Identification of proximal SUMO-dependent interactors using SUMO-ID

Abstract

The fast dynamics and reversibility of posttranslational modifications by the ubiquitin family pose significant challenges for research. Here we present SUMO-ID, a technology that merges proximity biotinylation by TurboID and protein-fragment complementation to find SUMO-dependent interactors of proteins of interest. We develop an optimized split-TurboID version and show SUMO interaction-dependent labelling of proteins proximal to PML and RANGAP1. SUMO-dependent interactors of PML are involved in transcription, DNA damage, stress response and SUMO modification and are highly enriched in SUMO Interacting Motifs, but may only represent a subset of the total PML proximal proteome. Likewise, SUMO-ID also allow us to identify interactors of SUMOylated SALL1, a less characterized SUMO substrate. Furthermore, using TP53 as a substrate, we identify SUMO1, SUMO2 and Ubiquitin preferential interactors. Thus, SUMO-ID is a powerful tool that allows to study the consequences of SUMO-dependent interactions, and may further unravel the complexity of the ubiquitin code.

Fig. 1. Identification of SUMO-dependent interactions: the SUMO-ID strategy.

Schematic representation of the SUMO-ID strategy (a) and the constructs used (b). BLAST^R blasticidin resistant cassette, CTurboID C-terminal TurboID, DOX doxycycline, GSQ repetitive linker, NTurboID N-terminal TurboID, P2A 2A self-cleaving peptide, PURO^R puromycin resistant cassette, S SUMO, SIM SUMO Interacting Motif, SDI SUMO-dependent interactor, SUMOnc SUMO non-cleavable, Strep streptavidin. The sequences of representative constructs are provided in the Source Data file.

Fig. 2. T194/G195 Split-TurboID is suitable for SUMO-ID studies.

a Structure of the E. coli BirA with bound biotin (PDB ID: 1HXD^,), depicting the T194/G195 split point and the BirA-Biotin interaction. T194/G195 split point breaks the 193-199 loop that connects the biotin-interacting β8 and β9 strands (see Supplementary Note 1). b Western blot of HEK293FT cells that were transiently transfected with combinations of the FLAG-N or MYC-C fused to PML, RANGAP1 or SUMO1/2 and treated with 50 μM of biotin for 16 h. Black arrowheads indicate SUMO-ID activity derived from MYC-C-SUMOylated FLAG-N-substrates. White squares indicate biotinylated free MYC-C-SUMOs. Neither FLAG-N nor MYC-C showed any detectable background biotinylating activity. Data are representative of two independent transfection experiments with similar results. Source data are provided in the Source Data file. c Immunostainings of transiently transfected U2OS cells treated with 50 μM of biotin for 16 h, showing the fragment-complementation dependency of SUMO-ID and its correct localization within the cell, enriched at PML NBs as expected for SUMOylated PML. Nuclei are stained with DAPI (blue) and biotinylated material with fluorescent streptavidin (Strep, magenta). BirA antibody recognizes both N and C (green). Black and white panels show the single green and magenta channels. Scale bar: 5 μm. Images are representative of two independent transfection experiments performed on cover slips. d Western blot of HEK293FT stable cells for N-FRB in combination with C-FKBP, treated or not with 1 μg/mL of rapamycin and 50 μM of biotin at indicated time-points. BirA antibody recognizes both N and C. White squares and circles indicate N-FRB and C-FKBP, respectively. Self-biotinylating activity of the reconstituted TurboID was measured and normalized to expression levels (BirA blot). Bar plots show the mean and standard deviation of three independent experiments. Statistical analyses were performed by 2-way ANOVA: *p < 0.05; **p < 0.01; ****p < 0.0001. Molecular weight markers are shown to the left of the blots in kDa. Source data and the exact p values are provided in the Source Data file.

Fig. 3. SUMO-ID is specific for SUMO-dependent interactions.

a Western blot of HEK293FT FLAG-N-PML stable cell line transfected with different combinations of MYC-C and SUMO^WT or SUMO^ΔGG. Cells were treated or not with 1 μM of ATO for 2 h and 50 μM of biotin at indicated time points. White square indicates biotinylation of unconjugated MYC-C- SUMO^ΔGG. White arrowhead points to SUMO-SIM interaction mediated SUMO-ID. Black arrowhead shows PML-SUMOylation derived SUMO-ID. b Western blot of HEK293FT transfected with constitutive MYC-C-SUMO1/2 or doxycycline-inducible and isopeptidase-cleavage resistant (nc) MYC-C-SUMO1/2nc or MYC-C-Ubnc. Doxycycline was added or not at 1 μg/mL for 24 h. White squares point to free/unconjugated MYC-C-SUMOs. Dotted line indicates a cut in the same blot. c Western blot of U2OS double stable cell lines for FLAG-N-PML^WT or the SUMO/SIM mutant FLAG-N-PML^3MAS together with doxycycline-inducible TRIPZ-MYC-C-SUMO2nc. Doxycycline was added or not at 1 μg/mL for 24 h. 50 μM of biotin was added at indicated time-points. PML SUMO-ID showed a high PML/SUMO interaction dependency. d Confocal microscopy of the same cells as in c, treated or not with doxycycline (1 μg/mL, 24 h), biotin (50 μM, 2 h) and ATO (1 μM, 2 h). Nuclei are stained with DAPI (blue) and biotinylated material with fluorescent streptavidin (Strep, magenta). BirA antibody shows N-PML staining (green). Black and white panels show the single green and magenta channels. Colocalization of the streptavidin and N-PML^WT signal is observed within PML NBs, that depends on PML-SUMO interaction. Scale bar: 5 μm. Images are representative of three independent experiments. a–c are representative of 2 biological replicates with similar results. Molecular weight markers are shown to the left of the blots in kDa. Source data are provided in the Source Data file.

Fig. 4. SUMO-ID identifies SUMO-dependent interactors of PML.

a Volcano plot of LC-MS analysis comparing streptavidin pull-downs of U2OS double stable cell lines for TRIPZ-MYC-C-SUMO2nc together with FLAG-N-PML^WT or FLAG-N-PML^3MAS. Cells were treated with 1 μg/mL of doxycycline for 24 h and 50 μM of biotin for 2 h. 59 high-confidence SUMO-dependent PML interactors were defined. Asterisk (*) indicates that IRF2BP2 was detected with one peptide but further validated by Western blot and immunofluorescence. Statistical analyses were performed by two-sided Student’s t test. Data, including proteins identified with 1 peptide, are provided as Supplementary Data 1. b Western blot validations of PML SUMO-dependent interactors identified by SUMO-ID in a. Blots 1/2 represent two independent experiments. UBC9 was added as an expected positive control. Dots indicate endogenous carboxylases that are biotinylated constitutively by the cell. Black arrowheads point to specific PML SUMO-ID biotinylating activity. Dotted lines indicate different exposures of the same blot. IN: input; St PD: streptavidin pull-down. Molecular weight markers are shown to the left of the blots in kDa. Source data are provided in the Source Data file. c STRING network analysis of the 59 SUMO-dependent interactors of PML identified in a. A highly interconnected cluster related to protein SUMOylation/ubiquitylation, transcriptional regulation, DNA repair and RNA stability proteins is depicted. Color, transparency and size of the nodes were discretely mapped to the Log2 enrichment value as described. The border line type was discretely mapped to the p value as described. d Gene ontology analysis of the 59 SUMO-dependent interactors of PML identified in a. Biological processes and molecular functions related to SUMOylation/ubiquitylation, stress response, DNA repair, transcription and RNA stability were significantly enriched. Dotted line represents the threshold of the p value (0.05). Data are provided as Supplementary Data 2.

Fig. 5. SUMO-dependent interactors of PML localize to PML NBs.

Confocal microscopy analysis of PML SUMO-ID identified proteins in U2OS YFP-PML knock-in cell line. UBC9 was added as an expected positive control. Yellow arrowheads indicate colocalization events. Dotted line-squares show the selected colocalization events for digital zooming and the signal profile plotting shown to the right. Nuclei are stained with DAPI (blue), YFP-PML is shown in green and the indicated proteins in magenta. Black and white panels show the single green and magenta channels. Images are representative of three independent stainings performed into the same YFP-PML cell line. Further colocalization analysis using the Colocalization Colormap and JACoP is provided as Supplementary Fig. 8. Arb. units Arbitrary units. Scale-bar: 5 μm.

Fig. 6. Characterization of the whole PML NBs proteome.

a Volcano plot of LC-MS analysis comparing streptavidin pull-downs of U2OS stable cell lines for TurboID-PML^WT or TurboID alone. Cells were treated with 50 μM of biotin for 2 h. High-confidence PML proteome composed of 271 proteins is shown as blue dots. Statistical analyses were performed by two-sided Student’s t test. Data are provided as Supplementary Data 3. b STRING network analysis of the whole PML NBs proteome defined in a shows a high interconnected network composed of the 73.6% of the proteins. Highly interconnected sub-clusters were characterized using MCODE. Color, transparency and size of the nodes were discretely mapped to the Log2 enrichment value as described. The border line type was discretely mapped to the p value as described. c Gene ontology analysis of the whole PML NBs proteome defined in a. Depicted biological processes, molecular functions and cellular components were significantly enriched. Dotted line represents the threshold of the p value (0.05). Data are provided as Supplementary Data 4.

Fig. 7. Proteins identified by PML SUMO-ID are a subset of the SUMO-dependent PML NBs proteome and are enriched in SIMs.

a Volcano plot of LC-MS analysis comparing streptavidin pull-downs of U2OS stable cell lines for TurboID-PML^WT or TurboID-PML^3MAS. Cells were treated with 50 μM of biotin for 2 h. Proteins enriched in TurboID alone compared to TurboID-PML^WT were previously removed for the comparison. PML SUMO-ID identified proteins (including 1 peptide identified proteins) are highlighted in orange. LC-MS data on the effect of the ATO treatment (1 μM; 2 h) for TurboID-PML^WT enriched proteins is represented with symbols as described. Statistical analyses were performed by two-sided Student’s t test. Data are provided as Supplementary Data 3. b WB validation of the effect of ATO treatment (1 μM; 2 h) on TRIM24 by PML SUMO-ID. Cells were treated with 1 μg/mL of doxycycline for 24 h and 50 μM of biotin for 2 h. After streptavidin pulldown, decreased levels of SUMO-PML interacting TRIM24 upon ATO treatment is observed. Dotted lines indicate different exposures of the same blot. Data are representative of three independent pull-down experiments. Molecular weight markers are shown to the left of the blots in kDa. IN: input; St PD: streptavidin pull-down. c Violin plots comparing the percentage of single SIM and multiple SIM presence in 1000 random lists and PML SUMO-ID list. Horizontal solid and dotted lines represent the median and quartiles (Q1, Q3), respectively. The 1000 random lists contain the same number of proteins (59) as the SUMO-ID list. d SIM presence was normalized by the length of the proteins to obtain the value of SIMs per thousand of amino acids (STAA). Gaussian distribution of STAA median values of the random lists was validated (d’Agostino and Pearson normality test, p value = 0.15), and PML SUMO-ID SIM enrichment factor with its corresponding empirical p value was calculated. The dotted black line represents the median STAA value of random lists. The dotted blue line represents the STAA value of the PML SUMO-ID list. Source data for b, c and d are provided in the Source Data file.

Fig. 8. SUMO-ID identifies interactors of SUMOylated SALL1.

a WB of HEK293FT stable cell lines for TRIPZ-MYC-C-SUMO1nc/SUMO2nc transfected with FLAG-N-SALL1^WT or the SUMO site mutant FLAG-NTurboID¹⁹⁴-SALL1^ΔSUMO. Cells were treated or not with 1 μg/mL of doxycycline for 24 h and 50 μM of biotin at indicated time points. Efficient SALL1 SUMO-ID biotinylating activity was detected for SUMO1nc and SUMO2nc (black arrowhead). Dots indicate endogenous carboxylases that are biotinylated constitutively by the cell. Results are representative of two independent transfection experiments on the same stable cell lines. Source data are provided in the Source Data file. b Confocal microscopy of U2OS stable cell line for TRIPZ-MYC-C-SUMO2nc transfected with FLAG-N-SALL1^WT or the SUMO site mutant FLAG-N-SALL1^ΔSUMO. Cells were treated or not with 1 μg/mL of doxycycline for 24 h and 50 μM of biotin for 4 h. Nuclei are stained with DAPI (blue) and biotinylated material with fluorescent streptavidin (Strep, magenta). BirA antibody shows N-SALL1 staining (green). Black and white panels show the single green and magenta channels. Nuclear colocalization of FLAG-N-SALL1^WT and streptavidin signal was observed. Images are representative of three independent transfection experiments performed on cover slips on the same stable cell line. Scale bar: 10 μm. c Volcano plot of LC-MS analysis comparing streptavidin pull-downs of HEK293FT TRIPZ-MYC-C-SUMO2nc stable cell line transfected with FLAG-N-SALL1^WT or the SUMO site mutant FLAG-N-SALL1^ΔSUMO. Cells were treated with 1 μg/mL of doxycycline for 24 h and 50 μM of biotin for 4 h. Potential interactors of SUMOylated SALL1 are depicted. Statistical analyses were performed by two-sided Student’s t test. Data are provided as Supplementary Data 5. d Western blot validations of SUMOylated SALL1 interactors found in c. NuRD complex proteins GATAD2B, MTA2 and RBBP4 were confirmed. Black arrowheads point to SUMOylated SALL1 signal. Dots indicate endogenous carboxylases that are biotinylated constitutively by the cell. Dotted lines indicate different exposures of the same blot. Data are representative of three independent transfection experiments. IN input, St PD streptavidin pull-down. Source data are provided in the Source Data file. e STRING network analysis of the SALL1 SUMO-ID list and MCODE clustering identifies the NuRD complex as a highly interconnected subcluster. Gene ontology analysis also identified the NuRD complex as an enriched term. Color, transparency and size of the nodes were discretely mapped to the Log2 enrichment value as described. The border line type was discretely mapped to the p value as described. Data are provided as Supplementary Data 6. Molecular weight markers are shown to the left of the blots in kDa in a and d.

Fig. 9. SUMO-ID identifies UbL and SUMO-paralogue preferential interactors of TP53.

a Western blot of HEK293FT double stable cell lines for FLAG-N-TP53 together with doxycycline-inducible TRIPZ-MYC-C-SUMO1nc, -SUMO2nc or -Ubnc. Doxycycline was added or not at 1 μg/mL for 24 h. 50 μM of biotin was added for 2 h. TP53 SUMO1-ID, SUMO2-ID and Ub-ID showed specific biotinylation patterns corresponding to each modification (black arrowhead). Dots indicate endogenous carboxylases that are biotinylated constitutively by the cell. Dotted lines indicate different exposures of the same blot. Results are representative of three independent pull-down experiments. Molecular weight markers are shown to the left of the blots in kDa. IN: input; St PD: streptavidin pull-down. Source data are provided in the Source Data file. Volcano plots of LC-MS analysis of (b) TP53 SUMO1-ID and SUMO2-ID, (c) SUMO1-ID or SUMO2-ID vs Ub-ID and (d) SUMO1-ID vs SUMO2-ID, from samples in a. SUMO-ID and Ub-ID of TP53 identified preferential interactors of each type of modification. Statistical analyses were performed by two-sided Student’s t test. Data are provided as Supplementary Data 7.