RNF169 limits 53BP1 deposition at DSBs to stimulate single-strand annealing repair

Abstract

Unrestrained 53BP1 activity at DNA double-strand breaks (DSBs) hampers DNA end resection and upsets DSB repair pathway choice. RNF169 acts as a molecular rheostat to limit 53BP1 deposition at DSBs, but how this fine balance translates to DSB repair control remains undefined. In striking contrast to 53BP1, ChIP analyses of AsiSI-induced DSBs unveiled that RNF169 exhibits robust accumulation at DNA end-proximal regions and preferentially targets resected, RPA-bound DSBs. Accordingly, we found that RNF169 promotes CtIP-dependent DSB resection and favors homology-mediated DSB repair, and further showed that RNF169 dose-dependently stimulates single-strand annealing repair, in part, by alleviating the 53BP1-imposed barrier to DSB end resection. Our results highlight the interplay of RNF169 with 53BP1 in fine-tuning choice of DSB repair pathways.

Keywords: 53BP1; DNA damage; DNA double-strand breaks; RNF169; single-strand annealing repair.

Fig. 1.

Spatial distribution of RNF169 and other DDR factors at DSBs. (A) Schematic illustration of the DIvA platform integrated with a doxycycline (Dox)-inducible RNF169 expression cassette. (B and C) Representative SR-SIM images reveals the juxtaposed orientation of eRNF169 and 53BP1 (B) and colocalization of eRNF169 and RAP80 (C) at AsiSI-induced DSBs. DIvA-eRNF169 cells were treated with 0.02 μg/mL doxycycline for 24 h, and 4-OHT was added 4 h before immunostaining experiments using indicated antibodies. (D) Representative STORM image shows the juxtaposition of eRNF169 and 53BP1 at AsiSI-induced DSBs. (E) Schematic illustration of the two AsiSI sites (Chr1_6 and Chr1_12) on Chromosome 1 used for ChIP-qPCR analysis. Each arrow represents a pair of primers employed for qPCR analysis. (F) ChIP-qPCR analysis of distribution of γH2AX, 53BP1, RAP80, and eRNF169 on one side of chromatin flanking each of the two AsiSI-induced DSBs. DIvA-eRNF169 cells were treated with or without 0.02 μg/mL doxycycline for 24 h, and 4-OHT was added 4 h before cells were processed for ChIP experiments using indicated antibodies. Data represents mean ± SEM (of two technical repeats) derived from one representative experiment (n = 3); (G) Graphical illustration of DSB spatial distribution of DDR factors characterized in this study. (Scale bars: 0.5 μm.)

Fig. 2.

Characterization of RNF169 end-proximal distribution. (A) RNF169 preferentially loads on RAD51-bound DSBs. DIvA-eRNF169 cells were treated as in Fig. 1F. ChIP-qPCR analyses were performed against γH2AX, RAD51, RPA-1, and Flag (eRNF169) at RAD51-bound and RAD51-unbound DSBs. Box and whisker plots are derived from one representative experiment (n = 3) of three technical replicates. **P < 0.01, ***P < 0.001. (B) Schematic illustrating flow of experiment to define genetic requirement of DSB end-proximal accumulation of eRNF169. (C and D) CtIP promotes RNF169 accumulation at DSB end-proximal regions. DIvA-eRNF169 cells pretreated with indicated siRNAs were incubated with 1.0 μg/mL doxycycline for 24 h. 4-OHT was added to cells for 4 h. Cells were subsequently processed for ChIP experimentations using anti-Flag antibodies (eRNF169). qPCR analysis was performed to determine eRNF169 enrichments at Chr1_6 and Chr1_12. Data represents mean ± SEM (of three technical repeats) derived from one representative experiment (n = 3). *P < 0.05, **P < 0.01. (D) Western blotting experiment was performed to assess RNAi-mediated CtIP knockdown efficiency. (E) CtIP depletion impairs RNF169 accumulation at RAD51-bound DSBs. ChIP-qPCR experimentations and analyses were performed as in C.

Fig. 3.

RNF169 promotes DSB resection and homology-directed repairs. (A) Schematic for quantitative DNA resection assay based on the DIvA system. (B and C) Quantitative measurement of ssDNA generation by 5′ end resection at two AsiSI-induced DSBs. DIvA cells pretreated with indicated siRNAs were incubated with 4-OHT for 4 h. Genomic DNA was extracted and digested with either BsrGI (B) or BanI (C). Percentage of ssDNA intermediates at indicated sites was measured by qPCR using primers indicated in A after restriction enzyme digestion. Data represents mean ± SEM (of two technical repeats) from three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001; ns, not significant. (D) Western blotting experiment was performed to assess RNAi-mediated knockdown efficiency in cells used in B and C. (E–H) RNF169 deficiency impairs resection-dependent DSB repair. Schematic representation of the DR-GFP, SA-GFP, EJ2-GFP, and EJ5-GFP reporters to analyze the repair of I-SceI-induced DSBs by HR, SSA, aNHEJ, and total NHEJ events (Top). U2OS cells stably expressing DR-GFP (E), SA-GFP (F), EJ2-GFP (G) and EJ5-GFP (H) were transfected with indicated siRNAs and were electroporated with plasmid encoding the I-SceI endonuclease. (Middle) Flow cytometric analysis of GFP-positive cell population was performed 48 h after electroporation. Data represents mean ± SEM from three independent experiments, **P < 0.01, ***P < 0.001; ns, not significant. (Bottom) Knockdown efficiencies were determined by Western blotting.

Fig. 4.

RNF169 stimulates SSA repair by counteracting 53BP1. (A) Ectopic expression of RNF169 but not RNF169MIU2 stimulates SSA repair. SA-U2OS-eRNF169 or SA-U2OS-eRNF169ΔMIU2 cells were electroporated with plasmid encoding the I-SceI endonuclease. Cells were cultured with or without 2 μg/mL doxycycline for 48 h before cells were harvested for flow cytometric analysis. Mock electroporation (no I-SceI) was used as negative control. Data represents mean ± SEM from three independent experiments, ***P < 0.001, ns, not significant. (B) Western blotting analysis showing expression of RNF169 (wild-type and ΔMIU2 mutant). (C) 53BP1 inactivation promotes SSA repair. SA-U2OS (vector control and 53BP1 KO) cells were electroporated with I-SceI expression construct and percentage of cells positive for GFP was analyzed 48 h after electroporation. Data represents mean ± SEM from three independent experiments, ***P < 0.001. (D) Western blotting analysis to determine 53BP1 expression. (E) RNF169-driven SSA repair was alleviated in 53BP1 KO cells. Parental SA-U2OS-eRNF169 cells (Vector) or its 53BP1 KO derivative were treated and processed as described in A. Data represents mean ± SEM from three independent experiments, *P < 0.05, ***P < 0.001. (F) Western blotting analysis to determine RNF169 expression in cells used in E. (G) 53BP1 deficiency restores SSA repair in RNF169-inactivated cells. Parental SA-U2OS cells (Vector) or its 53BP1 KO derivative were transfected with indicated siRNAs. Cells were treated and processed as described in C. Data represents mean ± SEM from three independent experiments, **P < 0.01. (H) Western blotting analysis was performed to assess RNAi-mediated knockdown efficiency in cells used in G.

Fig. 5.

RNF169 stimulates SSA repair in HR-deficient cells. (A and B) Ectopic expression of RNF169 stimulates SSA repair in BRCA1-deficient, PALB2-deficient, and BRCA2-deficient cells. SA-U2OS-eRNF169 cells pretreated with indicated siRNAs were treated and processed as described in Fig. 4A. Data represents mean ± SEM from four independent experiments, **P < 0.01, ***P < 0.001. (B) Western blotting experiment was performed to assess RNAi-mediated knockdown efficiencies. (C and D) RNF169 is required for hyperactive SSA in PALB2-deficient and BRCA2-deficient cells. SA-U2OS cells were transfected with indicated siRNAs and processed as described in Fig. 4C. Data represents mean ± SEM from four independent experiments, *P < 0.05, ***P < 0.001; ns, not significant. (D) Western blotting experiment was performed to assess RNAi-mediated knockdown efficiencies.