Fig 2: RNF4 accumulates at stalled replication forks in a SIM-dependent manner. (A) Schematic illustration of the iPOND assay. (B) Western blot analysis of the input and iPOND samples with the indicated antibodies. HEK293T cells were pulse-labeled with 10 μM EdU for 15 min followed with or without 10 μM thymidine chase for 1 h (Thd). Cells were then treated with 4 mM HU for 3 h prior to crosslinking with 1% formaldehyde. No Clk, no-click samples; Click rxn, Click reaction. (C) Analysis of RNF4 recruitment by PLA. HeLa cells stably expressing HF-tagged wild-type RNF4 or its mutants were pulse-labeled with 10 μM EdU for 15 min, left untreated or treated with 4 mM HU for 3 h, and then subjected to PLA with anti-Flag and anti-biotin antibodies. TOP: representative images of PLA foci. Scale bar, 10 μm. Bottom left: quantification of PLA foci number per focus positive cell. Data are means ± SEM of three independent experiments. At least 300 cells were counted in each individual experiment. *P <0.05, ****P <0.0001, one-way ANOVA test. Bottom right: western blot analysis of RNF4 expression. (D, F) Depletion of ZATT (D) or TOP2A (F) does not affect RNF4 recruitment to stalled forks. HeLa cells stably expressing HF-tagged wild-type RNF4 were transfected with the indicated siRNAs, pulse-labeled with 10 μM EdU for 15 min, left untreated or treated with 4 mM HU for 3 h, and then subjected to PLA with anti-Flag and anti-biotin antibodies. Top: representative images of PLA foci. Scale bar, 10 μm. Bottom: quantification of PLA foci number per focus positive cell. Data are means ± SEM of three independent experiments. At least 300 cells were counted in each individual experiment. ns, not significant; ****P <0.0001, one-way ANOVA test. (E, G) Knockdown efficiency of ZATT (E) or TOP2A (G) was confirmed by Western blotting.

Fig 3: RNF4 targets SUMOylated TOP2A for ubiquitination and degradation. (A) SUMOylation of TOP2A stimulates its ubiquitination. HEK293T cells stably expressing His-tagged wild-type ubiquitin or the ΔGG mutant were transfected with HA-tagged wild-type SUMO2 or the ΔGG mutant. 24 h post-transfection, the cells were pre-treated with 10 μM MG132 for 1 h followed by treatment with or without 4 mM HU for 3 h. The cells were then harvested and lysed under denaturing conditions, and ubiquitinated proteins were purified by cobalt agarose resin. The isolated proteins were analyzed by Western blotting with the indicated antibodies. (B) RNF4, Ubc9, or ZATT depletion inhibits HU-induced TOP2A ubiquitination. HEK293T cells stably expressing His-tagged ubiquitin were transfected with the indicated plasmids/siRNAs. 24 h post-transfection, the cells were pre-treated with 10 μM MG132 for 1 h followed by treatment with or without 4 mM HU for 3 h. The cells were then harvested and lysed under denaturing conditions, and ubiquitinated proteins were purified by cobalt agarose resin. The isolated proteins were analyzed by Western blotting with the indicated antibodies. (C) Mutation of the SUMOylation sites of TOP2A impairs its ubiquitination. HEK293T cells stably expressing His-tagged ubiquitin were transfected with the indicated plasmids. 24 h post-transfection, the cells were pre-treated with 10 μM MG132 for 1 h followed by treatment with or without 4 mM HU for 3 h. The cells were then harvested and lysed under denaturing conditions, and ubiquitinated proteins were purified by cobalt agarose resin. The resulting isolated proteins were analyzed by Western blotting with the indicated antibodies. (D) Schematic illustration of the combined in vitro SUMOylation and ubiquitination assay procedures. (E) RNF4 ubiquitinates SUMOylated TOP2A in vitro. High-salt-purified GFP-TOP2A proteins were subjected to in vitro SUMOylation assays and then subjected to in vitro ubiquitination assays in the presence of bacterially expressed GST-RNF4-WT, GST-RNF4-ΔSIM, or GST-RNF4-CS, as indicated. The ubiquitination and SUMOylation of TOP2A were analyzed by Western blotting with the indicated antibodies. (F) RNF4 depletion increases the levels of SUMOylated TOP2A. HEK293T cells stably expressing His-tagged wild-type SUMO2 or the ΔGG mutant were transfected with the indicated siRNAs, followed by treatment with or without 4 mM HU for 3 h. The cells were then harvested and lysed under denaturing conditions, and SUMOylated TOP2A were purified by cobalt agarose resin. Immunoblotting was performed with the indicated antibodies.

Fig 4: Model for the regulation of NELF condensation upon stress such as heat shockThe interaction between NELFA and NELFE tentacles of NELF complex are essential drivers of the NELF phase separation. The integration of two stress-dependent pathways—sequestration of P-TEFb complex and activation of SUMO E3 ligase ZNF451—results in NELF condensation in nuclei of stressed cells.

Fig 5: RNF4 forms a complex with TOP2A and ZATT. (A) Schematic illustration of RNF4 and its mutants used in this study. (B, C) List of proteins identified by mass spectrometry. Bait proteins are indicated in bold letters. HEK293T Cells stably expressing SFB-tagged wild-type RNF4 (RNF4-WT) or the catalytically- inactive mutant form of RNF4 (RNF4-CS) were treated with 4 mM HU for 3 h. Cells were then harvested and lysed with NETN buffer at 4°C for 30 min. Crude lysates were centrifuged at 4°C, 14 000 rpm for 10 min and the resulting supernatants were incubated with streptavidin-conjugated beads at 4°C for 3 h. Beads were washed three times with NETN buffer, and bounded proteins were eluted with NETN buffer containing 1 mg/ml biotin. Elutes were then incubated with S-protein beads at 4°C for 2 h. After washing three times with NETN buffer, the protein mixtures were subjected to mass spectrometry analysis. (D) HEK293T cells transiently transfected with plasmids encoding SFB-tagged RNF8, RNF4-WT, or RNF4-CS were treated with 4 mM HU for 3 h. Coprecipitation was carried out using S-protein beads, and immunoblotting was performed with the indicated antibodies. (E) HEK293T cells transiently transfected with plasmids encoding SFB-tagged RNF8, RNF4-WT or RNF4-CS were pre-treated with 10 μM MG132 for 1 h followed by treatment with 4 mM HU for 3 h. Coprecipitation was carried out using S-protein beads, and immunoblotting was performed with the indicated antibodies. (F) Association of endogenous RNF4 with TOP2A and ZATT. HeLa cells were pre-treated with 10 μM MG132 for 1 h followed by treatment with 4 mM HU for 3 h. Whole-cell lysates were immunoprecipitated with either anti-RNF4 antibodies or control IgG antibodies, followed by immunoblotting with anti-TOP2A, anti-ZATT and anti-RNF4 antibodies