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

Abstract

Somatic hypermutation (SHM) and class-switch recombination (CSR) of Ig genes are dependent upon activation-induced cytidine deaminase (AID)-induced mutations. The scaffolding properties of proliferating cell nuclear antigen (PCNA) and ubiquitylation of its residue K164 have been suggested to play an important role organizing the error-prone repair events that contribute to the AID-induced diversification of the Ig locus. We generated knockout mice for PCNA (Pcna(-/-)), which were embryonic lethal. Expression of PCNA with the K164R mutation rescued the lethal phenotype, but the mice (Pcna(-/-)tg(K164R)) displayed a meiotic defect in early pachynema and were sterile. B cells proliferated normally in Pcna(-/-)tg(K164R) mice, but a PCNA-K164R mutation resulted in impaired ex vivo CSR to IgG1 and IgG3, which was associated with reduced mutation frequency at the switch regions and a bias toward blunt junctions. Analysis of the heavy chain V186.2 region after NP-immunization showed in Pcna(-/-)tg(K164R) mice a significant reduction in the mutation frequency of A:T residues in WA motifs preferred by polymerase-eta (Poleta), and a strand-biased increase in the mutation frequency of G residues, preferentially in the context of AID-targeted GYW motifs. The phenotype of Pcna(-/-)tg(K164R) mice supports the idea that ubiquitylation of PCNA participates directly in the meiotic process and the diversification of the Ig locus through class-switch recombination (CSR) and somatic hypermutation (SHM).

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).

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).

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.

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.

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.