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SMARCB1 is required for widespread BAF complex–mediated activation of enhancers and bivalent promoters

Nature Genetics volume 49, pages 1613–1623 (2017)Cite this article

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Abstract

Perturbations to mammalian SWI/SNF (mSWI/SNF or BAF) complexes contribute to more than 20% of human cancers, with driving roles first identified in malignant rhabdoid tumor, an aggressive pediatric cancer characterized by biallelic inactivation of the core BAF complex subunit SMARCB1 (BAF47). However, the mechanism by which this alteration contributes to tumorigenesis remains poorly understood. We find that BAF47 loss destabilizes BAF complexes on chromatin, absent significant changes in complex assembly or integrity. Rescue of BAF47 in BAF47-deficient sarcoma cell lines results in increased genome-wide BAF complex occupancy, facilitating widespread enhancer activation and opposition of Polycomb-mediated repression at bivalent promoters. We demonstrate differential regulation by two distinct mSWI/SNF assemblies, BAF and PBAF complexes, enhancers and promoters, respectively, suggesting that each complex has distinct functions that are perturbed upon BAF47 loss. Our results demonstrate collaborative mechanisms of mSWI/SNF-mediated gene activation, identifying functions that are co-opted or abated to drive human cancers and developmental disorders.

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Figure 1: BAF47 confers BAF complex stability on chromatin without affecting intra-complex subunit stability.
Figure 2: Rescue of BAF47 drives a genome-wide gain in BAF complex chromatin occupancy.
Figure 3: Gain of BAF complex occupancy drives widespread enhancer activation.
Figure 4: Enhancer activation upon BAF47 rescue is specific to BAF complexes.
Figure 5: Resolution of bivalent promoters to activation by BAF complex–mediated opposition of Polycomb-mediated repression.
Figure 6: Collaborative gene activation by BAF complex–mediated enhancer activation and Polycomb opposition at bivalent promoters.
Figure 7: BAF47 restores BAF complex affinity and functional regulation of chromatin.

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Acknowledgements

We thank members of the Molecular Biology Core Facility at the Dana-Farber Cancer Institute, especially Z. Herbert, for expertise and technical assistance with ChIP-seq and RNA-seq data sets. We thank G.R. Crabtree, A. Kuo, and W. Wang for anti-BRG1 (J1) and anti-BAF155 rabbit polyclonal antibodies used in ChIP-seq experiments. The authors are grateful to T.J. Triche (Children's Hospital Los Angeles), P. Houghton (The University of Texas Health Science Center), H. Kawashima (Niigata University Graduate School of Medical and Dental Sciences), H. Iwasaki (Fukuoka University), H. Gotoh (Kanagawa Children's Medical Center), and T. Tsukahara (Sapporo Medical University) for providing the MRT and EpS cell lines. This work was supported in part by awards from the NIH DP2 New Innovator Award 1DP2CA195762-01 (C.K.), the American Cancer Society Research Scholar Award RSG-14-051-01-DMC (C.K.), the Pew-Stewart Scholars in Cancer Research Grant (C.K.), the Alex's Lemonade Stand Foundation Young Investigator Award (C.K.), the Rare Cancer Research Foundation (C.K.), the WWW.W Foundation (QuadW) (R.T.N.), and the Uehara Memorial Foundation (R.T.N.). Additionally, this work was supported by the US National Institutes of Health (NIH) grant 5 T32 GM095450-04 (A.M.V.) and National Institute of General Medical Sciences (NIGMS) award T32GM007753 (S.H.C.). The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIGMS or the NIH.

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Author notes

  1. Robert T Nakayama, John L Pulice and Alfredo M Valencia: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Pediatric Oncology, Dana-Farber Cancer Institute and Harvard Medical School, Boston, Massachusetts, USA

    Robert T Nakayama, John L Pulice, Alfredo M Valencia, Matthew J McBride, Zachary M McKenzie, Robert T Williams, Seth H Cassel, He Qing, Christian J Widmer & Cigall Kadoch

  2. Department of Medical Oncology, Ludwig Center at Dana-Farber/Harvard and Center for Sarcoma and Bone Oncology, Harvard Medical School, Boston, Massachusetts, USA

    Robert T Nakayama & George D Demetri

  3. Broad Institute of MIT and Harvard, Cambridge, Massachusetts, USA

    John L Pulice & Cigall Kadoch

  4. Program in Chemical Biology, Harvard University, Cambridge, Massachusetts, USA

    Alfredo M Valencia & Matthew J McBride

  5. Institute for Systems Biology, Seattle, Washington, USA

    Mark A Gillespie & Jeffrey A Ranish

  6. Systems Biology Center, NHLBI, National Institutes of Health, Bethesda, Maryland, USA

    Wai Lim Ku, Kairong Cui & Keji Zhao

  7. Department of Biostatistics and Computational Biology, Dana-Farber Cancer Institute, Boston, Massachusetts, USA

    Mingxiang Teng & Rafael A Irizarry

  8. Medical Scientist Training Program, Harvard Medical School, Boston, Massachusetts, USA

    Seth H Cassel

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Contributions

R.T.N., J.L.P., A.M.V., and C.K. conceived of and designed the experiments. R.T.N., A.M.V., M.J.M., Z.M.M., R.T.W., S.H.C., H.Q., and C.J.W. performed experiments. J.L.P. performed all bioinformatics analyses and statistical calculations. W.L.K., K.C., and K.Z. performed and analyzed chromatin capture experiments. M.A.G., J.R., and C.K. performed and analyzed 2D-LC-MS experiments. M.T. and R.A.I. provided advice for bioinformatics analyses. G.D.D. provided valuable conceptual advice and guidance. J.L.P., R.T.N., and C.K. wrote the paper.

Corresponding author

Correspondence to Cigall Kadoch.

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Competing interests

C.K. is a scientific founder, shareholder and consultant of Foghorn Therapeutics, Inc. (Cambridge, Massachusetts).

Integrated supplementary information

Supplementary Figure 1 Loss of BAF47 does not significantly impair BAF complex assembly or stability.

(a) Total nuclear protein input and IgG and BRG1 IPs in BT16 cells containing either empty vector or BAF47. (b) Silver stain analysis of IgG control, anti-BRG1, and anti-BAF250A (ARID1A) IPs in G401 empty vector and BAF47 conditions. (c) Density sedimentation analyses using 10-30% glycerol gradients in G401 cells with empty vector and BAF47 immunoblotted for BRG1, BAF47, and BAF57. (d) Urea denaturation analyses using anti-BRG1 IPs reflect similar intra-complex stability in empty vector and BAF47 conditions in G401 cells. HC, antibody heavy chain. (e-f) IgG, anti-BRG1, anti-BAF155 (SMARCC1), and anti-BAF250A IPs performed from (NH4)2SO4- precipitated nuclear extracts incubated in RIPA buffer (containing 0.1% SDS and 0.5% sodium deoxycholate) or IP Buffer in TTC1240 empty vector and BAF47 conditions subjected to (e) silver stain and (f) western blot analyses.

Supplementary Figure 2 BAF47 contributes to BAF complex affinity on chromatin.

(a) Schematic of differential salt extraction protocol for determining chromatin affinity of chromatin-binding proteins. (b) Differential salt extraction experiment in G401 empty and BAF47 conditions with western blot analysis for BAF complex subunits, biological replicate of experiment shown in Fig. 1h. (c) Densitometry of BAF155 relative to total. Error bars = mean ± SEM for n=2 biological replicates. (d) Differential salt extraction experiment in empty and BAF47 conditions in G401 cells with western blot analysis for PRC2 subunits, GAPDH, and H3. (e-f) Differential salt extraction experiments in naïve and BAF47-knockout (BAF47Δ/Δ) HEK293T cells with western blot analysis of (e) BAF complex subunits, (f) PRC2 subunits, and TBP. (g) Densitometry of BRG1 in HEK293T naïve and BAF47Δ/Δ conditions relative to total.

Supplementary Figure 3 Reintroduction of BAF47 drives genome-wide increases in BAF complex occupancy.

(a) Venn diagram of BRG1 and BAF155 peaks in (left) empty vector and (right) BAF47 conditions in TTC1240 cells. (b) Correlation plot of log2(fold change) for BRG1 and BAF155 over all BRG1-BAF155 sites in empty or BAF47 conditions in TTC1240 cells. (c-e) Example tracks for BRG1, BAF155, and RNA-seq at (c) LAMB1, (d) FBN1, and (e) HEG1 loci in TTC1240 empty vector and BAF47 conditions. (f) Venn diagram of BAF complex (BRG1-BAF155 overlapping) sites in TTC1240 cells. (g) Distance to closest transcription start site (TSS) for conserved (Empty-BAF47) and gained (BAF47-only) BRG1 and BAF155 sites in TTC1240 cells. (h) Centrimo plots for top four centrally enriched motifs at conserved (Empty-BAF47) BRG1-BAF155 sites in TTC1240 cells. (i-j) Average sequence conservation (PhyloP) in conserved and gained BRG1-BAF155 sites in TTC1240 cells separated by (i) promoter-proximal (≤2kb from TSS) and (j) promoter-distal (>2kb from TSS) sites.

Supplementary Figure 4 BAF47 rescue suppresses growth of MRT and AT/RT cell lines, but not all EpS cell lines.

(a) Total nuclear protein input blots confirm re-expression of BAF47 in A204, G402, HS-ES-2M, and BT12 cell lines. (b-d) Proliferation analyses of (b) MRT, (c) EpS, and (d) AT/RT cell lines in empty vector and BAF47 conditions. Error bars = mean ± SEM for n=3 experiments. p-values are for two-tailed t-test.

Supplementary Figure 5 BAF47 rescue drives genome-wide increases in BAF complex occupancy across MRT and EpS cell lines.

(a) Venn diagram of BRG1 and BAF155 peaks in (left) empty vector and (right) BAF47 conditions in G401. (b) Venn diagram of BAF155 peaks in empty vector and BAF47 conditions in G401 cells. (c) Heatmaps of BRG1 and BAF155 occupancy in G401 empty vector and BAF47 conditions over all BRG1-BAF155 shared sites in the G401+BAF47 condition. (d) Example tracks reflecting BRG1 and BAF155 gain of occupancy at LAMB1 locus. (e) Venn diagram of BRG1 and BAF155 peaks in (left) empty vector and (right) BAF47 conditions in HS-ES-2M. (f) Venn diagram of BAF155 peaks in empty vector and BAF47 conditions in HS-ES-2M cells. (g) Heatmaps of BRG1 and BAF155 occupancy in HS-ES-2M empty vector and BAF47 conditions over all BRG1-BAF155 shared sites in the HS-ES-2M+BAF47 condition. (h) Example tracks reflecting BRG1 and BAF155 gain of occupancy at NRP1 locus. (i) Venn diagram of BRG1 and BAF155 peaks in (left) empty vector and (right) BAF47 conditions in VA-ES-BJ cells. (j) Venn diagram of BAF155 peaks in empty vector and BAF47 conditions in VA-ES-BJ cells. (k) Heatmaps of BRG1 and BAF155 occupancy in VA-ES-BJ empty vector and BAF47 conditions over all BRG1 sites in the VA-ES-BJ+BAF47 condition. (l) Example tracks reflecting BRG1 and BAF155 gain of occupancy at the TGM2 locus. (m-o) Distance to closest transcription start site (TSS) for conserved (Empty-BAF47) and gained (BAF47-only) BRG1 and BAF155 sites in (m) G401, (n) HS-ES-2M, and (o) VA-ES-BJ cells.

Supplementary Figure 6 Gain of BAF complex occupancy drives widespread enhancer activation in both MRT and EpS lines.

(a) Heatmaps of BRG1, BAF155, and H3K27ac in G401 empty vector and BAF47 conditions over all BRG1-BAF155 shared sites in the G401+BAF47 condition. (b) Example tracks of BRG1, BAF155, and H3K27ac at LAMA3 in G401 empty vector and BAF47 conditions. (c) Heatmaps of BRG1, BAF155, and H3K27ac in HS-ES-2M empty vector and BAF47 conditions over all BRG1-BAF155 shared sites in the HS-ES-2M+BAF47 condition. (d) Example tracks of BRG1, BAF155, and H3K27ac at MMP2 in HS-ES-2M empty vector and BAF47 conditions. (e) Heatmaps of BRG1, BAF155, and H3K27ac in VA-ES-BJ empty vector and BAF47 conditions over all BRG1 sites in the VA-ES-BJ+BAF47 condition. (f) Example tracks of BRG1, BAF155, and H3K27ac at the BMP1 locus in VA-ES-BJ empty vector and BAF47 conditions.

Supplementary Figure 7 BAF47 mediates activation of both typical enhancers and super-enhancers in MRT cell lines.

(a) Metagene plots of BRG1-BAF155 sites in TTC1240+BAF47 split by (left) promoter-proximal (≤2kb from TSS) and (right) promoter-distal (>2kb from TSS) for Input occupancy. (b-c) Correlation plot of log2(fold change) for (b) BRG1 and H3K4me1 and (c) BRG1 and H3K4me3, over all BRG1-BAF155 sites in empty or BAF47 conditions. (d) Gained promoter-distal BAF complex sites were assigned to nearest gene, and genes were categorized based on number of gained distal sites. Log2(fold change) in expression was plotted for genes in each category. (e) Analysis of typical and super-enhancers over all H3K27ac sites in empty or BAF47 conditions. Enhancers and super-enhancers are specific to a condition if they have more than 10-fold greater read density in one condition, as denoted by dashed lines. (f) Example tracks of BRG1, BAF155, H3K4me3, H3K27ac, H3K4me1, and RNA-seq at the CDKN1A locus.

Supplementary Figure 8 BAF47 rescue does not significantly alter global 3D genomic architecture.

(a) Heatmap of Hi-C read density in (left) empty vector and (right) BAF47 in VA-ES-BJ over chr6:103,150,001-143,150,000. Bin size = 50kb. (b) Difference heatmap over region in (a). (c) Heatmap of Hi-C read density in (left) empty vector and (right) BAF47 in VA-ES-BJ over chr6:123,150,001-128,150,000. (d) Difference heatmap over region in (c). (e) Distribution of topologically associated domain (TAD) size in empty and BAF47 conditions. (f) Tracks of BRG1, BAF155, H3K27ac, and Hi-C interactions in empty and BAF47 conditions at the CDKN1A locus in VA-ES-BJ cells.

Supplementary Figure 9 Enhancer activation upon BAF47 rescue is mediated by BAF, but not PBAF, complexes.

(a) Metagene plots of BRG1-BAF155 sites in TTC1240+BAF47 split by (left) promoter-proximal (≤2kb from TSS) and (right) promoter-distal (>2kb from TSS) for BAF155. (b) Correlation plot of log2(fold change) for SS18 and BAF200 over all BRG1-BAF155 sites in empty or BAF47 conditions in TTC1240 cells. (c-d) Correlation plot of log2(fold change) for (c) BRG1 vs. SS18 or (d) BRG1 vs. BAF200, over promoter-proximal (left) or promoter-distal (right) BRG1-BAF155 sites in empty or BAF47 conditions in TTC1240 cells.

Supplementary Figure 10 Promoter occupancy of BAF complexes correlates with gene expression.

(a-h) Expression analysis of (a) BRG1, (b) BAF155, (c) SS18, (d) BAF200, (e) H3K4me3, (f) H3K27ac, (g) H3K27me3, (h) SUZ12 over promoters in (top) empty and (bottom) BAF47 conditions. Promoters are binned by expression quintile or non-expressed genes (RPKM < 1). (i) Distribution of promoter categorization for all TTC1240 promoters. (j) Example tracks of H3K4me3-marked, H3K27me3-marked, and bivalent promoters in TTC1240 cells. (k) Distribution of promoter categorization for all G401 promoters. (l) Heatmap of H3K4me3, BRG1, H3K27ac, H3K27me3, and SUZ12 across all hg19 promoters in G401, ranked by H3K4me3 occupancy.

Supplementary Figure 11 BAF47 rescue increases BAF complex occupancy at bivalent promoters and significantly reduces H3K27me3 occupancy.

(a) Overlap of BAF155, SS18, and BAF200 target genes in empty and BAF47 conditions in TTC1240. (b) Distribution of BRG1 target genes in empty and BAF47 conditions, with y-axis indicating proportion of all genes in each category. (c) Metagene plots for BRG1, H3K27me3, and Input over all TTC1240+BAF47 BRG1-BAF155 sites. (d-e) Metagene plots of BRG1-BAF155 sites in TTC1240+BAF47 split by (left) promoter-proximal (≤2kb from TSS) and (right) promoter-distal (>2kb from TSS) for (d) H3K27me3, and (e) SUZ12 occupancy. (f) Metagene plot over all 3512 bivalent promoters in TTC1240 for BAF155, SUZ12, and input. (g-i) Correlation plot of log2(fold change) for (g) BRG1, (h) SS18, or (i) BAF200 vs. H3K27me3 over promoter-proximal BRG1-BAF155 sites in empty or BAF47 conditions in TTC1240 cells.

Supplementary Figure 12 BAF complexes resolve bivalent promoters to activation via opposition of Polycomb-mediated repression.

(a) Overlap of BRG1 target genes in empty and BAF47 conditions in G401. (b) Breakdown of (left) conserved and (right) gained BRG1 target genes in G401. (c) Breakdown of BRG1 target genes in empty and BAF47 conditions, with y-axis indicating proportion of all genes in each category. (d) Metagene plots for BRG1, H3K27me3, and Input over all G401+BAF47 BRG1-BAF155 sites. (e) Overlap of bivalent genes in empty and BAF47 conditions in TTC1240. (f) Metagene plots over all 3512 bivalent promoters in TTC1240 for BRG1, BAF155, H3K4me3, H3K27me3, SUZ12, and input.

Supplementary Figure 13 BAF complex opposition of polycomb-mediated repression is limited in BAF47-insensitive EpS cell lines.

(a) Heatmaps of H3K4me3, BRG1, H3K27ac, H3K27me3, and SUZ12 across all hg19 promoters in HS-ES-2M EpS cells, ranked by H3K4me3 occupancy. (b) Breakdown of promoter categorization for all HS-ES-2M promoters. (c) Metagene plots for BRG1, H3K27me3, and Input over all HS-ES-2M+BAF47 BRG1-BAF155 sites. (d) Heatmap of H3K4me3, BRG1, H3K27ac, H3K27me3, and SUZ12 across all hg19 promoters in VA-ES-BJ, ranked by H3K4me3 occupancy. (e) Breakdown of promoter categorization for all VA-ES-BJ promoters. (f) Metagene plots for BRG1, H3K27me3, and Input over all VA-ES-BJ+BAF47 BRG1 sites.

Supplementary Figure 14 BAF47 rescue mediates activation of bivalent genes and developmental pathways in MRT cell lines.

(a) Heatmap of pairwise correlation between all RNA-seq experiments in G401 and TTC1240. (b) Correlation plots for biological replicates of RNA-seq experiments in G401 and TTC1240. PCC = Pearson correlation coefficient. (c) Breakdown of significantly-regulated genes in G401 (d) Directional regulation of G401 significantly changed genes, with y-axis indicating proportion of all genes in each category. (e) GO term analysis of significantly downregulated genes in both G401 and TTC1240. (f-h) GSEA analysis of ranked genes in (top) G401 and (bottom) TTC1240 shows significant positive enrichment of (f-g) BAF complex gene sets identified in prior studies and (h) epithelial-mesenchymal transition gene sets. (i-j) RPKM values for previously-implicated genes (i) EZH2 and (j) CDKN2A in MRT in (left) G401 and (right) TTC1240. Error bars = Mean ± SEM for n=2 experiments. (k) Genes categorized by number of distal conserved (Empty-BAF47) BAF complex sites broken down by promoter status of genes in each category. n = number of genes in each group.

Supplementary Figure 15 BAF47 reintroduction increases BAF complex occupancy and decreases PRC2 occupancy at promoters of upregulated genes.

(a-f) Metagene analysis of promoter occupancy changes at genes (right) upregulated, (left) downregulated, or (center) unchanged upon rescue of BAF47 in TTC1240 cells. Metagene occupancies for (a) BRG1, (b) BAF155, (c) SS18, (d) BAF200, (e) H3K27me3, and (f) SUZ12 are plotted in empty and BAF47 conditions over promoters binned by differential expression analysis. Number indicates total number of genomic loci analyzed, all hg19 promoters for a gene annotation are included in analysis.

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Supplementary Figures 1–15 (PDF 4054 kb)

Life Sciences Reporting Summary (PDF 129 kb)

Supplementary Table 1

G401 anti-BRG1 proteomics, empty vector vs. BAF47. (XLSX 192 kb)

Supplementary Table 2

Cell line information. (XLSX 9 kb)

Supplementary Table 3

Antibody information. (XLSX 12 kb)

Supplementary Table 4

RNA-seq and ChIP-seq statistics and information. (XLSX 57 kb)

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Nakayama, R., Pulice, J., Valencia, A. et al. SMARCB1 is required for widespread BAF complex–mediated activation of enhancers and bivalent promoters. Nat Genet 49, 1613–1623 (2017). https://doi.org/10.1038/ng.3958

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