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Ectopic expression of RNF168 and 53BP1 increases mutagenic but not physiological non-homologous end joining.

Zong D, Callén E, Pegoraro G, Lukas C, Lukas J, Nussenzweig A - Nucleic Acids Res. (2015)

Bottom Line: Recently, it was shown that supra-physiological levels of the E3 ubiquitin ligase RNF168 results in the hyper-accumulation of 53BP1/BRCA1 which accelerates DSB repair.We find that the anti-resection activities of 53BP1 are rate-limiting for mutagenic NHEJ but not for physiological CSR.As heterogeneity in the expression of RNF168 and 53BP1 is found in human tumors, our results suggest that deregulation of the RNF168/53BP1 pathway could alter the chemosensitivity of BRCA1 deficient tumors.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Genome Integrity; National Cancer Institute; National Institutes of Health; Bethesda, MD 20892, USA.

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RNF168 and 53BP1 block RPA foci formation and RPA2 phosphorylation. (A) BRCA1Δ11/Δ11 MEFs stably transduced with retroviral vectors encoding RNF168WT or RNF168R57D were irradiated with 10 Gy and fixed 4 h later. Samples were processed for standard immunofluorescence. Left panel: cells were co-stained for RNF168 (green) and RPA2 (red) and imaged at 63× magnification. Note that the polyclonal anti-RNF168 antibody used in this study recognizes only the exogenously expressed human RNF168. A representative experiment is shown. Right panel: the percentage of cells that contain >10 RPA2 foci. (B) BRCA1Δ11/Δ11 MEFs stably transduced with retroviral vectors encoding 53BP1DB or 53BP1DN were irradiated and processed for standard immunofluorescence as in (A). Left panel: cells were co-stained for 53BP1 (green) and RPA2 (red) and imaged at 63x magnification. A representative experiment is shown. Right panel: the percentage of cells that contain >10 RPA2 foci. The right panels in A and B show mean ± SD of three independent experiments. At least 200 cells were scored for each sample and treatment condition. (C) Similar to (A), except cells were harvested at the indicated post-irradiation time points for western blot analysis. (D) Similar to (B), except cells were harvested at the indicated post-irradiation time points for western blot analysis. A representative blot is shown in (C) and (D). Experiments were repeated three times. For (A) and (B), statistical significance was determined with two-tailed unpaired Student's t-test; *, P < 0.05 compared to empty vector-transduced cells.
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Figure 4: RNF168 and 53BP1 block RPA foci formation and RPA2 phosphorylation. (A) BRCA1Δ11/Δ11 MEFs stably transduced with retroviral vectors encoding RNF168WT or RNF168R57D were irradiated with 10 Gy and fixed 4 h later. Samples were processed for standard immunofluorescence. Left panel: cells were co-stained for RNF168 (green) and RPA2 (red) and imaged at 63× magnification. Note that the polyclonal anti-RNF168 antibody used in this study recognizes only the exogenously expressed human RNF168. A representative experiment is shown. Right panel: the percentage of cells that contain >10 RPA2 foci. (B) BRCA1Δ11/Δ11 MEFs stably transduced with retroviral vectors encoding 53BP1DB or 53BP1DN were irradiated and processed for standard immunofluorescence as in (A). Left panel: cells were co-stained for 53BP1 (green) and RPA2 (red) and imaged at 63x magnification. A representative experiment is shown. Right panel: the percentage of cells that contain >10 RPA2 foci. The right panels in A and B show mean ± SD of three independent experiments. At least 200 cells were scored for each sample and treatment condition. (C) Similar to (A), except cells were harvested at the indicated post-irradiation time points for western blot analysis. (D) Similar to (B), except cells were harvested at the indicated post-irradiation time points for western blot analysis. A representative blot is shown in (C) and (D). Experiments were repeated three times. For (A) and (B), statistical significance was determined with two-tailed unpaired Student's t-test; *, P < 0.05 compared to empty vector-transduced cells.

Mentions: 53BP1 has been shown to promote mutagenic NHEJ in BRCA1-deficient cells by blocking end resection, which leads to PARPi hypersensitivity and tumorigenesis (10). For both 53BP1 and its immediate upstream regulator RNF168 we found copy number variations, genetic mutations and/or heterogeneous mRNA expression changes in a subset of BRCA1-deficient ovarian and breast tumors within the TCGA database (39–42(Figure 1). We therefore sought to determine whether modulating the chromatin loading of RNF168 and/or 53BP1 would alter PARPi sensitivity. To this end, we established mouse embryonic fibroblast (MEF) cell lines transduced with retroviral vectors encoding either wild-type RNF168 (RNF168WT) or a RNF168 mutant (RNF168R57D) that is incapable of 53BP1 recruitment (9) (Figure 2, Supplementary Figures S1A and Supplementary Figure S2). Since efficient transduction of MEFs with wild-type 53BP1 proved to be difficult due to its large size, we chose instead to overexpress 53BP1DB, a construct that lacks the extreme C-terminal BRCT domain but behaves like wild-type 53BP1 in terms of DSB end-protection (28), or a dominant negative fragment of 53BP1 (53BP1DN) (43) (Supplementary Figure S1B and C). As reported by Gudjonsson et al. (37), we found that overexpression of RNF168WT in BRCA1Δ11/Δ11 MEFs promoted the formation of conjugated ubiquitin and 53BP1 as well as RIF1 foci upon DNA damage caused by PARP inhibition or ionizing radiation (Figure 3A and Supplementary Figure S2). Interestingly, enlarged 53BP1 foci were also observed in BRCA1Δ11/Δ11 MEFs overexpressing 53BP1DB (Figure 4B). Thus, overexpression of 53BP1 itself can bypass the control of its spreading on damaged chromatin, which is normally limited by the availability of RNF168.


Ectopic expression of RNF168 and 53BP1 increases mutagenic but not physiological non-homologous end joining.

Zong D, Callén E, Pegoraro G, Lukas C, Lukas J, Nussenzweig A - Nucleic Acids Res. (2015)

RNF168 and 53BP1 block RPA foci formation and RPA2 phosphorylation. (A) BRCA1Δ11/Δ11 MEFs stably transduced with retroviral vectors encoding RNF168WT or RNF168R57D were irradiated with 10 Gy and fixed 4 h later. Samples were processed for standard immunofluorescence. Left panel: cells were co-stained for RNF168 (green) and RPA2 (red) and imaged at 63× magnification. Note that the polyclonal anti-RNF168 antibody used in this study recognizes only the exogenously expressed human RNF168. A representative experiment is shown. Right panel: the percentage of cells that contain >10 RPA2 foci. (B) BRCA1Δ11/Δ11 MEFs stably transduced with retroviral vectors encoding 53BP1DB or 53BP1DN were irradiated and processed for standard immunofluorescence as in (A). Left panel: cells were co-stained for 53BP1 (green) and RPA2 (red) and imaged at 63x magnification. A representative experiment is shown. Right panel: the percentage of cells that contain >10 RPA2 foci. The right panels in A and B show mean ± SD of three independent experiments. At least 200 cells were scored for each sample and treatment condition. (C) Similar to (A), except cells were harvested at the indicated post-irradiation time points for western blot analysis. (D) Similar to (B), except cells were harvested at the indicated post-irradiation time points for western blot analysis. A representative blot is shown in (C) and (D). Experiments were repeated three times. For (A) and (B), statistical significance was determined with two-tailed unpaired Student's t-test; *, P < 0.05 compared to empty vector-transduced cells.
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Figure 4: RNF168 and 53BP1 block RPA foci formation and RPA2 phosphorylation. (A) BRCA1Δ11/Δ11 MEFs stably transduced with retroviral vectors encoding RNF168WT or RNF168R57D were irradiated with 10 Gy and fixed 4 h later. Samples were processed for standard immunofluorescence. Left panel: cells were co-stained for RNF168 (green) and RPA2 (red) and imaged at 63× magnification. Note that the polyclonal anti-RNF168 antibody used in this study recognizes only the exogenously expressed human RNF168. A representative experiment is shown. Right panel: the percentage of cells that contain >10 RPA2 foci. (B) BRCA1Δ11/Δ11 MEFs stably transduced with retroviral vectors encoding 53BP1DB or 53BP1DN were irradiated and processed for standard immunofluorescence as in (A). Left panel: cells were co-stained for 53BP1 (green) and RPA2 (red) and imaged at 63x magnification. A representative experiment is shown. Right panel: the percentage of cells that contain >10 RPA2 foci. The right panels in A and B show mean ± SD of three independent experiments. At least 200 cells were scored for each sample and treatment condition. (C) Similar to (A), except cells were harvested at the indicated post-irradiation time points for western blot analysis. (D) Similar to (B), except cells were harvested at the indicated post-irradiation time points for western blot analysis. A representative blot is shown in (C) and (D). Experiments were repeated three times. For (A) and (B), statistical significance was determined with two-tailed unpaired Student's t-test; *, P < 0.05 compared to empty vector-transduced cells.
Mentions: 53BP1 has been shown to promote mutagenic NHEJ in BRCA1-deficient cells by blocking end resection, which leads to PARPi hypersensitivity and tumorigenesis (10). For both 53BP1 and its immediate upstream regulator RNF168 we found copy number variations, genetic mutations and/or heterogeneous mRNA expression changes in a subset of BRCA1-deficient ovarian and breast tumors within the TCGA database (39–42(Figure 1). We therefore sought to determine whether modulating the chromatin loading of RNF168 and/or 53BP1 would alter PARPi sensitivity. To this end, we established mouse embryonic fibroblast (MEF) cell lines transduced with retroviral vectors encoding either wild-type RNF168 (RNF168WT) or a RNF168 mutant (RNF168R57D) that is incapable of 53BP1 recruitment (9) (Figure 2, Supplementary Figures S1A and Supplementary Figure S2). Since efficient transduction of MEFs with wild-type 53BP1 proved to be difficult due to its large size, we chose instead to overexpress 53BP1DB, a construct that lacks the extreme C-terminal BRCT domain but behaves like wild-type 53BP1 in terms of DSB end-protection (28), or a dominant negative fragment of 53BP1 (53BP1DN) (43) (Supplementary Figure S1B and C). As reported by Gudjonsson et al. (37), we found that overexpression of RNF168WT in BRCA1Δ11/Δ11 MEFs promoted the formation of conjugated ubiquitin and 53BP1 as well as RIF1 foci upon DNA damage caused by PARP inhibition or ionizing radiation (Figure 3A and Supplementary Figure S2). Interestingly, enlarged 53BP1 foci were also observed in BRCA1Δ11/Δ11 MEFs overexpressing 53BP1DB (Figure 4B). Thus, overexpression of 53BP1 itself can bypass the control of its spreading on damaged chromatin, which is normally limited by the availability of RNF168.

Bottom Line: Recently, it was shown that supra-physiological levels of the E3 ubiquitin ligase RNF168 results in the hyper-accumulation of 53BP1/BRCA1 which accelerates DSB repair.We find that the anti-resection activities of 53BP1 are rate-limiting for mutagenic NHEJ but not for physiological CSR.As heterogeneity in the expression of RNF168 and 53BP1 is found in human tumors, our results suggest that deregulation of the RNF168/53BP1 pathway could alter the chemosensitivity of BRCA1 deficient tumors.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Genome Integrity; National Cancer Institute; National Institutes of Health; Bethesda, MD 20892, USA.

Show MeSH
Related in: MedlinePlus