Smarca4 ATPase mutations disrupt direct eviction of PRC1 from chromatin

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). This opposition underlies the tumor-suppressive role of BAF subunits and is expected to contribute to neurodevelopmental disorders. 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.

Fig. 1. Characteristic accumulation of PRC1 and PRC2 following BAF subunit deletion

(a) Genome-wide increases of PRC1 occupancy by Ring1b ChIP-seq in conditional Smarca4 knockout mESCs. Each point on the plot represents an individual Ring1b peak. Sites that are increased (orange), decrease (blue), or remain unchanged (gray) are labeled using the criteria described in the Methods section. (b) Genome-wide increases of PRC2 occupancy by Suz12 ChIP-seq in conditional Smarca4 knockout mESCs. (c) Ring1b ChIP-seq in conditional Arid1a knockout mESCs. (d) Suz12 ChIP-seq in conditional Arid1a knockout mESCs. (e) Example track of PRC1/2 upon knockout of BAF subunits, aligned to histone mark tracks from the mouse ENCODE project. Additional tracks and analysis are presented as Supplemental Figures. (f) Pearson correlation heatmap of genome-wide PRC1/2 fold changes following Smarca4 or Arid1a knockout (top left); heatmap of all peaks with altered Polycomb occupancy. PRC1 and PRC2 are highly coupled, and changes arising from Arid1a knockout are a subset of changes arising from knockout of Smarca4 (right). (g) Comparison of RNA-seq data from wild-type and conditional Smarca4 knockout mESCs. Altered gene expression upon Smarca4 knockout is consistent across replicates. Comparison of changes of gene expression to the changes of (h) Ring1b peaks, or (i) Suz12 peaks found at TSSs. Following knockout of Smarca4, the expression levels of genes are negatively correlated with changes of Ring1b and Suz12 over their TSSs. Correlation and P values obtained from Pearson’s product moment correlation test.

Fig. 2. Mutations of the Smarca4 ATPase domain induce increased PRC1 occupancy at CpG-rich promoters

(a) The proportion of BAF-subunit mutation types varies continuously from truncating mutations to deleterious missense mutations. Smarca4 mutations are frequently deleterious missense mutations, rather than truncating mutations. (b) Mutated positions (black) of the Smarca4 ATPase domain summed across primary tumors and cancer cell lines overlap with conserved sequence motifs. Residue mutation frequency (blue) coincides with residue conservation scores (gray). (c) Heatmap of all fold changes of Ring1b peaks between wild-type and ATPase mutant Smarca4. ATPase mutants predominantly induce Ring1b increases. (d) Fold changes of Ring1b occupancy are correlated between Smarca4 ATPase mutants. Pearson correlation values of genome-wide fold changes are presented as a heatmap. (e) Observed changes between wild-type and Smarca4 mutants are consistent across independent replicates. (f) Heatmap of overlap enrichment of sites where Smarca4 mutants induce increased Ring1b occupancy based on genomic annotation. Values reflect enrichment of increased sites compared to unchanged sites.

Fig. 3. Chromatin features define sites predisposed to Polycomb increases upon expression of Smarca4 ATPase mutants

(a) Fisher’s linear discriminant shows that combinations of chromatin features define response to Smarca4 ATPase mutants. Increased and decreased sites have distinct combinations of features, with increased sites having elevated H3K4me3 and other positive factors/marks. (b) Regression weights relating each of the above chromatin features to the fold change of Ring1b at a given site obtained via Lasso multivariate regression, presented as a heatmap. The consistent positive regression weights for H3K4me3, its methylase Kmt2b, and demethylase Jarid1a, show that high levels of these marks in wild-type cells are associated with increased Polycomb occupancy upon expression of Smarca4 ATPase mutants.

Fig. 4. Smarca4 ATPase mutations result in increased H3K27me3 levels near sites of Ring1b increases

(a) Example genome tracks of Ring1b increases with adjacent H3K27me3 increases caused by E861K Smarca4. (b) Genome-wide changes of H3K27me3 peaks between wild-type and E861K Smarca4. Sites that are increased (orange), decrease (blue), or remain unchanged (gray) are labeled using the criteria described in the Methods section. (c) Heatmap of global H3K27me3 changes between cells expressing wild-type and mutant Smarca4. (d) Pearson correlation heatmap of H3K27me3 changes between all mutants and wild-type Smarca4. (e) H3K27me3 peaks occur adjacent to CpG islands; sites that increase H3K27me3 levels upon expression of mutant Smarca4 have increased Ring1b occupancy over the adjacent CpG island. (f) Meta-gene plots of H3K27me3 and H3K4me3 marking levels classified by Ring1b status [decreased (N=66 sites), increased (N=716 sites), or unchanged (N=3,078 sites) in E861K compared to wild-type Smarca4]. Sites with increased Ring1b have increased H3K27me3 marking ~2 kb away; however, sites show no change in the levels of H3K4me3.

Fig. 5. The ATPase of Smarca4 directly regulates the PRC1 complex

(a) Workflow for immunoprecipitation (IP) experiments. Nuclear protein was isolated for analysis from the soluble portion of nuclear lysates. (b) Co-IP of BAF components with PRC1 subunits (N=3; two cell-culture replicates). (c) Reciprocal co-IP of PRC1 components with BAF subunits. Co-IP of Ring1b, Rybp, and Smarcc1 was observed with antibodies directed against the dedicated BAF subunit Smarcb1 (N=3; two cell-culture replicates). (d) BAF binding of PRC1 is unaffected by the addition of DNase I, consistent with a direct interaction which does not require mediation through chromatin. See Figure S3F regarding the lack of staining of some factors in the input samples in (c) and (d). (e) Interaction of Smarca4 and Smarcc1 with PRC1 subunit Rybp is disrupted by the addition of 10 mM ATP (N=5; three cell-culture replicates). (f) Release of PRC1 is inefficient in the presence of ATP analogs that inhibit hydrolysis. ATP leads to reduced co-IP of BAF and PRC1 compared to AMPPNP [t(2)=4.5, p=0.046] and ATPγS [t(2)=6.2, p=0.025; two-sample t-tests]. As observed in other ATPases, ATPγS is weakly hydrolyzed, leading to partial release. Error bars are SEM (N=3; two cell-culture replicates). (g) Interaction of BAF and PRC1 is disrupted by ATPase mutants of Smarca4 (N=3; two cell-culture replicates). (h) Densitometry of individual replicates in (g). Mutant Smarca4 shows reduced interaction with PRC1.

Fig. 6. Chemical-induced proximity of BAF causes rapid loss of Polycomb occupancy on intact chromatin

(a) Schematic representation of chemical-induced recruitment experiments in live cells. Details are provided in main text. ZF, Zinc finger; FKBP, FK506 binding protein; Frb, FKBP-rapamycin binding domain of mTOR. (b) ChIP-qPCR enrichment profile of BAF subunit Smarcc1 following addition of rapamycin. (c) ChIP-qPCR profile of PRC1 subunit Ring1b following addition of rapamycin. (d) Time course for loss of Ring1b and Suz12 at recruitment site following addition of rapamycin. Differential loss occurs at the recruitment site, confirming that Polycomb occupancy is locally and not globally reduced, consistent with eviction activity on chromatin. For all subfigures, data shown are the means of N=3 cell-culture replicates; all error bars are SEM.