SMC5/6-mediated plasmid silencing is directed by SIMC1–SLF2 and antagonized by the SV40 large T antigen
- Department of Molecular and Cellular Biology, The Scripps Research Institute, United States
- San Diego Biomedical Research Institute, United States
Figures
Figure 1 with 1 supplement
SIMC1–SLF2 composite surface patch is required for SMC6 association.
(A) The cryo-EM structure of the SIMC1–SLF2 complex (PDB ID: 7T5P) (Oravcová et al., 2022). Residues mutated for interaction analysis are shown with side chains. The arrow at the bottom indicates the viewing angle for (B). (B) Conservation mapping on the surface of SIMC1–SLF2 complex, showing the N-terminus of SIMC1 and the C-terminus of SLF2. Conservation scores obtained from the Consurf server (Ashkenazy et al., 2016) are shown by the color graduation as indicated. Amino acids mutated in SIMC1combo (R473D/N477A/E480K/E481K in α1 of SIMC1), SLF2 mut1 (E1121A/K1122A/K1125D/C1126R/E1130K in α16 of SLF2), and SLF2 mut2 (T1138R/K1141E/D1142R/A1145R/G1149E in α17 of SLF2) are labeled. (C) Western blot of GFP-Trap immunoprecipitation from HEK293 cells transfected with plasmids expressing GFP-SIMC1, Myc-SMC6, and FLAG-tagged C-terminal domain (CTD) of SLF2, either WT or containing mutations (mut1, mut2). Input and GFP PD were detected with anti-GFP, FLAG, or SMC6 antibody. Full and unedited blots provided in Figure 1—source data 1.
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Figure 1—source data 1
Full and unedited blots corresponding to panel C.
- https://cdn.elifesciences.org/articles/106815/elife-106815-fig1-data1-v1.zip
Figure 1—figure supplement 1
SIMC1 combo control mutant and SLF2635-1160 retain interaction with SMC6.
(A) Western blot of GFP-trap immunoprecipitation from HEK293 cells transiently transfected, respectively, with GFP-SIMC1, GFP-SIMC1 combo mutant, or GFP-SIMC1 combo control mutant in combination with FLAG-SLF2CTD and Myc-SMC6. Signals were detected using FLAG, SMC6, and GFP antibodies. Full and unedited blots provided in Figure 1—figure supplement 1—source data 1. (B) Western blot of GFP-trap immunoprecipitation from HEK293 cells transiently transfected with either FLAG-SLF2CTD or FLAG-SLF2635-1160 co-transfected with GFP-SIMC1 and Myc-SMC6. Signals were visualized using FLAG, Myc, and GFP antibodies. Full and unedited blots provided in Figure 1—figure supplement 1—source data 2.
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Figure 1—figure supplement 1—source data 1
Full and unedited blots corresponding to panel A.
- https://cdn.elifesciences.org/articles/106815/elife-106815-fig1-figsupp1-data1-v1.zip
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Figure 1—figure supplement 1—source data 2
Full and unedited blots corresponding to panel B.
- https://cdn.elifesciences.org/articles/106815/elife-106815-fig1-figsupp1-data2-v1.zip
Figure 2 with 1 supplement
SIMC1–SLF2 subcomplex contacts the neck region of SMC6.
(A) The AlphaFold-Multimer model of the SIMC1Nse5–SLF2Nse6–SMC6 complex. The disordered regions in the N- and C-termini, the hinge domain, and the majority of the coiled-coil that do not contact SIMC1–SLF2 are omitted for clarity. The entire model and the predicted aligned error (PAE) plot of the prediction are shown in Figure 2—figure supplement 1. Residues mutated for interaction analysis are shown with side chains. (B) A close-up view of the interface between SMC6’s neck and the composite patch of SIMC1–SLF2. (C) Western blot of GFP-Trap immunoprecipitation from HEK293 cells transfected with plasmids expressing 2Myc-SIMC1, FLAG-SLF2CTD, and WT or mutant GFP-SMC6 (SIMC1-facing: K942A, D946A, K957A; SLF2-facing: R940A, Y944A, N947A; groove-facing: L939A, R940A, K942A, L943A). Input and GFP PD were detected with anti-GFP, FLAG, or Myc antibody. Full and unedited blots provided in Figure 2—source data 1. (D) Schematic of yeast (left) and human (right) SMC5/6 complex illustrating positions of its subunits within the complex and Nse5/Nse6 and SIMC1/SLF2 cofactors, respectively, interacting with the neck region of SMC6.
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Figure 2—source data 1
Full and unedited blots corresponding to panel C.
- https://cdn.elifesciences.org/articles/106815/elife-106815-fig2-data1-v1.zip
Figure 2—figure supplement 1
Structural model and interface analyses of the SMC6–SIMC1–SLF2 complex.
(A) The AlphaFold-Multimer model of the SIMC1Nse5 (425–872)–SLF2Nse6 (733–1173)–SMC6 (1–1091) complex. The entire model is shown, colored according to pLDDT values (left) or in the same manner as in Figure 2A. (B) The predicted aligned error (PAE) plot of the AlphaFold-Multimer prediction. Close-up views of the SMC6 neck-SIMC1 (C) and SMC6 neck-SLF2 (D) interfaces.
Figure 3 with 1 supplement
SIMC1/SLF2–SMC6 interaction is critical for plasmid silencing.
(A) Expression of a GFP reporter transiently transfected into U2OS WT and SIMC1-/- or SLF2-/- cell lines was measured by reverse transcription-quantitative PCR 72 hr after transfection. GFP expression was normalized to the expression of beta-actin (mean ± s.d. from n = 4 independent experiments, two-tailed unpaired t-test; **p < 0.005). Primary data provided in Figure 3—source data 1. (B) U2OS WT, SIMC1-/-, or SLF2-/- cells with integrated empty vector or vector expressing SIMC1 or SLF2 variants (WT, mut1), respectively, were transiently transfected with GFP reporter. After 72 hr, GFP intensity was measured by FACS and is displayed relative to GFP intensity measured in WT cells. Data are the average ± s.d. from n = 3 independent experiments, two-tailed unpaired t-test; **p < 0.005. No significant difference (p > 0.05) was found between WT and SIMC1-/- + pSIMC1 (left panel), or between WT and SLF2-/- + pSLF2 (right panel). Primary data provided in Figure 3—source data 2.
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Figure 3—source data 1
GFP transcripts qPCR corresponding to panel A.
- https://cdn.elifesciences.org/articles/106815/elife-106815-fig3-data1-v1.xlsx
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Figure 3—source data 2
GFP FACS corresponding to panel B.
- https://cdn.elifesciences.org/articles/106815/elife-106815-fig3-data2-v1.xlsx
Figure 3—figure supplement 1
Plasmid silencing defect in SLF2-/- cells is not caused by increased plasmid copy number and is lost upon plasmid integration into the genome.
(A) DNA from U2OS WT or SLF2-/- cells harboring either integrated or transiently transfected GFP plasmid was isolated 72 hr after transfection, GFP content was analyzed by quantitative PCR and normalized to beta-actin (mean ± s.d. from n = 3 independent experiments, two-tailed unpaired t-test; p > 0.05). Primary data provided in Figure 3—figure supplement 1—source data 1. (B) Expression of endogenous and integrated SLF2 WT or SLF2mut1 in U2OS WT or SLF2-/- cells was measured by reverse transcription-quantitative PCR and normalized to beta-actin (mean ±s.d. from n = 5 independent experiments). Primary data provided in Figure 3—figure supplement 1—source data 2. (C) GFP intensity from integrated or transfected GFP vector was measured by flow cytometry 72 hr after reporter transient transfection in U2OS WT cells or SLF2-/- cells. Data are the average ± s.d. from n = 3 independent experiments, two-tailed unpaired t-test; **p-value <0.005. Primary data provided in Figure 3—figure supplement 1—source data 3.
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Figure 3—figure supplement 1—source data 1
GFP DNA qPCR corresponding to panel A.
- https://cdn.elifesciences.org/articles/106815/elife-106815-fig3-figsupp1-data1-v1.xlsx
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Figure 3—figure supplement 1—source data 2
SLF2 transcripts qPCR corresponding to panel B.
- https://cdn.elifesciences.org/articles/106815/elife-106815-fig3-figsupp1-data2-v1.xlsx
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Figure 3—figure supplement 1—source data 3
GFP FACS corresponding to panel C.
- https://cdn.elifesciences.org/articles/106815/elife-106815-fig3-figsupp1-data3-v1.xlsx
Figure 4
SLF1 is involved in SMC5/6 DNA damage repair, not plasmid silencing.
(A) Representative immunofluorescence images of U2OS WT, SIMC1-/-, SLF1-/- cells exposed to laser microirradiation upon treatment with 50 µM angelicin and 1 µg/ml Hoechst 33342 for 30 min. One hour after microirradiation, cells were pre-extracted, fixed, and stained with gamma-H2A.X (phosphorylation of histone H2A.X Ser139; green), SMC6 (red) antibodies and DAPI (blue). Scale bar 10 μm. (B) GFP intensity was measured by flow cytometry 72 hr after reporter transient transfection in U2OS WT cells or SLF1-/- cells with integrated empty vector or stably expressing SLF1. Data are the average ± s.d. from n = 3 independent experiments, two-tailed unpaired t-test (p-value >0.05). Primary data provided in Figure 4—source data 1.
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Figure 4—source data 1
GFP FACS corresponding to panel B.
- https://cdn.elifesciences.org/articles/106815/elife-106815-fig4-data1-v1.xlsx
Figure 5 with 3 supplements
Plasmid silencing depends on the SUMO pathway and LT, not PML NBs.
(A) U2OS WT, SIMC1-/-, or SLF2-/- cells were transiently transfected with GFP reporter and treated with 100 nM TAK-981 (SUMOi) or DMSO at the same time. After 72 hr, GFP intensity was determined by FACS. Data are normalized to WT cells treated with DMSO and represent the average ± s.d. from n = 4 independent experiments, two-tailed unpaired t-test; **p < 0.005. No significant difference was found between WT and SIMC1-/- or SLF2-/- when treated with SUMOi (p > 0.05). Primary data provided in Figure 5—source data 1. (B) U2OS WT or PML-/- cells with integrated empty vector or vector expressing PML, respectively, were transiently transfected with GFP reporter. Intensity of GFP was measured by FACS 72 hr after transfection. Data are the average ± s.d. from n = 3 independent experiments, two-tailed unpaired t-test; *p < 0.05. No significant difference (p > 0.05) was found between WT and PML-/- + pPML. Primary data provided in Figure 5—source data 2. (C) Western blot of GFP-trap immunoprecipitation from HEK293 cells transiently transfected with either GFP-SMC5 or GFP alone in combination with SV40 vector expressing LT and Myc-SMC6. Signals were visualized using GFP, LT, and SMC6 antibodies. Full and unedited blots provided in Figure 5—source data 3. (D) GFP intensity measured by FACS in U2OS WT, SIMC1-/-, or SLF2-/- cells with integrated empty vector, vector expressing large T antigen (LT) or LT-HR684 variant, respectively, that were transiently transfected with GFP reporter for 72 hr. Data are the average ± s.d. from n = 4 independent experiments. *p < 0.05; ***p < 0.0005; n.s., p > 0.05 (two-tailed unpaired t-test). Primary data provided in Figure 5—source data 4.
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Figure 5—source data 1
GFP FACS corresponding to panel A.
- https://cdn.elifesciences.org/articles/106815/elife-106815-fig5-data1-v1.xlsx
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Figure 5—source data 2
GFP FACS corresponding to panel B.
- https://cdn.elifesciences.org/articles/106815/elife-106815-fig5-data2-v1.xlsx
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Figure 5—source data 3
Full and unedited blots corresponding to panel C.
- https://cdn.elifesciences.org/articles/106815/elife-106815-fig5-data3-v1.zip
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Figure 5—source data 4
GFP FACS corresponding to panel D.
- https://cdn.elifesciences.org/articles/106815/elife-106815-fig5-data4-v1.xlsx
Figure 5—figure supplement 1
Arbitrary luminescence units of U2OS (A) or RPE (B) cells expressing transfected pLuc reporter.
Cells were treated with TAK-981 or DMSO at the time of transfection. Means and error bars (s.d.) were derived from a minimum of n = 3 independent transfections representing biological replicates. Two-tailed unpaired t-test was performed (**p < 0.005; ***p < 0.0005). Primary data provided in Figure 5—figure supplement 1—source data 1, Figure 5—figure supplement 1—source data 2, respectively. (C) U2OS wt and SLF2-/- cells stably expressing GFP were treated with 100 nM TAK-981 (SUMOi) or DMSO, and GFP intensity was determined by FACS 72 hr later. Data normalized to WT cells in DMSO represent the average ± s.d. from n = 3 independent experiments. Primary data provided in Figure 5—figure supplement 1—source data 3.
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Figure 5—figure supplement 1—source data 1
Luciferase measurement corresponding to panel A.
- https://cdn.elifesciences.org/articles/106815/elife-106815-fig5-figsupp1-data1-v1.xls
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Figure 5—figure supplement 1—source data 2
Luciferase measurement corresponding to panel B.
- https://cdn.elifesciences.org/articles/106815/elife-106815-fig5-figsupp1-data2-v1.xlsx
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Figure 5—figure supplement 1—source data 3
GFP FACS corresponding to panel C.
- https://cdn.elifesciences.org/articles/106815/elife-106815-fig5-figsupp1-data3-v1.xlsx
Figure 5—figure supplement 2
Representative immunofluorescence images of U2OS WT or SLF2-/- cells fixed and stained with SMC6 (green) and PML (red) antibodies along with DAPI (blue).
Scale bar 10 μm.
Figure 5—figure supplement 3
LT does not promote expression from integrated GFP reporter.
(A) Immunoblot from U2OS WT, SIMC1-/-, or SLF2-/- cells stably expressing large T antigen (LT), LT-HR684 variant, or have an empty vector integrated, respectively. PSTAIR serves as a loading control. Full and unedited blots provided in Figure 5—figure supplement 3—source data 1. (B) U2OS wt and SLF2-/- cells stably expressing GFP were transiently transfected with vector expressing LT. After 72 hr, GFP intensity was measured by FACS and data normalized to non-transfected WT cells (average ± s.d. from n = 3 independent experiments). Primary data provided in Figure 5—figure supplement 3—source data 2.
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Figure 5—figure supplement 3—source data 1
Full and unedited blots.
- https://cdn.elifesciences.org/articles/106815/elife-106815-fig5-figsupp3-data1-v1.zip
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Figure 5—figure supplement 3—source data 2
GFP FACS corresponding to panel B.
- https://cdn.elifesciences.org/articles/106815/elife-106815-fig5-figsupp3-data2-v1.xlsx
Figure 6
SIMC1–SLF2 and SLF1/2 subcomplexes direct SMC5/6 to separate pathways of plasmid silencing and DNA lesion repair.
SIMC1–SLF2 and SMC5/6-mediated plasmid silencing is facilitated by the SUMO pathway and antagonized by SV40 LT. PML NBs are not involved in plasmid silencing. S stands for SUMO; U for ubiquitin. Both SIMC1–SLF2 and SLF1/2 likely contact SMC6 directly through their conserved Nse5/6-like domains, but the SIMC1 SIMs and SLF1 BRCT/ARD domains contact distinct posttranslational modifiers to direct the complex to its separate roles.
Tables
Author response table 1
| mutant | % |
|---|---|
| GFP-SMC6 blot | |
| wt-SMC6 | 100 |
| SLF2 only | 120,0891 |
| Myc-SIMC1 blot | |
| wt-SMC6 | 100 |
| SLF2 only | 43,88612 |
| Flag-SLF2 blot | |
| wt-SMC6 | 100 |
| SLF2 only | 21,28111 |
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SMC5/6-mediated plasmid silencing is directed by SIMC1–SLF2 and antagonized by the SV40 large T antigen
eLife 14:RP106815.
https://doi.org/10.7554/eLife.106815.3
