Abstract
Whereas oncogenes can potentially be inhibited with small molecules, the loss of tumour suppressors is more common and is problematic because the tumour-suppressor proteins are no longer present to be targeted. Notable examples include SMARCB1-mutant cancers, which are highly lethal malignancies driven by the inactivation of a subunit of SWI/SNF (also known as BAF) chromatin-remodelling complexes. Here, to generate mechanistic insights into the consequences of SMARCB1 mutation and to identify vulnerabilities, we contributed 14 SMARCB1-mutant cell lines to a near genome-wide CRISPR screen as part of the Cancer Dependency Map Project1,2,3. We report that the little-studied gene DDB1–CUL4-associated factor 5 (DCAF5) is required for the survival of SMARCB1-mutant cancers. We show that DCAF5 has a quality-control function for SWI/SNF complexes and promotes the degradation of incompletely assembled SWI/SNF complexes in the absence of SMARCB1. After depletion of DCAF5, SMARCB1-deficient SWI/SNF complexes reaccumulate, bind to target loci and restore SWI/SNF-mediated gene expression to levels that are sufficient to reverse the cancer state, including in vivo. Consequently, cancer results not from the loss of SMARCB1 function per se, but rather from DCAF5-mediated degradation of SWI/SNF complexes. These data indicate that therapeutic targeting of ubiquitin-mediated quality-control factors may effectively reverse the malignant state of some cancers driven by disruption of tumour suppressor complexes.
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Data availability
The ChIP–seq, ATAC–seq and RNA-seq data supporting the findings of this study have been deposited in the GEO database under accession number GSE215025. MS-based proteomics raw data files are provided in Supplementary Tables 1–3 and 6–8, and are available at PRIDE under the following dataset identifiers: PXD046276 (Supplementary Table 1), PXD046275 (Supplementary Tables 2 and 3), PXD046273 (Supplementary Table 6) and PXD04646 (Supplementary Tables 7 and 8). Coordinates for DDB1ΔB–DDA1–DCAF5 have been deposited at the PDB under accession number 8TL6. The cryo-EM volume data are available at the Electron Microscopy Data Bank under accession number EMD-41363. Source data are provided with this paper.
Code availability
The code for analysing the data and the relax_density_cart.xml has been deposited at GitHub (https://github.com/jamyers2358/SWISNF.DCAF5.Dependency)85.
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Acknowledgements
This work was supported by grants from the National Cancer Institute (NCI) (R01 CA113794, R01 CA273455 and R01 CA172152) to C.W.M.R.; CURE AT/RT Now to C.W.M.R.; the Garrett B. Smith Foundation to C.W.M.R.; and the St Jude Children’s Research Hospital Collaborative Research Consortium on Chromatin Regulation in Pediatric Cancer to C.W.M.R. S.R.-J. is supported by the St Jude Graduate School of Biomedical Sciences and the Ruth L. Kirschstein National Research Service Award (F31 CA261150); B.N. by the National Cancer Institute (NCI) (K22 CA258805); E.S.F. by grants from the National Cancer Institute (NCI) (R01 CA262188); J.P. by the National Institute on Aging (RF1AG068581). We thank members of the Roberts laboratory and S. Throm for discussions; G. Riddihough for assistance with editing this manuscript; C. Guy for assistance with microscopy; N. Thomä for the recombinant cBAF complex plasmids; the staff at Harvard Center for Cryo-Electron Microscopy for their support during grid screening and data collection; the members of the SBGrid Consortium for assistance with software and high-performance computing; and the members of the following St Jude core facilities: the Peptide Synthesis Core, the Protein Production Facility for Cas9, the Animal Resources Center for animal care, the Transgenic Core, the Flow Cytometry and Cell Sorting Shared Resource for FACS sorting, the Hartwell Center for Biotechnology for sequencing and peptide synthesis, the Center for In Vivo Imaging and Therapeutics for in vivo imaging and the Vector Development and Production Core for virus preparation. The St Jude Core facilities are supported by National Cancer Institute Cancer Center Support Grant (NCI CCSG 2 P30 CA021765) and by American Lebanese Syrian Associated Charities (ALSAC) of St Jude Children’s Research Hospital. The Center for Applied Bioinformatics is supported by the National Cancer Institute, Cancer Center Support Grant P30 CA21765 and ALSAC. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
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Authors and Affiliations
Contributions
S.R.-J. conceived the study, designed and performed the experiments, analysed the data, interpreted the results and wrote the manuscript. H.Y. performed DCAF5 structural studies, in vitro ubiquitylation assays, analysed results, provided intellectual input and wrote the manuscript. J.A.M. performed computational analysis, interpreted the results, assisted with manuscript writing and wrote computational methods. R.D.C. performed cellular experiments, assisted with mouse experiments, analysed the data and interpreted the results. A.N.R. performed cellular experiments, analysed the data and assisted with mouse experiments. P.M. assisted with growth assays, mouse experiments and colony management. Z.Z. assisted with ChIP–seq experiments and optimization. B.S.H. generated the CRISPR-edited pools and analysed the CRISPR fitness assay results. K.A.D. performed TMT profiling and AP-MS processing, analysed the data and interpreted the results. M.H. assisted with DCAF5 structural experiments and data processing. W.R. performed computational analysis, interpreted the results and wrote computational methods. Z.W. and M.G.M. assisted with ubiquitinome analysis and data processing. S.S.R.B. assisted with AlphaFold predictions and Rosetta refinement for the DCAF5 structure. A.M.S. performed LC–MS processing and analysed the data. N.M. performed TMT-profiling. S.A.B. assisted in DCAF5 antibody design and validation. R.J.M. performed SMARCB1 CUT&RUN and created SMARCB1 re-expression cell lines. J.F.P. provided intellectual input. E.A.S. assisted with in vivo study design and data analysis. S.M.P.-M. designed the CRISPR fitness assay and guides. B.N. and N.S.G. synthesized and provided the dTAGV-1 in vitro and in vivo molecules and assisted in dTAG-DCAF5 design. J.P. assisted with ubiquitinome analysis, data processing and data interpretation. E.S.F. supervised the DCAF5 structural studies and in vitro ubiquitylation assays, analysed the TMT-profiling data, interpreted the results, provided intellectual input and wrote the manuscript. C.W.M.R. conceived the study, designed the experiments, interpreted the results, wrote the manuscript, and supervised and funded the study. All of the authors read, reviewed and approved the final manuscript.
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Competing interests
C.W.M.R. is a scientific advisory board member of Exo Therapeutics, unrelated to this Article. E.S.F. is a founder, scientific advisory board member and equity holder of Civetta Therapeutics, Neomorph (also board member) and Proximity Therapeutics, scientific advisory board member and equity holder in Avilar Therapeutics and Photys Therapeutics, equity holder in Lighthorse Therapeutics and is a consultant to Novartis, Sanofi, EcoR1 capital, Ajax Therapeutics and Deerfield. The E.S.F. laboratory receives or has received research funding from Astellas, Novartis, Ajax, Voronoi, Interline and Deerfield on topics unrelated to this manuscript. B.N. is listed as an inventor on patent applications related to the dTAG system (WO/2017/024318, WO/2017/024319, WO/2018/148440 and WO/2018/148443). B.N. and N.S.G. are inventors on a patent related to the dTAG system and molecules described in this Article (WO/2020/146250). N.S.G. is a founder, science advisory board member and equity holder in Syros, C4 Therapeutics, Allorion, Lighthorse, Voronoi, Inception, Matchpoint, CobroVentures, GSK (scientific advisory board member), Larkspur (board member), Shenandoah (board member) and Soltego (board member). The N.S.G. laboratory receives or has received research funding from Novartis, Takeda, Astellas, Taiho, Jansen, Kinogen, Arbella, Deerfield and Sanofi on topics unrelated to this Article. K.A.D. is a consultant to Kronos Bio and Neomorph. The other authors declare no competing interests.
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Extended data figures and tables
Extended Data Fig. 1 DCAF5 dependence is specific to SMARCB1-mutant cancers independent of mRNA expression and tissue type.
a, Two-class comparison of n = 14 biologically independent Rhabdoid Tumour cell lines compared to other SWI/SNF mutant cancer cell lines (n = 190) or other non-SWI/SNF mutant cancer cell lines (n = 607) (****P = 1.22 × 10−16 and 7.16 × 10−21 respectively; two-tailed Student’s t test, release CERES 21Q1). The box plot indicates the median (centre line), the third and first quartiles (box limits) and 1.5 × interquartile range (IQR) above and below the box (whiskers). b, Box plot showing DCAF5 RNA expression across n = 1332 biologically independent cancer cell lines from different tumour types in the Cancer Cell Line Encyclopedia (CCLE) database. RT cell lines are shaded in red. The box plot indicates the median (centre line), the third and first quartiles (box limits) and 1.5 × interquartile range (IQR) above and below the box (whiskers). c, Bar plot demonstrating normalized expression (nTPM) levels of DCAF5 for n = 55 tissue types, created by combining the HPA and GTEx transcriptomics datasets using the Human Protein Atlas internal normalization pipeline. Colour-coding is based on tissue groups. d, Effects on proliferation upon DCAF5 shRNA knockdown in SMARCB1-mutant cell lines. Solid lines (shCTRL) and dotted lines (shDCAF5). Graphs show mean values from n = 8 technical replicates per cell line condition from one independent biological experiment. e, Western blot analysis of TTC549 RT cell line at Day 0 and Day 8 of IncuCyte proliferation assay. Band intensities were quantified by the Licor Image Studio Lite software and then the normalized DCAF5 level was calculated relative to Actin and normalized to shControl signal ± s.e.m (n = 3 independent biological replicates). ***P = 0.001; Two-Way ANOVA. f, DCAF5 immunoprecipitation in G401 and BT16 RT cell line demonstrates interaction of DCAF5 with E3 Ub Ligase machinery. Input is 1% of the protein used for the IP (n = 2 independent biological replicates).
Extended Data Fig. 2 Cryo-EM processing workflow for the DDB1∆B-DDA1-DCAF5 structure.
a, Raw micrograph (low pass-filtered to 10 Å, scale bar indicated). b, Representative 2D classes. c, Overview of processing workflow from raw micrograph. All processing steps were conducted in cryoSPARC. Particles belonging to coloured volumes were taken for the final map (EMD-41363). The final map is contoured at 0.134, and local resolution mapped onto the final reconstruction is shown. d, FSC plot for the deposited map (EMDB-41363). e, Viewing distribution plot. f, Directional resolution histogram and directional FSC plot. g, Model-to-map FSC for the deposited structure (PDB: 8TL6), value given for FSC (model)=0.5. h, Density example for the DCAF5 WD40 domain. i, Density for the DCAF5-motif in the DDB1ΔB binding site.
Extended Data Fig. 3 Details of DDB1∆B -DDA1-DCAF5 structure, evolutionary analysis and AlphaFold prediction.
a, Detailed view of DCAF5 and DDB1ΔB interaction shown in cartoon representation. The N-terminal α-helix of DCAF5 tightly inserts into the pocket of DDB1. b, Charge complementarity between DCAF5 and DDB1 at the interface. c, The N terminus of DDA1 inserts into DDB1, while the C terminus of DDA1 binds DCAF5 tightly with a hydrophobic interaction. DCAF5 surface is shown with hydrophobic and hydrophilic colour coding. d, Plot of the ConSurf conservation score versus the amino acid residue of full-length DCAF5 with domain annotations. e, ConSurf conservation scores are mapped onto DCAF5 with orange-white-purple colour scale in increasing conservation order. Top view and bottom view of the WD40 domain are shown. f, AlphaFold predictions for the DCAF5 aa 1-601 and SMARCC1 interaction. In the domain bar, DCAF5 is represented in green, with the WD40 domain specifically highlighted. SMARCC1 is depicted in magenta. g, The AlphaFold predicted binding mode of DCAF5 and SMARCC1 is shown. DCAF5 is represented in green, SMARCC1 is depicted in magenta, and DDB1-DDA1 is represented in grey.
Extended Data Fig. 4 DCAF5 loss upregulates protein levels of SWI/SNF members and alters SWI/SNF complex integrity.
a, Western blot analysis of SWI/SNF subunits in TTC549 SMARCB1-inducible RT cells treated with shCTRL or shDCAF5 after 72 h selection in the presence or absence of SMARCB1. b, RNA-Seq analysis in G401 RT cells treated with shCTRL or shDCAF5 after 72 h selection evaluating log2 fold change of mRNA for SWI/SNF in shDCAF5 versus shCTRL. ns = not significant; ** log2FC = −0.68, FDR = 0.02. Significance was determined by two-sided Empirical Bayes test for differential expression with FDR adjusted p-values. c, Left: Cycloheximide Chase (0-24 h with 50 ug/mL cycloheximide) in G401 shCTRL or shDCAF5 evaluating SWI/SNF subunit levels and control protein c-myc. Right: Graphical representation of the cycloheximide experimental data for the mean relative protein amount ± s.e.m of ARID1A (****P = < 0.0001), SMARCA4 (**P = < 0.0022), SMARCC1 (*P = < 0.0180), PBRM1(****P = < 0.0001) and c-myc (P = ns:not significant); Two-Way ANOVA. d, Glycerol gradient (10–30% glycerol) analysis of SMARCB1-deficient BT16 RT cells treated with either shCTRL or shDCAF5 after 72 h selection (top panel). SMARCB1 has been re-expressed in the cells in the bottom panel. e, SMARCA4 co-immunoprecipitation in G401 shCTRL and shDCAF5 conditions demonstrates that the SWI/SNF complex is maintained in the absence of DCAF5. Lamin A/C is a negative control. Input is 1% of the protein used for the IP. f, SMARCA4 co-immunoprecipitation in G401-dTAG-DCAF5 cells treated with DMSO and V-1 demonstrates retained SWI/SNF complex interactions in the absence of DCAF5. Lamin A/C is a negative control. Input is 1% of the protein used for the IP. g, SMARCA4 co-immunoprecipitation in G401 RT cells demonstrates interaction with DCAF5 and SWI/SNF subunits. Lamin A/C is a negative control. Input is 1% of the protein used for the IP. h, Two-class comparison of n = 14 biologically independent Rhabdoid Tumour cell lines compared to n = 789 biologically independent other cancer cell lines in DepMap analysing L3MBTL3 and LSD1 dependency (P = 0.907 and 0.701 respectively and is non-significant (ns); two-tailed Student’s t test, release CERES 21Q1). The box plots indicate the median (centre line), the third and first quartiles (box limits) and 1.5 × interquartile range (IQR) above and below the box (whiskers). i, L3MBTL3 co-immunoprecipitation in G401 RT cells detects no interaction with DCAF5 or SWI/SNF subunits. j, Western blot analysis of SWI/SNF subunits in BT16 and G401 RT cells treated with shCTRL or shL3MBTL3 after 72 h selection. k, Western blot analysis of SWI/SNF subunits in BT16 and G401 RT cells treated with shCTRL or shLSD1 after 72 h selection. Data are representative of three (c) or two (d,e,f,g,i,j and k) independent biological experiments.
Extended Data Fig. 5 In vitro and in vivo analyses of CRL4-DCAF5 and SWI/SNF substrates.
a, In vitro ubiquitylation assay screening of 13 E2-conjugating enzymes for CUL4-DDB1-RBX1-DCAF5 (CRL4DCAF5) ligase autoubiquitylation(n = 2). FL= full-length. b, In vitro ubiquitylation assay screening E2-conjugating enzymes for ubiquitylation of full-length (FL) SMARCC1 by FL-DCAF5 and DCAF5_ aa1-601). The combination of UBE2D3 + UBE2G1 has previously been identified as a canonical E2 pair for CRL4 ligases. c, In vitro ubiquitylation assay of SMARCC1 with 3 different CUL4DCAF5 constructs: DCAF5_aa 1-477 (which contains only the putatively active WD40 domain), DCAF5_aa 1-601 (which contains an extended region), and FL-DCAF5, alongside the CRL4DCAF11 complex (another ring E3 ligase) as a negative control and the whole recombinant SWI/SNF complex for ubiquitylation. The UBE2D3/UBE2G1 combination is chosen as the E2 pair for this assay and the following ubiquitylation assays. d, In vitro ubiquitylation assay of SMARCA4 (left) and ARID1A (right) in recombinant cBAF complex with CUL4DCAF5_aa 1-477 (which contains only the putatively active WD40 domain) complex. The experiment has been performed once. e, In vitro ubiquitylation assay of SMARCA4 (left) and ARID1A (right) in recombinant cBAF complex with CUL4DCAF5_aa 1-601 (which contains an extended region) complex. f, In vitro ubiquitylation assay of SMARCA4 (left) and ARID1A (right) in recombinant cBAF complex with full-length CUL4DCAF5_FL complex. The experiment has been performed once. g, Workflow of ubiquitylome analysis in G401 shCTRL and shDCAF5 RT cells. h, Comparison of global MS intensities in whole proteome and ubiquitylome (n = 2 biological replicates). Similar log2 values of intensities indicate minimal sample loading bias in both datasets. The boxplots of ubiquitinome and proteome were from n = 44,752 Peptide Spectrum Matches (PSMs) and n = 390,548 PSMs respectively. The box plots indicate the median (centre line), the third and first quartiles (box limits) and 1.5 × interquartile range (IQR) above and below the box (whiskers). i, MS intensities of two DCAF5 peptides indicate significant downregulation of DCAF5 protein in G401 shDCAF5 samples. Data are representative of two (a,c, and e) or three (b) independent biological experiments.
Extended Data Fig. 6 CRISPR-mediated knockout of DCAF5 SWI/SNF substrates rescues the lethal phenotype.
a, Indel toxicity assay evaluating selection against ARID1A out-of-frame alleles (containing ARID1A knockout) either in BT16 SMARCB1-deficient RT cells or in BT16 SMARCB1-deficient RT cells in which residual SWI/SNF subunits ARID1A, PBRM1, SMARCC1 and DCAF5 have been inactivated by CRISPR guides. CRISPR knockout of ARID1A is tolerated in both instances. b, Indel toxicity assay evaluating selection against SMARCC1 out-of-frame alleles (containing SMARCC1 knockout) either in BT16 SMARCB1-deficient RT cells or in BT16 SMARCB1-deficient RT cells in which residual SWI/SNF subunits SMARCC1, PBRM1, ARID1A and DCAF5 have been inactivated by CRISPR guides. CRISPR knockout of SMARCC1 is tolerated in both instances. c, Indel toxicity assay evaluating selection against PBRM1 out-of-frame alleles (containing PBRM1 knockout) either in BT16 SMARCB1-deficient RT cells or in BT16 SMARCB1-deficient RT cells in which residual SWI/SNF subunits PBRM1, SMARCC1, ARID1A and DCAF5 have been inactivated by CRISPR guides. CRISPR knockout of PBRM1 is tolerated in both instances. d, Western blot analysis in BT16-SMARCB1 deficient RT cells at Day 3 versus Day 21 in which residual SWI/SNF subunits ARID1A, PBRM1, SMARCC1 and DCAF5 have been inactivated by CRISPR guides. WT are wildtype cells. Data are representative of three independent biological experiments. Diagrams in a,b, and c were created using BioRender (https://biorender.com/).
Extended Data Fig. 7 SWI/SNF binding increases upon DCAF5 loss at enhancer regions.
a, Peak centred heatmaps +/−2 kb of averaged normalized coverage for significant, differentially bound regions defined as FC > 2 and FDR < 0.05 for ARID1A (n = 3 independent biological replicates) upon DCAF5 loss in G401 RT cells. b, Peak centred heatmaps +/−2 kb of averaged normalized coverage for significant, differentially bound regions defined as FC > 2 and FDR < 0.05 for SMARCC1 (n = 3 independent biological replicates) upon DCAF5 loss in G401 RT cells. c, Peak centred heatmaps +/−2 kb of averaged normalized coverage for significant, differentially bound regions defined as FC > 2 and FDR < 0.05 for SMARCA4 (n = 2 independent biological replicates) upon DCAF5 loss in G401 RT cells. d, Venn Diagram of gained regions (FC > 2 and FDR < 0.05) for ARID1A, SMARCC1, and SMARCA4. Peak centred heatmap +/−2 kb of averaged normalized coverage at each set of regions defined within the Venn Diagram. e, Sample locus depicting gains in averaged normalized coverage of SWI/SNF subunits and various histone marks in shDCAF5 treated G401 RT cells compared to control. f, Peak centred heatmaps +/−2 kb of averaged normalized coverage at 3,195 promoters for BRD9 in shCTRL (n = 2 independent biological replicates) and shDCAF5 (n = 2 independent biological replicates). g, Peak centred heatmaps +/−2 kb of averaged normalized coverage for SWI/SNF subunits at significant, differentially bound regions defined as FC > 2 and FDR < 0.05 for SMARCC1 in G401 RT cells. h, Peak centred heatmaps +/−2 kb of averaged normalized coverage for SWI/SNF subunits at significant, differentially bound regions defined as FC > 2 and FDR < 0.05 for SMARCA4 in G401 RT cells. i, Peak centred heatmaps +/−2 kb of averaged normalized coverage for SWI/SNF subunits (n = 1 independent biological replicate per mark) and H3K27ac (n = 2 independent biological replicates) 4 h after DCAF5 degradation with V-1 (FC > 0) at a previously defined subset of differentially bound regions. j, Genomic feature distribution of the entire genome (All) and ARID1A, SMARCC1, and SMARCA4 gained regions upon DCAF5 loss (FC > 2 and FDR < 0.05). k, Western blot analysis of p300 levels in G401 RT cells treated with shCTRL or shDCAF5 (n = 2 independent biological replicates). l, Peak centred metaplot of normalized, average coverage for p300 (n = 3 independent biological replicates) centred (+/−2 kb) on regions significantly gaining ARID1A upon loss of DCAF5 in G401 RT cells. Gains of p300 coincide with gains of H3K27ac upon loss of DCAF5.
Extended Data Fig. 8 Following DCAF5 loss, increased SWI/SNF binding results in transcriptional activation.
a, Peak centred metaplots of ARID1A gained regions (FC > 2, FDR < 0.05) +/−2 kb of averaged normalized nucleosome free coverage from ATAC-Seq for G401 shCTRL (n = 3 independent biological replicates) and shDCAF5 (n = 3 independent biological replicates) treated cells (left) compared to G401 −/+ SMARCB1 inducible cells (right) (n = 3 independent biological replicates). b, Motif enrichment analysis at regions gaining accessibility at SWI/SNF bound regions in SMARCB1 re-expressed cells, within the sites gained in both SMARCB1 addback and DCAF5 loss conditions and in shDCAF5 cells. P-values were calculated with a cumulative binomial distribution (one-sided) with Benjamini multiple test correction. c, Alignment of the position weight matrix (PWM) for the most significantly enriched de novo motif with the known AP-1 PWM (MA0099.2). d, Peak centred, +/−2 kb heatmaps at previously defined 4 h SWI/SNF gained regions (FC > 0) of averaged normalized nucleosome free coverage for G401-dTAG-DCAF5 DMSO treated (n = 3 independent biological replicates) and V-1 (n = 3 independent biological replicates) treated cells. e, Motif enrichment analysis at regions gaining SWI/SNF binding 4 h after DCAF5 degradation in G401-dTAG-DCAF5 cells. P-values were calculated with a cumulative binomial distribution (one-sided) with Benjamini multiple test correction. f, Relationship between transcriptional regulation (RNA-Seq) and gained binding of SMARCC1 and SMARCA4 upon loss of DCAF5 (ChIP-Seq) in G401 RT cells by Binding and Expression Target Analysis (BETA). Red and blue lines represent activated and repressed genes respectively and the dashed line represents an unchanging gene set. P-values calculated with one-tailed Kolmogorov-Smirnov test and compare the activated and repressed genes to the unchanging set. Predicted SMARCC1 and SMARCA4 target genes are upregulated upon DCAF5 loss. g, Top: Relationship between transcriptional changes (RNA-Seq) shDCAF5 vs. shCTRL log2FC y-axis and differential binding of shDCAF5 vs. shCTRL ARID1A, SMARCC1, and SMARCA4 (ChIP-Seq) log2FC x-axis. Bottom: GSEA results comparing gene sets of the top 500 ARID1A, SMARCC1, and SMARCA4 putative enhancer gene targets bound in shDCAF5 treated G401 cells, defined based on log2FC, −log10(p-value), and log10(Mean Enrichment +1, to transcriptional changes upon loss of DCAF5 in G401 RT cells, p-value: 0.002, 0.002, 0.002 and normalized enrichment score (NES): 2.37, 2.08, 2.08, respectively. Nominal P-value estimated using an empirical gene set permutation test. h, Venn diagram of predicted ARID1A, SMARCC1, and SMARCA4 upregulated target genes (predicted by BETA). i, GSEA results comparing a gene set of upregulated genes upon loss of DCAF5 in G401 RT cells (log2FC > 0 and adjusted p-value < 0.05) to the expression changes upon SMARCB1 re-expression in G401 RT cells (GSE71506) p-value = 0.001 and NES = 2.32. Nominal P-value estimated using an empirical gene set permutation test. j, Significantly enriched Gene Ontology (GO) terms ranked on Fold Enrichment (binomial over/under representation test with Bonferroni correction), based on genes significantly upregulated upon loss of DCAF5 in G401 RT cells (log2FC > 0 and adjusted p-value < 0.05). Pathways labelled in red are also upregulated upon SMARCB1 re-expression.
Extended Data Fig. 9 Generation and validation of G401-dTAG-DCAF5-YFP-dLuc cells.
a, Schematic of YFP-luciferase integration into G401-dTAG-DCAF5 cells. b, Flow cytometry plots and gating strategy for sorting G401-dTAG-DCAF5-YFP-dLuc cells that are YFP + . c, Immunofluorescence confirmation of YFP expression in G401-dTAG-DCAF5-YFP-dLuc cells compared to HeLa YFP negative control cells. Scale bar 100 μm. The experiment has been performed once. d, Western blot analysis confirming DCAF5 degradation of G401-dTAG-DCAF5-YFP-dLuc cells after treatment with 50 nM or 500 nM of dTAGV-1 at 4 h and 24 h. The experiment has been performed once. e, Weight comparisons from 8-week-old Dcaf5 female mice (n = 5 mice per genotype). WT (wildtype), Het. (heterozygous) and KO (knockout) P = ns (non-significant); Two-way ANOVA. The diagram in a was created using BioRender (https://biorender.com/).
Supplementary information
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Supplementary Tables 1–11.
Supplementary Video 1
Dcaf5 -knockout mice are viable. Germline Dcaf5-knockout mice were viable and indistinguishable from littermate controls at least through the most recent timepoint of 12 weeks of age.
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Radko-Juettner, S., Yue, H., Myers, J.A. et al. Targeting DCAF5 suppresses SMARCB1-mutant cancer by stabilizing SWI/SNF. Nature 628, 442–449 (2024). https://doi.org/10.1038/s41586-024-07250-1
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DOI: https://doi.org/10.1038/s41586-024-07250-1
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