Genomic instability, defective spermatogenesis, immunodeficiency, and cancer in a mouse model of the RIDDLE syndrome - PubMed (original) (raw)

. 2011 Apr;7(4):e1001381.

doi: 10.1371/journal.pgen.1001381. Epub 2011 Apr 28.

Miyuki Bohgaki, Renato Cardoso, Stephanie Panier, Dimphy Zeegers, Li Li, Grant S Stewart, Otto Sanchez, M Prakash Hande, Daniel Durocher, Anne Hakem, Razqallah Hakem

Affiliations

Genomic instability, defective spermatogenesis, immunodeficiency, and cancer in a mouse model of the RIDDLE syndrome

Toshiyuki Bohgaki et al. PLoS Genet. 2011 Apr.

Abstract

Eukaryotic cells have evolved to use complex pathways for DNA damage signaling and repair to maintain genomic integrity. RNF168 is a novel E3 ligase that functions downstream of ATM,γ-H2A.X, MDC1, and RNF8. It has been shown to ubiquitylate histone H2A and to facilitate the recruitment of other DNA damage response proteins, including 53BP1, to sites of DNA break. In addition, RNF168 mutations have been causally linked to the human RIDDLE syndrome. In this study, we report that Rnf168(-/-) mice are immunodeficient and exhibit increased radiosensitivity. Rnf168(-/-) males suffer from impaired spermatogenesis in an age-dependent manner. Interestingly, in contrast to H2a.x(-/-), Mdc1(-/-), and Rnf8(-/-) cells, transient recruitment of 53bp1 to DNA double-strand breaks was abolished in Rnf168(-/-) cells. Remarkably, similar to 53bp1 inactivation, but different from H2a.x deficiency, inactivation of Rnf168 impairs long-range V(D)J recombination in thymocytes and results in long insertions at the class-switch junctions of B-cells. Loss of Rnf168 increases genomic instability and synergizes with p53 inactivation in promoting tumorigenesis. Our data reveal the important physiological functions of Rnf168 and support its role in both γ-H2a.x-Mdc1-Rnf8-dependent and -independent signaling pathways of DNA double-strand breaks. These results highlight a central role for RNF168 in the hierarchical network of DNA break signaling that maintains genomic integrity and suppresses cancer development in mammals.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Figure 1

Figure 1. Impaired DNA damage response in Rnf168 deficient cells.

(A) Cells were subjected to IR or UV, and the extent of cell death was determined 24 hours later. Three independent experiments were performed. Data are presented as the mean ± SEM. *p<0.05. (B) Primary MEFs (passage 2) either untreated or irradiated (2 Gy) were stained with anti-phospho-histone H3 (pHH3) antibody and PI at 1 hour post IR. The percentage of mitotic cells was determined by FACS. Three independent experiments were performed. Data are presented as the mean ± SEM. *p<0.05. (C) Cell cycle progression post IR (10 Gy) treatment of WT and Rnf168−/− primary MEFs (Passage 2) was examined using BrdU/PI assay and FACS. Representative data are shown for three independent experiments. (D) Accumulation at the G2 phase of WT and Rnf168−/− primary MEFs (passage 2) post IR (10 Gy). Cell cycle profiles were examined by PI staining and representative data are shown from three independent experiments.

Figure 2

Figure 2. Rnf168 is required for the recruitment of 53bp1 to DNA damage sites.

(A) Representative micrographs of MEFs stained with anti-53bp1 antibody and DAPI. Rnf168−/− and WT MEFs were untreated or exposed to 5 Gy of IR and fixed at the indicated time post IR. Bars, 20 µm. (B) Representative micrographs of MEFs stained with anti-53bp1 antibody and DAPI are shown. Rnf168−/−, Rnf8−/− and WT MEFs were untreated or exposed to 5 Gy of IR and fixed at the indicated time post IR. Three independent experiments were performed. Bars, 20 µm. (C) Recovery of 53bp1 IRIF formation in Rnf168−/− MEFs complemented with full length RNF168. Immortalized Rnf168−/− MEFs were mock-transfected or transfected with GFP-tagged RNF168 expression vectors and cultured for 24 hours. Cells were fixed at 1 hour post-IR (5 Gy) and processed for immunofluorescence staining with anti-53bp1 antibodies. Three independent experiments were performed. Bars, 20 µm. (D) Clonogenic assay was performed to examine radiosensitivity of Rnf168−/− MEFs complemented with exogenous Rnf168 (left panel). Expression level of Rnf168 is shown for the MEFs used for the clonogenic assay (right panel). Data shown is representative of three independent experiments and is presented as the mean ± SEM. *p<0.05. (E) Representative micrographs of MEFs stained with anti-Brca1 antibody and DAPI. Rnf168−/− and WT MEFs were either untreated or exposed to 5 Gy of IR and fixed at the indicated time after IR. Bars, 2 µm.

Figure 3

Figure 3. Atm signaling pathway is not affected in the absence of Rnf168.

(A–C) IB analysis of untreated or irradiated WT and Rnf168−/− thymocytes (A and B) or MEFs (C). Cells were irradiated as indicated and examined 1 hour post-IR by immunoblotting using the indicated antibodies. Representative data are shown from three independent experiments. (D) Representative micrographs of MEFs stained with anti γ-H2a.x antibody and DAPI. Rnf168−/− and WT MEFs were untreated or exposed to 5 Gy of IR and fixed at the indicated time after IR. Bars, 20 µm. Three independent experiments were performed. (E) Representative micrographs of MEFs stained with anti-Mdc1 antibody and DAPI. Rnf168−/− and WT MEFs were untreated or exposed to 5 Gy of IR and fixed at the indicated times post IR. Three independent experiments were performed. Bars, 20 µm. (F) Representative micrographs of MEFs treated with DNA-PK inhibitors and stained with anti γ-H2a.x antibody and DAPI. Rnf168−/− and WT MEFs were treated with DNA-PK inhibitors (NU7026 or NU7441) and cultured for 1 hour. MEFs were left untreated or exposed to 5 Gy of IR and fixed 2 hours post-IR. Three independent experiments were performed. Bars, 20 µm.

Figure 4

Figure 4. Impaired long-term spermatogenesis in Rnf168−/− mice.

(A) Average litter sizes from intercrosses of 8-week-old mice with the indicated genotypes are shown [WT (F)×WT (M); n = 4, Het (F)×Het (M); n = 5, KO (F)×WT (M); n = 5, WT (F)×KO (M); n = 4, KO (F)×KO (M); n = 3. F = female, M = male, Het = Heterozygote (Rnf168+/−), KO = knockout (Rnf168−/−)]. (B) Twelve-month-old WT or Rnf168−/− males were mated with 8-week-old females. Number of pups from breeding 4 pairs of each genotype is shown. (C) Comparison of testes size in 8-week-old and 12-month-old WT or Rnf168−/− males. Bar, 3 mm. (D) Sections of testes stained with hematoxylin-eosin (left panels). Arrowheads indicate Leydig cells. Bar, 100 µm. The numbers of empty tubules per 100 seminiferous tubules are graphed (right panel). Data are presented as the mean ± SEM (WT n = 3; Rnf168−/− n = 3). *p<0.05. (E) Sections of epididymis stained with hematoxylin-eosin. Bar, 100 µm. Three mice of each genotype were analyzed.

Figure 5

Figure 5. Impaired class switch recombination in Rnf168−/− mice.

(A) Serum immunoglobulin levels in 6–8-week-old and 9–12-month-old Rnf168−/− mice. The data are presented as the mean ± SEM (n = 3–5). (B and C) FACS analysis of IgG1 (B) or IgG3 (C) and IgM expression on B220+ B-cells (left panels) and average percentages of IgG1+ or IgG3+ B-cells (right panels) from Peyer's patches. Three independent experiments were performed. (D) Secreted immunoglobulins were analyzed in the supernatants of B-cell cultures after 4 days of in vitro stimulation with LPS with or without IL-4. The data are presented as the mean ± SEM from three independent experiments. (E) FACS analysis of IgG1 expression on CFSE labeled B-cells stimulated with anti-CD40 plus IL-4 for 4 days (left panels) and average percentages of IgG1 switched cells (right panel). Five independent experiments were performed. (F) CFSE staining profiles of B-cells stimulated with anti-CD40 antibody plus IL-4 for 4 days (upper panel) and percentages of IgG1+ cells that had undergone the indicated number of cell divisions (lower panel). Representative data are shown from three independent experiments. (G) Schematic representations of WT and mutants Rnf168 cloned into the MSCV-IRES-GFP vector. (H) B-cells infected with the indicated ecotropic retroviruses [MSCV-mutated or full-length (FL) Rnf168-IRES-GFP] were stimulated with LPS plus IL-4 for 4 days, and the level of CSR to IgG1 was examined by FACS. Data are presented as the mean ± SEM of four independent experiments. * indicates p<0.05 compared to WT. (I) Representative DC-PCR for Sμ-Sγ1 recombination is shown from three independent experiments. nAchR served to normalize for the amount of input DNA. Fivefold serial dilutions were used as templates. H2O: no input DNA.

Figure 6

Figure 6. Analysis of the effect of Rnf168 inactivation on Sμ-Sγ1CSR junctions.

(A) Analysis of Sμ-Sγ1 CSR junctions. Overlap was determined by identifying the longest region at the switch junction of perfect uninterrupted donor/acceptor identity. No significant differences were observed. Representative data are shown from more than thirty clones in three independent experiments. (B) Purified splenic B cells were stimulated with LPS and IL-4 for 4 days. Genomic DNA was amplified by PCR and Sμ-Sγ1 junctions were sequenced. The percentage of junctions with the indicated nucleotide overlap is indicated (37 sequences from three WT mice and 39 sequences from three Rnf168−/− mice were analyzed). (C) Mutations in B-cells stimulated with LPS plus IL-4 for 4 days. Mutations near the Sμ-Sγ1 junctions (±50 bp) and frequencies of mutations are shown (37 sequences from three WT mice and 39 sequences from three Rnf168−/− mice were analyzed). The numbers of observed mutations are indicated in the periphery of the circular charts. (D) Sμ/Sγ1 junctions with unusual insertions obtained from Rnf168−/− B-cells. Sμ/Sγ1 sequences are shown in bold. The Sμ and Sγ1 [NT_114985.2 (strain 129/SvJ)] germline sequences are shown above or below each junction sequence. Lower-case letters indicate insertions. (|) indicates identity between nucleotides. Homology at the junctions is boxed. Two clones, 26L10.7 and 24L4.1, were obtained from independent experiments. 37 sequences from three WT mice and 39 sequences from three Rnf168−/− mice were analyzed.

Figure 7

Figure 7. Effect of Rnf168 deficiency on thymocyte development, TCRβ expression, and long-range V(D)J recombination.

(A) Flow cytometric analyses of DN thymocytes from WT and Rnf168−/− mice and average number of CD4−CD8−CD44−CD25+ (DNIII) cells. Data are presented as the mean ± SEM (n = 13 for each genotype). *p<0.05. (B) Expression levels of TCRβ in total thymocytes and CD4+CD8+ (DP) cells. (C) Histograms of the mean number of BrdU+ cells in each subpopulation of DN thymocytes from WT (n = 3) or Rnf168−/− (n = 3) mice. Data are presented as the mean ± SEM. (D) Relative frequency of _Tcr_δ locus rearrangements in total thymocytes. Quantitative assessment of genomic DNA rearrangements of Dδ1 to Dδ2, Dδ2 to Jδ1, and Vδ4 and Vδ5 to (D)Jδ1 genes were performed by real-time quantitative PCR and normalized to the signal of the non-rearranging DNA 3′ of Jδ2. Data are presented as the mean ± SEM (WT n = 9; Rnf168−/− n = 7). **p<0.005. (E) Representative primary PCR data for genomic DNA rearrangements of Dδ1 to Dδ2, Dδ2 to Jδ1, and Vδ4 and Vδ5 to (D)Jδ1.

Figure 8

Figure 8. Rnf168 maintains genomic integrity and suppresses cancer.

(A and B) Metaphase spread analysis of Rnf168−/− and WT B-cells. Representative data (A) and the percentage of aberrations (B) are shown. Three independent experiments were performed. A minimum of 40 metaphase spreads of untreated or irradiated Rnf168−/− and WT cells were analyzed. *p<0.05. f = acentric fragment, r = ring, rb = Robertsonian translocation, df = double acentric fragment. (C) Kaplan Meier tumor-free survival analysis for cohorts of WT (n = 56), Rnf168−/− (n = 50), p53−/− (n = 18) and Rnf168−/−p53−/− (n = 11) mice. A statistically significant difference was observed between the tumor-free survival of Rnf168−/−p53−/− and p53−/− mice (p = 0.0096, log-rank test). (D and E) H&E staining (D) and FACS analysis (E) of a thymoma from an Rnf168−/−p53−/− mouse. (F and G) H&E staining (F) and FACS analysis (G) of a B-cell lymphoma (B220+) from an Rnf168−/−p53−/− mouse. (H) Chromosomal translocations observed in an Rnf168−/−p53−/− lymphoma. Clonal reciprocal chromosomal translocations t(12;15) and t(15;12) are shown. Scale Bars: 50 µm; (D and F).

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