53BP1 regulates DSB repair using Rif1 to control 5' end resection

Abstract

The choice between double-strand break (DSB) repair by either homology-directed repair (HDR) or nonhomologous end joining (NHEJ) is tightly regulated. Defects in this regulation can induce genome instability and cancer. 53BP1 is critical for the control of DSB repair, promoting NHEJ, and inhibiting the 5' end resection needed for HDR. Using dysfunctional telomeres and genome-wide DSBs, we identify Rif1 as the main factor used by 53BP1 to impair 5' end resection. Rif1 inhibits resection involving CtIP, BLM, and Exo1; limits accumulation of BRCA1/BARD1 complexes at sites of DNA damage; and defines one of the mechanisms by which 53BP1 causes chromosomal abnormalities in Brca1-deficient cells. These data establish Rif1 as an important contributor to the control of DSB repair by 53BP1.

Figure 1. Rif1 recruitment requires the S/TQ ATM/ATR target sites of 53BP1

(A) Detection of 53BP1 and Rif1 at dysfunctional telomeres in Cre-treated SV40-LT immortalized TRF2^F/-53BP1^-/- MEFs expressing 53BP1 mutant alleles (shown in (C)). IF for 53BP1 and Rif1 (red) was combined with telomeric TTAGGG FISH (green). Blue: DAPI DNA stain. (B) Quantification of 53BP1 and Rif1 Telomere Dysfunction Induced Foci (TIFs; (21)) detected as in (A). Data represent means of 3 experiments ±SDs (≥70 cells/experiment). ** indicates p value <0.05 (two-tailed paired Student's t-test). (C) Schematic of the 53BP1 mutant alleles and the role of the N-terminal S/TQ sites in the recruitment of Rif1.

Figure 2. Rif1 promotes telomeric NHEJ without affecting telomere mobility

(A) Metaphase chromosomes of Cre-treated SV40-LT immortalized TRF2^F/FRif1^+/+ and TRF2^F/FRif1^F/F MEFs showing NHEJ-mediated telomere fusions detected by CO-FISH. Telomeres synthesized by leading-end DNA synthesis are in red; lagging-end telomeres in green. (B) Quantification of telomere fusions as determined in (A) at 96 and 120 h post Cre. Data represent means of three independent experiments ± SDs (> 3000 telomeres/experiment). ** indicates p value <0.01 based on two-tailed paired Student's t-test. (C) Distributions of telomere fusions per metaphase at 96 h after Cre for experiments shown in (B). (D) Distribution of cumulative distances traveled by mCherry-53BP1^1220-1711 foci in the indicated cell lines. Red lines represent medians. ** indicates p values < 0.0001 (two-tailed Mann-Whitney test).

Figure 3. Rif1 blocks 5’ end resection at dysfunctional telomeres

(A) Telomeric overhang assays on TRF2^F/FRif1^+/+, TRF2^F/FRif1^F/F and TRF2^F/-53BP1^-/- MEFs. Native in-gel hybridization of MboI/AluI digested DNA with end-labeled [AACCCT]₄ (top) and re-hybridization with the same probe after denaturation in situ (bottom). Dashed lines represent the bulk of free (unfused) telomeres used for quantification. (B) Quantification of overhang assays as in (A). Overhang signals in no Cre samples was set at 100%. (C, D) Overhang assays on TRF2^F/FRif1^F/+Lig4^-/-, TRF2^F/FRif1^F/FLig4^-/- and TRF2^F/-53BP1^-/- MEFs and quantification as in (B). (E, F) Overhang assays on TRF1^F/FTRF2^F/FRif1^+/+ and TRF1^F/FTRF2^F/FRif1^F/F MEFs and quantification. (G, H) Overhang assays to measure dependency on CtIP, BLM, and Exo1 and quantification. Cells infected with either pMX or pSR with or without the indicated shRNAs and treated with Cre for 96 h. Samples with empty vectors and no Cre (ref.) were used as references. Data in (B,D,F,H) represent means of ≥3 experiments ±SDs. ** indicates p values <0.05 (two-tailed paired Student's t-test). MEFs are SV40-LT immortalized.

Figure 4. Rif1 inhibits resection at DSBs and promotes radial formation

(A) IF for γ-H2AX (red) and MYC-RPA32 (green) in Cre-treated SV40-LT immortalized Rif1^F/F and Rif1^F/F53BP1^-/- cells expressing MYC-RPA32 treated with zeocin (100 ug/ml, for 1 h; 2 h prior to analysis). (B) Percentage of γ-H2AX positive cells in experiments as in (A). (C) Percentage of cells (as in A) scored positive when containing at least five γ-H2AX foci co-localizing with RPA. (D) IF for γH2AX (green) and BARD1 (red) in Rif1^F/F and Rif1^F/F53BP1^-/- MEFs. Cells and treatment as in (A). (E) Percentage of γ-H2AX positive cells in experiments in (D). (F) Percentage of cells in (D) containing >5 BARD1/γ-H2AX colocalizing foci. (G) Examples of mis-rejoined and radial chromosomes (arrowheads) in BRCA1sh/PARPi-treated Rif1^F/F cells with or without Cre. (H) Percentages of chromosomes that are mis-rejoined in the indicated genotypes and treatments. Data in (B,C), (E,F) and (H) are means of 3-5 experiments ±SDs. ** indicates p values <0.05 (two-tailed paired Student's t-test).