Perturbations to mammalian switch/sucrose non-fermentable (mSWI/SNF) chromatin remodeling complexes have been widely implicated as driving events in cancer1. One such perturbation is the dual loss of the SMARCA4 and SMARCA2 ATPase subunits in small cell carcinoma of the ovary, hypercalcemic type (SCCOHT)2,3,4,5, SMARCA4-deficient thoracic sarcomas6 and dedifferentiated endometrial carcinomas7. However, the consequences of dual ATPase subunit loss on mSWI/SNF complex subunit composition, chromatin targeting, DNA accessibility and gene expression remain unknown. Here we identify an ATPase module of subunits that is required for functional specification of the Brahma-related gene–associated factor (BAF) and polybromo-associated BAF (PBAF) mSWI/SNF family subcomplexes. Using SMARCA4/2 ATPase mutant variants, we define the catalytic activity–dependent and catalytic activity–independent contributions of the ATPase module to the targeting of BAF and PBAF complexes on chromatin genome-wide. Finally, by linking distinct mSWI/SNF complex target sites to tumor-suppressive gene expression programs, we clarify the transcriptional consequences of SMARCA4/2 dual loss in SCCOHT.
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Code to generate figures is available at https://github.com/joshbiology/sccoht/.
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We thank members of the Kadoch Laboratory for helpful conceptual and experimental advice throughout the development of this study. We thank the DFCI Molecular Biology Core Facility, particularly Z. Herbert, for library preparation and sequencing, G. Boulay for advice regarding ChIP-seq optimization and the Taplin Mass Spectrometry Facility for mass-spectrometry analysis and data processing. We thank B. Vanderhyden (Ottawa Hospital Research Institute) and R. Hass (Hannover Medical School) for providing the BIN-67 and SCCOHT-1 cell lines, respectively. This work was supported in part by funding from the National Science Foundation Graduate Research Fellowship (No. 2015185722) and NIH T32 Training Grant in Genetics and Genomics to J.P.; the NIH DP2 New Innovator Award (No. 1DP2CA195762-01) to C.K.; the American Cancer Society Research Scholar Award (No. RSG-14-051-01-DMC) to C.K.; and the Pew–Stewart Scholars in Cancer Research Grant to C.K.
C.K. is a Scientific Founder, fiduciary Board of Directors member, Scientific Advisory Board member, consultant and shareholder of Foghorn Therapeutics, Inc.
Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Integrated supplementary information
Supplementary Figure 1 Biochemical and genomic characterization of residual mSWI/SNF complexes in SMARCA4/2-deficient cancer cell lines.
a. Immunoblot for mSWI/SNF subunits performed on whole cell lysates shows loss of SMARCA4 and SMARCA2 in BIN-67 and SCCOHT-1 cell lines. 293T and SW-13 cell lines included as positive and negative controls, respectively. See also Supplementary Fig. 7c. b. Density sedimentation and immunoblot performed on nuclear extracts isolated from the BIN-67 cell line recapitulate results in the SCCOHT-1 cell line. See also Supplementary Fig. 7d. c. Density sedimentation and Western blotting of nuclear extract from the mSWI/SNF-intact lung cell line, BEAS-2B. See also Supplementary Fig. 7e. d. Immunoprecipitation using a panel of mSWI/SNF antibodies in BIN-67 cell nuclear extracts. SMARCC2, SMARCC1, SMARCD1, ARID1A and ARID2 subunits exhibit binding as part of the residual complex. ARID1A and ARID2 remain mutually exclusive, binding BAF and PBAF complexes, respectively. See also Supplementary Fig. 7f. e. SDS-PAGE/silver stain performed on purified BAF complexes (using HA-DPF2 and HA-SMARCD1 as overexpressed baits) in 293T and SW-13 cell lines. Identified subunits are labeled. Subunits common to all purifications are colored in black and subunits absent from the SW-13 purification are colored in purple. f. Replicate ChIP-seq experiments (n=3) performed in control BIN-67 cells. Spearman correlation was used to evaluate ChIP-seq replicate agreement. g. Combined gene expression, accessibility, and mSWI/SNF promoter occupancy for all 22,000+ genes in BIN-67 and SCCOHT-1 cell lines is shown. Each column in the heatmap is a gene, rank ordered from highest to lowest expression. A gene is annotated as mSWI/SNF-occupied if there is a ChIP-seq peak within 4kb of the TSS. A peak set with shuffled genomic coordinates (random) is included as a comparison. h. Differential essentiality (CERES score) of BAF and PRC2 subunits in the presence and absence of SMARCA4/2. First and third quartiles are denoted by lower and upper hinges. The smallest and largest values within 1.5x the interquartile range are denoted by the lower and upper whiskers. Dual ATPase deficient lines (n=5), mSWI/SNF intact lines (n=386).
Supplementary Figure 2 mSWI/SNF subunit stability and complex binding on chromatin after rescue of the ATPase subunit.
a. Overexpression of SMARCA2 in SW-13 and BIN-67 cell lines increases nuclear protein abundance of ATPase module subunits (total cell lysate). See also Supplementary Fig. 7g. b. Time-course of doxycycline-induced expression of SMARCA4 in SW-13 cells shows a stabilization of SS18, PBRM1 and ACTL6A in total cell lysate. See also Supplementary Fig. 7h. c. (Left) Immunofluorescence performed on a mixed population of SW-13 cells with and without exogenous expression of SMARCA4. SS18 protein levels are restored only in those cells that overexpress SMARCA4. (Right) Scatterplot of SMARCA4 and SS18 fluorescent intensity at an individual cell level, normalized to DAPI intensity. Spearman correlation = 0.860. Scale bar = 50 uM. d. Immunoprecipitation using anti-SMARCA4 antibody in SW-13 nuclear extracts with and without SMARCA4 overexpression. See also Supplementary Fig. 7i. e. Immunoprecipitation using SMARCD1 and SS18 antibodies in SW-13 nuclear extracts with and without SMARCA4 overexpression. See also Supplementary Fig. 7j. f. Immunoprecipitation using SMARCA4 and two distinct PBRM1 antibodies in SW-13 nuclear extracts with and without SMARCA4 overexpression. See also Supplementary Fig. 7k. g. Differential salt extraction performed on SW-13 cells with and without rescue of SMARCA4 demonstrates higher chromatin affinity in the SMARCA4 rescue setting. Two nuclear proteins (CTCF, high affinity to chromatin; TBP, low affinity to chromatin) are shown as controls. See also Supplementary Fig. 7l. h. Number of significant MACS-called ChIP-seq peaks across ChIP-seq experiments in GFP and SMARCA4 reintroduction conditions in BIN-67 cell lines.
Supplementary Figure 3 Biochemical and chromatin binding characterization of the SMARCA4 p.Thr910Met and SMARCA4 p.Lys785Arg mutant variants.
a. Western blot confirmation of SMARCA4 WT, T910M and K785R expression in BIN-67 cells. See also Supplementary Figure 7m. b. Silver stain of HA-DPF2-purified BAF complexes from SMARCA4/2- deficient, SMARCA4 WT rescued and SMARCA4 T910M rescued complexes in 293T SMARCA4/2 double knockout cells. c. ATP consumption assays performed on purified SMARCA4 ATPase protein variants shows differential activity between SMARCA4 WT and K785R and T910M mutants (for each group, n = 3). Central bar denotes the mean and error bars denote standard deviation. d. Western blot of differential salt extraction performed in SW-13 cell with overexpression of SMARCA4 WT, K785R and T910M mutants. See also Supplementary Fig. 7n. e. Scatterplots of ChIP-seq read density for SMARCA4 and SMARCC1 antibodies performed over three distinct SMARCA4 reintroduction conditions. Pearson correlation was used to assess similarity in peaks called for each mSWI/SNF antibody. f. Log-scale scatterplot showing the normalized read counts from SS18 and ARID2 ChIP-seq across a union set of SS18 and ARID2 peaks upon SMARCA4 T910M and K785R rescue.
a. Venn diagram of overlap between BAF activity-dependent peaks and two histone marks correlated with transcriptional repression, H3K27me3 and H3K9me3. Both histone markes were profiled in the BIN-67 control condition. b. Metagene plot of H3K4me1 occupancy over TSS’s. c. Heatmap of histone marks and ARID2/SS18 occupancy over ARID2 activity-dependent peaks.
Supplementary Figure 5 Gene expression phenotypes after rescue of SMARCA4 wild-type and mutant variants in SCCOHT cell lines.
a. Volcano plots representing significantly changed genes upon each of the four reintroduction conditions. The Wald test followed by BH false discovery rate correction was used to report significance (implemented by the DESeq2 package). The number of genes with |log2FC| > 1 are annotated above each plot. b. Genome browser view of KRT6A/C and PSG3/8 loci, genes in the highly upregulated cluster that show clear regulation by activity-dependent peaks. c. Senescence staining (beta-galactosidase) in control and SMARCA4 WT rescued BIN-67 cells. Scale bar = 100 um. d. Enrichment of the hallmark epithelial-to-mesenchymal transition geneset among upregulated genes upon SMARCA4 WT rescue.
Schematic highlighting features of ATPase-active and ATPase-deficient mSWI/SNF complexes.
a. Western blots related to Fig. 1a. b. Western blots related to Fig. 2b. c. Western blots related to Supplementary Fig. 1a. d. Western blots related to Supplementary Fig. 1b. e. Western blots related to Supplementary Fig. 1c. f. Western blots related to Supplementary Fig. 1d. g. Western blots related to Supplementary Fig. 2a. h. Western blots related to Supplementary Fig. 2b. i. Western blots related to Supplementary Fig. 2d. j. Western blots related to Supplementary Fig. 2e. k. Western blots related to Supplementary Fig. 2f. l. Western blots related to Supplementary Fig. 2g. m. Western blots related to Supplementary Fig. 3a. n. Western blots related to Supplementary Fig. 3d.
Supplementary Figures 1–7
Mass-spectrometry performed on the SCCOHT-1 cell line.
Genetic interactions with dual loss of SMARCA4 and SMARCA2.
Differential expression upon rescue of SMARCA4, SMARCA2 and SMARCA4 mutants.
Oligonucleotides used in cloning the SMARCA4 T910M mutant.
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Pan, J., McKenzie, Z.M., D’Avino, A.R. et al. The ATPase module of mammalian SWI/SNF family complexes mediates subcomplex identity and catalytic activity–independent genomic targeting. Nat Genet 51, 618–626 (2019). https://doi.org/10.1038/s41588-019-0363-5
Nature Communications (2020)
Current Opinion in Endocrine and Metabolic Research (2020)
Annual Review of Pathology: Mechanisms of Disease (2020)
Small Cell Carcinoma of the Ovary, Hypercalcemic Type (SCCOHT) beyond SMARCA4 Mutations: A Comprehensive Genomic Analysis