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SMARCB1-mediated SWI/SNF complex function is essential for enhancer regulation

Abstract

SMARCB1 (also known as SNF5, INI1, and BAF47), a core subunit of the SWI/SNF (BAF) chromatin-remodeling complex1,2, is inactivated in nearly all pediatric rhabdoid tumors3,4,5. These aggressive cancers are among the most genomically stable6,7,8, suggesting an epigenetic mechanism by which SMARCB1 loss drives transformation. Here we show that, despite having indistinguishable mutational landscapes, human rhabdoid tumors exhibit distinct enhancer H3K27ac signatures, which identify remnants of differentiation programs. We show that SMARCB1 is required for the integrity of SWI/SNF complexes and that its loss alters enhancer targeting—markedly impairing SWI/SNF binding to typical enhancers, particularly those required for differentiation, while maintaining SWI/SNF binding at super-enhancers. We show that these retained super-enhancers are essential for rhabdoid tumor survival, including some that are shared by all subtypes, such as SPRY1, and other lineage-specific super-enhancers, such as SOX2 in brain-derived rhabdoid tumors. Taken together, our findings identify a new chromatin-based epigenetic mechanism underlying the tumor-suppressive activity of SMARCB1.

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Figure 1: Histone modification landscape in primary rhabdoid tumors and cell lines.
Figure 2: SMARCB1 is essential in maintaining SWI/SNF complex integrity.
Figure 3: SMARCB1 re-expression alters SWI/SNF complex targeting at typical enhancers.
Figure 4: Residual SWI/SNF complexes are specifically maintained at super-enhancers in SMARCB1-deficient rhabdoid tumors.
Figure 5: Working model.

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Acknowledgements

We thank members of the Roberts and Park laboratories for assistance and discussion. We thank J. Francois (Boston Children's Hospital), J. Roth (Children's Hospital of Philadelphia), M. Lear (St. Jude Children's Research Hospital), and J. Silterra (Broad Institute) for their help in acquiring and performing preliminary clinical analysis of primary tumor samples; R. Rubio (Dana-Farber Cancer Institute) and M. Uziel (Broad Institute) for their assistance in sequencing samples; N. Shoresh (Broad Institute) for assistance in accessing Roadmap Epigenomics data; and R. Tomaino (Harvard Medical School) for assistance in proteomic analysis. X.W. was supported by the Pathway to Independence Award from the US National Institutes of Health (K99CA197640), a postdoctoral fellowship from the Rally Foundation for Childhood Cancer Research and The Truth 365, and a research grant from St. Baldrick's Foundation. R.S.L. was partially supported by an NSF Graduate Research Fellowship. This work was supported by US National Institutes of Health grants R01CA172152 (C.W.M.R.), R01CA113794 (C.W.M.R.), and U54HG006991 (B.E.B.). The Avalanna Fund, the Cure AT/RT Now Foundation, the Garrett B. Smith Foundation, Miles for Mary, and ALSAC/St. Jude (C.W.M.R.) provided additional support.

Author information

Authors and Affiliations

Authors

Contributions

X.W., R.S.L., B.H.A., B.E.B., P.J.P., and C.W.M.R. conceived the experiments and study design. X.W., J.R.H., E.P.T., and E.C.T. performed all cell line experiments. R.S.L. and S.M.G. performed all primary tumor experiments. B.H.A., R.S.L., S.W., J.M., and Y.D. performed computational analyses of the data. X.W., R.S.L., B.H.A., and S.W. performed statistical analyses. T.C.A., S.L.P., and J.A.B. contributed primary tumor samples and clinical data. J.N.W. designed the SMARCB1 re-expression vector. X.W., R.S.L., B.H.A., J.R.H., J.M., Y.D., M.Y.T., B.E.B., P.J.P., and C.W.M.R. contributed to the interpretation of experiments. X.W., R.S.L., B.H.A., and C.W.M.R. wrote the manuscript with input from all co-authors.

Corresponding authors

Correspondence to Bradley E Bernstein, Peter J Park or Charles W M Roberts.

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Competing interests

The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Histone modification landscape in primary rhabdoid tumors and cell lines.

Rhabdoid tumors from different tissues show commonalities in H3K4me1 or H3K4me3 signal but are clearly distinct in terms of H3K27ac signal at enhancers. (a) Summary of primary rhabdoid tumor samples and cell lines used in this study and types of ChIP–seq or RNA–seq performed in these samples. (bd) The first two principal components of H3K4me3 (b), H3K4me1 (c), and H3K27ac (d) signal across the union of all promoters or enhancers across the samples.

Supplementary Figure 2 SMARCB1 is essential in maintaining SWI/SNF complex integrity.

(a) mRNA levels of the indicated SWI/SNF complex subunits upon SMARCB1 re-expression (G401, BT16) shown by RNA–seq. (b) Protein levels of the indicated SWI/SNF complex subunits upon MG132 treatment in BT16 cells. (ce) Immunoprecipitation (IP) of the SWI/SNF complex by SMARCC1, SMARCA4, or ARID1A from the nuclear extracts of A204 (b), TM87 (d), and ES-2 (e) cells, before or after Dox induction (SMARCB1 re-expression); immunoblotting was performed for SWI/SNF complex SMARCB1, SMARCC1, SMARCA4, ARID1A, ARID1B, SMARCC2, SMARCD1, SMARCE1, ACTL6A, and DPF2. A204 and TM87 are both SMARCB1-mutant rhabdoid tumor cell lines derived from soft tissues, and ES-2 is an ovarian cancer cell line with wild-type SMARCB1. (f) SMARCC1 immunoprecipitation and mass spectrometry results in BT16 cells with or without SMARCB1 re-expression. (g) Glycerol sedimentation (10–30%) assay of the SWI/SNF complex from SMARCB1-mutant BT16 cells without Dox (top) or with Dox (bottom) and immunoblotting for the indicated SWI/SNF complex subunits. (h) mRNA levels of the indicated SWI/SNF complex subunits upon SMARCB1 re-expression (TM87) or SMARCB1 loss (MEFs) shown by RNA–seq.

Supplementary Figure 3 Correlation of the changes in SMARCA4 and SMARCC1 binding upon re-expression of SMARCB1 in G401, BT16, and TTC549 cells.

(ac) Log2-tranformed fold change in SMARCA4 signal versus log2-transformed fold change in SMARCC1 signal at SWI/SNF binding sites upon SMARCB1 re-expression in G401 (a), BT16 (b), and TTC549 (c) cells. The two subunits show consistent changes under the two conditions. Therefore, data for the two subunits were used together to identify SWI/SNF binding sites. The different colors denote sites classified as ‘gained/strengthened’, ‘stable’, or ‘weakened/lost’.

Supplementary Figure 4 SMARCB1 re-expression alters the SWI/SNF complex targeting at typical enhancers in BT16 cells.

(a) Number of SWI/SNF (SMARCC1 and SMARCA4) binding sites in regions of enrichment for different histone marks in BT16 cells with or without SMARCB1 re-expression. (b) Average enrichment of SMARCC1 and SMARCA4 without versus with SMARCB1 re-expression in TSS-distal or TSS-proximal SWI/SNF binding sites. (c) Heat maps depicting SMARCC1, SMARCA4, H3K27ac, H3K4me1, and H3K4me3 signal intensities for TSS-distal SWI/SNF binding sites, grouped by change upon SMARCB1 re-expression. The rows show 9-kb regions, centered on SMARCC1/SMARCA4 peaks, ranked by overall signal intensities of SMARCC1/SMARCA4. The average profiles for each heat map are shown above, where different y-axis ranges are denoted as 1/2× or 1/4×. (d) Representative screenshot of SMARCC1, SMARCA4, H3K4me1, H3K4me3, and H3K27ac signal with or without SMARCB1 re-expression in G401 cells showing increased SMARCC1 and SMARCA4 binding upon SMARCB1 re-expression accompanied by increased and flanking H3K27ac and H3K4me1 marks at enhancers. (e) Correlation of gene expression changes with SMARCC1/SMARCA4 binding or H3K27ac signal at TSS-distal binding sites in BT16 cells upon Dox treatment. (f) GO analysis of genes proximal to enhancers with increased SMARCC1/SMARCA4 signal upon Dox treatment.

Supplementary Figure 5 SMARCB1 re-expression alters SWI/SNF complex targeting at typical enhancers in TTC549 cells.

(a) Number of SWI/SNF (SMARCC1 and SMARCA4) binding sites in regions of enrichment for different histone marks in TTC549 cells with or without SMARCB1 re-expression. (b) Average enrichment of SMARCC1 and SMARCA4 without versus with SMARCB1 re-expression in TSS-distal or TSS-proximal SWI/SNF binding sites. (c) Heat maps depicting SMARCC1, SMARCA4, H3K27ac, H3K4me1, and H3K4me3 signal intensities for TSS-distal SWI/SNF binding sites, grouped by change upon SMARCB1 re-expression. The rows show 9-kb regions, centered on SMARCC1/SMARCA4 peaks, ranked by overall signal intensity of SMARCC1/SMARCA4. The average profiles for each heat map are shown above, where different y-axis ranges are denoted as 1/2× or 1/4×. (d) Representative screenshot of SMARCC1, SMARCA4, H3K4me1, H3K4me3, and H3K27ac signal with or without SMARCB1 re-expression in G401 cells showing increased SMARCC1 and SMARCA4 binding upon SMARCB1 re-expression accompanied by increased and flanking H3K27ac and H3K4me1 marks at enhancers. (e) Correlation of gene expression changes with SMARCC1/SMARCA4 binding or H3K27ac signal at TSS-distal binding sites in TTC549 cells upon Dox treatment. (f) GO analysis of genes proximal to enhancers with increased SMARCC1/SMARCA4 signal upon Dox treatment.

Supplementary Figure 6 RNA–seq analysis of six different rhabdoid tumor cell lines upon SMARCB1 re-expression.

k-means clustering of log2 (fold change) values (Dox/no Dox) for rhabdoid tumor cell lines to ten clusters. Selected enriched GO terms for each cluster (FDR < 0.0001 or ‘development’ terms with FDR < 0.01) are shown.

Supplementary Figure 7 Gene set enrichment analysis of different rhabdoid tumor cell lines upon SMARCB1 re-expression.

GSEA gene sets most enriched in upregulated and downregulated differential expression patterns.

Supplementary Figure 8 Re-expression of SMARCB1 has little effect on the number and structure of super-enhancers in three different rhabdoid tumor cell lines.

(ac) Number of H3K27ac RoEs present under each condition (called in the sample or having greater than 2/3 signal relative to the other condition) in different RoE categories and input-subtracted H3K27ac ChIP signal in stitched H3K27ac regions, excluding signal within H3K4me3 regions, with or without SMARCB1 re-expression in the G401 (a), BT16 (b), and TTC549 (c) cell lines. Red dots indicate super-enhancer calls. Middle plots show ranked values, whereas right plots compare the two conditions on a log scale.

Supplementary Figure 9 Residual SWI/SNF complexes are specifically maintained at super-enhancers in SMARCB1-mutant rhabdoid tumors.

(a,b) Heat maps depicting SMARCC1, SMARCA4, H3K27ac, H3K4me1, and H3K4me3 signal intensities for SMARCC1/SMARCA4 bound within super-enhancers in BT16 (a) and TTC549 (b) cells. (c,d) Scatterplots showing changes in average SMARCC1/SMARCA4 signal for TSS-proximal binding sites or TSS-distal ones split into those outside and inside super-enhancers in BT16 (c) and TTC549 (d) cells. (e,f) Representative screenshots for BT16 (e) and TTC549 (f) cells showing limited changes in SMARCC1, SMARCA4, H3K27ac, or H3K4me1 upon SMARCB1 re-expression inside super-enhancers, in contrast to outside super-enhancers.

Supplementary Figure 10 Residual SWI/SNF complex binds to super-enhancers and regulates super-enhancer gene expression in G401 cells.

(a) Residual SWI/SNF complex binding at a typical enhancer is increased upon SMARCB1 re-expression and is accompanied by increased H3K27ac marks at the gene QKI enhancer. (b) Residual SWI/SNF complex binding at super-enhancers, as well as H3K27ac marks, is only modestly increased upon SMARCB1 re-expression but is largely reduced upon further loss of residual complex subunit SMARCC1 or SMARCD1, as shown at selected loci for two super-enhancer genes (SALL4 and SPRY1). (c,d) The expression of super-enhancer genes is significantly reduced upon loss of residual complex subunit SMARCC1 (c) or SMARCD1 (d).

Supplementary Figure 11 Screenshots of H3K27ac super-enhancers in primary rhabdoid tumors and cell lines.

(a) Super-enhancers are found at the SPRY1 locus in all primary rhabdoid tumors and cell lines. The one sample in which a super-enhancer is not called using our algorithm (RT262) still shows significant H3K27ac signal that overlaps with the peaks observed in the other samples. (b) Super-enhancers are found at the SALL4 locus in all primary rhabdoid tumors and all cell lines. (c) Super-enhancers are found at the HMGA2 locus in six primary rhabdoid tumors and all cell lines. (d) Super-enhancers are found at the SOX2 locus in all primary rhabdoid tumors and all cell lines.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–11. (PDF 2475 kb)

Supplementary Table 1

Rhabdoid tumor primary GO terms. (XLSX 53 kb)

Supplementary Table 2

Rhabdoid tumor primary super-enhancers. (XLSX 209 kb)

Supplementary Table 3

Mass spectrometry. (XLSX 162 kb)

Supplementary Table 4

Cell line GO terms. (XLS 140 kb)

Supplementary Data

Full scans of blots. (PDF 30341 kb)

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Wang, X., Lee, R., Alver, B. et al. SMARCB1-mediated SWI/SNF complex function is essential for enhancer regulation. Nat Genet 49, 289–295 (2017). https://doi.org/10.1038/ng.3746

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