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Smc5/6 silences episomal transcription by a three-step function

Abstract

In addition to its role in chromosome maintenance, the six-membered Smc5/6 complex functions as a restriction factor that binds to and transcriptionally silences viral and other episomal DNA. However, the underlying mechanism is unknown. Here, we show that transcriptional silencing by the human Smc5/6 complex is a three-step process. The first step is entrapment of the episomal DNA by a mechanism dependent on Smc5/6 ATPase activity and a function of its Nse4a subunit for which the Nse4b paralog cannot substitute. The second step results in Smc5/6 recruitment to promyelocytic leukemia nuclear bodies by SLF2 (the human ortholog of Nse6). The third step promotes silencing through a mechanism requiring Nse2 but not its SUMO ligase activity. By contrast, the related cohesin and condensin complexes fail to bind to or silence episomal DNA, indicating a property unique to Smc5/6.

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Fig. 1: Only Smc5/6, not cohesin or condensin, binds to and silences episomal templates.
Fig. 2: Smc5 and Smc6 ATP binding and hydrolysis are required for episomal DNA restriction.
Fig. 3: Nse4b-containing Smc5/6 complexes support cell viability but fail to silence episomal DNA.
Fig. 4: Nse2 but not its SUMO ligase activity is essential for episomal restriction but not for Smc5/6 DNA binding and colocalization with PML-NBs.
Fig. 5: SLF2 promotes silencing by recruiting Smc5/6 to PML-NBs.

Data availability

Source data are provided with this paper. All other data are available from corresponding authors on reasonable request.

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Acknowledgements

We thank T. Gligoris and S. Gruber for help in designing the Smc5 and Smc6 ATPase mutants; O. Fernandez-Capetillo for providing the anti-Nse2 antibody; and S. Kassem and J. Curran for critical reading of the manuscript. This work was supported by grants from the Swiss National Science Foundation (310030-149626 and 310030-175781) to M.S., by the Canton of Geneva and by Gilead Sciences.

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Contributions

F.A., R.K.B., S.P.F. and M.S. conceived the study and designed the experiments. D.R. performed the PHH experiments shown in Extended Data Fig. 1a,b. D.R. and D.K. performed the confocal microscopy experiments shown in Fig. 4d. R.K.B. and D.K. performed the confocal microscopy experiments shown in Fig. 5d. F.A. performed all the other experiments. B.B. contributed to several figures. A.D. contributed to the revision of the manuscript. All authors interpreted the results. F.A. and M.S. wrote the manuscript with input from all authors.

Corresponding author

Correspondence to Michel Strubin.

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Competing interests

This study was partly funded by Gilead Sciences, Inc., and D.R., D.K., R.K.B. and S.P.F are employees of Gilead Sciences, Inc. The other authors declare no competing interests.

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Extended data

Extended Data Fig. 1 Smc5/6 is the only SMC complex to silence expression of the episomal HBV genome. Related to Fig. 1.

a, PHH were transfected with a non-targeting control siRNA (siCtrl) or with siRNA targeting the indicated Smc subunits. Cells were then incubated for 3 days and infected with wild-type (WT) or an HBx-deficient (ΔX) HBV. HBeAg secretion, a marker for HBV gene expression, was measured 14 days later. HBeAg levels are expressed as a percentage of those in control cells infected with HBV(WT) (grey bars) or in siSmc6-treated cells infected with HBV(ΔX) (black bars). Data are means ± SEM of three independent experiments with samples from one PHH donor. The schematic diagram summarizing the design of the study is shown at the top. b, The mRNA levels of the indicated Smc proteins in the samples analyzed in a were determined by real-time RT–PCR and normalized to β-actin. Values are expressed as a percentage of those in control cells (siCtrl). Data are means ± SEM of three independent experiments with one PHH donor. c, Luciferase assay for the ChIP experiment presented in Fig, 1c. Data are means ± SEM of 2 independent experiments. d, HA-Smc2 assembles into a condensin complex. Whole extracts prepared from HepG2 cells expressing the indicated HA-tagged SMC protein in a CRISPR/Cas9 knockout background were immunoprecipitated with anti-HA antibodies. The amounts recovered and the presence of Smc5, CAP-H and Nse4 in the eluates were assessed by Western blotting. CAP-H and Nse4 are the kleisin subunits of, respectively, condensin (Smc2) and the Smc5/6 complex. See Fig. 1a. Actin serves as a negative control. The experiment was repeated twice independently with similar results.

Source data

Extended Data Fig. 2 Effect of single-subunit depletion on Smc5/6 complex integrity and restriction activity.

HepG2 cells were transduced with lentiviral constructs expressing Cas9 alone (Ctrl) or Cas9 together with sgRNA targeting the indicated Smc5/6 subunits, or with lentiviruses encoding GFP or GFP-HBx, as indicated. After selection, cells were transfected with a luciferase reporter plasmid. Luciferase assay and Western blot analysis were performed 5 days later. Hsp90 serves as a loading control. Unessential lanes were removed from the original blot images. Luciferase activity is relative to that measured in cells expressing HBx, which was set to 10. Data are means ± SEM of 3 independent experiments.

Source data

Extended Data Fig. 3 Involvement of Smc5 and Smc6 ATP binding and hydrolysis in Smc5/6 episomal DNA binding and restriction, and degradation by HBx. Related to Fig. 2.

a, Luciferase assay for the ChIP experiment presented in Fig. 2c. Data are means ± SEM of 3 independent experiments. b, Smc6 ATP binding mutants normally assemble into Smc5/6 complexes. Whole extracts prepared from HepG2 cells depleted of Smc6 and expressing HA-GFP or the indicated HA-tagged SMC protein were immunoprecipitated with anti-HA antibodies. The amounts recovered and the presence of other Smc5/6 subunits in the eluates were assessed by Western blotting. Hsp90 serves as a negative control. The experiment was repeated twice independently with similar results. c, Luciferase assay for the ChIP experiment presented in Fig, 2d. Data are means ± SEM of 3 independent experiments. d, HepG2 cells depleted of Smc6 and expressing GFP or the indicated wild-type or mutant Smc6 proteins from lentiviral vectors exactly as in Fig. 2b were also transduced with GFP or GFP-HBx. Western blot analysis was performed as before. GAPDH serves as a loading control. The experiment was repeated thrice independently with similar results.

Source data

Extended Data Fig. 4 Nse4a performs an essential function in Smc5/6 restriction for which Nse4b cannot substitute. Related to Fig. 3.

a, Schematic diagram of Nse4a (long), the shorter isoform of Nse4a, Nse4b and the Nse4b variant bearing the N-terminal region unique to Nse4a. White boxes indicate Nse4a sequences and the region of the short Nse4a protein common to both splicing isoforms. Hatched boxes indicate regions of Nse4b showing homology to Nse4a. The grey box indicates a region with no homology. Highlighted in black are the highly conserved N-terminal and C-terminal kleisin domains that have the potential to form helix-turn-helix and winged-helix motifs and are involved in Nse4 interaction with Smc6 and Smc5, respectively (Palecek et al., 2006; Vondrova et al., 2020). b, Luciferase assay for the ChIP experiment presented in Fig. 3b. Data are means ± SEM of 2 independent experiments. c, Same experiment as in Fig. 3a but including an Nse4b chimeric protein carrying the N-terminal region unique to Nse4a (Nse4a-b; see panel a). Expression of Nse4b and Nse4a-b was inferred from their stabilization effect on the other Smc5/6 subunits (lane 8). Data are means ± SEM of 2 independent experiments.

Source data

Extended Data Fig. 5 Nse1 and Nse3 DNA-binding mutants are functional in vivo.

a,b, Control HepG2 cells (black bars) and cells depleted of Nse1 (a) or Nse3 (b; grey bars) were transfected with a luciferase reporter plasmid and shortly after transduced with GFP or the corresponding wild-type or DNA-binding mutant protein as indicated (Zabrady et al., 2016). Luciferase assay and Western blot analysis were as before. Data are means ± SEM of 3 independent experiments.

Source data

Extended Data Fig. 6 HBx triggers Smc5/6 degradation in the absence of Nse2. Related to Fig. 4.

a, Control HepG2 cells (black bars) and cells depleted of Nse2 (grey bars) were transfected with a reporter gene and transduced with GFP or Flag-tagged Nse2 (F-Nse2). Cells were then split and further transduced with GFP or GFP-HBx. Luciferase assay and Western blot analysis were as before. Data are expressed as mean ± SEM of 2 independent experiments. b, Luciferase assay for the ChIP experiment presented in Fig. 4b. Data are expressed as mean ± SEM of 3 independent experiments. c, Single channel confocal images of middle right panels merged images presented in Fig. 4d.

Source data

Extended Data Fig. 7 HBx triggers Smc5/6 degradation in the absence of SLF2. Related to Fig. 5.

a, Control HepG2 cells (black bars) and cells depleted of SLF2 (grey bars) were transfected with a reporter gene and shortly after transduced with GFP, GFP-HBx and/or SLF2 in the indicated combinations. Luciferase assay and Western blot analysis were performed as before. Data are expressed as mean ± SEM of 2 independent experiments. b, Luciferase assay for the ChIP experiment presented in Fig. 5b. Data are means ± SEM of 2 independent experiments. c, Single channel confocal images of middle right panels merged images presented in Fig. 5d. d, Luciferase assay for the ChIP experiment presented in Fig. 5e. Data are means ± SEM of 4 independent experiments.

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Supplementary information

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Abdul, F., Diman, A., Baechler, B. et al. Smc5/6 silences episomal transcription by a three-step function. Nat Struct Mol Biol 29, 922–931 (2022). https://doi.org/10.1038/s41594-022-00829-0

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