Rif1 Prevents Resection of DNA Breaks and Promotes Immunoglobulin Class Switching

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Science  08 Feb 2013:
Vol. 339, Issue 6120, pp. 711-715
DOI: 10.1126/science.1230624


DNA double-strand breaks (DSBs) represent a threat to the genome because they can lead to the loss of genetic information and chromosome rearrangements. The DNA repair protein p53 binding protein 1 (53BP1) protects the genome by limiting nucleolytic processing of DSBs by a mechanism that requires its phosphorylation, but whether 53BP1 does so directly is not known. Here, we identify Rap1-interacting factor 1 (Rif1) as an ATM (ataxia-telangiectasia mutated) phosphorylation-dependent interactor of 53BP1 and show that absence of Rif1 results in 5′-3′ DNA-end resection in mice. Consistent with enhanced DNA resection, Rif1 deficiency impairs DNA repair in the G1 and S phases of the cell cycle, interferes with class switch recombination in B lymphocytes, and leads to accumulation of chromosome DSBs.

The DNA damage response factor p53 binding protein 1 (53BP1) is a multidomain protein containing a chromatin-binding tudor domain, an oligomerization domain, tandem breast cancer 1 (BRCA1) C-terminal (BRCT) domains, and an N-terminal domain with 28 SQ/TQ potential phosphorylation sites for phosphatidylinositol 3-kinase–related kinases [PIKKs, ataxia-telangiectasia mutated (ATM)/ATM and Rad3-related/DNA-dependent protein kinase catalytic subunit (DNA-PKcs)] (13). 53BP1 contributes to DNA repair in several ways: This protein facilitates joining between intrachromosomal double-strand breaks (DSBs) at a distance (synapsis) (47), it enables heterochromatic DNA repair through relaxation of nucleosome compaction (2, 3), and it protects DNA ends from resection and thereby favors repair of DSBs that occur in G1 phase by nonhomologous end joining (NHEJ) (4, 5, 8). Consistent with its role in DNA-end protection, 53BP1 is essential for class switch recombination (CSR) in B lymphocytes (9, 10).

Structure-function studies indicate that, besides the recruitment of 53BP1 to DNA ends, protection requires 53BP1 phosphorylation (4), but how this protective effect is mediated is unknown. To identify phosphorylation-dependent interactors of 53BP1, we applied stable isotope labeling by amino acids in cell culture (SILAC). Trp53bp1−/− (Trp53bp1 encodes 53BP1) B cells were infected with retroviruses encoding a C-terminal deleted version of 53BP1 (53BP1DB) or a phosphomutant in which all 28 N-terminal potential PIKK phosphorylation sites were mutated to alanine (53BP1DB28A) (4), in media containing isotopically heavy (53BP1DB) or light (53BP1DB28A) lysine and arginine (fig. S1, A to C) (11).

Most proteins coprecipitating with 53BP1DB and 53BP1DB28A displayed a H/(H + L) ratio of ~0.5 (H, heavy; L, light), which is characteristic of phospho-independent association (average of 0.57 ± 0.09, peptide count: at least four) (Fig. 1 and table S1). Many of these proteins are nonspecific contaminants, but others such as KRAB-associated protein 1 (KAP-1), dynein light chain LC8-type 1 (Dynll1), Nijmegen breakage syndrome 1 (Nbs1), and H2AX represent authentic phospho-independent 53BP1-interacting proteins (fig. S1D). Three proteins displayed an abundance ratio that was more than four standard deviations (SDs) above the mean, indicating that these proteins interact specifically with phosphorylated 53BP1: Pax interaction with transcription-activation domain protein-1 (Paxip1, or PTIP; 0.95), PTIP-associated protein 1 (Pa1; 0.97), and Rap1-interacting factor 1 (Rif1) (0.96) (Fig. 1 and figs. S1D and S2). PTIP was known to interact with 53BP1 in a phospho-dependent manner (12), whereas Pa1 and Rif1 were not.

Fig. 1

Identification of phospho-dependent 53BP1 interactors. The graph shows the H/(H + L) ratio distribution of proteins identified by SILAC. Error bars represent the SD of the H/(H + L) mean value for all of the peptides identified for each individual protein (only proteins with at least four peptides were included). Embedded Imageand σ are the mean (0.57) and SD (0.09) of the distribution, respectively.

Rif1 was originally identified in budding yeast as a protein with a key role in telomere length maintenance (13). However, in mammalian cells, Rif1 is not essential for telomere homeostasis, but has been assigned a number of different roles in maintaining genome stability, including participation in the DNA damage response (1416), repair of S-phase DNA damage (17, 18), and regulation of origin firing during DNA replication (19, 20). However, the mechanism by which Rif1 might contribute to DNA repair and maintenance of genome stability is not known.

To confirm that Rif1 interaction with 53BP1 is dependent on phosphorylation, we performed Western blot analysis of Flag immunoprecipitates from lysates of irradiated Trp53bp1−/− B cells infected with retroviruses encoding 53BP1DB or 53BP1DB28A. Whereas Dynll1, a phospho-independent 53BP1 interactor (SILAC ratio: 0.55) (fig. S1D), coimmunoprecipitated with 53BP1DB and 53BP1DB28A to a similar extent (Fig. 2A), only 53BP1DB coimmunoprecipitated with Rif1. We conclude that the interaction between 53BP1 and Rif1 is dependent on phosphorylation of 53BP1.

Fig. 2

Rif1 interaction with 53BP1 is dependent on phosphorylation, DNA damage, and ATM. (A) Western blot analysis of anti-Flag immunoprecipitates (IP) from irradiated (IR) Trp53bp1−/− B lymphocytes infected with empty vector (vec), 53BP1DB, or 53BP1DB28A virus. Triangles indicate threefold dilution. Data are representative of two independent experiments. (B) Western blot analysis of anti-Flag immunoprecipitates from Trp53bp1−/− B cells infected with empty vector or 53BP1DB. Cells were either left untreated or irradiated [50 gray (Gy), 45-min recovery] in the presence or absence of the ATM kinase inhibitor KU55933 (ATMi). Triangles indicate threefold dilution. Data are representative of two independent experiments. (C) Immunofluorescent staining for 53BP1 (Flag) and Rif1 in irradiated Trp53bp1−/− iMEFs reconstituted with 53BP1DB or 53BP1DB28A retroviruses (4). Magnification, 100×; scale bars, 5 μm. Data are representative of two independent experiments. DAPI, 4′,6-diamidino-2-phenylindole.

Ataxia-telangiectasia mutated phosphorylates 53BP1 in response to DSBs (1, 3). To determine whether ATM induces DNA damage–dependent association between Rif1 and 53BP1, we compared irradiated and nonirradiated B cells in coimmunoprecipitation experiments. Although we detected small amounts of Rif1 in 53BP1DB immunoprecipitates from unirradiated cells, this was increased by a factor of >3 after irradiation, and the increase was abrogated by treatment with the ATM inhibitor KU55933 (Fig. 2B). We conclude that Rif1 preferentially interacts with phosphorylated 53BP1 in a DNA damage- and ATM-dependent manner.

Rif1 is recruited to DNA damage foci by 53BP1 (15). To determine whether 53BP1 phosphorylation is required for Rif1 focus formation, we tested Rif1 foci in irradiated Trp53bp1−/− immortalized mouse embryonic fibroblasts (iMEFs), which were stably transduced with either 53BP1DB or 53BP1DB28A. Rif1 foci were readily detected and colocalized with 53BP1DB (Fig. 2C). In contrast, although 53BP1DB28A formed normal-appearing foci, Rif1 foci were rare and did not colocalize with 53BP1 (Fig. 2C). Furthermore, Rif1 recruitment to ionizing radiation–induced foci (IRIF) and colocalization with 53BP1 were abrogated in ATM-deficient but not DNA-PKcs–deficient iMEFs (fig. S3) (15). We conclude that Rif1 recruitment to DNA damage response foci is dependent on ATM-mediated 53BP1 phosphorylation.

The phosphorylation of 53BP1 is essential for CSR (4). To examine the role of Rif1 in joining DSBs during CSR, we conditionally ablated Rif1 in B cells using CD19Cre, which is expressed specifically in B cells (Rif1F/FCd19Cre/+ mice) (fig. S4, A to C). To induce CSR, B cells were activated with lipopolysaccharide (LPS) and interleukin-4 (IL-4) in vitro, and switching to immunoglobulin G1 (IgG1) or IgG3 was measured by flow cytometry. CSR to IgG1 and IgG3 was markedly reduced in Rif1F/FCd19Cre/+ B cells, but less so than in Trp53bp1−/− controls (Fig. 3, A and B, and fig. S5). Switch junctions from Rif1F/FCd19Cre/+ B cells were comparable to those from Trp53bp1−/− and wild-type controls (fig. S6) (7), which indicates that, similar to 53BP1 deficiency, absence of Rif1 does not alter the nature of productive CSR joining events. A similar CSR defect was also obtained by conditionally deleting Rif1 with 4-hydroxy-tamoxifen (4HT) in Rif1F/FROSA26Cre-ERT2/+ B cells (fig. S7). Finally, short hairpin RNA–mediated partial down-regulation of CtBP-interacting protein (CtIP), which interacts with Rif1 (fig. S8C) and has been implicated in processing of DNA ends (21, 22), resulted in a very small but reproducible increase in CSR (fig. S8, A and B). Thus, Rif1 is essential for normal CSR, and CtIP may not be the only factor that contributes to end processing in Rif1-deficient B cells.

Fig. 3

Rif1 deficiency impairs CSR and causes Igh and genome instability in primary B cells. (A) (Left) CSR to IgG1 96 hours after stimulation of B lymphocytes with LPS and IL-4. (Right) Summary dot plot for three independent experiments (n = three mice per genotype). Mean values are: 23.6% for Cd19Cre/+, 23.4% for Rif1F/+Cd19Cre/+, and 5.0% for Rif1F/FCd19Cre/+ (P < 0.008 with the paired Student's t test). (Bottom) B cell proliferation by carboxyfluorescein succinimidyl ester (CFSE) dilution. Data are representative of three independent experiments. (B) Same as in (A) but for CSR to IgG3 after stimulation with LPS alone. Mean values are: 3.2% for Cd19Cre/+, 3.4% for Rif1F/+Cd19Cre/+, and 0.5% for Rif1F/FCd19Cre/+ (P < 0.008). (C) (Left) Cell cycle analysis of primary B cells after stimulation with LPS and IL-4. BrdU, 5-bromo-2′-deoxyuridine; 7-AAD, 7-amino-actinomycin D. (Right) Summary histograms for S, G0/G1, and G2/M phase cells from two independent experiments (n = four mice per genotype). Error bars indicate SEM. * 0.01 < P < 0.05, ** 0.001 < P < 0.01, *** P < 0.001. WT, wild type. (D) (Left) Cell cycle analysis of LPS- and IL-4–stimulated splenocytes at the indicated times after irradiation (6 Gy). (Right) Summary graphs for S, G0/G1, and G2/M phase cells from two independent experiments (n = three mice per genotype). Error bars indicate SD. (E) Analysis of genomic instability in metaphases from B cell cultures. Chtid, chromatid; Chre, chromosome. Data are representative of two independent experiments (n = 50 metaphases analyzed per genotype per experiment). (F) Examples of Igh-associated aberrations in Rif1F/FCd19Cre/+ B cells. Chromosomes were hybridized with an Igh Cα probe (green; centromeric of Cγ1) and a telomere sequence-specific probe (red) and were counterstained with DAPI (dark blue/black). Arrows indicate Igh Cα/telomeric signal on chromosome 12. Magnification, 63×; scale bars, 1 μm. (G) Frequency of c-myc/Igh translocations in activated B cells. The graph shows combined results from three mice per genotype.

Class switch recombination requires cell division, activation-induced cytidine deaminase (AID) expression, and Igh germline transcription (23). There are conflicting reports that Rif1 is required for proliferation in MEFs, but not in DT40 B cells (17, 18). We found that cell division profiles of Rif1F/FCd19Cre/+ and 4HT-treated Rif1F/FROSA26Cre-ERT2/+ B cells were indistinguishable from controls (Fig. 3, A and B; and fig. S7, A, C, E, and G), indicating that Rif1 is dispensable for B cell proliferation in vitro. Finally, AID mRNA and protein expression and Igh germline transcription were not affected by Rif1 deletion (fig. S4, B and D).

We next examined the role of Rif1 in cell cycle progression in primary B cells. We found no major differences in the percentage of cells in G0/G1 and S phases (Fig. 3C). However, the number of cells in G2/M phase was increased approximately twofold in the absence of Rif1 (2.64-, 2.56-, and 1.91-fold at 48, 72, and 96 hours, respectively) (Fig. 3C). We obtained similar results with the use of Rif1F/FROSA26Cre-ERT2/+ B cells treated with 4HT (fig. S7, H and I). Furthermore, irradiation increases the accumulation of Rif1F/FCd19Cre/+ B cells in G2/M phase (Fig. 3D). In addition, Trp53bp1−/− iMEFs expressing 53BP1DB28A, which did not recruit Rif1 to IRIF (Fig. 2C), exhibited delayed progression through S phase following DNA damage with accumulation of cells in G2 phase after irradiation (fig. S9).

Accumulation of cells in G2/M phase may reflect the persistence of unrepaired DNA damage in a fraction of Rif1-deficient cells. To investigate this possibility, we analyzed metaphase spreads from B cells dividing in response to LPS and IL-4 in vitro. These cells express AID, which produces DSBs in Igh and, less frequently at off-target sites throughout the genome, in the G1 phase of the cell cycle (2426). Chromosomal aberrations were increased in Rif1F/FCd19Cre/+ B cells compared to controls (Fig. 3E), with many localized to the Igh locus (Fig. 3E). Consistent with the observation that Igh is targeted by AID in the G1 phase of the cell cycle, all of the Igh breaks were chromosome breaks (Fig. 3, E and F). Interestingly, the frequency of c-myc/Igh translocations is moderately increased in Rif1F/FCd19Cre/+ B cells; however, the breakpoint distribution was similar to the Cd19Cre/+ control (1.5 × 10−6 versus 1.0 × 10−6 in the control; P = 0.039) (Fig. 3G and fig. S10). We conclude that in the absence of Rif1, DSBs fail to be resolved efficiently in the G1, S, or G2 phases, which leads to increased levels of genomic instability, including chromosome breaks at Igh and translocations in dividing B cells.

In the absence of 53BP1, DSBs produced by AID at the Igh locus accumulate the single-stranded DNA-binding replication protein A complex (RPA) as a result of increased DNA-end resection (24). To determine if Rif1 is required for DNA-end protection by 53BP1, we performed RPA–chromatin immunoprecipitation followed by massive parallel sequencing (ChIP-seq) experiments on Rif1F/FCd19Cre/+ and control B cells. Ablation of Rif1 was indistinguishable from the loss of 53BP1 in that in its absence, RPA decorates the Igh locus asymmetrically, in a manner consistent with 5′-3′ resection (Fig. 4A) (27). In addition, absence of Rif1 also results in RPA accumulation at non-Igh genes, such as Il4ra and Pim1, that are damaged by AID in G1 phase (Fig. 4B) (24, 25). Rad51 is the recombinase that mediates repair of DSBs by homologous recombination in S/G2/M phase (22). To confirm that Rif1 prevents resection that takes place in S phase, we monitored Rad51 accumulation in activated B cells by ChIP-seq. Loss of Rif1 was indistinguishable from the loss of 53BP1 (27), in that it led to asymmetric Rad51 accumulation at sites of AID-inflicted DNA damage (Fig. 4, C and D). We conclude that in the absence of Rif1, AID-induced DSBs incurred in G1 phase persist and undergo extensive 5′-3′ DNA-end resection in S/G2/M phase, as measured by RPA and Rad51 accumulation.

Fig. 4

Rif1 prevents resection of DNA ends at sites of AID-induced DNA damage. (A to D) RPA and Rad51 occupancy at the Igh locus (A and C) and at non-Igh AID targets genes (B and D) in B cells activated to undergo class switching. ChIP-seq libraries were resolved into upper (+) and lower (-) DNA strands to show RPA and Rad51 association with sense and antisense strands. Within a specified genomic window, graphs have the same scale and show tag density. Deep-sequencing samples were normalized per library size, and tags per million values were calculated for each genic region, as indicated in the supplementary materials and methods and shown in parenthesis. Data are representative of two independent experiments for RPA ChIP-seq and one for Rad51. (E) Model of Rif1 recruitment and DNA-end protection at DSBs. DNA damage activates ATM, which phosphorylates many targets, including 53BP1. This event recruits Rif1 to 53BP1 at the DSB, where it inhibits DNA resection. The extensive resection in the absence of Rif1 impairs CSR at the Igh locus. P, phosphate.

A role for Rif1 in maintenance of genome stability and protection of DNA ends against resection is consistent with its phosphorylation-dependent recruitment to the N-terminal domain of 53BP1 (4). 53BP1 facilitates DNA repair and prevents DNA-end resection during CSR. In the absence of 53BP1, AID-induced DSBs are resolved inefficiently in G1 phase, leading to chromosome breaks, Igh instability, and resolution by alternative NHEJ or homologous recombination instead of classical NHEJ (4, 8, 27). Our experiments show that in the absence of Rif1, 53BP1 is insufficient to promote genomic stability or mediate efficient Igh repair, DNA-end protection, or CSR. Thus, these 53BP1 activities require Rif1 recruitment to the phosphorylated N terminus of 53BP1. Rif1 is likely to have additional functions beyond 53BP1, CSR, and DNA-end protection because although Trp53bp1−/− mice are viable, Rif1 deletion is lethal (17). Indeed, Rif1 is believed to play a role in the repair of S-phase DNA damage (17, 18), as well as in the regulation of replication timing (19, 20, 28). Analogously, additional CSR factor(s) may exist downstream of 53BP1, as class switching in Rif1-deficienct B cells is significantly higher than in Trp53bp1−/−.

In summary, our data are consistent with a model in which ATM-mediated phosphorylation of 53BP1 recruits Rif1 to sites of DNA damage, where it facilitates DNA repair in part by protecting DNA ends from resection (Fig. 4E). In the absence of Rif1, DNA breaks incurred in G1 phase fail to be repaired by NHEJ and undergo extensive 5′-3′ end resection, resulting in the accumulation of chromosome breaks and genome instability.

Supplementary Materials

Materials and Methods

Figs. S1 to S10

Table S1

References (2949)

References and Notes

  1. Materials and methods are available as supplementary materials on Science Online.
  2. Acknowledgments: We thank all members of the Nussenzweig laboratory for discussion, D. Bosque and T. Eisenreich for help in managing mouse colonies, A. Gazumyan for assistance with Igh germline and AID transcript levels analysis, and K. Yao for help with genotyping. We thank T. de Lange (The Rockefeller University, New York) for Rif1F/F mice; S. Buonomo (European Molecular Biology Laboratory Mouse Biology Unit, Monterotondo, Italy) for the anti-mouse Rif1 serum #1240; G. Gutierrez (NIAMS, NIH, Bethesda, MD) for Illumina sequencing; N. Zampieri (Columbia University, New York) for assistance with immunofluorescence image processing, and M. P. Rout, J. LaCava, S. Obado, and L. Hough (The Rockefeller University) for invaluable help, discussions, and protocols for cryolysis and magnetic bead-mediated immunoisolation. The data presented in the manuscript are tabulated in the main text and in the supplementary materials. Sequence data shown in Fig. 4 have been deposited in the Gene Expression Omnibus database (accession number GSE42298) at M.D.V. was a Fellow of the American Italian Cancer Foundation, and A.D.G. was supported by NIH Medical Scientist Training Program grant GM007739. This work was supported in part by NIH grants AI037526 (M.C.N.), RR022220 (B.T.C.), RR00862 (B.T.C.), and GM103314 (B.T.C.); and by the intramural program of NIAMS at the NIH (R.C.); and the intramural research program of NCI at the NIH and Center for Cancer Research (A.N. and E.C.). M.C.N. is an HHMI Investigator.

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