53BP1-RIF1-shieldin counteracts DSB resection through CST- and Polα-dependent fill-in

Abstract

In DNA repair, the resection of double-strand breaks dictates the choice between homology-directed repair-which requires a 3' overhang-and classical non-homologous end joining, which can join unresected ends^1,2. BRCA1-mutant cancers show minimal resection of double-strand breaks, which renders them deficient in homology-directed repair and sensitive to inhibitors of poly(ADP-ribose) polymerase 1 (PARP1)^3-8. When BRCA1 is absent, the resection of double-strand breaks is thought to be prevented by 53BP1, RIF1 and the REV7-SHLD1-SHLD2-SHLD3 (shieldin) complex, and loss of these factors diminishes sensitivity to PARP1 inhibitors^4,6-9. Here we address the mechanism by which 53BP1-RIF1-shieldin regulates the generation of recombinogenic 3' overhangs. We report that CTC1-STN1-TEN1 (CST)¹⁰, a complex similar to replication protein A that functions as an accessory factor of polymerase-α (Polα)-primase¹¹, is a downstream effector in the 53BP1 pathway. CST interacts with shieldin and localizes with Polα to sites of DNA damage in a 53BP1- and shieldin-dependent manner. As with loss of 53BP1, RIF1 or shieldin, the depletion of CST leads to increased resection. In BRCA1-deficient cells, CST blocks RAD51 loading and promotes the efficacy of PARP1 inhibitors. In addition, Polα inhibition diminishes the effect of PARP1 inhibitors. These data suggest that CST-Polα-mediated fill-in helps to control the repair of double-strand breaks by 53BP1, RIF1 and shieldin.

Extended Data Figure 1. Shieldin and CST counteract telomere hyper-resection

a-c, Effect of Shld2 on hyper-resection at telomeres lacking TPP1. a, Immunoblot for Chk1-P, an indicator of TPP1 deletion, in TPP1^F/F MEFs with and without bulk population treatment with an sgRNA to Shld2 and/or Cre (representative of three experiments). b, Quantitative analysis of telomere end resection as in Fig. 1c using the cells shown in (a). c, Quantification of the extent of resection detected in (c) as in Fig. 1d. Means (center bars) and SDs (error bars) from three independent experiments. Statistical analysis as in Fig. 1. d, FACS profiles of the indicated cells incubated with BrdU to measure (lack of) S phase effects of the Stn1 shRNA. Gating strategy for live cells and singlets is shown below the FACS profiles. Representative of two experiments. e, f, Experiments to verify that the ssDNA signal derives from a 3′ overhang. e, Immunoblot for Stn1 and γ-tubulin in TPP1^F/F (Rif1^F/+) cells treated with Stn1 shRNA and/or Cre. Representative of two experiments. f, Quantitative assay for telomeric overhangs as in Fig. 1c. Plugs in the ExoI lanes were treated with the 3′ exonuclease from E. coli. Representative of two experiments.

Extended Data Figure 2. Hyper-resection at telomeres lacking TPP1 is counteracted by CST and Shieldin

a, Immunoblots showing absence of Rev7 and reduction of Stn1 expression in the indicated TPP1^F/F and TPP1^F/F Rev7^−/− MEFs treated with either Ctc1 or Stn1 shRNA. Diminished Stn1 expression is used as a proxy for the efficacy of the Ctc1 shRNA. Representative of two experiments. b, Quantitative analysis of telomeric overhangs as in Fig. 1c. Representative of two experiments. c, Quantification of the effect of Ctc1 and Stn1 on resection at telomeres lacking TPP1 as in Fig. 1d. Data is obtained from two independent Rev7-proficient and two independent Rev7-deficient clones (light and dark shading).

Extended Data Figure 3. No effect of CST depletion on telomere hyper-resection when 53BP1 or Rif1 are absent

a, SV40LT-immortalized TPP1^F/F 53BP1^−/− cells were complemented with wt 53BP1 or a mutant 53BP1 lacking the ability to interact with Rif1, treated with a Stn1 shRNA as indicated, and analyzed by immunoblotting for 53BP1 and Stn1. Representative of four experiments. b, Quantitative analysis of telomeric overhangs as in Fig. 1c. c, Quantification of the resection at telomeres lacking TPP1 in four independent experiments performed as in Fig. 1d. d, Immunoblots showing loss of Rif1 and Stn1 in the indicated TPP1^F/F Rif1^F/+ and TPP1^F/F Rif1^F/F MEFs treated with Cre (96 h) as indicated and with or without Stn1 shRNA. Note diminished Rif1 levels after Cre due to heterozygosity in the TPP1^F/F Rif1^F/+ cells. e, Quantitative analysis of telomeric overhangs as in Fig. 1c. f, Quantification of the extent of resection detected as in (c), determined from three independent experiments (indicated by different shades of gray) showing means (center bars) and SDs (error bars). Each experiment involved all indicated samples analyzed in parallel. g, h, Experiments to verify that the ssDNA signal derives from a 3′ overhang. g, Immunoblot for Stn1 and γ-tubulin in TPP1^F/F Rif1^F/F cells treated with Stn1 shRNA and/or Cre. Representative of two experiments. h, Quantitative assay for telomeric overhangs as in Fig. 1c. Plugs in the ExoI lanes were treated with the 3′ exonuclease from E. coli. Representative of two experiments. All statistical analysis as in Fig. 1.

Extended Data Figure 4. Shld2 counteracts resection at telomeres lacking TRF2

a, Immunoblots for TRF2 deletion and Chk2 phosphorylation in TRF2^F/F Lig4^−/− MEFs with and without bulk population treatment with an sgRNA to Shld2 and/or Cre. Asterisk: non-specific band. Representative of three experiments. b, Quantitative analysis of telomere end resection as in Fig. 1c using the cells shown in (a). c, Quantification of the extent of resection detected in (b) as in Fig. 1d. Means (center bars) and SDs (error bars) from three independent experiments. All statistical analysis as in Fig. 1.

Extended Data Figure 5. CST interacts with Shieldin

a, Immunoprecipitation of individual mouse CST subunits or the three subunit complex (each subunit bearing a Myc-tag) with Flag-tagged mouse Shld1 co-expressed in 293T cells. Flag-tagged POT1b and POT1a serve as positive and negative controls for CST binding, respectively. Representative of two experiments. b, Two-hybrid analysis of CST-Shieldin interaction. Yeast cultures were grown overnight in synthetic complete medium lacking tryptophan and leucine to a density of 5*10⁷ cells/ml. Serial 10-fold dilutions were generated and 4 ul of each dilution was spotted on synthetic complete media lacking the nutrients tryptophan, leucine, adenine, histidine and containing 3-aminotriazole (3-AT) as indicated. Plates were then incubated for 5 days at 30°C before imaging. Representative of three experiments.

Extended Data Figure 6. Localization of CST and Polα to DSBs

a, Quantification of HA-Stn1 localization to FOKI-induced DSBs as in Fig. 3e. Means (center bars) and SDs (error bars) from 4-6 independent experiments (>80 induced nuclei for each condition in each experiment) are shown. b, IF for endogenous Polα in FOKI-LacI U2OS cells in S phase and after RO3306 treatment (G2). Dotted line: outline of the nucleus. Representative of two experiments. c, Examples of HA-Stn1 and Polα localization at FOKI-induced DSBs in G2-arrested FOKI-LacI U2OS cells (as in Fig. 3f). Representative of three experiments. d, Quantification of co-localization of Polα with FOKI-induced DSBs (as in Fig. 3f). Means (center bars) and SDs (error bars) from three independent experiments (>80 induced nuclei for each condition in each experiment) are shown. All statistical analysis as in Fig. 1.

Extended Data Figure 7. Effect of Stn1 knockdown on the intensity of IR-induced RPA foci

Quantification of myc-RPA32 intensity per nucleus in the experiments shown in Fig. 3g-h. Medians (center bars and numbers below) obtained from four independent experiments with >20 nuclei for each experimental condition in each experiment. Each symbol represents one nucleus. Statistical analysis as in Fig. 1.

Extended Data Figure 8. Effect of CST and Polα on PARPi treatment of BRCA1-deficient cells

a-f, Immunoblots on the MEFs used in Fig. 4a-e to verify the absence of deleted proteins and efficacy of the shRNAs. Reduction in Stn1 expression is used as a proxy for the efficacy of the Ctc1 shRNA since no antibody to mouse Ctc1 is available. Each immunoblot is representative of three experiments. g, Immunoblots for BRCA1 and Stn1 in the cells used in Fig. 4f. Representative of two experiments. h-j, Control experiment to assess that cells analyzed in Fig. 4f progressed through S phase during PARPi treatment. h, Experimental timeline as in Fig. 4f but with inclusion of BrdU in the media during PARPi treatment. i, Example of the assay for the presence of BrdU (IF) in metaphases harvested after the experimental timeline as in (h). j, Quantification of the BrdU incorporation into metaphase chromosomes as in (i) (one experiment with 10 metaphases per condition).

Figure 1. Shieldin and CST counteract resection at dysfunctioinal telomeres

a, Left: Schematic showing POT1b-bound CST counteracting resection of telomere ends. Right: Depiction of telomeres lacking TPP1, POT1a, and POT1b as a proxy for DSB resection. Telomeres lacking TPP1 undergo ATR-dependent hyper-resection that is repressed by 53BP1. b, Immunoblots showing loss of Rev7 and Stn1 in the indicated TPP1^F/F Rev7^+/+ MEFs and TPP1^F/F Rev7^−/− (CRISPR) clones treated with Cre (96 h) and/or Stn1 shRNA as indicated. Chk1-P serves as a proxy for TPP1 deletion. c, Quantitative analysis of telomere end resection in the cells shown in (b) using in-gel hybridization to detect the 3′ overhang (top) followed by rehybridization to the denatured DNA in the same gel (bottom) to determine the ratio of ss to total TTAGGG signal. Representative of four experiments. d, Quantification of resection detected as in (c), determined from four independent experiments (different shades of gray) showing means and SDs. Three independent Rev7 KO clones were used (distinct symbols). e, Telomeres lacking TRF2 as a model for resection upon ATM activation. f, Immunoblots showing Cre-mediated deletion of TRF2 from TRF2^F/F Lig4^−/− cells, CRISPR deletion of Rev7, shRNA-mediated reduction of Stn1, and Chk2 phosphorylation. Asterisk: non-specific. g and h, Telomere end resection analysis on the cells in (f) as in (c) and (d). Means and SDs from four independent experiments using two clones of each genotype. Note that the order of the samples is different in (h) versus (f) and (g). All data panels in the figure are representative of four experiments. All means are indicated with center bars and SDs with error bars. All statistical analysis based on two-tailed Welch’s t-test. *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001; ns, not significant.

Figure 2. 53BP1- and Shieldin-dependent localization of CST to dysfunctional telomeres

a, Left: Representative IF-FISH for 6myc-tagged Ctc1 (red) at telomeres (false-colored in green) in TPP1^F/F MEFs before and after Cre (96 h). Arrowheads: Ctc1 at telomeres. POT1b^−/− cells control for spurious telomere-Ctc1 co-localization. Right: The same nuclei showing γ-H2AX (red) at telomeres lacking TPP1. The γ-H2AX and Ctc1 signals are both false-colored in red. Arrows: telomeres with Ctc1 and γ-H2AX. b, Quantification of the % of telomeres co-localizing with Ctc1 detected as in (a). Each dot represents one nucleus from the indicated TPP1^F/F cell lines with and without Cre and/or ATRi. Means and SDs from three independent experiments. c, As in (b) but using TPP1^F/F cells treated with a Shld2 or a control sgRNA. Means and SDs as in (b). d, Immunoblots for POT1 deletion, ATR knockdown, and HA-Stn1 in conditional POT1 KO HT1080 cells. Asterisk: non-specific band. e, IF-FISH showing telomeric DNA co-localizing with Stn1 in cells as in (d) treated with Cre (96 h) and ATR shRNAs. f, Quantification of Stn1 localization at telomeres before and after POT1 deletion with or without ATR shRNAs as in (e). Means and SDs from three independent experiments. Each symbol represents one nucleus. g, Immunoprecipitation of human CST (each subunit Myc-tagged) with Flag-tagged human Shld1 or Rev7 co-expressed in 293T cells. h, Yeast 2-hybrid assay for interaction between CST and Shieldin subunits. All data panels in the figure are representative of three experiments. All means are indicated with center bars and SDs with error bars. All statistical analysis as in Fig. 1.

Figure 3. CST localizes to DSBs and represses ssDNA formation

a, IF for 53BP1 and HA-Stn1 in IR-treated HT1080 cells. b, Quantification of 53BP1/Stn1 co-localization as in (a) in cells with the indicated sgRNAs. Means and SDs from three independent experiments (>15 nuclei/experiment (symbols) for each experimental setting). c, Immunoblots for the indicated proteins in FOKI-LacI U2OS cells treated with the indicated sgRNAs. d, IF for mCherry-FOKI (red), and HA-Stn1 (green) in FOKI-LacI U2OS cells as in (c). e, Examples of HA-Stn1 co-localizing with FOKI foci in cells as in (d) treated with ATM and ATR inhibitors, or the indicated sgRNAs and quantification of Stn1/FOKI co-localization. Means and SDs from three independent experiments with >80 induced nuclei analyzed for each condition. f, As in (e) but monitoring Polα at DSBs in G2-arrested cells expressing HA-Stn1. g, Immunoblot for Stn1 knockdown in Myc-RPA32-expressing MEFs. h, IF for myc-RPA32 after 10 Gy IR (6 h). i, Quantification of cells with RPA foci as in (h) in >30 nuclei for each condition in three independent experiments (grey shading) with means and SDs. j and k, Immunoblots for IR-induced RPA phosphorylation (pS4/S8) after deletion of CTC1 from human cells (j) or after depletion of Stn1, Ctc1, or 53BP1 from MEFs (k). l, IF for Rad51/γH2AX co-localization at IR-induced DSBs in BRCA1-deficient cells treated with Ctc1 shRNA. m, Quantification of data as in (l). Means and SDs from four independent experiments (grey shading) (>60 nuclei/experiment). n, Immunoblot for Stn1 knockdown and TRF2 deletion from TRF2^F/F RosaCreER MEFs. Asterisk: non-specific. o, Effect of Stn1 shRNA knockdown on telomere-telomere fusions. Means and SDs from three independent experiments (>6000 telomeres each). All IF and immunoblots shown are representative of three experiments. All means are indicated with center bars and SDs with error bars. All statistical analysis as in Fig. 1.

Figure 4. CST and Polα affect the outcome of PARPi in BRCA1-deficient cells

a, Colonies detected in a PARPi survival assay using BRCA1^F/F MEFs with or without Cre and shRNAs to CST. b, Graphical representation of data as in (a) from three independent experiments. c, Epistasis analysis of PARPi resistance induced by absence of 53BP1 or Rev7 and depletion of CST subunits. Means (symbol) and SEMs (error bars) from three independent experiments. d, PARPi-induced radial chromosomes in BRCA1-deficient cells. Scale bar: 1 μm. e, Means (center bar) and SDs (error bars) of % of misrejoined (radial) chromosomes in >10 metaphases per experimental setting for each of three independent experiments. Each dot represents one metaphase. f, Effect of Polα inhibition on radial formation in PARPi-treated BRCA1^−/− cells using the experimental timeline shown. Means (center bar) and SDs (error bars) of % radial chromosomes in >10 metaphases per experimental setting for each of three independent experiments. Each dot represents one metaphase. g, Graphical representation of the similar mechanisms by which resection is counteracted at functional telomeres and at DSBs. Panels (a) and (d) are representative of three experiments. All statistical analysis as in Fig. 1.