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. 2019 Jul;26(7):1221-1234.
doi: 10.1038/s41418-018-0199-z. Epub 2018 Sep 20.

PP4 Deficiency Leads to DNA Replication Stress That Impairs Immunoglobulin Class Switch Efficiency

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Free PMC article

PP4 Deficiency Leads to DNA Replication Stress That Impairs Immunoglobulin Class Switch Efficiency

Ming-Yu Chen et al. Cell Death Differ. .
Free PMC article

Abstract

The serine/threonine phosphatase PP4 has been implicated in DNA damage repair and cell cycle regulation through its dephosphorylation of specific substrates. We previously showed that PP4 is required for mouse B cell development, germinal center (GC) formation and immunoglobulin (Ig) class switch recombination (CSR). Here, we investigate the mechanisms underlying this requirement and demonstrate that murine PP4-deficient B lymphocytes have a defect in cell proliferation. Strikingly, the DNA damage response pathway that involves ATM/p53 and is linked to cell cycle arrest and impaired cell survival is strongly induced in these mutant B cells. In response to LPS + IL-4, stimuli that trigger IgG1 production, these PP4-deficient B cells show inefficient phosphorylation of ATR, leading to reduced retention of γH2AX-NBS1 complexes at sites of DNA damage, and compromised switching to IgG1. However, beyond the cell proliferation phase, conditional deletion of PP4 under the control of AID/cre completely restores normal IgG1 production in mutant B cell cultures. In vivo, co-deletion of PP4 and p53 by AID/cre partially rescues switching to IgG1 in B cells of mice immunized with TNP-KLH. Our findings establish that PP4 is indispensable for preventing DNA replication stress that could interfere with CSR, thereby promoting antibody switching during the humoral immune response.

Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Fig. 1
Fig. 1
Reduced Ig class switch efficiency is associated with impaired cell division in PP4-deficient B cells. a FACS profiles of CFSE decay versus IgG1 expression by B220+IgM-IgD- gated B cells that were isolated from mice of the indicated genotypes (n = 3/group) and stimulated with LPS + IL-4 for 72 h in vitro. Numbers 0 to 6 indicate cell divisions. Data are representative of three independent experiments. b Quantitation of frequencies of IgG1+-switched B cells after the 2nd to 6th cell divisions among the B220+IgM-IgD- gated B cells in the experiment described in a. Data are the mean ± SD of triplicates. *p ≤ 0.05; **p ≤ 0.005. c Overlay of CFSE decay curve and proliferation curve as determined by FACS for the proliferating B cells in a. Data are representative of two independent trials. d Quantitation of the percentage of proliferating cells in the 1st to 6th cell divisions of the B cells in a. Data are the mean ± SD (n = 3/group). e Quantitation of the percentage of viable B cells stimulated with LPS + IL-4 for 72 h in a. Data are the mean ± SD (n = 3/group). f Left: Schematic illustration n of cell cycle phases in a FACS profile. Right: FACS profiles showing BrdU incorporation by purified B cells of the indicated genotypes that were stimulated with LPS + IL-4 for 72 h in vitro. Numbers in quadrants are frequencies of BrdU/7AAD-positive cells in each phase, as indicated. Data are representative of three independent trials. g Quantitation of the ratio of cells in G1 versus G0 phase (left) or in S versus G1 phase (right) in the experiment described in f. Data are the mean ± SD (n = 3/groups)
Fig. 2
Fig. 2
The ATM-p53 pathway is strongly induced in PP4-deficient B cells. a Immunoblots to detect phospho-p53 (p-p53) S15, total p53 and p84 (nuclear loading control) in the nuclear fraction of splenic B cells that were isolated from mice of the indicated genotypes (n = 3/group) and left unstimulated (0), or stimulated with LPS + IL-4 for 1 or 2 days. Data are representative of two independent trials. b Quantitation of the number of nuclear p-p53 S15-foci/cell in purified splenic B cells that were isolated from mice of the indicated genotypes and were left untreated (resting) or treated with 10 μM etoposide for 1 h in vitro. Data are values for individual mice (n = 3/group). Horizontal bars = mean values ± SD (cell number 39–126/group). *p ≤ 0.05. ***p ≤ 0.0005. c Representative confocal microscopy images of cells in the experiment described in b. Data are representative of two independent trials. d Immunoblots to detect phospho-ATM (p-ATM) S1987, total ATM, p-ATR S431, total ATR, and p84 in the nuclear fraction of B cells of the indicated genotypes that were left untreated (resting) or were stimulated with anti-CD40 or LPS + IL-4 for 4 days. Data are representative of two independent trials. e Curve overlay to show intracellular staining to detect p-ATM S1987 in B cells of the indicated genotypes that were left untreated (resting) or stimulated with LPS or LPS + IL-4 for the indicated times. Data are representative of two independent trials
Fig. 3
Fig. 3
Induction of p53 facilitates CSR in PP4-deficient mature B cells by promoting cell survival. a FACS profiles of IgG1 versus IgG3 expression by B220+IgM-IgD- gated B cells that were isolated from mice of the indicated genotypes (n = 3/group) and stimulated in vitro with LPS + IL-4 or LPS for 72 h. Data are representative of three independent trials. b Quantitation of the percentage of IgG1+- and IgG3+-switched B cells among total B cells stimulated by LPS + IL-4 (left) and LPS (right) from the data in a. Data are the mean ± SD (n = 3/group). c Curve overlay to show CFSE decay in B cells in (top) CD23/cre;PP4+/+ versus CD23/cre;PP4F/F, and (bottom) CD23/cre;p53F/F versus CD23/cre;PP4F/F/p53F/F cultures that were stimulated with LPS + IL-4 for 72 h. Numbers 0 to 6 indicate cell divisions. Data are representative of two independent trials. d Quantitation of percent viability as determined by 7AAD staining among GFP+ B cells that had been retrovirally transduced with pMSCV-PIG empty vector, or vectors encoding PP4 WT, PP4 R236L, p53 WT, p53 S15A, or p21, as indicated. Data are the mean ± SD (n = 4/group). e Quantitation of the percentage of IgG1-switched GFP+IgM-IgD- cells among the transduced B cells in d. Data are the mean ± SD (n = 4/group). f Quantitation of %VPD-positive cells at the 6th cell division among the transduced B cells in d. Data are the mean ± SD (n = 4/group)
Fig. 4
Fig. 4
Deletion of PP4 by CD23/cre reduces CSR efficiency. a Immunoblots to detect p-ATR S431, p-Chk1 S345, p-NBS1 S343, NBS1, γH2AX, H2AX, PP4, GAPDH and p84 in the cytosolic and nuclear fractions of B cells of the indicated genotypes that were left unstimulated (0), or stimulated with LPS + IL-4 for 24 h or 48 h. Data are representative of two independent trials. b Confocal microscopy images of immunofluorescent staining to detect RPA1 and quantitation of the intensity of RPA1 stainining in B cells of the indicated genotypes that were left untreated (resting) or stimulated with LPS + IL-4 for 24 h. c Confocal microscopy images of immunostaining to detect γH2AX (red) and quantitation of γH2AX-foci in B cells of the indicated genotypes that were left untreated (resting) or stimulated with LPS + IL-4 for 48 h. d Confocal microscopy images of immunostaining to detect NBS1 (green) and quantitation of NBS1-foci in the experiment described in c. e Confocal microscopy images of immunostaining to detect γH2AX overlaid with NBS1 and quantitation of colocalized γH2AX-NBS1 foci (index of correlation, Icorr) in the experiment described in c. Horizontal line = mean ± SD. Data from b–e are analyzed by MetaMorph. Values are for individual cells, and representative of two independent experiments. ns, not significant
Fig. 5
Fig. 5
Deletion of PP4 by AID/cre restores B cell proliferation and IgG1-switching in vitro. a Semi-quantitative RT-PCR analysis of pp4 mRNA expression in B cells of the indicated genotypes that were stimulated with LPS + IL-4 for 72 h in vitro. Extracts were diluted as indicated. hprt, loading control. Data are representative of two independent trials. b FACS profiles of CFSE decay versus IgG1 expression in B220+IgM-IgD- gated B cells of the indicated genotypes that were stimulated with LPS + IL-4 for 72 h in vitro. Numbers 1–6 indicate cell divisions. Data are representative of three independent experiments. c Quantitation of IgG1-switched B cells at the 2nd to 6th cell divisions among the B220+IgMIgD gated B cells in the experiment described in b. Data are the mean ± SD of triplicates. d CFSE overlay of proliferating B cells in the experiment described in b. Data are representative of three independent trials. e Quantitation of frequencies of proliferating B cells at the 1st to 6th cell divisions in the experiment described in b. Data are the mean ± SD (n = 4/group). f Quantitation of percent viability for the B cells in the experiment described in b. g FACS profiles of the kinetics of induction of IgG1 versus IgG3 expression by IgMIgD gated B cells of the indicated genotypes stimulated with LPS + IL-4 for 2 to 6 days in vitro, as indicated. Data are representative of two independent experiments. h FACS profiles of CD138 versus IgG1 expression by IgM-IgD- gated B cells that were isolated from mice of the indicated genotypes on day 14 post-immunization with TNP-KLH. Data are representative of two independent experiments. i Quantitation of the percentage of IgG1+ B cells in the experiment described in h. Data are the mean ± SD (n = 3–5/group)
Fig. 6
Fig. 6
p53 deficiency restores normal levels of germline transcript Cγ1. a RT-PCR analysis of B cells that were isolated from mice of the indicated genotypes (n = 4/group) and stimulated in vitro with LPS + IL-4 for 72 h. Extracts were diluted as indicated and analyzed to detect switched transcript Sμ-Sγ1, germline transcripts Cμ and Cγ1, and pp4, p53, aicda, and hprt mRNAs. Data are representative of two independent trials. b Quantitation of fold change in the levels of the transcripts in the experiment described in a
Fig. 7
Fig. 7
Schematic illustration of proposed roles for PP4 in B cell proliferation and Ig class switching. (Left) In WT activated B cells, LPS + IL-4 stimulation results in cell proliferation requiring DNA replication. In the presence of PP4, replication stress is prevented, S region transcripts are produced, CSR occurs with normal efficiency, and Ig class switching is normal. (Right) In activated PP4-deficient B cells, ATR activation is decreased and reduces the retention of γH2AX-NBS1 complexes on the DNA DSB sites needed for DNA replication and CSR. A sustained DNA damage response is triggered via the ATM-p53 axis that results in cell cycle arrest, promoting cell viability. On the other hand, p53 exerts a suppressive effect on the production of germline acceptor S region transcripts

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