Fig 2: VCP accumulates on chromatin and interacts with FAF1 and SUMOylated proteins upon USP7 inhibition(A) Immunofluorescence of VCP in U2OS cells treated with DMSO (Control), 5 µM NMS873 (VCPi), 50 µM P22077 (USP7i), or a combination of both (USP7i+VCPi) for 4 h. Soluble, nuclear material was removed by pre-extraction previous to fixation. Scale bar, 50 µm.(B) Analysis of the levels of VCP on chromatin by high-throughput microscopy in U2OS cells treated with DMSO (C), 5 µM NMS873 (VCPi), 50 µM P22077 (USP7i), or a combination of both (USP7i-VCPi) for 4 h.(C) WB analysis of the levels of VCP in the soluble nuclear (Nuc) and chromatin (Chr) fractions obtained from HCT116 cells treated with DMSO (C), 5 µM NMS873 (VCPi), 50 µM P22077 (USP7i), or a combination of both (USP7i-VCPi) for 4 h. CDK2 is shown as a loading control. The experiments in (A)–(C) were repeated 3 times, and one representative experiment is shown.(D) Mass spectrometry analysis of the pull-down of VCP after cross-linking in HCT116 cells treated with 10 µM NMS873 or 50 µM P22077 for 4 h. The enrichment of proteins in each condition in two independent experiments was compared to DMSO-treated cells and normalized to the total amount of VCP in the samples. Enrichment upon VCPi treatment is shown on the x axis and enrichment upon USP7i treatment is shown on the y axis. SUMO2/3 is shown in pink, proteasome components in orange, components of the CMG helicase in green, and known VCP adaptors in blue.(E) WB analysis of the pull-down of VCP in cells treated as in (D). The levels of VCP, SUMO2/3, FAF1, and CDK1 were analyzed with specific antibodies. 5% of the input material is shown (Input). A control immunoprecipitation with a non-specific IgG was performed (IgG) and compared to the pull-down of VCP (VCP). The experiment was repeated three times with equivalent results, and an additional blot is shown in Figure S2.

Fig 3: USP7 and VCPFAF1 functions in DNA replication converge into the SUMOylation pathway(A and B) Immunofluorescence analysis of chromatin-bound VCP (green) and SUMO2/3 (red) (A) or SUMO2/3 (green) and MCM3 (red).(B) Levels in U2OS cells that were either untreated (CONTROL) or after treatment with 50 µM USP7i for 4 h. DNA was stained with DAPI (blue). The overlay for the different staining is also shown. Scale bar, 10 µm. The intensity of the individual stainings was quantified along the line indicated in the figure to assess the co-localization of the analyzed proteins (right).(C and D) HeLa cells were transfected with a non-specific siRNA (Con, black) or siRNA against FAF1 (FAF1, orange), and the levels of SUMO2/3 (C) or VCP (D) on chromatin were measured by high-throughput microscopy 48 h after transfection. Cells were treated with DMSO (Control) or 25 µM P22077 (USP7i) for the indicated times.(E and F) Representative images taken at 4 h of treatment are shown. The experiment was repeated 3 times, and one representative result is shown.(G) Western blot analysis of the inputs and eluates of an in vitro pull-down using GFP and SMO-1-GFP as bait. UBXN-3 or a deletion variant of the N-terminal UBA (?UBA) domain were used along with increasing amounts of tetra-Ubiquitin chains linked via Lysine 48 (4Ub(K48)). Note, that detection of Ubiquitin revealed cross-reactivity of the antibody with GFP. SMO-1-GFP-bound UBXN-3 can efficiently recover 4Ub(K48) without affecting UBXN-3 binding to SMO-1. UBXN-3(?UBA) interacts with SMO-1-GFP, while it is deficient in ubiquitin binding.(H) Schematic representation of quantitative analysis of UBXN-3 (green) and SMO-1 (red) spots in C. elegans embryonic nuclei. Raw images were masked for embryonic tissue and nuclei, before nuclear spots were defined for subsequent analysis. Images show a representative of npl-4-depleted embryos, immune labeled with UBXN-3 and SMO-1 antibodies.(I) Quantification of co-localized UBXN-3 (green) and SMO-1 (red) spot volume in embryos depleted for indicated gene products relative to green or red total spot volume, respectively. Whisker plots of the 10–90th percentile are shown, and statistical significance was interrogated using one-way ANOVA Sidak’s multiple-comparison test and is indicated by asterisks (*p < 0.05, **p < 0.01, ****p < 0.0001).Number of independent images analyzed: nempty(RNAi) = 25, nubc-9(RNAi) = 34, nnpl-4(RNAi) = 38. Scale bars, 10 µm in (A) and (B) and 5 µm in (H).

Fig 4: p97 R155H disease mutation and conditional p97 knock out in mice recapitulates the p97-UBXD8 loss of function effect on ERMCS.a Split luciferase assay to measure contacts in mouse embryonic fibroblasts with heterozygous or homozygous p97 R155H mutation. Cells were supplemented with indicated concentrations of oleic acid and palmitic acid for 4 h. (For a: n = 5, 4, 4, 5, 4, 4, 5, 4, and 4 biologically independent samples from left to right, respectively). b Representative SREBP1 and SCD1 staining from CA1 regions of 1 month-old control (CAMK2α) and p97 cKO mice (scale bar is 25 μm). c Quantification of fluorescence intensity of images in b. Individual points represent mean ROI intensity from each mouse, 3 or 4 animals per group. d Representative immunoblot for SREBP1 and SCD1 from cortical brain lysates of 12-month-old control (C57), or 6- and 12-month-old p97R155C/WT mice (n = 4 for each group, except for p97 cKO in SREBP1 panel (n = 3)). Pan 14-3-3 was used as housekeeping control. e Quantification of d. The ratio of mature SREBP1 to total SREBP1 is shown. SCD1 intensities are normalized to 14-3-3 levels in each lane. Individual points represent each mouse, 4 animals per group. Data are means ± SEM (*, **, ***P < 0.05, 0.01, 0.0001, respectively. Significance was analyzed by one-way ANOVA with Tukey’s multiple comparison test (a) or Dunnett’s multiple comparison (e) or two-tailed Student’s t test (c). f Model: Schematic of SREBP pathway activation. At ERMCS, UBXD8 recruits p97 to mediate the extraction and degradation of INSIG1 when it is ubiquitylated by the E3 ligase GP78. Loss of INSIG1, which is a negative regulator of SREBP1, allows translocation of cleaved SREBP1 transcription factor into the nucleus to activate expression of lipid desaturases, notably SCD1. Loss of p97-UBXD8 leads to decrease in SCD1 levels and loss of lipid desaturation resulting in membrane order and increased contacts. Source data are provided as a Source data file.

Fig 5: siRNA-mediated repression of p97 and UBXD8 results in increased ERMCS.a Top: Domain organization of p97 and indicated mutations, Middle: Split luciferase assay to measure contacts in HEK293T cells transfected with siRNAs to p97 and indicated N-Myc siRNA-resistant rescue constructs, RLU relative luminescence unit, Bottom: Immunoblot of indicated proteins (n = 9, 7, 8, 5, 3, 3, 3, and 3 biologically independent samples from left to right, respectively). b Top: Domain organization of UBXD8 and indicated mutations, Middle: Split luciferase assay to measure contacts in HEK293T cells transfected with siRNAs to UBXD8 and indicated C-HA/FLAG siRNA-resistant rescue constructs, RLU relative luminescence unit, Bottom: Immunoblot of indicated proteins. UBA ubiquitin associated, UAS upstream activating sequence, UBX ubiquitin X. (n = 10, 6, 10, 7, 4, 6, & 4 biologically independent samples from left to right, respectively). c Top: Split luciferase assay to measure contacts in HEK293T cells transfected with the indicated siRNAs against the p97 adapters. (n = 8, 6, 3, 3, 5, 3, and 8 biologically independent samples from left to right, respectively). Bottom: Immunoblot of HEK293T cells transfected with indicated siRNAs. d Top: Split luciferase assay to measure contacts in HEK293T cells transfected with two independent siRNAs against HRD1 or in HEK293 WT or GP78 KO cells. (n = 3 biologically independent samples of siControl, siHRD1#3, and siHRD1#4; n = 4 biologically independent samples of WT and GP78 KO). Bottom: Immunoblot of HEK293T cells transfected with indicated siRNAs. Data are means ± SEM. *, **, ***P < 0.05, 0.01, 0.0001 respectively, ns: not significant, one-way ANOVA with Tukey’s multiple comparison test (a, b, d: siControl vs siHRD1); one-way ANOVA with Dunnett’s multiple comparisons test (c); Two-tailed paired t test for d: HEK293 WT vs GP78 KO #1B. Source data are provided as a Source data file.