Dominant-negative SMARCA4 mutants alter the accessibility landscape of tissue-unrestricted enhancers

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.

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Fig. 1: Heterozygous SMARCA4 mutations contribute to many cancers.
Fig. 2: SMARCA4 cancer mutations affect functional surfaces and induce distinct dynamic defects.
Fig. 3: Convergent effects of SMARCA4 cancer mutants on the DNA-accessibility landscape.
Fig. 4: Accessibility losses and PRC1 changes do not directly overlap.
Fig. 5: Heterozygous SMARCA4 mutation induces loss of accessibility and H3K27ac at active enhancers and superenhancers from many tissues.
Fig. 6: Chromatin organization influences dominant-negative effects of SMARCA4 mutants on the open chromatin landscape.
Fig. 7: SMARCA4 ATPase mutants can induce pro-oncogenic gene expression changes.

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Acknowledgements

We thank C. Weber, E. Chory, K. Cui, G. Hu, and E. Son for helpful discussions and dedicate this manuscript to the lasting memory of Joseph P. Calarco. We gratefully acknowledge the assistance of the DNA Sequencing and Genomics Core facility of NHLBI, the Stanford Cell Sciences Imaging Facility (S10OD01227601), and the Stanford BioX3 cluster (S10RR02664701). Ring1a −/− ;Ring1b fl/fl mESCs were a generous gift from H. Koseki (RIKEN, Japan). This work was supported by the Simons Foundation Autism Research Initiative (G.R.C.), NIH grants R37NS046789 (G.R.C.), R01CA163915 (G.R.C.), T32CA009151 (J.G.K.), R00CA187565 (H.C.H.), the Division of Intramural Research of the NHLBI/NIH (K.Z.)., the Czech Science Foundation grant 16-06357S (V.V.), the Cancer Prevention & Research Institute of Texas grant RR170036 (H.C.H.), and the Howard Hughes Medical Institute (G.R.C.).

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H.C.H. and B.Z.S. conceived of and performed experiments and wrote the paper. K.C., H.C.H., and V.V. developed the homology model. H.C.H, C.-Y.C., and E.L.M. performed analyses. B.Z.S., H.C.H., C.-Y.C., J.G.K., and W.L.K. prepared materials. K.Z. and G.R.C. designed experiments and wrote the paper.

Corresponding authors

Correspondence to Keji Zhao or Gerald R. Crabtree.

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The authors declare no competing financial interests.

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Integrated supplementary information

Supplementary Figure 1 The SMARCA4 ATP cleft and DNA groove are frequently mutated in cancer.

(a) Surface rendering of the SMARCA4 homology model viewed from the DNA-binding surface. Details of homology model are described in the Methods section. Surface residues are colored based on mutation frequency of deleterious missense mutations at the time of analysis in cBioPortal, for residue positions 758–1335. DNA is shown in yellow. (b) Rotation of the view in (a) 90° around the x-axis to show the ATP-binding cleft. ATP is shown in blue. (c) Rotation of the view in (a) 180° around the y-axis. (d) Rotation of the view in (c) –90° around the x-axis reveals few other surface mutation hotspots.

Supplementary Figure 2 Construction of a Smarca4-Dendra2 fluorescent knockin mouse.

(a) A 15.5-kbp homology template was used to target the last exon of SMARCA4 to create a SMARCA4-Dendra2 fusion. Thymidine kinase from herpes simplex virus (HSV-tk) was used for negative selection with gancyclovir, and positive selection from neo was achieved by selecting for G418-resistant colonies. (b) Southern blot of resulting heterozygous knockin mESC colony using the probe shown in panel (a). (c) Western blotting of the heterozygous knockin mESC colony shows a new band with higher molecular weight that also stains positive using anti-Dendra2 antibodies (Evrogen). (d) Fluorescent knockin SMARCA4 constructs assemble with endogenous BAF and PBAF subunits to form natural complexes. (e) Confocal image of resulting fluorescent mESC colony. (f) Homozygous mice obtained through breeding are phenotypically normal. Strain has been deposited for cryopreservation at Jackson Lab (stock #27901). PCR primers for genotyping are provided in the Methods section.

Supplementary Figure 3 SMARCA4-Dendra2 is highly stable and undergoes dynamic changes in interaction with chromatin throughout the cell cycle.

(a) Following photoconversion with 405-nm light using a custom-made LED lamp, SMARCA4-Dendra2 irreversibly photoswitches from green to red. (b) Photoswitching is readily detectable using flow cytometry. (c) The persistence of red fluorescence provides a measure of the stability of the protein fusion. Red fluorescence persists with a half-life of 11.8 ± 1.2 h, comparable to the canonical 12-hour half-life of mESCs. This result indicates that fluorescent fusions of SMARCA4 are stable. (d) Live-cell imaging of mitosis. Mitotic cells are readily distinguishable from interphasic cells due to the altered distribution of fluorescence intensity. (e) Fluorescence recovery after photobleaching (FRAP) of an individual mESC colony. (f) FRAP recovery times of SMARCA4-Dendra2 fusions are faster in mitosis, when the complex is excluded from condensed mitotic chromatin (g) Statistical comparison of FRAP recovery times of SMARCA4-Dendra2 fluorescent fusions. Mitotic cells show significantly increased FRAP recovery times (p<0.002, KS test). (h) Custom LED array for bulk photoconversion of cells using 405-nm LEDs (DigiKey 492-1349-ND).

Supplementary Figure 4 ATPase-dead SMARCA4 shows altered interaction with chromatin during interphase.

(a) Fluorescence deconvolution image of fixed mESCs expressing lentivirally transduced SMARCA4-GFP. During mitosis, SMARCA4 is excluded from chromatin. (b) Live-cell imaging of SMARCA4-GFP and DNA (imaged using DRAQ5) shows comparable results. (c) ATPase-dead K785R mutant (K798R in some citations), a cancer mutation found in the ATP-binding cleft) shows a dramatic dynamic defect, consistent with failure to release immobile chromatin. (d) Wild-type SMARCA4-GFP and SMARCA4-Dendra2 have indistinguishable dynamics in FRAP assays, while K785R mutants show a significant increase in the FRAP recovery times during interphase (p<2.2e-16). (e) During mitosis, mutant and wild-type complexes show similar dynamics, consistent with a model whereby ATP-dependent dynamics predominate during interphase when the complex engages chromatin (Figure 2g).

Supplementary Figure 5 Genome-wide alterations of ATAC-seq following acute Smarca4 knockout and relationship with Ring1b ChIP-seq.

(a) Heat maps and plot showing sites that lose accessibility following acute deletion of SMARCA4. (b) Heat maps and plot showing sites that do not change accessibility following acute deletion of SMARCA4. (c) Heat maps and plot showing sites that gain accessibility following acute deletion of SMARCA4. The criteria for classification is provided in the Methods section. (d) Genome-wide signal of ATAC-seq and Ring1b ChIP-seq, in relation to other chromatin features from the Mouse ENCODE Project. ATAC-seq is highly correlated with permissive marks, while Ring1b shows a high degree of correlation with H3K27me3, the mark of Polycomb Repressive Complex 2. (e) Enrichment of enhancers, transcription start sites (TSSs), transcription termination sites (TTSs), and intergenic regions in ATAC-seq datasets by class. ATAC-seq peaks are enriched at enhancers and TSSs. Small deviations between the knockout and WT/G784E expression reflect variability in genome-wide peak calling. (f) Enrichment of enhancers, transcription start sites (TSSs), transcription termination sites (TTSs), and intergenic regions in Ring1b ChIP-seq datasets by class. Ring1b peaks are enriched at TSSs.

Supplementary Figure 6 Analysis of altered chromatin signals and consistency of results across independent cell-culture replicates.

All fold changes below refer to changes that arise in G784E/WT mESCs compared to WT/WT mESCs. (a) MA plot of fold change of ATAC read density at individual sites across the genome. (b) Reproducibility of ATAC changes across independent cell-culture replicates. (c) MA plot of fold change of H3K4me1 read density at individual sites across the genome. (d) Reproducibility of H3K4me1 changes across independent cell-culture replicates. (e) MA plot of fold change of H3K27ac read density at individual sites across the genome. (f) Reproducibility of H3K27ac changes across independent cell-culture replicates. (g) MA plot of fold change of RNAP2 read density at individual sites across the genome. (h) Reproducibility of RNAP2 changes across independent cell-culture replicates.

Supplementary Figure 7 Examples of enhancer changes across the genome.

(a) Example of enhancer accessibility loss coinciding with H3K27ac loss but not H3K4me1 loss on chromosome 8. (b) Example of enhancer accessibility loss coinciding with H3K27ac loss but not H3K4me1 loss on chromosome 10. (c) Example of enhancer accessibility loss coinciding with H3K27ac loss but not H3K4me1 loss on chromosome 2. These changes are representative of the dominant-negative effects of SMARCA4 mutations on the enhancer landscape.

Supplementary Figure 8 Summary of ChIP-seq data at sites with increased and decreased accessibility measured by ATAC-seq.

(a) Summary of individual H3K4me1 ChIP-seq tracks at ATAC-seq sites identified as increased or decreased. (b) Summary of individual RNAP2 ChIP-seq tracks at ATAC-seq sites identified as increased or decreased. (c) Summary of individual H3K27ac ChIP-seq tracks at ATAC-seq sites identified as increased or decreased. (d) Mean RNA polymerase 2 (RNAP2) ChIP-seq read density at TSSs and enhancers in wild-type and heterozygous mutant cells.

Supplementary Figure 9 SMARCA2 (BRM) is repressed in wild-type and mutant SMARCA4 mESCs.

Expression of SMARCA2 compared to other genes in mutant and wild-type mESCs. Little to no SMARCA2 is expressed in mESCs, either in wild-type or mutant cells. Counting of transcripts reveals that SMARCA2 is expressed at less than 1% compared to SMARCA4, hence confounding effects due to SMARCA2 expression can be excluded. Plotted values are mean normalized expression counts (a.u.), error bars are 95% confidence intervals from independent cell-culture replicates (n=2).

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Hodges, H.C., Stanton, B.Z., Cermakova, K. et al. Dominant-negative SMARCA4 mutants alter the accessibility landscape of tissue-unrestricted enhancers. Nat Struct Mol Biol 25, 61–72 (2018). https://doi.org/10.1038/s41594-017-0007-3

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