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Smarca4 ATPase mutations disrupt direct eviction of PRC1 from chromatin

Nature Genetics volume 49, pages 282–288 (2017)Cite this article

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Abstract

Trithorax-group proteins and their mammalian homologs, including those in BAF (mSWI/SNF) complexes, are known to oppose the activity of Polycomb repressive complexes (PRCs)1,2,3,4,5. This opposition underlies the tumor-suppressive role of BAF subunits3,5,6,7 and is expected to contribute to neurodevelopmental disorders8,9. However, the mechanisms underlying opposition to Polycomb silencing are poorly understood. Here we report that recurrent disease-associated mutations in BAF subunits induce genome-wide increases in PRC deposition and activity. We show that point mutations in SMARCA4 (also known as BRG1) mapping to the ATPase domain cause loss of direct binding between BAF and PRC1 that occurs independently of chromatin. Release of this direct interaction is ATP dependent, consistent with a transient eviction mechanism. Using a new chemical-induced proximity assay, we find that BAF directly evicts Polycomb factors within minutes of its occupancy, thereby establishing a new mechanism for the widespread BAF–PRC opposition underlying development and disease.

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Figure 1: Characteristic accumulation of PRC1 and PRC2 after BAF subunit deletion.
Figure 2: Mutations mapping to the SMARCA4 ATPase domain induce increased PRC1 occupancy at CpG-rich promoters.
Figure 3: Chromatin features define sites predisposed to increased Polycomb occupancy upon expression of SMARCA4 ATPase mutants.
Figure 4: Mutations mapping to the SMARCA4 ATPase domain result in increased H3K27me3 levels near sites of increased RING1B occupancy.
Figure 5: The ATPase activity of SMARCA4 directly regulates the PRC1 complex.
Figure 6: Chemical-induced proximity of BAF causes rapid loss of Polycomb occupancy on intact chromatin.

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Acknowledgements

This paper is dedicated to the memory of Joseph P. Calarco, a great friend and passionate scientist. We apologize to our colleagues whose work we could not cite owing to space constraints. We thank G. Hu, W. Jin, E. Chory, J. Bradner, and D. Hargreaves for helpful discussions and E. Miller for sharing curated GEO data sets. Arid1a conditional deletion cells were a gift from D. Hargreaves (Salk Institute). All libraries were sequenced by the DNA Sequencing and Genomics Core facility of NHLBI. Analysis was performed using the Stanford BioX3 cluster, supported by NIH S10 Shared Instrumentation Grant 1S10RR02664701. This work was also supported by the SFARI Foundation (G.R.C.), NIH grants R37NS046789 (G.R.C.) and R01CA163915 (G.R.C.), and the Division of Intramural Research, NHLBI/NIH (K.Z.). G.R.C. is an HHMI Investigator. S.M.G.B. is supported by a Swiss National Science Foundation (SNSF) postdoctoral fellowship. C.H. is supported by NCI career transition award K99CA187565.

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Author notes

  1. Benjamin Z Stanton and Courtney Hodges: These authors contributed equally to this work.

Authors and Affiliations

  1. Departments of Pathology and Developmental Biology, Stanford University School of Medicine, Stanford, California, USA

    Benjamin Z Stanton, Courtney Hodges, Joseph P Calarco, Simon M G Braun & Gerald R Crabtree

  2. Systems Biology Center, National Heart, Lung, and Blood Institute, US National Institutes of Health, Bethesda, Maryland, USA

    Benjamin Z Stanton, Wai Lim Ku & Keji Zhao

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

    Cigall Kadoch

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

    Cigall Kadoch

  5. Howard Hughes Medical Institute, Chevy Chase, Maryland, USA

    Gerald R Crabtree

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  1. Benjamin Z Stanton
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Contributions

B.Z.S. and C.H. designed and conducted the experiments, performed analyses, and wrote the manuscript. J.P.C., S.M.G.B., and C.K. performed experiments. W.L.K. performed analyses. K.Z. and G.R.C. designed experiments and wrote the manuscript.

Corresponding authors

Correspondence to Keji Zhao or Gerald R Crabtree.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Genomic changes for ChIP–seq of Ring1b upon knockout of BAF subunits.

(a) Fold changes in genomic Ring1b sites 72 h after conditional knockout of Smarca4 (Brg). (b) Fold changes in Ring1b sites 72 h after conditional knockout of Arid1a. For a and b, each point is a site, colored as unchanged (black), increased (orange), or decreased (blue), using the criteria in the Online Methods. Each plot represents four independent ChIP–seq experiments (two wild-type samples and knockout samples). (c) mRNA levels of core Polycomb factors do not increase upon knockout of Smarca4, indicating that the increased occupancy we observe across the genome does not arise from non-specific increased expression of core Polycomb subunits. Complete tabulated RNA–seq data are supplied as a source data file for Figure 1. (d) Venn diagram of PRC1-increased genes in the Smarca4 knockout, as compared to genes previously annotated as Polycomb targets28. Most PRC1-increased genes are normal Polycomb target genes (odds ratio (OR) = 9.72, Fisher’s exact test P < 1 × 10–30). For individual sites, 1,583 of 1,634 (~97%) PRC1-increased sites in the Smarca4 knockout directly overlap with existing sites in wild-type cells, but some new sites arise. (e) Distribution of gene targets by PRC annotation. Sites with increased PRC1 occupancy include several subclasses of Polycomb sites, sites that are normally active, and sites that are inactive or were not previously annotated. (f) Expression of pluripotency markers remains high upon Smarca4 knockout. Mean expression values from RNA–seq are plotted; error bars are 95% confidence intervals from n = 2 cell culture replicates. (g) The mean density of Ring1b changes across each class of PRC promoter. Positive distance is defined by the direction of transcription. (h) Top 20 Gene Ontology (GO) terms for genes with increased PRC1 occupancy upon Smarca4 knockout, ranked by hypergeometric P value.

Supplementary Figure 2 ChIP–qPCR validation of ChIP–seq changes.

(a) ChIP–qPCR validation of Ring1b, Suz12, and Cbx7 ChIP data for select targets confirm the changes observed with ChIP–seq analysis upon Smarca4 knockout. Together, occupancy of Polycomb factors is increased in the knockout as compared to wild type at validation sites (Fabp3: ANOVA P = 0.006, F[1,7] = 15.1; Esrrb: ANOVA P = 6 × 10–4, F[1,7] = 34.9), but not at control sites, confirming the conclusions of our ChIP–seq analysis. (b) Genome-wide distribution of Ring1b changes identified via ChIP–seq for the Smarca4 knockout, with Fabp3 labeled. (c) ChIP–qPCR validation of the Ring1b, Cbx7, and H3K27me3 changes observed with ChIP–seq analysis comparing ATPase-mutant G784E to wild-type protein. Together, the levels of Cbx7, Ring1b, and H3K27me3 at the Esrrb validation site are increased in the ATPase mutant in comparison to wild-type protein (ANOVA P = 0.014, F[1,8] = 9.7), but not at control sites, confirming the conclusions of our ChIP–seq analysis. Error bars are s.e.m. from n = 3 cell culture replicates. (d) Genome-wide distribution of Ring1b changes for Smarca4 mutation, with Esrrb labeled. Sequences for the primers used in this study are provided in Supplementary Table 3.

Supplementary Figure 3 Protein levels upon knockout of Smarca4 and Arid1a or expression of Smarca4 mutants.

Immunoblots of the PRC1 and PRC2 components Ring1b, Rybp, and Ezh2 show no significant changes in protein levels. (a) Blots from Arid1a conditional knockout mESCs 72 h after treatment with ethanol (EtOH) or tamoxifen (Tam). (b) Blots from Smarca4 conditional knockout mESCs 72 h after treatment with ethanol or tamoxifen. (c) Expression levels of the PRC1 ad PRC2 core subunits Ring1b and Ezh2 do not change when different ATPase mutants of Smarca4-V5 are expressed in place of wild-type Smarca4-V5. (d) Lentiviral transduction results in expression of Smarca4-GFP and Smarca4-V5 at levels comparable to endogenous Smarca4 levels in unmodified mESCs. (e) Consistency of expression of Smarca4-V5 and Smarca4-GFP across variants. All Smarca4-V5 variants (including wild type) derive from the same Smarca4-GFP parent line; hence, Smarca4-GFP serves as a built-in loading control. The relative expression of Smarca4-GFP and Smarca4-V5 shows comparable expression of Smarca4 across all variants. (f) Calibration of sensitivity to protein abundance in input samples. Because of differences in abundances and antibody sensitivity, proteins do not stain uniformly in input samples. At lower concentrations (shown as percent input for immunoprecipitation samples), staining for some PRC1 subunits is less intense than that for BAF subunits. At concentrations where the staining for PRC1 subunits is more intense, staining for BAF subunits results in increased non-specific staining. Immunoprecipitation alters the relative abundance of these factors.

Supplementary Figure 4 Examples of Ring1b sites upon expression of Smarca4 ATPase mutants.

(a) Ring1b occupancy is generally unchanged across the scale of the entire HoxA cluster. (b) Ring1b occupancy is unchanged in the CpG island Gapdh promoter. (c) Ring1b occupancy increases at Fgf11 in the presence of Smarca4 ATPase mutants. (d) Detail of Ring1b increases in the Camk2n1 promoter. Loci with increased Ring1b occupancy showed broadly similar ~2-fold increases in read density at bivalent CpG island promoters.

Supplementary Figure 5 Genome-wide comparison of Smarca4 knockout and Smarca4 ATPase mutants.

(a) Comparison of global Ring1b changes in wild-type replicates, Smarca4 knockout (KO) cells, and cells expressing mutant Smarca4 relative to those with wild-type protein. In comparison to wild-type cells, Smarca4 knockout or expression of Smarca4 mutants leads to general increases in Ring1b occupancy over the genome. (b) Comparison of the effects of Smarca4 knockout and expression of E861K Smarca4 at unique sites that directly overlap in both data sets. Increases co-occur at more sites than expected by random chance (overlap odds ratio (OR) = 2.72, Fisher’s test P = 1.0 × 10–30). (c) Ring1b sites affected by knockout of Smarca4 are enriched at bivalent sites in wild-type cells (sites with high levels of both H3K4me3 and H3K27me3). (d) Ring1b sites affected by expression of E861K Smarca4 are enriched at bivalent sites but also include some sites with high levels of H3K4me3 in wild-type cells. (e) Like Smarca4 knockout, expression of ATPase-mutant Smarca4 leads to increased PRC1 occupancy across several classes of Polycomb promoters (compare with Supplementary Fig. 1g).

Supplementary Figure 6 Effect of expression of Smarca4 mutants in comparison to wild-type Smarca4 in A549 cells.

A549 cells, which have inactivated SMARCA4, were transduced with lentivirus to express either wild-type or E861K Smarca4. ChIP–seq analysis using antibodies directed against Ring1b shows increases in PRC1 occupancy at CpG island promoters and 5′ regulatory regions, similar to the effect we observed in mESCs. (a) MA plot showing the effects of E861K Smarca4 in comparison to wild-type Smarca4. Far more sites increase than decrease when E861K Smarca4 is expressed than when wild-type Smarca4 is expressed. (b) Genomic annotations were enriched similarly as in mESCs, with high CpG island enrichment in sites that have increased PRC1/Ring1b occupancy. (c) Top 20 Gene Ontology (GO) terms for genes with increased PRC1 occupancy upon expression of E861K Smarca4 as compared to wild-type Smarca4, ranked by hypergeometric P value.

Supplementary Figure 7 PRC1 sites with Smarca4 and robust H3K4me3 have increased PRC1 occupancy upon expression of Smarca4 ATPase mutants.

(a) Rank ordering of chromatin features at PRC1 sites, classified by Ring1b alteration (blue, decreased; orange, increased; yellow, unchanged; in cells expressing E861K as compared to wild-type Smarca4). Bars are the 99% confidence interval for the mean of each class. (b) Sites are presented ranked by fold change in PRC1 occupancy upon expression of E861K Smarca4 (mut) as compared to wild-type (WT) Smarca4. Sites that increase PRC1 occupancy in the mutant typically have the highest levels of H3K4me3, while H3K27me3 shows little trend. (c) Sites that increase PRC1 intensity upon expression of E861K Smarca4 have higher mean occupancy of Smarca4. (d) Cumulative distribution function (CDF) of Smarca4 ChIP–seq intensities for each class of PRC1 site. Sites with increased intensity have significantly higher Smarca4 density (KS test P < 1 × 10–30). (e) Sites that have increased PRC1 intensity have a higher proportion of genomic peaks overlapping with Smarca4.

Supplementary Figure 8 Expression of the ATPase-dead K785R Smarca4 mutant leads to changes similar to those observed for other mutants.

(a) MA plot of H3K27me3 levels showing the effect of K785R Smarca4 as compared to wild-type Smarca4. (b) Example browser track at Aff1 showing that expression of K785R Smarca4 also leads to increased Ring1b occupancy at Aff1 (similar to the E861K data presented in the main text), with nearby increased levels of H3K27me3. (c) H3K27me3 changes in cells expressing K785R mirror the changes seen with E861K expression, presented in the main text, although the magnitude of the change is less. (d) Enrichment of wild-type relative to K785R Smarca4 during coimmunoprecipitation with Rybp.

Supplementary Figure 9 Smarca4 also regulates Cbx7-containing PRC1 complexes.

(a) Example browser tracks of Ring1b and Cbx7 occupancy in Smarca4 knockout (KO) and wild-type (WT) cells. Changes in Ring1b and Cbx7 occupancy are coupled throughout the genome. (b) Example browser tracks of changes in Ring1b and Cbx7 occupancy between cells expressing wild-type and G784E Smarca4. (c) Summary of unique genome-wide ChIP–seq overlap between sites that have increased Ring1b occupancy and sites that have increased Cbx7 occupancy. Across all sites, sites have greater overlap than expected by random chance: overlap odds ratio (OR) = 59.5, Fisher’s test P < 1 × 10–30. Using the criteria described in the Online Methods, more sites are detected for Cbx7, but we cannot rule out differences in antibody efficiency as the origin for this observation. Nevertheless, the sites that have increased Cbx7 occupancy upon Smarca4 knockout nearly all also have increased occupancy in Ring1b ChIP–seq data upon Smarca4 knockout. The slight differences in numbers between these values and those in Supplementary Figure 1d arise because this analysis is of individual ChIP–seq peaks whereas Supplementary Figure 1d reports analysis for genes. The numbers differ somewhat because some genes have more than one peak. Furthermore, a negligible deviation from the total number reported in the text arises because a single peak in one ChIP–seq data set can overlap with more than one peak in the other data set. The numbers reported here correspond to unique overlaps. (d) ATP-dependent coimmunoprecipitation of Cbx7-containing complexes with Smarca4 and BAF155 observed with antibodies directed against Cbx7. We find a similar interaction with Rybp-containing PRC1 complexes as presented in the main text. See also the note in Supplementary Figure 3f regarding the lack of input staining of Cbx7.

Supplementary Figure 10 ATP-dependent binding of PRC1 by BAF.

(a) Putative free energy diagram for the reversible process of BAF–PRC1 binding. In this model, binding is freely reversible upon ATP-dependent BAF activity but is kinetically trapped after the hydrolysis cycle is completed. Subsequent addition of ATP permits release of PRC1 from BAF. (b) Example diagram of the process described in a. Reducing the energy barrier between the bound and free states catalyzes both the forward and reverse reactions.

Supplementary Figure 11 Chemical-induced recruitment of the Snf2-like remodeler Lsh1 does not lead to Polycomb eviction.

(a) Schematic of the chemical-induced recruitment experiments with Lsh1 in live cells. Rapamycin-mediated recruitment of Lsh1 occurs through an Lsh1-Frb-V5 fusion. ZF, zinc finger; FKBP, FK506-binding protein; Frb, FKBP-rapamycin-binding domain of mTOR. (b) ChIP–qPCR enrichment profile of V5 following addition of rapamycin, showing induced recruitment of Lsh1. (c) ChIP–qPCR profile of the PRC1 subunit Ring1b following addition of rapamycin. (d) ChIP–qPCR profile of the PRC2 subunit Suz12 following addition of rapamycin. In contrast to recruitment of the BAF complex, recruitment of HELLS/Lsh1 does cause major decreases in Polycomb occupancy, confirming that Polycomb eviction is not a general property of all Snf2-like remodelers. For all subfigures, data shown are the means of n = 3 independent biological replicates; all error bars are s.e.m.

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Stanton, B., Hodges, C., Calarco, J. et al. Smarca4 ATPase mutations disrupt direct eviction of PRC1 from chromatin. Nat Genet 49, 282–288 (2017). https://doi.org/10.1038/ng.3735

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