer for ogg1 wt GTACCGTGCCCGC-TATGTA; CH598, 39 primer for ogg1 wt CCTCTCGTACGCT-CAGTGT; CH593, 59 primer for ogg1 ko, CCTTTAG-GCGTCCTTTCAGTGT; CH218, 39 primer from 39 end of neo gene, GCAGCGCATCGCCTTCTAT (C. Hollander, NIH, unpublished).

Splenic B cell cultures and analysis of CSR
Splenic B cells were isolated and induced to undergo CSR as previously described [9], except we add LPS at 25 mg/ml, anti-IgD dextran at 25 ng/ml, and human BLyS at 50 ng/ml for all CSR cultures. Also, our treatment for inducing switching to IgA has been changed to retinoic acid (10 nM), LPS (25 mg/ml), TGFb (2 ng/ml), IL-5 (30 ng/ml), anti-IgD dextran (25 ng/ml), and human BLyS (50 ng/ml). Retinoic acid was shown to increase IgA CSR [21], and we have found that it specifically increases IgA CSR and no other isotype (unpublished).