EMBO/soda-nel huggingface dataset:
FET family fusion oncoproteins target the SWI/SNF chromatin remodeling complex (2019)
Figure 3

Figure 3. Co-immunoprecipitation of SWI/SNF and FET proteins in sarcoma cell lines.

A. Immunoblot analysis (IB) of proteins co-immunoprecipitated with nuclear extracted SWI/SNF from MLS cell line 402‑91 and EWS cell line TC-71. Antibodies against BRG1, DDIT3 (FUS-DDIT3), the C-terminal parts of normal FUS and EWSR1, and FLI1 (EWSR1-FLI1) were used for detection. In order to directly quantify the fraction of bound and non-bound protein, relative amounts of protein for each IP‑sample were loaded on the gel, with consideration taken for dilutions during the immunoprecipitation procedure. I: input of nuclear extract, B: bound proteins, NB: proteins not bound. One representative immunoblot is shown. Immunoblots from all replicates are shown in the source data.

B. Graphs showing the percentage of bound to total (bound + non‑bound) signal intensities from immunoblots for FUS‑DDIT3, EWSR1 and FUS in MLS 402-91 cell line and EWSR1-FLI1, EWSR1 and FUS in EWS TC-71 cell line. Mean ± SEM is shown with individual replicates indicated by circles (n=3). Student's t-test, *p<0.05, **p<0.01, ***p<0.001. Original data for all quantifications, including p-values, are shown as source data.

C. Immunoblot analysis of proteins co-immunoprecipitated with nuclear extracted SWI/SNF from the model cell line HT1080 with overexpressed FUS-DDIT3-dsRED, EWSR1-FLI1-EGFP or dsRED are shown as representative immunoblots. Antibodies against BRG1, DDIT3 (FUS-DDIT3-dsRED), the C‑terminal parts of normal FUS and EWSR1, EZH2 and FLI1 (EWSR1-FLI1-EGFP) were used for detection. In order to directly quantify the fraction of bound and non-bound protein, relative amounts of protein for each IP‑sample were loaded on the gel, with consideration taken for dilutions during the immunoprecipitation procedure. I: input of nuclear extract, B: bound proteins, NB: proteins not bound. Immunoblots from all replicates are shown in the source data (with either dsRED- or EGFP‑tagged proteins expressed).

D. Graphs showing the percentage of bound to total (bound + non‑bound) signal intensities from immunoblots for the fusion protein (FUS‑DDIT3 or EWSR1-FLI1), EWSR1, FUS and EZH2 in the model cell line HT1080 with overexpressed FUS-DDIT3-dsRED/-EGFP (n=4, circles), EWSR1-FLI1-EGFP (n=3, squares) or dsRED/EGFP (n=4, triangles). Mean ± SEM is shown with individual replicates indicated. Student's t-test, no significant changes, p>0.05. Original data for all quantifications, including p-values, are shown as source data.

E. Four alternative models for binding of normal and oncogenic FET proteins to SWI/SNF. (I): FET oncoproteins bind directly to SWI/SNF. (II): normal FET proteins form homo- and hetero-complexes and mediate binding of FET oncoproteins. (III): FET oncoproteins and normal FET proteins bind to different variants of the SWI/SNF complex with distinct biochemical compositions. (IV): FET oncoproteins and normal FET proteins bind to different sites on the SWI/SNF complex.
FET family fusion oncoproteins target the SWI/SNF chromatin remodeling complex (2019)
Figure 4

Figure 4. FET-oncogene-induced changes in H3K27 trimethylation and downstream effects.

A. Immunoblot analysis (IB) of histone modifications in stably transfected HT1080 cell lines expressing EGFP, FUS‑DDIT3‑EGFP or EWSR1‑FLI1‑EGFP. Antibodies against H3K27Ac, H3K27me3, H3K4me3 and histone loading control H4 for detection of histone modifications and antibodies against EZH2 and loading control GAPDH to evaluate catalytic PRC2 amount. Immunoblot analysis with GFP antibody is shown to verify expression of FET fusion oncoproteins and EGFP. One representative immunoblot is shown. More immunoblots including a second stable biological replicate and analysis of histone modifications after transient transfection (24 and 48 hours) are shown in Figure EV2A-D.

B. Graphs showing amount of protein (H3K27me3, H3K4me3, ratio H3K27me3/H3K4me3, H3K27Ac, and EZH2) quantified from immunoblots relative each corresponding loading control H4 or GAPDH, normalized to parental HT1080. Mean ± SEM is shown with individual replicates indicated by circles from two experiments with stable transfection (see Figure 4A, EV2A and F) and by squares from two experiments with transient transfection (24h and 48h, see Figure EV2B and G), n=4. Student's t-test, ns= not significant, *p<0.05, **p<0.01, ***p<0.001. Original data for all quantifications, including p-values, are shown as source data.

C. SWI/SNF opposes the Polycomb complex PRC2. The catalytic subunit of PRC2, EZH2, catalyzes the trimethylation of Lys27 on histone H3 (H3K27me3), a chromatin modification associated with closed chromatin, leading to downregulation of polycomb-regulated genes. FET-fusion binding to SWI/SNF might compromise the function of SWI/SNF, including polycomb opposition. In particular, genes downregulated when FUS-DDIT3 is overexpressed in HT1080 cells (RNA-seq data, n=3, compared to control, n=4) overlap with upregulated gene-sets after EZH2 knockdown, NIPP1 knockdown and HDAC knockdown, and BAF57 (SMARCE1) reconstitution in a SMARCE1 null cell line. RNA-seq data of HT1080 wt (n=4), HT1080 EGFP (n=4), HT1080 FUS-DDIT3-EGFP (n=3) and HT1080 EWSR1-FLI1-EGFP (n=4) were generated and genes > two-fold regulated were analyzed using the molecular signature database (MSigDB). Gene lists were compared to the gene-set collection "chemical and genetic perturbations" and top 20 gene sets for each comparison are shown in the source data. q‑values (FDR-adjusted p‑values) are indicated in parentheses.

D. Schematic presentation of FET family of fusion oncoproteins, targeted DNA sequences and associated tumors. Left: FET N-terminal fusion partners and binding to SWI/SNF. Center: C-terminal transcription factor fusion partners and their DNA target sequences (JASPAR database). Right: Tumor types caused by respective fusion oncogene. Note tumor type specificity for each fusion oncoprotein and that the FET NTDs replace each other as fusion partners in some entities. Only a selection of the FET family fusion-oncoproteins and tumors is shown as more members are continuously discovered.

Expanded view Figure Legends
FBXO2/SCF ubiquitin ligase complex directs xenophagy through recognizing bacterial surface glycan (2021)
Figure 2

Figure 2 - GlcNAc affects ubiquitination of GAS.

A Expression levels of GlcNAc in WT GAS, ΔgacI, and ΔgacI::gacI were measured as the sWGA fluorescence intensity (normalized to WT GAS).

B, C Accumulation of LC3-II. (B) Representative western blot of LC3-II during non-infection (NI), WT GAS, ΔgacI, and ΔgacI::gacI. (C) LC3-II intensity normalized to NI.

D, E Recruitment of ubiquitin in GAS-infected cells. HeLa cells were infected with WT GAS, ΔgacI, and ΔgacI::gacI for 4 h, fixed, and immunostained for ubiquitin (FK2: magenta). Cellular and bacterial DNA were stained with DAPI (cyan). (D) Representative confocal images and (E) percentage of cells with ubiquitin-positive GAS. Scale bar, 10 μm.

F Immunoblot of the indicated GAS isolated from host cells. Lysate of GAS-infected HeLa cells were precipitated with anti-lipoteichoic acid antibody to isolate GAS, and the immunoprecipitates were analyzed by immunoblotting.

G, H Localization of ubiquitin in sWGA-treated GAS. HeLa WT were infected with the indicated GAS strains masked with sWGA for 4 h, fixed, and immunostained for ubiquitin (magenta). (G) Representative confocal images and (H) percentages of cell with ubiquitin-positive GAS. Scale bar, 10 μm.

I, J Recruitment of p62 to gacI mutants. HeLa cells were infected with WT GAS, ΔgacI, and ΔgacI::gacI for 4 h, fixed, and immunostained for p62 (magenta). Cellular and bacterial DNA were stained with DAPI (cyan). (I) Representative confocal images and (J) percentage of cells with p62-positive GAS. Scale bar, 10 μm.

K Bacterial viability rates of GAS mutants in cells (CFU recovered at 6 h post-infection/CFU at 2 h post-infection).

Data information: Data are shown as the mean ± SEM of (A) nine and (C, E, H, J, and K) three independent experiments. Asterisks indicate significant differences (*, p > 0.05 **, p > 0.01) as determined by Student's t-test.
FBXO2/SCF ubiquitin ligase complex directs xenophagy through recognizing bacterial surface glycan (2021)
Figure 3

Figure 3 - FBXO2 targets GAS and is involved in xenophagy.

A, B Co-localization of GFP-FBXO2 and mCherry-LC3 in GAS-infected cells. HeLa cells transfected with the indicated EmGFP-FBXO (green) and mCherry-LC3 (magenta) were infected with WT GAS for 2 h; scale bar, 10 μm. Representative confocal images (A) and (B) percentages of FBXO-positive cells.

C Enlarged image of GAS-associated FBXO2 of (A). Graphs show the signal intensities of EmGFP-FBXO2 and mCherry-LC3 measured along the arrows in the merged image. Green and red arrows indicate the signal peaks observed in EmGFP-FBXO2 and mCherry-LC3, respectively. White arrow indicates bacteria that are targeted by FBXO2 but not by LC3. Scale bar, 10 μm.

D Time course of FBXO2 and LC3 recruitment during GAS infection.

E, F Localization of EmGFP-FBXO2 in GAS-infected cells. HeLa cells transfected with GFP-FBXO2 (green) were infected with the indicated GAS strains for 2 or 4 h at an MOI of 100; scale bar, 10 μm. Representative confocal images at 4 h of WT GAS, ΔgacI, and ΔgacI::gacI (E) and (F) percentages of FBXO2‐positive cells.

Data information: Data are shown as the mean ± SEM of (B-D, F) (n > 200 cells per condition) three independent experiments. Asterisks indicate significant differences (**, p > 0.01) as determined by Student's t-test.
FBXO2/SCF ubiquitin ligase complex directs xenophagy through recognizing bacterial surface glycan (2021)
Figure 4

Figure 4 - FBXO2 targets intracellular GAS through sugar-binding motifs.

A Schematic representation of wild-type FBXO2 and domain deletion mutants of FBXO2. Numbers above the constructs represent the amino acid positions of FBXO2. PEST (residues 1-54) and F-box domains (residues 55-95), linker sequence (residues 96-124), and sugar-binding domain (residues 125-297) are indicated by P, FBD, linker, and SBD, respectively.

B, C HeLa cells transfected the indicated GFP-FBXO2 mutants (green) were infected with WT GAS for 4 h; scale bar, 10 μm. Representative confocal images (B) and percentages of each FBXO2 mutants localized to GAS (C).

D EmGFP intensities bound to GAS cells in vitro. GAS cells were incubated cell lysate expressing EmGFP, EmGFP-FBXO2, or EmGFP-FBXO2Y279A/W280A, and precipitated EmGFP with GAS cells.

E, F Recruitment of FBXO2 to intracellular GAS in SKP1/CUL1/ROC1 knockdown cells. HeLa cells transfected with GFP-FBXO2 (green) and the indicated siRNAs were infected with GAS, fixed at 4 h; scale bar, 10 μm. Representative confocal images (E) and percentages of FBXO2 localized to GAS (F).

Data information: Data are shown as the mean ± SEM of three independent experiments. Asterisks indicate significant differences (**, p > 0.01) as determined by Student's t-test.
FBXO2/SCF ubiquitin ligase complex directs xenophagy through recognizing bacterial surface glycan (2021)
Figure 5

Figure 5 - FBXO2 is required for GAS xenophagy.

A Wester blotting of FBXO2 in human cell lines.

B Bacterial invasion rates into host cells (CFU recovered at 2 h post-infection/CFU at 1 h post infection) which were normalized to that in wild-type cells.

C Intracellular bacterial survival rates (CFU recovered at 6 h post-infection/CFU at 2 h post-infection) which were normalized to that in wild-type cells.

D HeLa WT or FBXO2-KO cells transfected with mCherry-Gal3 (magenta) were infected with WT GAS for 2 or 4 h, and percentages of Gal3-positive GAS-containing cells were quantified.

E - J Localization of ubiquitin, LC3 and p62 in FBXO2 KO cells. HeLa WT or FBXO2-KO cells were infected with WT GAS for 4 h, fixed, and immunostained for ubiquitin (FK2: magenta) or LC3 (magenta) or p62 (magenta). Representative confocal images (E, G, and I) and percentages of cells with ubiquitin, LC3, or p62 -positive GAS (F, H, and J).

K, L Accumulation of LC3-II during GAS infection. (K) Representative western blot of LC3 during infection. (L) LC3-II intensities normalized to non-infection condition of wild-type cells.

M, N GlcNAc recognition of FBXO2 required for GAS xenophagy. Non-transfected HeLa WT cells and HeLa FBXO2-KO cells transfected GFP, GFP-FBXO2, GFP-FBXO2 YW/AA were infected with GAS WT for 4 h, fixed, and immunostained for ubiquitin. Percentages of cells with LC3 (M) or ubiquitin (N) -positive GAS were quantified.

Data information: Data are shown as the mean ± SEM of more than three independent experiments (B-D, F, H, J, and L-N), n > 200 cells per condition. Asterisks indicate significant differences (* p > 0.05, ** p > 0.01) as determined by Student's t-test.
FBXO2/SCF ubiquitin ligase complex directs xenophagy through recognizing bacterial surface glycan (2021)
Figure 6

Figure 6 - SCF complex is involved in ubiquitination and xenophagy against GAS infection.

A-C Recruitment of LC3 and ubiquitin to intracellular GAS in SKP1/CUL1/ROC1 knockdown cells. HeLa cells transfected with mCherry-LC3 (magenta) and the indicated siRNAs were infected with GAS for 4 h. Cells were immunostained for ubiquitin (FK2); scale bar, 10 μm. Representative confocal images (A) and percentages of cells with ubiquitin (B) and LC3 (C) -positive GAS.

D, E Neddylated cullin inhibitor: MLN4924-treated HeLa cells were infected with WT GAS, and fixed at 4 h. Cells were immunostained for ubiquitin (FK2: magenta) or LC3 (magenta); scale bar, 10 μm. Representative images and quantification of cells with ubiquitin-positive GAS (D) and LC3-positive GAS (E).

F Lysates from HeLa cells infected with GAS were immunoprecipitated with an anti-lipoteichoic acid antibody and Protein G-Magnetic Beads. Immunoprecipitates were analyzed by immunoblotting.

G Proposed model for FBXO2-mediated recognition of bacterial glycan in xenophagy. GAS internalized via endocytosis enters the cytosol, and then GAS escapes into the cytoplasm by SLO secretion. Escaped GAS is targeted by FBXO2 via recognition of GlcNAc and is ubiquitinated by the SCF complex. Finally, LC3 is recruited to ubiquitinated GAS for autophagy formation.

Data information: Data are shown as the mean ± SEM of three independent experiments (B-E), n > 200 cells per condition. Asterisks indicate significant differences (** p > 0.01) as determined by Student's t-test.

Expanded View Figure Legends
FIP200, a ULK-interacting protein, is required for autophagosome formation in mammalian cells. (2008)
Figure 1.

ULK1 and 2 localize to the isolation membrane (phagophore) under starvation conditions. (A and B) NIH3T3 cells stably expressing GFP-ULK1 (A) and -ULK2 (B) were cultured in complete or starvation medium for 30 min. They were then cultured in fresh complete medium for an additional 30 min (starvation→complete). (C and D) NIH3T3 cells stably expressing GFP-ULK1 (C) and -ULK2 (D) were cultured in starvation medium for 120 min. The cells were fixed, permeabilized, and subjected to immunofluorescence microscopy using anti-Atg16L1 antibody and Alexa Fluor 660–conjugated secondary antibody. More than 90% of GFP-ULKs dots were positive for Atg16L1. (E–H) Wild-type (E and G) and Atg5−/− (F and H) MEFs were transfected with retroviral vectors encoding GFP-ULK1 and -ULK2. MEFs stably expressing GFP-ULK1 (E and F) and -ULK2 (G and H) were cultured in complete or starvation medium for 120 min. The cells were fixed and examined by fluorescence microscopy. Bars, 20 μm.
FIP200, a ULK-interacting protein, is required for autophagosome formation in mammalian cells. (2008)
Figure 2.

The kinase-dead ULK mutants inhibit autophagy. (A) In vitro kinase assay of endogenous ULK1. MEFs were cultured in complete or starvation medium for 60 min. ULK1 kinase activity was determined as described in Materials and methods. Relative kinase activity is shown. Data are the mean ± SE of five independent experiments. (B and C) NIH3T3 cells were transiently transfected with HA-ULK1, HA-ULK2, or their kinase-dead mutants and subjected to immunofluoresence microscopy using monoclonal anti-HA and polyclonal anti-Atg16L1 antibodies for primary staining and Alexa Fluor 488–conjugated goat anti–mouse IgG and Alexa Fluor 568–conjugated goat anti–rabbit IgG antibodies for secondary antibodies. Transfected cells are indicated by arrows. Bars, 20 μm. (D) NIH3T3 cells stably expressing GFP-ULK1K46N and GFP-ULK2K39T were cultured in starvation medium for 120 min. The cells were subjected to immunofluorescence microscopy using anti-Atg16L1 antibody. Bar, 20 μm. (E) NIH 3T3 cells were transfected with the retroviral vectors encoding HA-ULK1K46N or with the corresponding empty retrovirus (mock). They were cultured in complete or starvation medium for 120 min. Cell lysates were then analyzed by immunoblot analysis with the indicated antibodies.
FIP200, a ULK-interacting protein, is required for autophagosome formation in mammalian cells. (2008)
Figure 3.

ULK1 interacts with FIP200. (A) HEK293T cells were cotransfected with FLAG-FIP200 and HA-ULKs. Cell lysates were subjected to immunoprecipitation (IP) using antibodies against FLAG. The resulting precipitates were examined by immunoblot (IB) analysis with the indicated antibodies. The asterisk indicates nonspecific band. (B) Lysates of MEFs were immunoprecipitated with anti-ULK1 or anti-FIP200 antibody or preimmune rabbit serum, and the resulting precipitates were subjected to immunoblot analysis with antibodies against ULK1 and FIP200. (C) Schematic representation of ULK1 mutants used in D. (D) HEK293T cells were cotransfected with FLAG-FIP200 and various ULK1 mutants and analyzed as in A using anti-HA and anti-FLAG antibodies. (E) NIH3T3 cells stably expressing GFP-ULK1 (left) and GFP-ULK1ΔC (right) were cultured in starvation medium for 120 min. Bar, 20 μm.
FIP200, a ULK-interacting protein, is required for autophagosome formation in mammalian cells. (2008)
Figure 4.

FIP200 localizes to phagophore after starvation treatment. (A) NIH3T3 cells stably expressing GFP-FIP200 were cultured in complete or starvation medium for 120 min and the GFP signal was observed. (B) NIH3T3 cells stably expressing GFP-FIP200 were cultured in starvation medium for 120 min and then subjected to immunofluorescence microscopy using anti-Atg16L1 antibody and Alexa Fluor 568–conjugated secondary antibody. Bars, 20 μm. More than 90% of GFP-FIP200 dots were positive for Atg16L1. (C) NIH3T3 cells stably expressing GFP-ULK1 were starved for 120 min and then subjected to immunofluorescence microscopy using anti-FIP200 antibody and Alexa Fluor 568–conjugated secondary antibody. Black squares indicate the enlarged areas shown in insets. Bar, 20 μm.
FIP200, a ULK-interacting protein, is required for autophagosome formation in mammalian cells. (2008)
Figure 5.

FIP200 is required for autophagy. (A) FIP200+/+, FIP200−/−, Atg5+/+, and Atg5−/− MEFs were cultured in complete or starvation medium for 60 min. In the recovery experiments, starved MEFs were cultured in fresh complete medium for an additional 60 min (replenishment). The cell lysates were subjected to immunoblot analysis with the indicated antibodies. (B) Wild-type and FIP200−/− MEFs were cultured in the complete or starvation medium for indicated time with or without 100 nM bafilomycin A1. The cell lysates were subjected to immunoblot analysis with anti-LC3 antibody. (C) Wild-type and FIP200−/− MEFs were transfected with retroviral vectors encoding GFP-Atg5 or GFP-LC3. Resulting cells were cultured in the starvation medium for 120 min. The cells were fixed and examined by fluorescence microscopy. Bar, 20 μm. (D–G) Wild-type (D and E) and FIP200−/− (F and G) MEFs were cultured in complete (D and F) or starvation (E and G) medium for 120 min and then fixed and subjected to EM analysis. Autophagosome-like structures (open arrowheads), and autolysosomes (closed arrowheads) are indicated. Bar, 1 μm. (H) The ratio of total area of autophagosomes (AP) and autolysosomes (AL) to total cytoplasmic area in D–G was determined by morphometric analysis.
FIP200, a ULK-interacting protein, is required for autophagosome formation in mammalian cells. (2008)
Figure 6.

FAK is not required for autophagy. (A) FAK+/− and FAK−/− MEFs were cultured in the complete or starvation medium for the indicated times with or without 100 nM bafilomycin A1. The cell lysates were subjected to immunoblot analysis with the indicated antibodies. (B) FAK+/− and FAK−/− MEFs stably expressing GFP-LC3 were cultured in complete or starvation medium for 120 min. The cells were fixed and examined by fluorescence microscopy. (C) Wild-type MEFs stably expressing GFP-FAK were cultured in complete or starvation medium for 120 min. The cells were fixed and subjected to immunofluorescence microscopy using anti-Atg16 antibody. Black squares indicate the enlarged areas shown in insets. Bars, 20 μm.
FIP200, a ULK-interacting protein, is required for autophagosome formation in mammalian cells. (2008)
Figure 7.

FIP200 functions downstream of mTOR in autophagosome formation. (A) Wild-type and FIP200−/− MEFs were treated with 100 ng/ml rapamycin (rapa) or vehicle (DMSO) for 120 min in the presence or absence of 100 nM bafilomycin A1. The cell lysates were subjected to immunoblot analysis with anti-LC3 antibody. (B) Wild-type and FIP200−/− MEFs stably expressing GFP-Atg5 or GFP-LC3 were cultured in the presence of 100 ng/ml rapamycin for 120 min. The formation of GFP-Atg5 (top) and GFP-LC3 (bottom) puncta was examined by fluorescence microscopy. Bar, 20 μm. (C) Wild-type and FIP200−/− MEFs were treated with 10 mM lithium chloride for 24 h or 100 μM C2-ceramide for 2 h.
FIP200, a ULK-interacting protein, is required for autophagosome formation in mammalian cells. (2008)
Figure 8.

FIP200 is required for membrane targeting and proper function of ULK. (A) FIP200+/− and FIP200−/− MEFs stably expressing GFP-ULK1 or -ULK2 were cultured in starvation medium for 120 min. The cells were fixed and examined by fluorescence microscopy. Bar, 20 μm. (B) FIP200+/+ and FIP200−/− MEFs were cultured in complete medium. The cell lysates were subjected to immunoblot analysis with antibodies against ULK1 or HSP90 (loading control). (C) Phosphatase sensitivity of ULK1. FIP200+/+ and FIP200−/− MEFs were cultured in complete medium. ULK1 was immunoprecipitated from cell lysates and treated with λ phosphatase for 30 min at 30°C in the absence or presence of phosphatase inhibitors (1 mM Na2VO4, 50 mM KF, 15 mM Na4P2O7, and 1 mM EGTA).