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Science 339 (6120): 711-715

Copyright © 2013 by the American Association for the Advancement of Science

Rif1 Prevents Resection of DNA Breaks and Promotes Immunoglobulin Class Switching

Michela Di Virgilio1, Elsa Callen3,*, Arito Yamane4,*, Wenzhu Zhang5,*, Mila Jankovic1, Alexander D. Gitlin1, Niklas Feldhahn1, Wolfgang Resch4, Thiago Y. Oliveira1,6,7, Brian T. Chait5, André Nussenzweig3, Rafael Casellas4, Davide F. Robbiani1, and Michel C. Nussenzweig1,2,{dagger}

1 Laboratory of Molecular Immunology, The Rockefeller University, New York, NY 10065, USA.
2 Howard Hughes Medical Institute (HHMI), The Rockefeller University, New York, NY 10065, USA.
3 Laboratory of Genome Integrity and Center for Cancer Research, National Cancer Institute (NCI), National Institutes of Health (NIH), Bethesda, MD 20892, USA.
4 Genomics and Immunity and National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS), NCI, NIH, Bethesda, MD 20892, USA.
5 Laboratory of Mass Spectrometry and Gaseous Ion Chemistry, The Rockefeller University, New York, NY 10065, USA.
6 Department of Genetics, Faculty of Medicine, University of São Paulo, Ribeirão Preto, Brazil.
7 National Institute of Science and Technology for Stem Cells and Cell Therapy, Ribeirão Preto, Brazil.


Figure 1
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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). Formulaand {sigma} are the mean (0.57) and SD (0.09) of the distribution, respectively.

 

Figure 2
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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, 100x; scale bars, 5 μm. Data are representative of two independent experiments. DAPI, 4',6-diamidino-2-phenylindole.

 

Figure 3
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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 C{gamma}1) 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, 63x; 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.

 

Figure 4
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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.

 


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