PLoS Genet

To generate highly specific and adapted immune responses, B cells diversify their antibody repertoire through mechanisms involving the generation of programmed DNA damage. Somatic hypermutation (SHM) and class switch recombination (CSR) are initiated by the recruitment of activation-induced cytidine deaminase (AID) to immunoglobulin loci and by the subsequent generation of DNA lesions, which are differentially processed to mutations during SHM or to double-stranded DNA break intermediates during CSR. The latter activate the DNA damage response and mobilize multiple DNA repair factors, including Parp1 and Parp2, to promote DNA repair and long-range recombination. We examined the contribution of Parp3 in CSR and SHM. We find that deficiency in Parp3 results in enhanced CSR, while SHM remains unaffected. Mechanistically, this is due to increased occupancy of AID at the donor (Smu) switch region. We also find evidence of increased levels of DNA damage at switch region junctions and a bias towards alternative end joining in the absence of Parp3. We propose that Parp3 plays a CSR-specific role by controlling AID levels at switch regions during CSR

Phenotypic characterization of Parp-1 and Parp-2 deficient mice and cells

Poly(ADP-ribosyl)ation is a post-translational modification of proteins mediated by Poly(ADP-ribose) polymerases (Parps), a family of 17 members. Among them, Poly(ADP-ribose) polymerase-1 (Parp-1) and Parp-2 are so far the sole enzymes whose catalytic activity has been shown to be induced by DNA strand breaks. The generation and characterization of Parp-1 and Parp-2 deficient cellular and animal models have largely contributed to describe both proteins as active players of the base excision repair/single-strand break repair (BER/SSBR) process with both redundant and more specific functions. Double Parp-1(-/-)Parp-2(-/-) embryos die at gastrulation demonstrating the crucial role of poly(ADP-ribosyl)ation during embryonic development, whereas a specific female lethality related to X chromosome instability is associated with the Parp-1(+/-)Parp-2(-/-) genotype. Finally, recent research discovered emerging unique functions of Parp-2 in physiological processes including spermatogenesis, T-cell maturation, and adipogenesis although with distinct mechanisms. In this chapter, we describe standard operating procedures used to genotype and phenotype both mouse lines and the derived mouse embryonic fibroblasts

Parg is dispensable for recovery from transient replicative stress but required to prevent detrimental accumulation of poly(adp-ribose) upon prolonged replicative stress

Poly(ADP-ribosyl)ation is involved in numerous biological processes including DNA repair, transcription and cell death. Cellular levels of poly(ADP-ribose) (PAR) are regulated by PAR polymerases (PARPs) and the degrading enzyme PAR glycohydrolase (PARG), controlling the cell fate decision between life and death in response to DNA damage. Replication stress is a source of DNA damage, leading to transient stalling of replication forks or to their collapse followed by the generation of double-strand breaks (DSB). The involvement of PARP-1 in replicative stress response has been described, whereas the consequences of a deregulated PAR catabolism are not yet well established. Here, we show that PARG-deprived cells showed an enhanced sensitivity to the replication inhibitor hydroxyurea. PARG is dispensable to recover from transient replicative stress but is necessary to avoid massive PAR production upon prolonged replicative stress, conditions leading to fork collapse and DSB. Extensive PAR accumulation impairs replication protein A association with collapsed forks resulting in compromised DSB repair via homologous recombination. Our results highlight the critical role of PARG in tightly controlling PAR levels produced upon genotoxic stress to prevent the detrimental effects of PAR over-accumulation

PARG is dispensable for recovery from transient replicative stress but required to prevent detrimental accumulation of poly(ADP-ribose) upon prolonged replicative stress

Poly(ADP-ribosyl)ation is involved in numerous bio-logical processes including DNA repair, transcription and cell death. Cellular levels of poly(ADP-ribose) (PAR) are regulated by PAR polymerases (PARPs) and the degrading enzyme PAR glycohydrolase (PARG), controlling the cell fate decision between life and death in response to DNA damage. Replication stress is a source of DNA damage, leading to transient stalling of replication forks or to their collapse followed by the generation of double-strand breaks (DSB). The involvement of PARP-1 in replicative stress response has been described, whereas the consequences of a deregulated PAR catabolism are not yet well established. Here, we show that PARG-deprived cells showed an enhanced sensitivity to the replication inhibitor hydroxyurea. PARG is dispensable to recover from transient replicative stress but is necessary to avoid massive PAR production upon prolonged replicative stress, conditions leading to fork collapse and DSB. Extensive PAR accumulation impairs replication protein A association with collapsed forks resulting in compromised DSB repair via homologous recombination. Our results highlight the critical role of PARG in tightly controlling PAR levels produced upon genotoxic stress to prevent the detrimental effects of PAR over-accumulation.Science Foundation IrelandCentre National de la Recherche ScientifiqueLigue contre le CancerElectricité de FranceAgence Nationale de la RechercheFrench governmen

PARG is dispensable for recovery from transient replicative stress but required to prevent detrimental accumulation of poly(ADP-ribose) upon prolonged replicative stress

Poly(ADP-ribosyl)ation is involved in numerous bio-logical processes including DNA repair, transcription and cell death. Cellular levels of poly(ADP-ribose) (PAR) are regulated by PAR polymerases (PARPs) and the degrading enzyme PAR glycohydrolase (PARG), controlling the cell fate decision between life and death in response to DNA damage. Replication stress is a source of DNA damage, leading to transient stalling of replication forks or to their collapse followed by the generation of double-strand breaks (DSB). The involvement of PARP-1 in replicative stress response has been described, whereas the consequences of a deregulated PAR catabolism are not yet well established. Here, we show that PARG-deprived cells showed an enhanced sensitivity to the replication inhibitor hydroxyurea. PARG is dispensable to recover from transient replicative stress but is necessary to avoid massive PAR production upon prolonged replicative stress, conditions leading to fork collapse and DSB. Extensive PAR accumulation impairs replication protein A association with collapsed forks resulting in compromised DSB repair via homologous recombination. Our results highlight the critical role of PARG in tightly controlling PAR levels produced upon genotoxic stress to prevent the detrimental effects of PAR over-accumulation

Switch region transcription, AID expression and AID sub-cellular localization are not affected by Parp3-deficiency.

<p><b>(A)</b> Real-time qRT-PCR analysis for germline transcripts (Ix-Cx) at donor and acceptor switch regions in wild-type and <i>Parp3</i>^<i>-/-</i> splenic B lymphocytes cultured for 72 h with LPS alone or with LPS + IL-4 or LPS + IFN-γ. Expression is normalized to <i>CD79b</i> and is presented relative to expression in wild-type B cells, set as 1. Mean and SD of triplicate samples are shown, ratio SD were calculated following the rules for error propagation while calculating a ratio. Statistical analysis was performed using two-tailed Student’s t test. Data are representative of four experiments with two mice per genotype. (B) ChIP-qPCR analysis for RNA polymerase II occupancy across the μ region on chromatin prepared from wild-type and <i>Parp3</i>^<i>-/-</i> B cells cultured <i>in vitro</i> with LPS + IL-4 for 60 h. RNA polymerase II-ChIP values (mean of triplicate samples ± SD) were normalized to the input control and are expressed as percent input. Data are representative of two independent experiments. <b>(C)</b> ChIP-qPCR analysis for Spt5 occupancy at the Sμ switch region (with Sμ -1 and Sμ -2 primer pairs) on chromatin prepared from wild-type and <i>Parp3</i>^<i>-/-</i> B cells cultured <i>in vitro</i> with LPS + IL-4 for 60 h. For each sample, Spt5-ChIP values (mean ± SD) were normalized to the input control and expressed as fold-change relative to the wild-type condition. Error bars are indicative of the variation between the different PCRs. Ratio SD were calculated following the rules for error propagation while calculating a ratio. <b>(D)</b> Real-time qRT-PCR analysis for AID mRNA level in wild-type and <i>Parp3</i>^<i>-/-</i> activated B lymphocytes. Expression is normalized to <i>CD79b</i> and is presented relative to expression in wild-type B cells, set as 1. Mean and SD of triplicate samples are shown, ratio SD were calculated following the rules for error propagation while calculating a ratio. Statistical analysis was performed using two-tailed Student’s t test. Data are representative of four experiments with two mice per genotype. <b>(E)</b> Western blot analysis for AID and β-actin from protein extracts of wild-type and <i>Parp3</i>^<i>-/-</i> splenic B lymphocytes stimulated with LPS + IL-4 for 72 h. Theoretical molecular masses are indicated in kilodaltons (kDa). Numbers below the panels reflect the intensity of bands representing AID relative to wild-type after normalization to β-actin. Extracts from three animals from each genotype from two independent experiments are shown. <b>(F)</b> Western blot analysis for AID, Gapdh and Nbs1 from nuclear (N) and cytoplasmic (C) protein fractions from wild-type and <i>Parp3</i>^<i>-/-</i> splenic B lymphocytes stimulated with LPS + IL-4 for 72 h. Numbers below the panel reflect the intensity of bands representing AID relative to wild-type after normalization to GAPDH for cytoplasmic fraction or Nbs1 for nuclear fraction. Note that the nuclear fraction corresponds to four cell equivalents of the cytoplasmic extracts and that AID recovery yields are not equivalent between cytoplasmic and nuclear fractions. Data are representative of two experiments.</p

AID binding to Sμ is enhanced <i>Parp3^-/-</i> B cells but enhanced DNA damage in the absence of Parp3 is restricted to the IgH locus.

<p><b>(A)</b> ChIP-qPCR analysis for AID occupancy at the Sμ switch region on chromatin prepared from wild-type, <i>Parp3</i>^<i>-/-</i> and <i>AID</i>^{<i>Cre/Cre</i>} B cells cultured <i>in vitro</i> with LPS + IL-4 for 60 h, assessed with two polyclonal anti AID antibodies (anti AID 1 and anti AID-2) with two primer pairs (Sμ-1 and Sμ-2). Normalized AID-ChIP qPCR data for each primer set and antibody from a representative experiment is shown. For each sample, AID-ChIP values (mean ± SD) were normalized to the input control and expressed as fold-change relative to the wild-type condition. Error bars are indicative of the variation between the different PCRs. Ratio SD were calculated following the rules for error propagation while calculating a ratio. Statistical significance versus wild-type was determined by a two-tailed Student’s t test. *, p ≤ 0.05; ***, p ≤ 0.001. Data are from four independent experiments. <b>(B)</b> ChIP-qPCR analysis for AID occupancy at the Sμ switch region on chromatin prepared from wild-type, <i>Parp3</i>^<i>-/-</i> and <i>AID</i>^{<i>Cre/Cre</i>} B cells cultured <i>in vitro</i> with LPS + IL-4 for 48h and 60 h, assessed with anti-AID 1 with two primer pairs (Sμ-1 and Sμ-2). Normalized AID-ChIP qPCR data for each primer set and antibody from a representative experiment are shown. For each sample, AID-ChIP values (mean ± SD) were normalized to the input control and expressed as fold-change relative to the wild-type condition. Error bars are indicative of the variation between the different PCRs. Ratio SD were calculated following the rules for error propagation while calculating a ratio. Statistical significance versus wild-type was determined by a two-tailed Student’s t test. *, p ≤ 0.05; ***, p ≤ 0.001. Data are from two independent experiments. <b>(C)</b> B cells obtained from wild-type and <i>Parp3</i>^-/- mice were cultured <i>in vitro</i> with LPS + IL-4 and transduced with a control retrovirus expressing GFP and a non-target shRNA (control shRNA) or with retroviruses expressing an shRNA targeting AID and expressing GFP, or expressing double-tagged AID (AID^Flag-HA) and GFP. Representative flow cytometry profiles showing IgG1 surface expression in stimulated and transduced wild-type and <i>Parp3</i>^-/- B cells. Plots are gated on GFP^hi cells. The percentage of switched cells is indicated in each plot. The data are representative of two independent experiments and two independent retroviral transductions are shown for <i>Parp3</i>^-/- B cells. <b>(D)</b> Frequency of IgH/<i>c-myc</i> translocation in stimulated <i>Parp3</i>^<i>-/-</i> and control B cells as determined by long-range PCR and Southern blot. Number of identified translocations (T) and individual assays done (n, with template DNA corresponding to 10⁵ cells) are as follow: wild-type, T = 7, n = 188; <i>Parp3</i>^<i>-/-</i>, T = 7, n = 188; <i>AID</i>^{<i>Cre/Cre</i>}, T = 0, n = 188. ND: Not detected. Statistical significance was determined by the two-tailed Fisher’s exact test. NS: Not significant. Data are from four independent experiments.</p

Parp3 is dispensable for SHM and affinity maturation.

<p><b>(A)</b> Flow cytometry analysis of wild-type and <i>Parp3</i>^<i>-/-</i> germinal center B cells in the lymph nodes of NP-CGG immunized animals at day 10. Plots are gated on B220⁺ cells. The percentage of germinal center B cells (B220⁺ Fas⁺ GL-7⁺) is indicated above each gate. <b>(B)</b> Mutation analysis was performed in J_H4 intron sequences amplified from germinal center B cells (B220⁺ Fas⁺ GL-7⁺) obtained from the lymph nodes of <i>Parp3</i>^-/- and wild-type mice 10 days post-immunization. Spatial distribution of mutations in the 550-bp sequence of the J_H4 intron comparing wild-type (top panel) and <i>Parp3</i>^<i>-/-</i> (bottom panel) sequences. The numbers of mutations at each nucleotide position are shown as a percentage of total mutations. <b>(C)</b> Pie charts show the proportion of J_H4 intron sequences carrying different number of mutations. Segment sizes are proportional to the frequency of sequences carrying the number of the mutations indicated in the periphery. Mutation frequency (F) per base pair is shown below and the number of sequences analyzed is indicated in the center. p value was determined using two-tailed Student’s t-test. Sequences were obtained from two independent immunization experiments. See also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005240#pgen.1005240.s004" target="_blank">S1</a> and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005240#pgen.1005240.s005" target="_blank">S2</a> Tables. <b>(D)</b> Affinity maturation analysis of NP-specific IgM and IgG in the serum from NP-CGG immunized wild-type and <i>Parp3</i>^<i>-/-</i> mice at the indicated time points. The relative binding affinity of NP-specific antibodies was calculated as the ratio of NP(4) binding titers to NP(23) binding titers.</p