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. 2018 Dec;16(6):428-438.
doi: 10.1016/j.gpb.2018.07.004. Epub 2018 Dec 4.

RNF126 Quenches RNF168 Function in the DNA Damage Response

Lianzhong Zhang  1 , Zhenzhen Wang  2 , Ruifeng Shi  3 , Xuefei Zhu  4 , Jiahui Zhou  2 , Bin Peng  4 , Xingzhi Xu  5
Affiliations

RNF126 Quenches RNF168 Function in the DNA Damage Response

Lianzhong Zhang et al. Genomics Proteomics Bioinformatics. 2018 Dec.

Abstract

DNA damage response (DDR) is essential for maintaining genome stability and protecting cells from tumorigenesis. Ubiquitin and ubiquitin-like modifications play an important role in DDR, from signaling DNA damage to mediating DNA repair. In this report, we found that the E3 ligase ring finger protein 126 (RNF126) was recruited to UV laser micro-irradiation-induced stripes in a RNF8-dependent manner. RNF126 directly interacted with and ubiquitinated another E3 ligase, RNF168. Overexpression of wild type RNF126, but not catalytically-inactive mutant RNF126 (CC229/232AA), diminished ubiquitination of H2A histone family member X (H2AX), and subsequent bleomycin-induced focus formation of total ubiquitin FK2, TP53-binding protein 1 (53BP1), and receptor-associated protein 80 (RAP80). Interestingly, both RNF126 overexpression and RNF126 downregulation compromised homologous recombination (HR)-mediated repair of DNA double-strand breaks (DSBs). Taken together, our findings demonstrate that RNF126 negatively regulates RNF168 function in DDR and its appropriate cellular expression levels are essential for HR-mediated DSB repair.

Keywords: DNA damage response; DNA repair; RNF126; RNF168; RNF8; Ubiquitination.

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Figures

Figure 1
Figure 1
RNF126 is recruited to the UV laser micro-irradiation-induced DNA damage stripes A. and B. Both wild-type (WT) RNF126 and catalytically-inactive RNF126(CC229/232AA) are recruited to DNA damage stripes. U2OS cells transiently expressing GFP-RNF126 or GFP-RNF126(CC229/232AA) were irradiated with a 365-nm UV laser beam (white dashed line). Images were collected every 30 sec after irradiation and representative images are shown (A). The recruitment kinetics of GFP-RNF126 (WT) and GFP-RNF126(CC229/232AA) were assessed in terms of signal intensity at DNA damage stripes relative to the un-irradiated area in three independent experiments (B). Data represent the mean ± SD. C. Diagram depicting the domain structure of RNF126 and its truncation mutants containing the N-terminus (amino acid residues 1–100), middle region (amino acid residues 101–200), and C-terminus (amino acid residues 201–311), respectively. D. and E. U2OS cells expressing GFP-tagged RNF126 truncation mutants were subjected to UV laser micro-irradiation and the recruitment of GFP fusion proteins to the DNA damage stripes were monitored in live cells. Representative images are shown (D). The percentage of cells positive for GFP fusion protein enrichment at DNA damage stripes was determined by analyzing >100 GFP-positive cells for each GFP fusion protein (E). Data represent the mean ± SD. A two-way ANOVA was performed. **P < 0.01. Scale bar, 10 μm. RING, really interesting new gene; RNF, ring finger protein.
Figure 2
Figure 2
RNF126 interacts with RNF8 and RNF168 A. Endogenous RNF126 interacts with both RNF8 and RNF168. Total lysates from HEK293T cells were immunoprecipitated with an anti-RNF126 antibody, and the immunocomplexes were exposed to the indicated antibodies. B. Epitope-tagged RNF126 interacts with RNF8 and RNF168. A GFP-RNF126 expression construct was co-transfected with FLAG-RNF8 or FLAG-RNF168 in HEK293T cells. Total lysates were harvested 48 h after transfection and subjected to immunoprecipitation with anti-FLAG beads followed by immunoblotting with the indicated antibodies. C. The RNF126 N-terminus (amino acid residues 1–100) interacts with RNF8. FLAG-RNF8 was co-expressed with GFP-RNF126 or its truncation mutants in HEK293T cells. D. The FHA domain-containing N-terminus of RNF8 (amino acid residues 1–141) interacts with RNF126. HA-RNF126 was co-expressed with GFP-RNF8 or its truncation mutants in 293T cells. E. The RNF126 N-terminus interacts with RNF168. FLAG-RNF168 was co-expressed with GFP-RNF126 or its truncation mutants in HEK293T cells. F. The UMI and MIU1 domains-containing region of RNF168 (amino acid residues 90–210) interact with RNF126. GFP-RNF126 was co-expressed with HA-RNF168 or its truncation mutants in HEK293T cells. G. Both UMI domain and MIU1 domain of RNF168 are essential for its interaction with RNF126. GFP-RNF126 was co-expressed with wild-type HA-RNF168, the UMI point mutant HA-RNF168(LL149/150AA), or the MIU1 point mutant HA-RNF168(A179G) in HEK293T cells. In panels C–G, total cell lysates were harvested 48 h after transfection and subjected to immunoprecipitation and immunoblotting with the indicated antibodies. IB, immunoblot; IP, immunoprecipitation; FHA, forkhead-associated; MIU1, motif interacting with ubiquitin 1; UMI, ubiquitin interacting motif and MIU-related ubiquitin binding domain.
Figure 3
Figure 3
RNF126 recruitment to DNA damage sites is RNF8-dependent A. The siRNF8 knockdown efficiency was determined by immunoblotting using the indicated antibodies. B. Representative images of GFP-RNF126 recruitment to DNA damage stripes (white dashed line). C. Percentage of cells exhibiting GFP-RNF126 recruitment to DNA damage stripes, quantitated from three independent experiments, for each of which at least 100 GFP-positive cells were evaluated. Data represent the mean ± SD. A two-way ANOVA was performed. **P < 0.01. In panels A–C, U2OS cells expressing GFP-RNF126 were transfected with a control siRNA oligo (siCTR) or a siRNA oligo specifically targeting RNF8 [siRNF8 (3′UTR)-1)]. D. Inhibition of RNF126 expression has no obvious impact on RNF168 recruitment to DNA damage sites. U2OS cells expressing GFP-RNF168 were transfected with a control siRNA oligo (siCTR) or a siRNA oligo specifically targeting RNF126 (siRNF126-A) and subjected to UV laser micro-irradiation 48 h after transfection. Representative images of GFP-RNF168 recruitment to DNA damage stripes are shown. IB, immunoblot. Scale bar, 10 μm.
Figure 4
Figure 4
RNF126 suppresses recruitment of RNF168 and downstream DDR factors to sites of DNA damage A. Over-expression of wild-type RNF126, but not catalytically-inactive RNF168(CC229/232AA), compromises the recruitment of RNF168 but not RNF8 to DNA damage stripes. U2OS cells expressing RFP-RNF8 or RNF168 were transfected with GFP vector, GFP-RNF126, or GFP-RNF126(CC229/232AA) and subjected to UV laser micro-irradiation 48 h after transfection. The percentage of RFP-positive DNA damage stripes compared to cells dually positive for RFP and GFP was quantitated in three randomly-selected fields on the condition that each field had >100 cells dually positive for GFP and RFP. B. Over-expression of wild-type RNF126, but not catalytically-inactive RNF168(CC229/232AA), compromises the recruitment of RNF168 downstream factors to bleomycin-induced DNA damage sites. U2OS cells expressing GFP vector, GFP-RNF126 or GFP-RNF126(CC229/232AA) were subjected to bleomycin treatment (10 μg/ml) for 1 h before immunofluorescence staining with the indicated antibodies. The percentage of cells with >5 (for RAP80 and 53BP1) or >10 foci (for FK2, MDC1, and γH2AX) over GFP-positive cells was quantitated in three randomly-selected fields on the condition that each field had >100 GFP-positive cells. Data represent the mean ± SD. A two-way ANOVA was performed. **P < 0.01; ns, not significant; IB, immunoblot; RAP80, receptor-associated protein 80; 53BP1, TP53-binding protein 1; MDC1, mediator of DNA damage checkpoint protein 1; γH2AX, phosphorylated H2A histone family member X.
Figure 5
Figure 5
RNF126 suppresses UBC13 binding to RNF168 and RNF168-mediated H2AX ubiquitination A. RNF126 directly interacts with RNF168. GST pulldown assays were performed using bacterially-produced GST-RNF126 to pull down FLAG-RNF168 present in the total cell lysate of HEK293T cells transiently expressing FLAG-RNF168 or bacterially-produced His-RNF168. B. RNF126 negatively modulates the interaction between RNF168 and UBC13. HEK293T cells transiently co-expressing HA-RNF168 and FLAG-UBC13 and GFP-RNF126 or GFP-RNF126(CC229/232AA) were subjected to mock treatment or bleomycin treatment for 1 h. Total cell lysates were extracted with SDS-containing lysis buffer and boiled for 10 min before subjected to immunoprecipitation with anti-FLAG beads followed by immunoblotting with the indicated antibodies. C. RNF126 ubiquitinates RNF168 in vitro. In vitro ubiquitination assays were performed by incubating bacterially-produced wild-type GST-RNF126 or catalytically-inactive GST-RNF126(CC229/232AA) with wild-type His-RNF168 or catalytically-inactive His-RNF168(CC16/19SS) in the presence of UBE1, UbcH5b, and HA-ubiquitin at 30 °C for 1 h. The reactions were analyzed by immunoblotting with the indicated antibodies. D. Over-expression of wild-type RNF126, but not catalytically-inactive RNF126(CC229/232AA), compromises H2AX ubiquitination. Total cell lysates derived from HEK293T cells co-expressing Myc-RNF168 and FLAG-H2AX and GFP-RNF126 or GFP-RNF126(CC229/232AA) were subjected to immunoprecipitation with anti-FLAG beads followed by immunoblotting with the indicated antibodies. IB, immunoblot; IP, immunoprecipitation; UBC13, E2 ubiquitin-conjugating enzyme N; UBE1, ubiquitin-like modifier-activating enzyme 1; UbcH5b, ubiquitin-conjugating enzyme E2 D2.
Figure 6
Figure 6
Maintaining proper levels of RNF126 is essential for HR-mediated DSB repair A. and B. Inhibition of RNF126 expression compromises HR-mediated DSB repair. Endogenous expression of RNF126 in DR-U2OS cells was inhibited by transfection with siRNF126 (3′UTR-1) that specifically targeted the 3′UTR of RNF126. Wild-type RNF126 or catalytically-inactive RNF126(CC229/232AA) were re-introduced into RNF126-depleted cells by retroviral infection. A. Expression levels of RNF126 were determined by immunoblotting with an anti-RNF126 antibody. B. HR-mediated DSB repair efficiency was based on the percentage of GFP-positive cells measured by flow cytometry. Three independent experiments were performed. C. Over-expression of RNF126 compromises HR-medicated DSB repair. DR-U2OS cells expressing mCherry vector or mCherry-RNF126 was infected with lentiviral particles encoding I-SceI. The percentage of GFP-positive cells over mCherry-positive cells was determined by flow cytometry to indicate the HR repair efficiency. Three independent experiments were performed. Data represent the mean ± SD. A two-way ANOVA was performed. **P < 0.01. IB, immunoblotting; siCTR, siRNA control; HR, homologous recombination; ns, not significant.
Supplementary Figure S1
Supplementary Figure S1
RNF126 recruitment to the DNA damage stripe is dependent on PARP1 and ATM A. U2OS cells transiently expressing GFP-RNF126 were pre-treated with mock control (DMSO), ATM inhibitor KU55933, ATR inhibitor NU6027, PARP inhibitor olaparib, or DNA-PKcs inhibitor NU7026 for 1 h before subjected to micro-irradiation with a 365-nm UV laser beam (white dashed line). Images were collected every 30 s after irradiation and representative images are shown. B. The percentage of cells positive for GFP-RNF126 enrichment at DNA damage stripes was determined by analyzing >100 GFP-positive cells for each treatment. Data represent the mean ± SD. A two-way ANOVA was performed. **P < 0.01. ns, not significant. Scale bar, 10 μm. ATM, ataxia telangiectasia mutated; ATR, ataxia telangiectasia and Rad3-related protein; DNA-PKcs, DNA-dependent protein kinase catalytic subunit; PARP1, poly [ADP-ribose] polymerase 1.
Supplementary Figure S2
Supplementary Figure S2
RNF126 interacts with RNF8 and RNF168 A. Diagram depicting the domain structure of RNF8 and its truncation mutants. B. Diagram depicting the domain structure of RNF168 and truncation mutants. C. Both the UMI and MIU1 domains of RNF168 are essential to mediate the interaction between RNF168 and RNF126. GFP-RNF126 was co-expressed with wild-type HA-RNF168(90−210), the UMI point mutant HA-RNF168(90−210)(LL149/150AA), or the MIU1 point mutant HA-RNF168(90−210)(A179G) in HEK293T cells. Total cell lysates were harvested 48 h after transfection and subjected to immunoprecipitation and immunoblotting with the indicated antibodies. IB, immunoblot; FHA, forkhead-associated; MIU1, motif interacting with ubiquitin; UMI, ubiquitin interacting motif and MIU-related ubiquitin binding domain.
Supplementary Figure S3
Supplementary Figure S3
RNF126 recruitment to DNA damage sites is RNF8-dependent U2OS cells were transfected with a control siRNA oligo (siCTR) or a siRNA oligo specifically targeting RNF8 [siRNF8(3’UTR-1)]. A. The siRNF8 knockdown efficiency was determined by immunoblotting with the indicated antibodies. B. Representative images of DNA damage-induced RNF126 foci and γH2AX. The transfected cells were treated with bleomycin (10 mg/ml) for 1 h before co-immunofluorescence staining with anti-RNF126 (green) and anti-γH2AX (red). Genomic DNA was stained with DAPI (blue). C. The percentage of cells with >10 RNF126 foci was quantitated from three randomly selected fields on the condition that each field had >100 cells. Data represent the mean ± SD. A two-way ANOVA was performed. **P < 0.01. IB, immunoblot. Scale bar, 10 μm.
Supplementary Figure S4
Supplementary Figure S4
RNF126 recruitment to DNA damage sites is RNF168-dependent U2OS cells stably expressing GFP-RNF126 were transfected with a control siRNA oligo (siCTR) or a siRNA oligo specifically targeting RNF168 (siRNF168-A). A. The siRNF168 knockdown efficiency was determined by immunoblotting with the indicated antibodies. B. Representative images of UV laser micro-irradiation-induced RNF126 stripes were shown. C. The percentage of cells positive for GFP-RNF126 enrichment at DNA damage stripes was determined by analyzing >100 GFP-positive cells for each treatment. Data represent the mean ± SD. Scale bar, 10 μm.
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