SIMC1-SLF2 composite surface patch is required for SMC6 association.

(A) The cryo-EM structure of the SIMC1-SLF2 complex (PDB ID: 7T5P) (33). Residues mutated for interaction analysis are shown with side chains. (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 (35) 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.

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 coilded-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 only: K942A, D946A, K957A; SLF2 only: R940A, Y944A, N947A; Mixed: L939A, R940A, K942A, L943A). Input and GFP PD were detected with anti-GFP, FLAG, or Myc antibody. (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.

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 quantitative PCR 72 hours 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). (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 hours, 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).

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 uM angelicin and 1 ug/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).

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 hours, 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). (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 hours 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. (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. (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 hours. 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).

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.

(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. (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.

(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. (C, D) Close-up views of the SMC6 neck-SIMC1 (C) and SMC6 neck-SLF2 (D) interfaces.

HEK293 WT or SIMC1-/- cells were transiently transfected with GFP reporter and GFP intensity was assessed 72 hours later by FACS.

Data are the average ± s.d. from n=6 independent experiments, two-tailed unpaired t-test; (***) p<0.0005).

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.

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).

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.