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

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.

Figure 1

BAF47 confers BAF complex stability on chromatin without affecting intra-complex subunit stability. (a) Schematic for rescue experiments in BAF47-deficient cell lines. (b) Nuclear extract inputs and anti-BRG1 IP from G401 nuclear extracts in empty vector and BAF47 conditions. (c) Nuclear extract input and IPs for IgG, BRG1, and BAF47 in control and BAF47Δ/Δ (knockout) HEK293T cells. (d) Silver stain analysis of control IgG and anti-BRG1 IPs in G401 empty vector and BAF47 conditions. (e) anti-BRG1 IP-mass spectrometry proteomics in G401 empty vector and BAF47 conditions for BAF complex subunits. (f-g) Density sedimentation analyses using 10-30% glycerol gradients (10m; 0.5ml/ fx) on nuclear extracts from G401 MRT cells in (f) the empty vector control and (g) BAF47 conditions. (h) (left) Schematic for differential salt extraction experiments in G401 empty vector or BAF47 conditions; (right) Immunoblot analysis of BAF complex subunits in differential salt extraction experiments. (i) Relative densitometry from differential salt extraction demonstrates gained stability of BAF complexes on chromatin in the BAF47 condition. Error bars = mean ± SEM for n=2 biological replicates.

Figure 2

Rescue of BAF47 drives a genome-wide gain in BAF complex chromatin occupancy (a) Input blot for TTC1240 and G401 cell nuclear extracts in empty vector and BAF47 conditions. (b) Venn diagram of (left) BRG1 and (right) BAF155 peaks in empty vector and BAF47 conditions in TTC1240 cells. (c) Heatmaps of BRG1 and BAF155 occupancy in TTC1240 empty vector and BAF47 conditions over all BRG1-BAF155 shared sites in the TTC1240+BAF47 condition. (d) Example BRG1, BAF155, and RNA-seq tracks at CDKN1A enhancers in TTC1240 cells. (e) Distance to closest transcription start site (TSS) for conserved (empty-BAF47) and gained (BAF47-only) BRG1-BAF155 sites in TTC1240 cells. (f) Centrimo plots for top four centrally enriched motifs at gained BRG1-BAF155 sites in TTC1240 cells. (g) Average sequence conservation (PhyloP) of conserved and gained BRG1-BAF155 sites. (h) Proliferation analyses of MRT, EpS, and AT/RT cell lines; values shown are relative proliferation between BAF47 and empty vector conditions at noted days. (i-k) Venn diagrams of BRG1 peaks in empty vector and BAF47 conditions in (i) G401, (j) HS-ES-2M, and (k) VA-ES-BJ cell lines.

Figure 3

Gain of BAF complex occupancy drives widespread enhancer activation. (a) Heatmaps of BRG1, H3K4me3, H3K4me1, and H3K27ac sites in TTC1240 empty vector and BAF47 conditions over all BRG1-BAF155 shared sites in the TTC1240+BAF47 condition. Heatmaps are ranked by BRG1 occupancy in empty condition. (b-d) Metagene plots of BRG1-BAF155 sites in the TTC1240+BAF47 condition split by (left) promoter-proximal (≤2kb from TSS), and (right) promoter-distal (>2kb from TSS) for (b) H3K4me3, (c) H3K4me1, and (d) H3K27ac occupancy. (e) Correlation plot of log2(fold change) for BRG1 and H3K27ac over all BRG1-BAF155 sites (70777) in empty or BAF47 conditions. (f) Gained promoter-distal BAF complex sites assigned to nearest gene (genes were categorized based on number of gained distal sites) versus log2(fold change) in expression. (g) Example tracks of BRG1, BAF155, H3K4me3, H3K4me1, H3K27ac, and RNA-seq at the TGM2 locus in TTC1240 cells.

Figure 4

Enhancer activation upon BAF47 rescue is specific to BAF but not PBAF complexes. (a) Schematic of BAF and PBAF complex subunits with subunits targeted for ChIP-seq in respective colors. (b) Input and IPs for IgG control, BAF155, BAF250A, and BAF200 from HEK293T nuclear extracts in naive and BAF47Δ/Δ conditions. (c) Heatmap of BRG1, BAF155, SS18, and BAF200 in TTC1240 empty vector and BAF47 conditions over all BRG1-BAF155 shared sites in the TTC1240+BAF47 condition. (d-f) Metagene plots of BRG1-BAF155 shared sites in TTC1240+BAF47 split by (left) promoter-proximal (≤2kb from TSS), and (right) promoter-distal (>2kb from TSS) for (d) BRG1, (e) SS18, and (f) BAF200 occupancy. (g-h) Correlation plot of log2(fold change) for (g) BRG1 and SS18, and (h) BRG1 and BAF200, over all TTC1240 BRG1-BAF155 sites in empty or BAF47 conditions. (i) Example ChIP-seq tracks for BRG1, SS18, BAF200, H3K27ac, and RNA-seq at the VIM locus in TTC1240 cells.

Figure 5

Resolution of bivalent promoters to activation by BAF complex-mediated opposition of polycomb-mediated repression. (a) Heatmaps of H3K4me3, BRG1, SS18, BAF200, H3K27ac, H3K27me3, and SUZ12 across all hg19 promoters in empty vector condition in TTC1240 cells, ranked by H3K4me3 occupancy. (b) GO term analysis of bivalent genes in TTC1240 cells. (c) Overlap of bivalent genes in G401 and TTC1240 cells. (d) Overlap of BRG1 target genes in empty and BAF47 conditions in TTC1240 cells. (e) Distribution of (left) conserved and (right) gained BRG1 target genes in TTC1240 cells. (f) Overlap of bivalent genes in empty and BAF47 conditions in TTC1240 cells. (g-h) Metagene plots of (g) H3K4me3 and H3K27me3, as well as (h) BRG1, SS18, and BAF200, over all 3512 bivalent promoters in TTC1240 cells. (i) Example tracks at the LAMB1 bivalent promoters demonstrate resolution of bivalent promoters to activation upon gain of BAF complex occupancy in TTC1240 cells.

Figure 6

Collaborative gene activation by BAF complex-mediated enhancer activation and polycomb opposition at bivalent promoters (a) Distribution of significantly-regulated genes in TTC1240 cells. (b) Directional regulation of significantly changed genes in TTC1240 cells, with y-axis indicating proportion of all genes in each category. (c) (left) Overlap of significantly-changed genes in G401 and TTC1240 cell types in empty vector and BAF47 conditions; (right) genes significantly-regulated in both cell lines show significant concordance (p < 2.2e-16, Fisher exact test). (d) Heatmap of 642 significantly changed genes in both G401 and TTC1240 cells. Right bar indicates promoter status of each gene in each cell line using colors from (a). (e) Heatmap of selected genes that are significantly-upregulated by BAF47 in both G401 and TTC1240 cells. (f) GO term analysis of significantly upregulated genes in both G401 and TTC1240. (g) Genes categorized by number of distal gained (BAF47-only) BAF complex sites, broken down by promoter status of genes in each category. n = number of genes in each group. (h-i) Example ChIP-seq tracks of BRG1, SS18, BAF200, H3K27ac, H3K4me3, H3K27me3, and RNA-seq for (h) CTGF and (i) FN1 shows collaborative gene activation via enhancer activation and polycomb opposition at bivalent promoters.

Figure 7

BAF47 restores BAF complex affinity and functional regulation of chromatin (a) BAF47 rescues the biochemical association of BAF complexes with chromatin, absent major changes in subunit composition or intra-complex stability. (b) Rescue of BAF47 leads to a widespread gain in BAF complex occupancy, mediating enhancer activation and opposition of polycomb-mediated repression at bivalent promoters.