Ubiquitylated PCNA plays a role in somatic hypermutation and class-switch recombination and is required for meiotic progression

Results

Rescue of Lethal Knockout Phenotype in Mice Expressing PCNA^K164R Transgene (tg^K164R).

To investigate the role of ubiquitylation of PCNA in the diversification of the Ig locus, we generated a transgenic mouse that expresses PCNA with a lysine-to-arginine mutation at residue 164 (K164R) in the absence of any endogenous wild-type (WT) PCNA (Pcna^−/−tg^K164R). We first generated a null mutation of the Pcna gene by deleting exons 2, 3, and 4 through homologous recombination of one allele in embryonic stem (ES) cells [supporting information (SI) Fig. S1 A and C]. These heterozygous ES cells were used to generate Pcna^+/− mice which, when intercrossed, resulted in an early embryonic cell lethal phenotype in Pcna^−/− progeny consistent with the critical importance of PCNA in orchestrating DNA replication (13).

Mice that were transgenic for PCNA with the K164R mutation were made by carrying out site-directed mutagenesis of exon 4 in a genomic fragment of the mouse Pcna gene that contained ≈3 kb of the 5′ promoter region, all of the exon and intron sequences, and 875 bp of the 3′ untranslated region (Fig. S1 B and C). This mutated transgene was introduced by pronuclear micro-injection into FVB embryos to generate Pcna^+/+tg^K164R transgenic mice. The Pcna^+/+tg^K164R mice were then bred to the heterozygous Pcna^+/− mice to obtain transgenic mice that were homozygous for the null mutation at the endogenous Pcna locus and expressed the mutant transgene (Pcna^−/−tg^K164R). The mutant PCNA (tg^K164R) rescued the embryonic lethal phenotype of the PCNA-deficient mice, demonstrating that PCNA that cannot be ubiquitylated at residue K164 is able to mediate DNA replication and repair and allows the apparently normal growth and development of the mouse.

Real-time PCR showed an approximately two- to eightfold increase of steady-state PCNA mRNA in Pcna^−/−tg^K164R splenic B cells compared to Pcna^+/+ cells (Fig. S2). Western blot analysis revealed higher amounts of unmodified PCNA protein in whole cell lysates from mutant B cells (Fig. 1A). Furthermore, modified PCNA at position K164, which has been described to be the only site to accommodate ubiquitylation (19), could not be detected in Pcna^−/−tg^K164R mice (Fig. 1B).

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Fig. 1.

PCNA protein expression in wild-type and Pcna^−/−tg^K164R mice. Western blot analysis of splenic B cell extracts using anti-PCNA and anti-β-actin antibodies. When compared to WT, a greater amount of unmodified PCNA protein is detected in Pcna^−/−tg^K164R mice (A). Post-translational modification of lysine 164 is not detected in mutant PCNA protein (B).

Meiotic Defect in Transgenic Pcna^−/−tg^K164R Mice.

The Pcna^−/−tg^K164R mice develop normally, but they are sterile. In males, testes size is reduced to <40% of wild-type testes (Fig. S3A). Histopathological analysis revealed that early meiotic progression in Pcna^−/−tg^K164R males appears normal as indicated by the presence of spermatogonia and early spermatocytes (Fig. S3B). However, meiotic progression is disrupted at early pachynema resulting in the loss of spermatocytes at this stage. Meiotic chromosomes in Pcna^−/−tg^K164R mice can undergo complete synapsis and form a functional synaptonemal complex as indicated by the synaptonemal complex protein 1 (SYCP1) immunofluorescence, a marker for transversal filament formation (Fig. 2A and 2B) (20). Pcna^−/−tg^K164R mice also display the localization of Rad51 on meiotic univalent chromosomes at the initial stages of the synaptonemal complex formation during zygonema-to-pachynema transition (data not shown), suggesting that double-strand breaks are being processed and meiotic recombination is initiated. In addition, Mlh1, a key component of meiotic nodules, localized normally in Pcna^−/−tg^K164R mice during mid pachynema, suggesting crossover formation (Fig. 2 C and D). However, most meiotic chromosomes in mutant mice displayed elongated chromosome axes, suggesting either that meiosis is arrested at an early stage in pachynema in most nuclei or that structural defects in the synaptonemal complex occur (Fig. 2 B and D).

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Fig. 2.

Localization of SYCP1 and MLH1 on meiotic chromosomes during zygonema/pachynema of prophase I in wild-type and Pcna^−/−tg^K164R mice. Left panel (A and C) wild type, right panel (B and D) Pcna^−/−tg^K164R. (A and B) Colocalization of SYCP1 (red) and SYCP3 (green) indicates complete synapsis (yellow) of meiotic chromosomes in Pcna^−/−tg^K164R mice during pachynema. Note the elongated chromosome axis of the mutant chromosomes (B) compared to wild-type chromosomes (A). (C and D) Formation of late recombination nodules during pachynema in wild-type (C) and mutant mice (D) is indicated by MLH1 foci (red). Centromers are detected with anti-CREST antibodies (blue).

Impaired ex Vivo Class-Switch Recombination in Transgenic Pcna^−/−tg^K164R Mice.

To examine whether the K164R mutation of PCNA has an effect on CSR, we purified splenic B cells from Pcna^−/−tg^K164R mice and their Pcna^+/+ littermates and stimulated them ex vivo with LPS to induce switching from IgM to IgG3, and with LPS plus IL-4 to induce switching to IgG1 (21). The stimulated B cells from individual mice were cultured for 4 days and analyzed by flow cytometry for surface expression of IgG3 and IgG1. Six different experiments were done with two groups of mice: one that consisted of 3-month-old mice that had undergone NP-immunization for the SHM experiments described below, and a second group of unimmunized 8-month-old mice. The groups from the two sets of mice showed similar efficiencies of switching to IgG1 or to IgG3 within each genotype (Fig. S4), so we combined the results from NP-immunized and unimmunized mice (Fig. 3). A representative FACS profile from a PCNA mutant and a wild-type mouse is presented in Fig. S5. The combined results (Fig. 3) show that in Pcna^−/−tg^K164R mice there is a ≈50% reduction in switching to IgG3 and a ≈25% reduction in switching to IgG1 when compared to their Pcna^+/+ littermate controls (P < 0.0001).

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Fig. 3.

Reduced ex vivo class-switch recombination in PCNA^−/−tg^K164R mice. Relative switching to IgG3 and to IgG1 in a total of eight wild-type and eight transgenic mice from six different experiments. The efficiency of switching in the wild-type group within each experiment was defined as 100% and two replicates were assayed for each stimulation. The data shown represent relative mean efficiency of switching ± SEM. P-values were calculated using two-tailed unpaired Student's t tests.

To test if the differences in isotype switching could be the result of a defect in the replication and proliferation of the stimulated B cells expressing the mutated PCNA, the doubling time of the stimulated cells was analyzed by staining with 5,6-carboxyfluorescein diacetate succinimidyl ester (CFSE) and IgG1 expression was measured at each division (Fig. S6). Throughout the 4 days of stimulation with LPS plus IL-4, the percentage of Pcna^−/−tg^K164R B cells in each generation was not significantly different from Pcna^+/+ controls (Fig. S6A). Consistent with the lack of a defect in replication, the body weight, size of the spleen, and the number of Peyer's patches in Pcna^−/−tg^K164R mice were comparable to those in Pcna^+/+ mice (data not shown). Moreover, the decrease in switching to IgG1 accumulated after the second division, indicating that there was a decrease in switching at each generation (Fig. S6B). In addition, the frequency of B220⁺PNA^high GC B cells in the spleen and Peyer's patches was not significantly different between Pcna^+/+ and Pcna^−/−tg^K164R littermates (Fig. S7), suggesting that B cell activation and differentiation are normal in the transgenic Pcna^−/−tg^K164R mice. Together, these findings confirmed that the inability to ubiquitylate PCNA at residue 164 was not merely delaying the process of CSR, but rather was having a more direct effect on the switching process.

Altered Junctions and Fewer Mutations at the Sμ–Sγ3 Junctions in Pcna^−/−tg^K164R Mice.

We next asked whether the switching deficiency observed in Pcna^−/−tg^K164R mice was accompanied by any alteration in the switch junctions. The Sμ–Sγ3 junctional DNA segments from splenic B cells stimulated for 4 days with LPS were compared to Sμ and Sγ3 germline sequences (32 unique junctions from Pcna^+/+ and 25 from Pcna^−/−tg^K164R mice are shown in Fig. S8). There were relatively more blunt Sμ–Sγ3 junctions (P = 0.0285) and fewer microhomologies and insertions in Pcna^−/−tg^K164R mice when compared to Pcna^+/+ littermates (Fig. 4A). However, the remaining Sμ–Sγ3 junctions from Pcna^−/−tg^K164R mice showed an average length of microhomology (2.4 ± 0.44) similar to the Pcna^+/+ control mice (2.1 ± 0.36) (P = 0.6639). There was no significant difference between Pcna^+/+ and mutant mice in the utilization of GAGCT or GGGT switching consensus sequences or of WRC/GYW AID hotspots at the breakpoints (Fig. S9A). Furthermore, there was no difference in the parts of the donor μ or recipient γ3 switch regions that participated in the recombination to IgG3 (Fig. 4B).

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Fig. 4.

Bias toward blunt Sμ–Sγ3 switch junctions and normal distribution of breakpoints in PCNA^−/−tg^K164R mice. (A) A significant increase in the relative number of blunt junctions in PCNA^−/−tg^K164R mice. The graph depicts the relative frequency of junctions with blunt joins, small (1–9 bp) microhomologies (mh) or insertions (ins) at the junction. (B) Scatter analysis of the Sμ–Sγ3 breakpoints from in vitro LPS-stimulated splenic B cells. The x-axis indicates the position of the Sγ3 breakpoint and the y-axis of the Sμ breakpoint. Open circles denote breakpoints from PCNA^−/−tg^K164R mice, filled circles from their littermate controls.

There was also a significant decrease in the overall frequency of mutation at the donor Sμ region (1 × 10⁻³ mutations/base in the mutant mice vs. 6.8 × 10⁻³ in the WT; P < 0.001) and also at the acceptor Sγ3 region (0.8 × 10⁻³ mutations/base in the mutant mice vs. 2.7 × 10⁻³ in the WT mice; P = 0.0395) (Table 1). Furthermore, the few mutations detected at the Sμ and Sγ3 regions in the Pcna^−/−tg^K164R mice were almost exclusively at C:G pairs (Fig. S9 B and C).

Altered Pattern of V Region Somatic Hypermutation in Transgenic Pcna^{−/−tgK164R} Mice.

To investigate whether ubiquitylation of PCNA at residue K164 is involved in somatic hypermutation of the V regions of the Ig genes, four Pcna^−/−tg^K164R and three Pcna^+/+ mice were immunized with NP₃₀-CGG and boosted 4 weeks later. One week after boost, splenic B cells were analyzed for the pattern of somatic mutations in the rearranged V186.2 gene, which is a member of the J558 gene family that dominates the response to NP-immunization (22). cDNA was generated from the spleens of the immunized mice using a high-fidelity reverse transcriptase and the rearranged V186.2 genes, including the 5′ region of the Cγ1, were amplified using nested PCR, subcloned and sequenced. The vast majority of the sequences could be assigned to germline V186.2 gene, and the 15 sequences (of a total of 173) that showed higher homology to other members of the family (i.e., V24.8, V23, and V3 genes) were excluded from the mutation analysis.

Unique mutations in the nontranscribed strand of the V186.2 region (273 bp) were compiled from a total of 77 and 81 sequences from WT and Pcna^−/−tg^K164R mice, respectively. The experimental error rate because of reverse transcription and PCR was estimated by analysis of the first 39 bp from the Cγ1 segment adjacent to the V186.2 region to be 0.1 × 10⁻² mutations/base (9 mutations in 6,162 bp). The overall frequency of unique mutations at the V186.2 gene in the Pcna^−/−tg^K164R mice was 9.1 × 10⁻² mutations/base, which was significantly higher than the 7.2 × 10⁻² mutations/base detected in the Pcna^+/+ mice (P = 0.0152) (Table 2, Fig. 5 and Table S1). The overall frequencies of transitions and transversions, however, were similar in both mutant and WT mice (Table 2 and Table S1).

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Fig. 5.

Detailed mutations in the V186.2 region from WT and PCNA^−/−tg^K164R mice. Left panel, absolute number of unique mutations classified by base pair from (y-axis) → to (x-axis); middle panel, percentages of the total number of unique mutations; right panel, mutation frequency corrected for base composition (number of unique mutations/theoretical maximum number of unique mutations). For each category a contingency table was assigned and a χ² test was applied (see Table S1). Black boxes denote statistically significant increase of mutation frequency compared to WT; gray boxes denote significant decrease.

In agreement with earlier studies of V regions (1), the Pcna^+/+ group showed a similar frequency of unique mutations at C:G and A:T residues (Table 2). However, there were significantly more mutations at C:G than at A:T residues in the Pcna^−/−tg^K164R group. The reason for this imbalance was that the frequency of unique mutations at A:T was reduced by approximately twofold in the Pcna^−/−tg^K164R mice (2.9 × 10⁻² mutations/base in the mutant mice vs. 6.0 × 10⁻² in the WT, P < 0.0001) (Table 2 and Table S1), while the frequency at C:G was increased by approximately twofold in the Pcna^−/−tg^K164R mice (14.5 × 10⁻² mutations/base in the mutant mice vs. 8.3 × 10⁻² in the WT, P < 0.0001). This suggests that ubiquitylation of PCNA affects the regulation of the balance between the mechanisms that target A:T and C:G in the V region.

The WA motif, and its complementary TW (W = A or T), has been shown to be a target for Polη on the Ig locus (23, 24), with a preference for A-to-G substitutions on WA motifs of the nontranscribed strand (25). For the WA motif, there was a significant decrease of mutations in the Pcna^−/−tg^K164R mice compared to Pcna^+/+ (P = 0.0343) (Table 2), but no difference was detected for the frequency of mutation at the TW motif (P = 0.2527) that reflects WA targeting on the transcribed strand. Additional evidence for an impairment of Polη activity in the Pcna^−/−tg^K164R mouse became apparent when the different types of substitutions were examined (Fig. 5 and Table S1). A significant decrease in A-to-G and T-to-A mutations (P < 0.05), which are largely generated by Polη (7), was observed in the Pcna^−/−tg^K164R mice.

On the other hand, the increase in the frequency of C:G mutations in the Pcna^−/−tg^K164R mice was attributable to a significant increase in G-to-A and G-to-T substitutions (P ≤ 0.0001), whereas no difference was detected in the overall mutation frequency at C (P = 0.0940) (Fig. 5 and Table S1). This bias toward mutation of G residues was also observed when we examined the frequency of mutation occurring in WRC motifs (Table 2), and in the complementary GYW (R = A or G, Y = C or T) motifs, which have been described as hotspots for AID targeting (26, 27). A significant increase in mutability of the V186.2 nontranscribed strand at GYW motifs (P = 0.0002), which reflects the targeting of WRC on the transcribed strand, was not accompanied by the same increase in WRC mutations (P = 0.1641). A role for AID in the strand-biased targeting of C in hotspots in the transcribed strand was further supported by the absence of difference in the frequency of mutation of SYC/GRS AID coldspots (S = G or T) (P ≥ 0.2262) (Table 2). In summary, the K164R mutation of PCNA reveals a strand bias in C:G mutations and an excess of targeting of GYW motifs on the nontranscribed strand of the V186.2 region. This may be interpreted to mean that Cs within the context of WRC motifs on the transcribed strand are targeted more frequently for mutation when PCNA cannot be ubiquitylated.