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. 2018 Jan;25(1):61-72.
doi: 10.1038/s41594-017-0007-3. Epub 2017 Dec 11.

Dominant-negative SMARCA4 Mutants Alter the Accessibility Landscape of Tissue-Unrestricted Enhancers

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Free PMC article

Dominant-negative SMARCA4 Mutants Alter the Accessibility Landscape of Tissue-Unrestricted Enhancers

H Courtney Hodges et al. Nat Struct Mol Biol. .
Free PMC article

Abstract

Mutation of SMARCA4 (BRG1), the ATPase of BAF (mSWI/SNF) and PBAF complexes, contributes to a range of malignancies and neurologic disorders. Unfortunately, the effects of SMARCA4 missense mutations have remained uncertain. Here we show that SMARCA4 cancer missense mutations target conserved ATPase surfaces and disrupt the mechanochemical cycle of remodeling. We find that heterozygous expression of mutants alters the open chromatin landscape at thousands of sites across the genome. Loss of DNA accessibility does not directly overlap with Polycomb accumulation, but is enriched in 'A compartments' at active enhancers, which lose H3K27ac but not H3K4me1. Affected positions include hundreds of sites identified as superenhancers in many tissues. Dominant-negative mutation induces pro-oncogenic expression changes, including increased expression of Myc and its target genes. Together, our data suggest that disruption of enhancer accessibility represents a key source of altered function in disorders with SMARCA4 mutations in a wide variety of tissues.

Conflict of interest statement

Competing financial interests

The authors declare no competing financial interests.

Figures

Figure 1
Figure 1. Heterozygous SMARCA4 mutations contribute to many cancers
(a) Heat map illustrating the genomic states of 927 human tumor samples from different tissues with SMARCA4 mutations and deletions. Samples without genetic changes to SMARCA4 or with only silent mutations are not shown. (b) SMARCA4 has disproportionately more missense mutations than other tumor suppressors like ARID1A and RB1. Pie chart showing the proportion of tumor samples showing patterns of genetic aberrations. Fisher exact test comparing proportion of missense mutations compared to SMARCA4, *** p<2.2e-16. (c) Expression of SMARCA4 in tumor samples bearing missense mutations show comparable levels of SMARCA4, consistent with heterozygous expression.
Figure 2
Figure 2. SMARCA4 cancer mutations affect functional surfaces and induce distinct dynamic defects
(a) Homology model of SMARCA4 ATPase domains bound to a nucleosome, derived from Snf2 and Snf2-like family member structures from other organisms (details in main text and Methods). Three-dimensional coordinates of the SMARCA4 homology models are provided in PDB format as supplemental data. (b) Distribution of missense mutations of SMARCA4 across all cancers compiled by cBioPortal. 56% of mutations target the N- and C-terminal ATPase domain. (c) Solvent accessible surface area (SASA) for frequently mutated residue positions of SMARCA4 in human cancers. Buried and surface mutations are frequent sites of mutations, including the ATP-binding cleft, DNA-binding groove, and other sites with unknown function. (d) FRAP recovery curves for SMARCA4-GFP mutants of the ATP-binding cleft show slow photobleaching recovery. (e) FRAP recovery curves for SMARCA4-GFP mutants of the DNA-binding groove show fast photobleaching recovery. Curves for WT controls (dotted) are the same for all plots in panels (d) and (e). (f) Positions found within the ATP-binding cleft or DNA-binding groove show similar FRAP recovery kinetics. Two-sample KS test compared to WT, * p<0.05; ** p<0.01; *** p<0.001. Plotted values are mean t1/2 recovery times, error bars are SEM from n=10 cells. (g) Model of how SMARCA4 surface mutations alter dynamic engagement of chromatin. DNA-binding groove mutants impair engagement with DNA, while ATP-binding cleft mutants impair ATP hydrolysis, which may be required for transition to a mobile state.
Figure 3
Figure 3. Convergent effects of SMARCA4 cancer mutants on the DNA accessibility landscape
(a) Genome tracks showing loss of accessibility at an annotated enhancer downstream of Bcl11b. Heterozygous mutant SMARCA4 cells but not wild-type cells show similar loss of enhancer accessibility. (b) Heat map of Pearson correlation coefficients (PCC) of reads densities in peaks shows correlated genome-wide changes induced by heterozygous SMARCA4 mutants. Changes are uncorrelated with changes between wild-type cell-culture replicates. (c) Heat map showing the 2,000 genome-wide sites with the highest variance across datasets. All mutants show a similar trend of altered accessibility across these sites. (d) Genomic annotation enrichment datasets for decreased sites across mutant ATAC-seq. Decreased sites are enriched at non-coding regions such as introns and intergenic regions. (e) Lasso multivariate regression identifies a consistent set of features associated with positive or negative changes of accessibility in mutants compared to wild-type. Features associated with altered accessibility include several factors and marks associated with enhancers. (f) Characterization of ATAC-seq in sites with decreased accessibility in mutant cells, along with ChIP-seq of Smarcc1, Lsd1, and H3K4me1 in wild-type cells. (g) Characterization of ATAC-seq in sites with unchanged accessibility in mutant cells, along with ChIP-seq of Smarcc1, Lsd1, and H3K4me1 in wild-type cells. (h) Characterization of ATAC-seq in sites with increased accessibility in mutant cells, along with ChIP-seq of Smarcc1, Lsd1, and H3K4me1 in wild-type cells.
Figure 4
Figure 4. Accessibility losses and PRC1 changes do not directly overlap
(a) ATAC accessibility sites ranked by altered ATAC-seq read density in SMARCA4 knockout (left) and in hetereozygous G784E SMARCA4 (right). Genomic sites lacking both ATAC and Ring1b changes are not shown. Corresponding changes of Ring1b ChIP-seq read density at these same sites show that Ring1b is broadly unchanged at these sites, and shows no clear relationship to accessibility changes measured by ATAC. (b) Example genome track showing representative accessibility but not Ring1b changes. (c) Ring1b ChIP sites ranked by altered ATAC-seq read density in SMARCA4 knockout (left) and in heterozygous G784E SMARCA4 (right). Corresponding changes of Ring1b ChIP-seq read density at these same sites show that Ring1b is broadly unchanged at these sites, and shows no clear relationship to accessibility changes measured by ATAC. Genomic sites lacking both ATAC and Ring1b changes are not shown. (d) Example genome track showing representative Ring1b but not accessibility changes. (e) Genomic fold changes of enhancers (Enh) and transcription start sites (TSS) using ATAC-seq upon conditional Ring1b knockout. TSSs show few changes while enhancers show significantly greater variability (KS test p<2.2e-16). Center line of box plot is mean, box limits are 1st and 3rd quartiles, whiskers are limits + 1.5*IQR (inter-quartile range), points are actual values of outliers.
Figure 5
Figure 5. Heterozygous SMARCA4 mutation induces loss of accessibility and H3K27ac at active enhancers and superenhancers from many tissues
(a) Mean ATAC-seq read density at TSSs and enhancers in wild-type and heterozygous mutant cells. (b) Example genome track showing representative loss of accessibility and H3K27ac but preservation of H3K4me1 at an enhancer. (c) Relationship between H3K4me1 ChIP and ATAC fold changes from wild-type and mutant cells shows significant correlation with a small effect size (m). (d) Relationship between RNAP2 ChIP and ATAC fold changes between wild-type and mutant cells shows significant correlation with a small effect size. (e) Relationship between H3K27ac ChIP and ATAC fold changes between wild-type and mutant cells shows significant correlation with a large effect size. (f) Abundance of H3K4me1 and H3K27ac in wild-type cells, classified by whether site increased, decreased accessibility, or was unchanged. Sites with decreased accessibility cluster have elevated levels of H3K4me1 and H3K27ac, consistent with active enhancers. (g) Mean accessibility changes at the Oct4-Sox2-Tcf-Nanog combination transcription factor motif. (h) Heat map of 18 transcription factor motifs associated with reduced accessibility across cell-culture replicates in heterozygous G784E SMARCA4 cells. (i) Cumulative distribution of ATAC-seq fold change, classified by genomic annotation. TSSs show few changes, while enhancers and superenhancers show biased losses. Superenhancers show distributions of fold changes comparable to enhancers. (j) Analysis of sites with decreased accessibility that overlap with superenhancers from multiple tissue types. Color in heat map shows fractional overlap with known annotated superenhancers from tissues obtained from dbSUPER. (k) Distribution of the number of tissue types associated with each superenhancer site analyzed in (j). (l) Pie chart of the proportion of tissue-restricted superenhancers compared with superenhancers that identified in >1 tissue type. RPM, reads per million; Enh, enhancer; SE, superenhancer.
Figure 6
Figure 6. Chromatin organization influences dominant-negative effects of SMARCA4 mutants on the open chromatin landscape
(a) Demonstration of a line of heterozygous conditional deletion SMARCA4 cells. A single allele of SMARCA4 is converted to a null allele in the presence of 4-hydroxytamoxifen (Tam) but not ethanol control (EtOH). (b) Quantification of bands in (a), showing loss of half the SMARCA4 present caused by incubation with Tam. (c) Plot of genome-wide fold changes for all ATAC-seq sites following acute deletion of one allele of SMARCA4. (d) Mean profiles of ATAC-seq read density at TSSs and (e) enhancers following acute deletion of one allele of SMARCA4. The absence of changes demonstrates that the changes we observe in Figure 4 are not due to haploinsufficiency, but are a dominant-negative effect. (f) Heterozygous G784E SMARCA4 but not heterozygous null cells show clusters of reduced accessibility in chromosomal A compartments (described in more detail in the main text and Methods). (g) Quantification of genome-wide ATAC-seq site changes based on presence in A or B compartments in heterozygous null cells. (h) In heterozygous G784E SMARCA4 cells, ATAC-seq sites in A compartments have reduced accessibility compared to sites in B compartments. CDF, cumulative distribution function.
Figure 7
Figure 7. SMARCA4 ATPase mutants can induce pro-oncogenic gene expression changes
(a) Summary of genome-wide RNA expression changes by RNA-seq. Each point is a gene, colored by whether it is increased, decreased, or unchanged, using the criteria described in the Methods section. (b) Heat map of genes with altered expression based on whether they are annotated to be promoters targeted by Polycomb Repressive Complexes (PRCs) in mESCs, or are not annotated to have PRC-target promoters. (c) Expression of pluripotency factors remains elevated in heterozygous G784E SMARCA4 cells. Myc is expressed ~2-fold higher in mutant cells compared to wild-type cells. Plotted values are mean normalized expression values (a.u.), error bars are 95% confidence intervals from independent cell-culture replicates (n=2). (d) Gene set enrichment analysis shows significant enrichment of downstream targets of Myc (p<2.2e-16). (e) Model of dominant-negative effects following inactivation of SMARCA4 ATP hydrolysis or DNA/nucleosome binding. (f) Effect of SMARCA4 heterozygous mutation on the epigenetic landscape. Mutation and the ensuing compensatory changes shifts the epigenetic-phenotypic landscape, resulting in stabilization of a new phenotypic state that may adopt pathogenic patterns of gene expression.

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