EMBO/SourceData huggingface dataset:
Figure 3(A) Cell extracts were generated after the indicated treatments and then incubated with anti-GFP beads. Factors co-purifying with GFP-SLD5 were monitored by immunoblotting.(B) TAP-SLD5 cells were subjected to the indicated treatments. The level of LRR1 and VHL was assessed by immunoblotting. The asterisk denotes a non-specific band. (C) For the experiment described in (B), TAP-SLD5 was isolated from cell extracts and the indicated factors monitored by immunoblotting.(D) The activity of CUL2LRR1 was inhibited in GFP-SLD5 cells , by treatment with a combination of LRR1 siRNA and MLN4924 Fixed cells were analysed Arrows indicate the accumulation of GFP-SLD5 on constitutive heterochromatin (49 % cells, n = 125). (E) Cells were treated as in (D) and the localisation of GFP-SLD5 was compared with CLASPIN by immunofluorescence. Arrows indicate the co-localisation of GFP-SLD5 and CLASPIN on constitutive heterochromatin (in 90 % cells with GFP-SLD5 patches, n = 103).
Figure 4(A) Time-lapse analysis of mouse ES cells expressing GFP-SLD5 and mCherry-PCNA, following inhibition of CUL2LRR1. Arrows illustrate the persistence of GFP-SLD5 on heterochromatin after disappearance of mCherry-PCNA.(B) time-lapse analysis of mouse ES cells expressing GFP-SLD5 and mCherry-Histone H2B (from the CAG promoter at the ROSA26 locus after inhibition of CUL2LRR1. Arrows indicate heterochromatic patches of GFP-SLD5 that disappear during entry into mitosis (98 % cells, n = 97). (C) Equivalent data to (B) but for untreated cells (100 % of untreated cells lacked GFP-SLD5 on mitotic chromatin, n = 149).
Figure 5(A) TRAIP co-purifies with the CMG helicase from extracts of mouse ES cells. The asterisk indicates a non-specific band.(B) Mouse ES cells were processed Arrows in (i) indicate accumulation of GFP-SLD5 on heterochromatin during interphase, whereas arrows in (ii) denote GFP-SLD5 on chromatin during mitotic metaphase. Scale bars correspond to 10 µm.(C) Immunoblot illustrating the stable expression from the CAG promoter of the indicated forms of TRAIP at the ROSA26 locus in TRAIP -/- mouse ES cells. Asterisks indicate non-specific bands. (D) Quantification of the percentage of mitotic cells with GFP-SLD5 on mitotic chromosomes, after inhibition of CUL2LRR1 in cells with the indicated genotypes. Cells were fixed before analysis.
Figure 1B Quantified fluorescence of UbG76V-GFP normalized to mRFP in the hypodermis from animals of the indicated timepoint (in hours) after the L4 stage. Animals have been exposed to the indicated knockdown RNAi bacterial strains or a strain that only contains the empty RNAi vector. *P<0.001, Student t-test corrected for multiple comparisons at each time point using the Holm-Sidek method. N=20 animals per genotype and timepoint. Error bars indicate SEM.C,D Co-expression via the col-19 promoter of UbG76V-GFP (green) and mRFP (red) in C. elegans hypodermis from a single integrated transgene at L4+48 hours. Wild-type animals are shown. Bar, 50 microns. UbG76V-GFP fluorescence is barely detectable.E,F Co-expression via the sur-5 promoter of UbG76V-GFP (green) and mCherry (red) in C. elegans from two separate integrated transgenes (with focus on the intestine) at L4+48 hours. Wild-type animals are shown. Bar, 50 microns. UbG76V-GFP fluorescence is barely detectable.G,H Co-expression of UbG76V-GFP (green) and mRFP (red) in C. elegans hypodermis at L4+48 hours in dop-1(vs100) mutants. Bar, 50 microns. Abundant, stable UbG76V-GFP fluorescence is observed.I,J Co-expression of UbG76V-GFP (green) and mCherry (red) in C. elegans from the sur-5 promoter at L4+48 hours in dop-1(vs100) mutants. Bar, 50 microns. Abundant, stable UbG76V-GFP fluorescence is observed, particularly in the intestine.K Quantified fluorescence of UbG76V-GFP normalized to mRFP in the hypodermis from animals of the indicated timepoint (in hours) after the L4 stage and of the indicated genotype. *P<0.001, Student t-test corrected for multiple comparisons at each time point using the Holm-Sidek method. N=20 animals per genotype and timepoint. Error bars indicate SEM.L Quantified fluorescence of UbG76V-GFP normalized to mRFP in the hypodermis of animals at L4+48 hours and of then indicated genotypes. ***P<0.001, ANOVA with Dunnetts posthoc comparison to the wild-type control. N=20 animals per genotype and timepoint. Error bars indicate SEM.M Quantified fluorescence of UbG76V-GFP normalized to mCherry (both expressed by the sur-5 promoter) in the intestine of animals at L4+48 hours and of then indicated genotypes. ***P<0.001, **P<0.01, *P<0.05, ANOVA with Dunnetts posthoc comparison to the wild-type control. N=20 animals per genotype and timepoint. Error bars indicate SEM.
Figure 2A Western blot of lysed nematodes at the L4+48 hour stage, probed with antibodies recognizing, ubiquitin, GFP, RPN-11, multiple 20S proteasome subunits, or actin as a loading control. Animals express UbG76V-GFP protein in their hypodermis. The position of molecular weight markers is shown on the left of each blot. The position of UbG76V-GFP protein, as well as UbG76V-GFP with the indicated number of additional ubiquitin moieties, based on molecular weight, is indicated on the right of each blot. Twenty animals were loaded per lane for each indicated genotype (WT indicates wild type).B Western blot of lysed nematodes at the L4+48 hour stage, probed and annotated as in (A). Animals express UbG76V-GFP protein from the sur-5 promoter. Thirty animals were loaded per lane for each indicated genotype (WT indicates wild type).C Quantified fluorescence of UbG76V-GFP normalized to mRFP from lysates of transgenic nematodes at L4+48 hours that express these reporters in the hypodermis. Animals were exposed to bacteria containing the indicated RNAi clone or an empty RNAi vector as a control. *P<0.05, ANOVA with Dunnetts posthoc comparison to the empty RNAi vector control. N=3 trials. Error bars indicate SEM.D Quantified epoxomicin-sensitive proteasome activity (as measured through the turnover of fluorescent chymotrypsin substrate) from the same lysates and exposed to the same RNAi as in (C). *P<0.05, ANOVA with Dunnetts posthoc comparison to the empty RNAi vector control. N=3 trials. Error bars indicate SEM.E Quantified fluorescence of UbG76V-GFP normalized to mRFP from lysates of transgenic nematodes at L4+48 hours that express these reporters in the hypodermis. Animals were of the indicated genotype. ***P<0.001, **P<0.01, *P<0.05, ANOVA with Dunnetts posthoc comparison to wild type. N=4 trials. Error bars indicate SEM.F Quantified epoxomicin-sensitive proteasome activity (as measured through the turnover of fluorescent chymotrypsin substrate) from the same lysates as in (E). No statistical difference (P<0.05 cutoff) was detected by ANOVA. N=4 trials.G Percentage of live animals of the indicated genotype assayed after 6-hour exposure to heat shock. 100% of control animals of the same genotype but not given heat shock survived. *P<0.01, Student t-test. N=3 trials. Error bars indicate SEM.H Percent of animals of the indicated genotype containing aggregates of Poly(Q)44::YFP at the indicated time (in days) after L4 stage. *P<0.01, Student t-test. N=3 trials, 50-80 animals per trial per day. Error bars indicate SEM.
Figure 3A Relative abundance of mRNA for the indicated xenobiotic stress response gene (or UbG76V-GFP or mRFP) detected in wild type versus dop-1 mutants. Values are based on RNA-seq/EdgeR analysis, with the relative levels for each gene normalized to the value observed in the wild-type control. ****P<0.0001, **P<0.01, *P<0.05 FDR from EdgeR analysis using a Benjamini and Hochberg correction.B,C Quantified fluorescence of UbG76V-GFP normalized to (B) mRFP in the hypodermis from L4+48 hour animals exposed to feeding RNAi for either an empty vector control or the indicated xenobiotic stress gene. ****P<0.0001, ***P<0.001, **P<0.01, ANOVA, Tukey's multiple comparison test compared to control. N=20 animals per genotype and timepoint. Error bars indicate SEM.B,C Quantified fluorescence of UbG76V-GFP normalized to (C) mCherry in the intestine from L4+48 hour animals exposed to feeding RNAi for either an empty vector control or the indicated xenobiotic stress gene. ****P<0.0001, ***P<0.001, **P<0.01, ANOVA, Tukey's multiple comparison test compared to control. N=20 animals per genotype and timepoint. Error bars indicate SEM.D Percentage of live animals raised on the indicated feeding RNAi (or empty vector control) bacteria assayed after 6-hour exposure to heat shock. 100% of control animals of the same genotype but not given heat shock survived. ***P<0.001, **P<0.01, *P<0.05, ANOVA, Tukey's multiple comparison test compared to wild type. N=3 trials. Error bars indicate SEM.E Quantified fluorescence of UbG76V-GFP normalized to mRFP in the hypodermis from L4+48 hour animals of the indicated genotype. ****P<0.0001, ANOVA, Tukey's multiple comparison test compared to wild type. N=20 animals per genotype and timepoint. Error bars indicate SEM.F Percentage of live animals of the indicated genotype assayed after 6-hour exposure to heat shock. 100% of control animals of the same genotype but not given heat shock survived. **P<0.01, *P<0.05, ANOVA, Tukey's multiple comparison test compared to wild type. N=3 trials. Error bars indicate SEM.G Percent of animals of the indicated genotype containing aggregates of Poly(Q)44::YFP at the indicated time (in days) after L4 stage. *P<0.001, Student t-test corrected for multiple comparisons at each time point using the Holm-Sidek method. N=3 trials, 50-80 animals per trial per day. Error bars indicate SEM.
Figure 4A Quantified fluorescence of UbG76V-GFP normalized to mRFP in the hypodermis from L4+48 hour animals of the indicated genotype. ****P<0.0001, ANOVA, Tukey's multiple comparison test compared to wild type. N=20 animals per genotype. Error bars indicate SEM.B Quantified fluorescence of UbG76V-GFP normalized to mRFP in the hypodermis from either L4+24 hour animals (left half of graph) or L4+48 hour animals (right half of graph) of the indicated genotype. Animals were either continually grown on regular OP50 food (gray bars) or switched to PA14 food at the L4 stage (orange bars). ****P<0.0001, ***P<0.001, **P<0.01, ANOVA, Tukey's multiple comparison test compared to wild type. N=20 animals per genotype. Error bars indicate SEM.C Survival curves (percentage of live animals assayed each hour after PA14 exposure) for the indicated mutants at 20oC. Plog-rank<0.0001. Median survival time was 90 hours for wildtype (N=304), 72 hours for dop-1(vs100) (N=180), 78 hours for elt-3(gk121) (N=120), 78 hours for pqm-1(ok485) (N=116), 78 hours for nhr-28(gk568) (N=123), and 42 hours for skn-1(tm3411) (N=33). Eight wild-type animals and six dop-1 mutants were censored (the animals crawled up the side of the plate and dessicated). The media contained 50 µg/ml of 5-fluoro-2′-deoxyuridine (FUdR to prevent the growth of egg offspring during the experiment.D Quantified fluorescence of UbG76V-GFP normalized to mRFP in the hypodermis from either L4+48 hour animals (left half of graph) or L4+72 hour animals (right half of graph) of the indicated genotype. Animals were either continually grown on regular OP50 food (gray bars) or switched to NGM plates containing 1 mM paraquat at the L4 stage (orange bars). ****P<0.0001, ***P<0.001, ANOVA, Tukey's multiple comparison test compared to wild type. #P<0.001 ANOVA, Bonferoni multicomparison as indicated by the bracketed lines. N=20 animals per genotype. Error bars indicate SEM. No UbG76V-GFP fluorescence was detected in wild type at L4+72 hours on either OP50 or switched to NGM plates containing 1mM paraquat.
Figure 1A: Graph of the ratio of the median IC50 of the entire panel to that of each cell line generated from a screen of 23 pediatric cancer cell lines. Rhabdoid tumor cell lines (red) cluster towards the left of the graph indicating these cell lines are more sensitive to mithramycin. These results confirm a previously published screen (Osgood et al., 2016).B: Graph of the ratio of the median IC50 of the entire panel to that of each cell line generated from a published screen of 445 agents in 62 sarcoma cell lines (Teicher et al., 2015). The rhabdoid tumor cell line, G401 (red), appears on the left side of the graph indicating this cell line is more sensitive to mithramycin.C: Dose response curves of rhabdoid tumor and Ewing sarcoma cell lines. RT cell lines (black) are sensitive to mithramycin treatment with a similar IC50 value as TC32 ES cells (grey). RT cell lines are not sensitive to three broadly-active chemotherapeutic agents: etoposide, doxorubicin or SN38. Data represents mean with standard deviation derived from three independent experiments.
Figure 2A, B: Western blot showing concentration dependent increase in γH2AX following 8-hour exposure to etoposide in BT12 and G401 RT cells (A). Red bar indicates the measured IC50 (Figure 1C). Despite induction of DNA damage, 15 μM etoposide does not lead to apoptosis as indicated by live cell imaging in the presence of cleaved caspase 3,7 (CC3,7) reagent that fluoresces with caspase activation (B). Scale bar (lower left): 150μm.  C, D: Western blot showing concentration dependent increase in γH2AX following 8-hour exposure to mithramycin (MMA) in BT12 and G401 RT cells (C). Red bar indicates the measured IC50 (Figure 1C). 100 nM Mithramycin induces apoptosis at 8-hours as measured by CC3,7 fluorescence without the presence of DNA damage (D). Scale bar (lower left): 150μm.  E: BT12 cells treated with 20nM MMA show a different cellular phenotype than with 100nM. Beyond 24h of exposure, there is evidence of mesenchymal differentiation and the appearance of maturing adipocytes. Scale bar (lower left): 150μm.  F, G: Western blot showing concentration dependent increase in γH2AX following 8-hour exposure to EC8042 in BT12 and G401 RT cells. Red bar indicates the measured IC50 (Figure 1C). In contrast to apoptotic induction, 75nM EC8042 exhibits evidence of mesenchymal differentiation, similar to 20nM MMA (E,G). Please note, solvent control for (E) and (G) is the same although different fields are shown. Scale bar (lower left): 150μm.  H: Mesenchymal differentiation confirmed with oil red O staining of lipid deposits following 20nM MMA or 75nM EC8042 treatment for 24 and 48-hours. Please note, solvent control for 20nM MMA and 75nM EC8042 is the same. Scale bar (lower left): 150μm.  I: PPARγ mRNA expression is induced following 20nM mithramycin treatment as measured by qPCR fold-change relative to GAPDH (2ddCT). **, P = 0.002, ****, P = 0.0001. Data represents mean with standard deviation derived from three independent experiments. P-values were determined by one-way ANOVA using Dunnet test for multiple comparisons.
Figure 3 A, B: Mithramycin displaces SMARCC1 and SMARCE1 SWI/SNF subunits from chromatin in a time-dependent manner in BT12 rhabdoid tumor (A) but not U20S osteosarcoma (B) cells. Western blot analysis showing whole cell lysate (Total), cytoplasmic soluble (CS), nuclear soluble (NS), and chromatin bound (Chr) fractions collected after exposure to solvent (S) or 100nM mithramycin for 8 or 18h and probed for the SWI/SNF subunits (BRD9, SMARCC1 or SMARCE1) or H3 (chromatin fraction control) and GAPDH (soluble fraction control). C: SMARCC1 and SMARCE1 mRNA expression does not change following 100nM mithramycin treatment as measured by qPCR fold-change relative to GAPDH (2ddCT). Data represents mean with standard deviation derived from three independent experiments. D: Addition of the proteasome inhibitor (bortezomib) rescues the loss of protein expression following 8 or 18-hour mithramycin treatment. BT12 cells were treated for 8 or 18-hours with solvent (S), mithramycin (M, 100nM), bortezomib (PI, 2.5μM), or combination of 100nM mithramycin and 2.5μM bortezomib (C).  E: Loss of SWI/SNF occupancy at defined loci in the genome as measure by chromatin immunoprecipitation qPCR (ChIP-qPCR) at previously described SWI/SNF target genes, MYT1 (8-hour, P=0.0001; 18-hour P=0.0001) and CCND1 (8-hour, P=0.0001; 18-hour P=0.0001). ChIP quantitation is percent input (ng of immunoprecipitated DNA/input DNA *100) determined by absolute quantitation of sheared chromatin relative to a standard curve. Data represents mean with standard deviation derived from three independent experiments. P-values were determined by one-way ANOVA using Dunnet test for multiple comparisons.  F: Chromatin immunoprecipitation qPCR (ChIP-qPCR) of H3K27me3 at MYT1 (8-hour, P= 0.0001; 18-hour P=0.0001) and CCND1 (8-hour, P=0.02; 18-hour P=0.0001). H3K27me3 occupancy is increased in a time-dependent manner. ChIP quantitation is percent input (ng of immunoprecipitated DNA/input DNA *100) determined by absolute quantitation of sheared chromatin relative to a standard curve. Data represents mean with standard deviation derived from three independent experiments. P-values were determined by one-way ANOVA using Dunnet test for multiple comparisons. Western blot showing concentration-dependent increase in H3K27me3 following exposure to 100nM, 50nM, 25nM mithramycin for 18h in BT12 cells relative to loading control (H3). Western blot showing concentration-dependent increase in H3K27me3 following exposure to 100nM, 50nM, 25nM mithramycin for 18h in G401 cells relative to loading control (H3). Re-expression of SMARCB1 following doxycycline treatment inhibits the mithramycin-dependent effects on H3K27me3 amplification
Figure 4 A, B: Mithramycin displaces BRD9 and SMARCE1 SWI/SNF subunits from chromatin in a time-dependent manner in G401 (A) but only BRD9 with SMARCB1 re-expression in G401 (B) cells. Western blot analysis showing whole cell lysate (Total), cytoplasmic soluble (CS), nuclear soluble (NS), and chromatin bound (Chr) fractions collected after exposure to solvent (S) or 100nM mithramycin for 8 or 18h and probed for the SWI/SNF subunits (BRD9, SMARCB1 or SMARCE1) or H3 (chromatin fraction control) and GAPDH (soluble fraction control).  C: BT12 cells show a threshold of exposure that leads to irreversible growth inhibition. The cells were exposed to 100nM MMA for the indicated times followed by replacement with drug-free medium. Beyond 8h (red) of mithramycin exposure, the cells do not recover proliferative potential and exhibit a phenotype consistent with cell death. Data represents mean with standard deviation derived from three independent experiments.  D: Mithramycin leads to H3K27me3 amplification in a time-dependent manner that precedes the induction of apoptosis as measured by the cleavage of PARP. Western blot lysates collected at 2h, 4h, 6h, 8h, 12h, 18h, 24h of continuous 100nM mithramycin treatment.  E: Suppression of EZH2 expression and activity antagonizes mithramycin activity in BT12 rhabdoid tumor cells. Data represents dose response curves of mithramycin in BT12 cells following a 48h exposure in the presence of siRNA silencing of the PRC2 subunit EZH2 or treatment with the EZH2 inhibitor EPZ-6438 relative to mithramycin alone (media) or a non-targeting siRNA (siNeg). Data represents mean with standard deviation derived from three independent experiments.  F: Suppression of SMARCB1 sensitizes U2OS osteosarcoma cells (wild type SWI/SNF) to mithramycin. Data represents dose response curves of mithramycin in BT12 cells following a 48h exposure in the presence of siRNA silencing of the SWI/SNF subunit SMARCB1 relative to a non-targeting siRNA (siNeg). Data represents mean with standard deviation derived from three independent experiments.  G. Western blot showing concentration-dependent increase in H3K27me3 following exposure to 100nM, 50nM, 25nM mithramycin for 18h in SMARCB1- silenced U2OS cells relative to loading control (H3). Knockdown of SMARCB1 following siRNA suppression triggers mithramycin-dependent H3K27me3 amplification, while siNegative (control) does not have an effect on H3K27me3 following mithramycin exposure.
Figure 5A-C: Volcano plot showing gene expression trends at 8h MMA treatment. Dashed lines represent a 2 log2FC (drug over control) (A) or 1.5 log2FC (drug over control) (B) and 10e-7 q-value threshold. Quantification of induced and repressed genes in (C). Labels indicate names of genes that meet the logFC and q-value threshold.D-F: Volcano plot showing gene expression trends at 18h MMA treatment. Dashed lines represent a 2 log2FC (drug over control) (D) or 1.5 log2FC (drug over control) (E) and 10e-7 q-value threshold. Quantification of induced and repressed genes in (F). Labels indicate names of genes that meet the logFC and q-value threshold. G: SP1 protein expression is reduced following mithramycin exposure. Western blot showing suppression of SP1 expression compared to loading control (GAPDH) after 100nM mithramycin exposure for 1h, 4h, 8h, 12h, 18h.  The loss of SP1 expression is associated with a decrease in SWI/SNF occupancy of the SP1 promoter and an increase in H3K27me3. Data represents ChIP-qPCR analysis following 8-hours or 18-hours of 100nM mithramycin exposure and immunoprecipitation of SMARCC1 (8-hour, P= 0.0006; 18-hour P=0.0001) . ChIP quantitation is percent input (ng of immunoprecipitated DNA/input DNA *100) determined by absolute quantitation of sheared chromatin relative to a standard curve. Data represents mean with standard deviation derived from three independent experiments. P-values were determined by one-way ANOVA using Dunnet test for multiple comparisons.  I: The loss of SP1 expression is associated with a decrease in SWI/SNF occupancy of the SP1 promoter and an increase in H3K27me3. Data represents ChIP-qPCR analysis following 8-hours or 18-hours of 100nM mithramycin exposure and immunoprecipitation of H3K27me3 (8-hour, P= 0.009; 18-hour P=0.0001) ChIP quantitation is percent input (ng of immunoprecipitated DNA/input DNA *100) determined by absolute quantitation of sheared chromatin relative to a standard curve. Data represents mean with standard deviation derived from three independent experiments. P-values were determined by one-way ANOVA using Dunnet test for multiple comparisons.
Figure 6A: Heatmaps depicting ATAC-seq global chromatin structure following 8-hours (middle) and 18-hours (right) 100nM MMA treatment. A 2kb window is centered on the TSS. Chromatin accessibility clusters at the TSS and does not change globally relative to solvent. Quantification of genes that gain accessibility or reduce accessibility on the right. E: IGV tracks of rhabdoid tumor genes previously identified to be occupied by non-canonical SWI/SNF (Michel et al., 2018). ID3 and JUND3 decrease in H3K27ac occupancy and chromatin accessibility following exposure to mithramycin for 8-hours or 18-hours.  F: IGV tracks of rhabdoid tumor genes previously identified to gain H3K27me3 upon SWI/SNF loss (Erkek et al., 2019). CDK6 and CDK2 decrease in H3K27ac occupancy and chromatin accessibility following exposure to 8-hours and 18-hours mithramycin treatment.  G: IGV tracks of genes identified to have an increase in accessibility, H3K27ac and gene expression following mithramycin exposure. BMP1 and ADIPOR1 play crucial roles in bone and adipogenic differentiation, respectively.