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. 2018 Nov 15;175(5):1272-1288.e20.
doi: 10.1016/j.cell.2018.09.032. Epub 2018 Oct 18.

Modular Organization and Assembly of SWI/SNF Family Chromatin Remodeling Complexes

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Free PMC article

Modular Organization and Assembly of SWI/SNF Family Chromatin Remodeling Complexes

Nazar Mashtalir et al. Cell. .
Free PMC article

Abstract

Mammalian SWI/SNF (mSWI/SNF) ATP-dependent chromatin remodeling complexes are multi-subunit molecular machines that play vital roles in regulating genomic architecture and are frequently disrupted in human cancer and developmental disorders. To date, the modular organization and pathways of assembly of these chromatin regulators remain unknown, presenting a major barrier to structural and functional determination. Here, we elucidate the architecture and assembly pathway across three classes of mSWI/SNF complexes-canonical BRG1/BRM-associated factor (BAF), polybromo-associated BAF (PBAF), and newly defined ncBAF complexes-and define the requirement of each subunit for complex formation and stability. Using affinity purification of endogenous complexes from mammalian and Drosophila cells coupled with cross-linking mass spectrometry (CX-MS) and mutagenesis, we uncover three distinct and evolutionarily conserved modules, their organization, and the temporal incorporation of these modules into each complete mSWI/SNF complex class. Finally, we map human disease-associated mutations within subunits and modules, defining specific topological regions that are affected upon subunit perturbation.

Keywords: ATP-dependent chromatin remodeling; BAF complex; PBAF complex; SWI/SNF complex; cancer; cross-linking mass spectrometry; mutations; ncBAF complex; protein complex assembly; subunit organization.

Figures

Figure 1.
Figure 1.. Distinct mSWI/SNF Complexes and Their Intermediates Revealed through Affinity Purification
(A) Density sedimentation and immunoblot performed on HEK293T nuclear extracts. *, non-specific bands. (B) Silver stain performed on density sedimentation of HA-SMARCD1 mSWI/SNF complexes purified from HEK293T cells. (C) Silver stain performed on density sedimentation of HA-DPF2 BAF complexes purified from HEK293T cells. (D) Silver staining of the indicated HA-SMARCD1 gradient fractions from (B). Identified proteins are labeled. (E) Mass spectrometry analysis performed on selected fractions (fractions 3–18) collected from the HA-SMARCD1 density gradient in (B). Peptide proportion (0to1) representsthe fraction ofthe maximum number of peptides captured for each subunit overthe full gradient. Total spectral counts in fraction with highest peptide abundance for each subunit are indicated on the left. Colors distinguish mSWI/SNF complexes and modules. See also Figure S1.
Figure 2.
Figure 2.. Cross-Linking Mass Spectrometry of SWI/SNF Complexes Reveals Conserved Connectivity of Interacting Modules
(A) Matrix heatmap of the total crosslinks identified in combined HA-SS18 and HA-DPF2 BAF complex cross-linking mass spectrometry datasets. Individual subunits are divided into domains and ordered according to modules in (B). See also Figures S2B, S2J, and S2K. (B-D) Louvain modularity analysis performed on (B) mammalian cBAF complex cross-linking mass spectrometry datasets, (C) D. melanogaster D4 and BAP60 cross-linking mass spectrometry datasets, and (D) S. cerevisiae cross-linking mass spectrometry datasets (from Sen et al., 2017). (E) Correlations between mammalian and Drosophila BAF or BAP subunit domain and region interactions from cross-linking mass spectrometry datasets. See also Figures S2B and S2J. (F) Correlations between mammalian and yeast SWI/SNF subunit domain and region interactions from cross-linking mass spectrometry datasets. See also Figures S2B and S2K. See also Figure S2.
Figure 3.
Figure 3.. Identification and Characterization of the BAF Core Module: SMARCC, SMARCD, SMARCB1, and SMARCE1 Subunits
(A) Circle-plot analysis of the mammalian BAF complex cross-linking mass spectrometry dataset, with the BAF core module highlighted in blue. (B) Silver stain performed on density sedimentation of HA-SMARCC1 complexes purified from HEK293T cells (left) and clustered heatmap of mass spec-called peptides and spectral counts on selected fractions (right). (C) Distribution of inter-paralog crosslinks and self-crosslinks in the BAF cross-linking mass spectrometry dataset. (D) SMARCC self-crosslinks and SMARCC1 and SMARCC2 inter-paralog crosslinks from the BAF cross-linking mass spectrometry dataset. Line width is proportional to the number of crosslinks. (E) Heatmap depicting SMARCC crosslinks with BAF subunits from the BAF cross-linking mass spectrometry dataset. (F) Silver stain performed on density sedimentation of HA-SMARCE1 complexes purified from ∆SMARCD HEK293T cells (left) and clustered heatmap of mass spec-called peptides and spectral counts on selected fractions (right). (G) Silver stain performed on density sedimentation of HA-SMARCD1 complexes purified from ∆SMARCE1 HEK293T cells (left) and clustered heatmap of mass spec-called peptides and spectral counts on selected fractions (right). *, minimal SMARCE1 peptide abundance was detected despite no observed band (see also Table S2). (H) Schematic representation of initial steps of BAF core assembly. Subunit abbreviations are indicated. See also Figure S3.
Figure 4.
Figure 4.. ARID Subunits Dictate Specific Branches of BAF and PBAF Complex Assembly
(A) Circle-plot analysis of the mammalian cross-linking mass spectrometry dataset, with BAF core subunit crosslinks in blue and ARID module subunits in teal. (B) Clustered heatmap of cross-linking mass spectrometry data, highlighting crosslinks between ARID subunits and other complex components. (C) ARID1A, SMARCC1, and SMARCD1 crosslinks from the BAF cross-linking mass spectrometry dataset. Line width is proportional to the number of crosslinks. (D) Gradient and mass spectrometry heatmap of native HA-ARID1A C terminus-bound BAF complexes purified from HEK293T cells. (E-G) Native HA-SMARCD1 purification and gradient mass spectrometry in (E) ARID1Aand ARID1B-deficient, (F) ARID1A, ARID1B, and ARID2-deficient, and (G) SMARCA4/2-deficient HEK293T cells. (H) mSWI/SNF assembly branch points are initiated by ARID subunits. Subunit abbreviations are indicated. See also Figure S4.
Figure 5.
Figure 5.. The mSWI/SNF ATPases Recruit Accessory Subunits and Finalize BAF, PBAF, and ncBAF Complex Assembly
(A) Circle-plot analysis of the mammalian cross-linking mass spectrometry dataset with ATPase module subunits crosslinks in red and ATPase/ARID module crosslinks in yellow. (B) Clustered heatmap of the cross-linking mass spectrometry analysis of the mammalian BAF complex, highlighting the occurrence of crosslinks between SMARCA and other complex components. (C) Silver stain performed on density sedimentation of HA-SMARCA4-bound complexes purified from HEK293T cells. (D) Gradient mass spectrometry of selected fractions collected from the HA-SMARCA4 density gradient. Total spectral counts in fraction with highest peptide abundance for each subunit are indicated on the left. (E) Silver stain performed on density sedimentation analysis of FLAG-HA-SS18-bound BAF complexes purified from HEK293T cells (left) and clustered heatmap of mass spec-called peptides and spectral counts on selected fractions (right). (F) Clustered correlation heatmap performed on HA-SMARCD1, HA-SMARCB1, and HA-SMARCA4 density gradient mass spectrometry results from HEK293T cells. Experimentally determined complexes and subcomplexes are indicated. (G) Schematic of the assembly and incorporation of the BAF ATPase module. Subunit abbreviations are indicated. See also Figure S5.
Figure 6.
Figure 6.. Assembly of Alternative mSWI/SNF Complexes, PBAF and ncBAF, and the Full Assembly Pathway
(A) Silver stain performed on density sedimentation of HA-mARID2 PBAF complexes purified from HEK293T cells (left) and clustered heatmap of mass spec-called peptides and spectral counts on selected fractions (right). (B) Silver stain performed on density sedimentation of HA-PBRM1 PBAF complexes purified from HEK293T cells (left) and clustered heatmap of mass spec-called peptides and spectral counts on selected fractions (right). (C) Louvain network analysis of PBAF subunit (PHF10 and BRD7) cross-linking mass spectrometry datasets. (D) HA-GLTSCR1L-bound ncBAF complexes were purified from HEK293T, PAGE-separated, and silver stained. Individual identified proteins are indicated. (E) Silver stain performed on density sedimentation of HA-GLTSCR1L-bound ncBAF complexes purified from HEK293T cells (left) and clustered heatmap of mass spec-called peptides and spectral counts on selected fractions (right). *, non-specific contaminants in fraction 16. (F) Silver stain performed on density sedimentation of HA-BRD9 ncBAF complexes purified from HEK293T cells (left). Clustered heatmap of mass spec-called peptides and spectral counts on selected fractions (right). (G) Schematic of the full mSWI/SNF complex assembly pathway. Subunit abbreviations are indicated. Numbers indicate the steps in assembly (see text). See also Figure S6.
Figure 7.
Figure 7.. Disruption of mSWI/SNF Complex Assembly in Human Disease
(A) Frequency of mSWI/SNF gene mutations across human cancers (The Cancer Genome Atlas [TCGA]). Hypermutated samples were excluded from analysis. (B) Mass spectrometry analysis of mSWI/SNF complex subunit relative abundance in complexes purified from the indicated cell types (WT and subunit KO cells), normalized toWTSMARCC1 purifications. DSMARCDcomplexeswere purified using SMARCE1; ∆SMARCE1, ∆SMARCB1, ∆ARID1/2, ∆ARID1, and ∆SMARCA complexes were purified using HA-SMARCD1. (C) Correlation analysis reflectingtheeffect oftruncating mutationson mSWI/SNF subunit linkages. Subunits mostfrequentlytruncated exhibit higherproportions of inter-crosslinked sites lost. (D) Top-ranked cancer-associated missense mutations (TCGA). Mutations predicted to disrupt catalytic activity are shown in red. (E) Non-truncating mutations in ARID1A across human cancers mapped over intra-crosslinks. The hotspot mutation in the highly crosslinked C-terminal CBRB region of the protein is indicated. (F) Truncating mutations in ARIDIAacross human cancers mapped over crosslinks to other BAF subunits. The position ofthetruncating mutation Y2254* used in this study is indicated by the arrow. (G) Top: Representative cycloheximide chase experiment assessing the half-life of ARID1A WT and G2087R mutant C-terminal region variants. Bottom: quantification of western blot (WB) above, normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH). (H) MG-132 treatment (8 hr) of HEK293T cells expressing ARID1A WT and G2087R C-terminal regions. IP performed under denaturing conditions; immunoblot performed using indicated antibodies. (I) Silver stain performed on ARID1A WT, G2087R, and Y2254* BAF complexes purified from HEK293T cells. (J) Immunoblot of ARID1A WT, G2087R-, and Y2254*-bound BAF complexes purified from HEK293T cells. See also Figure S7.

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