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• W2895850607 abstract "•mSWI/SNF complexes assemble in an ordered, modular pathway•mSWI/SNF core module is a required platform for BAF, PBAF, and ncBAF formation•ARID/GLTSCR subunits define complex identity and facilitate ATPase module binding•Recurrent missense and nonsense mutations affect mSWI/SNF complex assembly 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. 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. ATP-dependent chromatin remodeling complexes are multimeric molecular assemblies that regulate chromatin architecture (Kadoch and Crabtree, 2015Kadoch C. Crabtree G.R. Mammalian SWI/SNF chromatin remodeling complexes and cancer: Mechanistic insights gained from human genomics.Sci. Adv. 2015; 1: e1500447Crossref PubMed Google Scholar, Masliah-Planchon et al., 2015Masliah-Planchon J. Bièche I. Guinebretière J.M. Bourdeaut F. Delattre O. SWI/SNF chromatin remodeling and human malignancies.Annu. Rev. Pathol. 2015; 10: 145-171Crossref PubMed Google Scholar, Wu et al., 2009Wu J.I. Lessard J. Crabtree G.R. Understanding the words of chromatin regulation.Cell. 2009; 136: 200-206Abstract Full Text Full Text PDF PubMed Scopus (274) Google Scholar). These complexes are grouped into four major families, including switching (SWI)/sucrose fermentation (sucrose non-fermenting [SNF]), INO80 (Conaway and Conaway, 2009Conaway R.C. Conaway J.W. The INO80 chromatin remodeling complex in transcription, replication and repair.Trends Biochem. Sci. 2009; 34: 71-77Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar), ISWI (imitation SWI; Bartholomew, 2014Bartholomew B. ISWI chromatin remodeling: one primary actor or a coordinated effort?.Curr. Opin. Struct. Biol. 2014; 24: 150-155Crossref PubMed Scopus (24) Google Scholar), and CHD/M-2 (chromodomain helicase DNA-binding) groups (Murawska and Brehm, 2011Murawska M. Brehm A. CHD chromatin remodelers and the transcription cycle.Transcription. 2011; 2: 244-253Crossref PubMed Scopus (62) Google Scholar), all of which contain Snf2-like ATPase subunits but differ substantially via the incorporation of distinct subunits and in their targeting and activity on chromatin (Clapier et al., 2017Clapier C.R. Iwasa J. Cairns B.R. Peterson C.L. Mechanisms of action and regulation of ATP-dependent chromatin-remodelling complexes.Nat. Rev. Mol. Cell Biol. 2017; 18: 407-422Crossref PubMed Scopus (505) Google Scholar). SWI/SNF complexes were originally discovered and characterized in yeast, later in Drosophila (Dingwall et al., 1995Dingwall A.K. Beek S.J. McCallum C.M. Tamkun J.W. Kalpana G.V. Goff S.P. Scott M.P. The Drosophila snr1 and brm proteins are related to yeast SWI/SNF proteins and are components of a large protein complex.Mol. Biol. Cell. 1995; 6: 777-791Crossref PubMed Google Scholar, Stern et al., 1984Stern M. Jensen R. Herskowitz I. Five SWI genes are required for expression of the HO gene in yeast.J. Mol. Biol. 1984; 178: 853-868Crossref PubMed Google Scholar), and most recently in mammals (Ho et al., 2009Ho L. Ronan J.L. Wu J. Staahl B.T. Chen L. Kuo A. Lessard J. Nesvizhskii A.I. Ranish J. Crabtree G.R. An embryonic stem cell chromatin remodeling complex, esBAF, is essential for embryonic stem cell self-renewal and pluripotency.Proc. Natl. Acad. Sci. USA. 2009; 106: 5181-5186Crossref PubMed Scopus (402) Google Scholar, Kadoch et al., 2013Kadoch C. Hargreaves D.C. Hodges C. Elias L. Ho L. Ranish J. Crabtree G.R. Proteomic and bioinformatic analysis of mammalian SWI/SNF complexes identifies extensive roles in human malignancy.Nat. Genet. 2013; 45: 592-601Crossref PubMed Scopus (823) Google Scholar). Mammalian SWI/SNF (mSWI/SNF) complexes are ∼1- to 1.5-MDa entities combinatorially assembled from the products of 29 genes, including multiple paralogs, generating extensive diversity in composition. All complexes contain an ATPase subunit, either SMARCA4 (BRG1) or SMARCA2 (BRM), that catalyzes the hydrolysis of ATP. The roles of most other accessory subunits in complex assembly and stability as well as targeting and function remain unknown. Over the past several years, mSWI/SNF complexes have emerged as a major focus of attention because of the striking mutational frequencies in the genes encoding their subunits across a range of human diseases, from cancer to neurologic disorders. Indeed, recent exome sequencing studies have revealed that over 20% of human cancers bear mutations in the genes encoding mSWI/SNF subunits (Kadoch et al., 2013Kadoch C. Hargreaves D.C. Hodges C. Elias L. Ho L. Ranish J. Crabtree G.R. Proteomic and bioinformatic analysis of mammalian SWI/SNF complexes identifies extensive roles in human malignancy.Nat. Genet. 2013; 45: 592-601Crossref PubMed Scopus (823) Google Scholar). In addition, heterozygous point mutations in mSWI/SNF genes have been implicated as causative events in intellectual disability and autism spectrum disorders (Bögershausen and Wollnik, 2018Bögershausen N. Wollnik B. Mutational Landscapes and Phenotypic Spectrum of SWI/SNF-Related Intellectual Disability Disorders.Front. Mol. Neurosci. 2018; 11: 252Crossref PubMed Google Scholar, López and Wood, 2015López A.J. Wood M.A. Role of nucleosome remodeling in neurodevelopmental and intellectual disability disorders.Front. Behav. Neurosci. 2015; 9: 100Crossref PubMed Scopus (51) Google Scholar) A major barrier to our understanding of the functions, tissue-specific roles, and effect of mutations on mSWI/SNF complex mechanisms lies in the lack of information regarding subunit organization, assembly, and 3D structure. Several factors pose major challenges to such studies. Individually expressed subunits are often unstable or incorrectly folded without their appropriate binding partners, and minimal complexes pieced together via in vitro co-expression may not represent endogenous, physiologically relevant complexes in cells. Large quantities of purified endogenous complexes with minimal heterogeneity are required for downstream analyses, and selection of appropriate purification strategies cannot be informed without understanding modular architecture and assembly order. For these reasons and others, to date only low-resolution maps have been achieved using cryoelectron microscopy (cryo-EM) approaches (Dechassa et al., 2008Dechassa M.L. Zhang B. Horowitz-Scherer R. Persinger J. Woodcock C.L. Peterson C.L. Bartholomew B. Architecture of the SWI/SNF-nucleosome complex.Mol. Cell. Biol. 2008; 28: 6010-6021Crossref PubMed Scopus (112) Google Scholar, Leschziner et al., 2007Leschziner A.E. Saha A. Wittmeyer J. Zhang Y. Bustamante C. Cairns B.R. Nogales E. Conformational flexibility in the chromatin remodeler RSC observed by electron microscopy and the orthogonal tilt reconstruction method.Proc. Natl. Acad. Sci. USA. 2007; 104: 4913-4918Crossref PubMed Scopus (81) Google Scholar), and X-ray crystallographic analyses have been successfully performed on only a few isolated domains (Yan et al., 2017Yan L. Xie S. Du Y. Qian C. Structural Insights into BAF47 and BAF155 Complex Formation.J. Mol. Biol. 2017; 429: 1650-1660Crossref PubMed Scopus (21) Google Scholar), including the recently reported yeast Snf2 ATPase domain (Liu et al., 2017Liu X. Li M. Xia X. Li X. Chen Z. Mechanism of chromatin remodelling revealed by the Snf2-nucleosome structure.Nature. 2017; 544: 440-445Crossref PubMed Scopus (140) Google Scholar, Xia et al., 2016Xia X. Liu X. Li T. Fang X. Chen Z. Structure of chromatin remodeler Swi2/Snf2 in the resting state.Nat. Struct. Mol. Biol. 2016; 23: 722-729Crossref PubMed Scopus (41) Google Scholar). To establish a comprehensive structural framework for mSWI/SNF complexes, we used a multifaceted series of approaches involving complex and subcomplex purification, mass spectrometry (MS), crosslinking mass spectrometry (CX-MS), systematic genetic manipulation of subunits and subunit paralog families, evolutionary analyses, and human disease genetics. These studies reveal that mSWI/SNF complexes exist in three non-redundant final form assemblies: BRG1/BRM-associated factor complexes (BAFs), polybromo-associated BAF complexes (PBAFs), and non-canonical BAFs (ncBAFs), for which we establish the assembly requirements and modular organization. We define the full spectrum of endogenous combinatorial possibilities and the effect of individual subunit deletions and mutations, including recurrent, previously uncharacterized missense and nonsense mutations, on complex architecture. These studies provide important insights into mSWI/SNF complex organization, structure, and function and the biochemical consequences of a wide range of human disease-associated mutations. To begin to probe the modular organization and assembly order of mSWI/SNF family complexes, we subjected HEK293T cell nuclear extracts to density sedimentation analyses using 10%–30% glycerol gradients, reasoning that such an approach could reveal the presence of distinct final-form SWI/SNF complexes as well as assembly pathway intermediates (Figure 1A). We identified a range of migration patterns, with subunits such as SMARCD1 and SMARCC1 exhibiting marked spreading across the gradient and complex-defining subunits migrating in a restricted set of fractions, such as DPF2 and ARID1A (fraction of [Fx] 13–14), marking canonical BAF (cB or BAF) complexes and ARID2, BRD7, and PBRM1 in higher-mass fractions, Fx 16–17, marking PBAF complexes. In addition, BRD9 and GLTSCR1/1L subunits, corresponding to a newly identified class of mSWI/SNF complexes we have termed ncBAF (Alpsoy and Dykhuizen, 2018Alpsoy A. Dykhuizen E.C. Glioma tumor suppressor candidate region gene 1 (GLTSCR1) and its paralog GLTSCR1-like form SWI/SNF chromatin remodeling subcomplexes.J. Biol. Chem. 2018; 293: 3892-3903Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar, Ho et al., 2009Ho L. Ronan J.L. Wu J. Staahl B.T. Chen L. Kuo A. Lessard J. Nesvizhskii A.I. Ranish J. Crabtree G.R. An embryonic stem cell chromatin remodeling complex, esBAF, is essential for embryonic stem cell self-renewal and pluripotency.Proc. Natl. Acad. Sci. USA. 2009; 106: 5181-5186Crossref PubMed Scopus (402) Google Scholar, Hohmann et al., 2016Hohmann A.F. Martin L.J. Minder J.L. Roe J.S. Shi J. Steurer S. Bader G. McConnell D. Pearson M. Gerstberger T. et al.Sensitivity and engineered resistance of myeloid leukemia cells to BRD9 inhibition.Nat. Chem. Biol. 2016; 12: 672-679Crossref PubMed Scopus (109) Google Scholar, Kadoch et al., 2013Kadoch C. Hargreaves D.C. Hodges C. Elias L. Ho L. Ranish J. Crabtree G.R. Proteomic and bioinformatic analysis of mammalian SWI/SNF complexes identifies extensive roles in human malignancy.Nat. Genet. 2013; 45: 592-601Crossref PubMed Scopus (823) Google Scholar, Sarnowska et al., 2016Sarnowska E. Gratkowska D.M. Sacharowski S.P. Cwiek P. Tohge T. Fernie A.R. Siedlecki J.A. Koncz C. Sarnowski T.J. The Role of SWI/SNF Chromatin Remodeling Complexes in Hormone Crosstalk.Trends Plant Sci. 2016; 21: 594-608Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar), exhibited distinct lower-molecular-weight migration patterns (Fx 9–10). Using these results, we developed a robust purification strategy to capture endogenous mammalian complexes at each of these extremes with over 95% purity (Figure S1A; Table S1). SMARCD1-based purifications were used to capture all forms of mSWI/SNF complexes (because SMARCD1 is present across the full gradient), and hemagglutinin (HA)-DPF2 was used to purify fully assembled BAF complexes, which do not contain PBAF or ncBAF complex components (Figures S1B and S1C). Remarkably, density sedimentation and silver staining of purified complexes revealed that SMARCD1-captured complexes spread across the gradient, whereas DPF2 complexes marked only complete BAF complexes with no detectable intermediates (Figures 1B–1D; Figures S1D and S1E; Table S2), highlighting the utility of this approach to detect specific complexes and intermediate modules. Analysis of spectral counts from mass spectrometry performed across SMARCD1 gradient fractions confirmed the silver stain results and further identified components with lower abundance, such as ncBAF and PBAF subunits (Figure 1E; Figure S1F; Table S2). Taken together, these data suggest a stepwise, modular assembly pathway for mSWI/SNF family complexes, resulting in three distinct final complex forms, each with their own combinatorial diversity. We next performed bis(sulfosuccinimidyl)suberate (BS3)-based cross-linking mass spectrometry using DPF2 and SS18 as baits to identify BAF subunit architecture and linkages. We generated high-density subunit crosslinking maps based on 1,560 identified spectra for inter-protein crosslinks and 2,373 identified spectra for intra-protein crosslinks with coverage across all BAF complex subunits, with the exception of SS18 (because of limited lysine residues) (Figures 2A and S2A; Table S3; STAR Methods). To comprehensively define regions of crosslinking between BAF complex subunits, we divided each subunit family (collapsed; i.e., SMARCD = SMARCD1, SMARCD2, or SMARCD3) into regions based on existing domain annotation, conservation, and newly defined domains stemming from this cross-linking mass spectrometry work (Figures 2A and S2B). The median distance between crosslinked residues within domains of known structure was 12.2 Å, close to the expected 11.4–30 Å distance for the BS3 crosslinking agent (Figure S2C; Table S4). In addition, the C-alpha distances between crosslinked residues mapped onto the structure of the Snf2 helicase were within expected distances for the nucleosome-bound and free conformations (Liu et al., 2017Liu X. Li M. Xia X. Li X. Chen Z. Mechanism of chromatin remodelling revealed by the Snf2-nucleosome structure.Nature. 2017; 544: 440-445Crossref PubMed Scopus (140) Google Scholar, Xia et al., 2016Xia X. Liu X. Li T. Fang X. Chen Z. Structure of chromatin remodeler Swi2/Snf2 in the resting state.Nat. Struct. Mol. Biol. 2016; 23: 722-729Crossref PubMed Scopus (41) Google Scholar; Figure S2D).Figure S2Purification and Cross-Linking Mass Spectrometry on Mammalian, Fly, and Yeast SWI/SNF Complexes, Related to Figure 2Show full caption(A) Silver stains of affinity-purified complexes from mammalian HEK293T cells expressing Flag-HA-SS18 or HA-DPF2.(B) Schematic representation of defined and newly-identified regions in mammalian SWI/SNF subunits used in representing inter-subunit crosslinks. Only one paralog of each subunit family is displayed.(C) Analysis of the distance between crosslinked residues in known 3D structures of mSWI/SNF complex subunit domains. Dashed line indicates the median distance calculated. Length of the BS3 crosslinker spacer is 11.4Å.(D) Structures of the Snf2 ATPase domain in nucleosome-bound (blue) and nucleosome-free (green) states. SMARCA4 crosslinks in dynamic regions are colored in purple and orange. Crosslinks in constant regions are colored in yellow.(E) Clustered distribution of the total crosslinks from mammalian BAF complex cross-linking mass spectrometry. Clustering indicates similarly strong correlations between SMARCC, SMARCD, and SMARCE subunits with ARID1, which bridges this module to the ATPases and their associated subunits (See also Figure 2B).(F) Silver stains of affinity-purified complexes from D. melanogaster S2 cells expressing D4-HA, BAP60-HA or mock control.(G) SWI/SNF subunit orthologs in S. cerevisiae, D. melanogaster and H. sapiens.(H) Clustered distribution of the total crosslinks from cross-linking mass spectrometry performed on D. melanogaster complexes.(I) Clustered distribution of the total crosslinks from cross-linking mass spectrometry performed on S. cerevisiae complexes.(J) Schematic representation of defined and newly-identified regions in D. melanogaster BAP subunits used in representing inter-subunit crosslinks.(K) Schematic representation of defined and newly-identified regions in S. cerevisiae SWI/SNF subunits used in representing inter-subunit crosslinks.(L) Matrix heatmap of the total crosslinks from S. cerevisiae SWI/SNF complex cross-linking mass spectrometry (Sen et al., 2017Sen P. Luo J. Hada A. Hailu S.G. Dechassa M.L. Persinger J. Brahma S. Paul S. Ranish J. Bartholomew B. Loss of Snf5 Induces Formation of an Aberrant SWI/SNF Complex.Cell Rep. 2017; 18: 2135-2147Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar).Individual subunits are divided into domains (per K) and ordered according to Figure 2D.(M) Matrix heatmap of the total crosslinks from D. melanogaster BAP complex cross-linking mass spectrometry performed as part of this study. Individual subunits are divided into domains (per J) and ordered according to Figure 2C.(N) Correlation analysis between D. melanogaster BAP and S. cerevisiae SWI/SNF subunit domain and region interactions from cross-linking mass spectrometry datasets.View Large Image Figure ViewerDownload Hi-res image Download (PPT) (A) Silver stains of affinity-purified complexes from mammalian HEK293T cells expressing Flag-HA-SS18 or HA-DPF2. (B) Schematic representation of defined and newly-identified regions in mammalian SWI/SNF subunits used in representing inter-subunit crosslinks. Only one paralog of each subunit family is displayed. (C) Analysis of the distance between crosslinked residues in known 3D structures of mSWI/SNF complex subunit domains. Dashed line indicates the median distance calculated. Length of the BS3 crosslinker spacer is 11.4Å. (D) Structures of the Snf2 ATPase domain in nucleosome-bound (blue) and nucleosome-free (green) states. SMARCA4 crosslinks in dynamic regions are colored in purple and orange. Crosslinks in constant regions are colored in yellow. (E) Clustered distribution of the total crosslinks from mammalian BAF complex cross-linking mass spectrometry. Clustering indicates similarly strong correlations between SMARCC, SMARCD, and SMARCE subunits with ARID1, which bridges this module to the ATPases and their associated subunits (See also Figure 2B). (F) Silver stains of affinity-purified complexes from D. melanogaster S2 cells expressing D4-HA, BAP60-HA or mock control. (G) SWI/SNF subunit orthologs in S. cerevisiae, D. melanogaster and H. sapiens. (H) Clustered distribution of the total crosslinks from cross-linking mass spectrometry performed on D. melanogaster complexes. (I) Clustered distribution of the total crosslinks from cross-linking mass spectrometry performed on S. cerevisiae complexes. (J) Schematic representation of defined and newly-identified regions in D. melanogaster BAP subunits used in representing inter-subunit crosslinks. (K) Schematic representation of defined and newly-identified regions in S. cerevisiae SWI/SNF subunits used in representing inter-subunit crosslinks. (L) Matrix heatmap of the total crosslinks from S. cerevisiae SWI/SNF complex cross-linking mass spectrometry (Sen et al., 2017Sen P. Luo J. Hada A. Hailu S.G. Dechassa M.L. Persinger J. Brahma S. Paul S. Ranish J. Bartholomew B. Loss of Snf5 Induces Formation of an Aberrant SWI/SNF Complex.Cell Rep. 2017; 18: 2135-2147Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). Individual subunits are divided into domains (per K) and ordered according to Figure 2D. (M) Matrix heatmap of the total crosslinks from D. melanogaster BAP complex cross-linking mass spectrometry performed as part of this study. Individual subunits are divided into domains (per J) and ordered according to Figure 2C. (N) Correlation analysis between D. melanogaster BAP and S. cerevisiae SWI/SNF subunit domain and region interactions from cross-linking mass spectrometry datasets. To elucidate potential crosslinking preferences between subunits, we performed Louvain two-nearest-neighbor analysis, where nodes are subunits (or paralog families) and edges are drawn between the top two crosslinking partners for each subunit based on the number of BAF crosslinks (Blondel et al., 2008Blondel V.D. Guillaume J.-L. Lambiotte R. Lefebvre E. Fast unfolding of communities in large networks.J. Stat. Mech. 2008; 2008: P10008Crossref Scopus (11160) Google Scholar). This clustering revealed three distinct network modules: a catalytic module containing the SMARCA ATPase subunit, β-actin, and ACTL6A, an associated module containing SMARCB1 and BCL7, and a module containing SMARCC, SMARCD, SMARCE1, and ARID1 (Figure 2B), recapitulating our inferred assembly of components. In addition, correlation analyses of total inter-subunit crosslinks for each subunit revealed similar results (Figure S2E). Arthropods represent a parallel evolutionary branch to metazoans that retain at least two classes of SWI/SNF complexes, namely BAP (BAF in mammals) and PBAP (PBAF in mammals). Hence, we isolated BAP complexes from D. melanogaster S2 cells using insect orthologs of DPF2 (D4) and SMARCD1 (BAP60) as baits and performed cross-linking mass spectrometry (Figures S2F and S2G). Similar to mammalian complexes, the ATPase module clustered with BAP55 (ACTL6A ortholog) and ACT2 (β-actin ortholog), and moira (mor) (SMARCC ortholog) formed a tight network with BAP60, BAP111 (SMARCE1 ortholog), and Osa (ARID1 ortholog), whereas Snr1 (SMARCB1 ortholog) and D4 separated as a distinct module (Figures 2C and S2H; Table S5). These cross-linking mass spectrometry results suggest conserved modularity for at least two complex modules: the BAF ATPase module and the “core module” that forms around SMARCC or mor subunits. Finally, using a recently published S. cerevisiae SWI/SNF cross-linking mass spectrometry dataset (Sen et al., 2017Sen P. Luo J. Hada A. Hailu S.G. Dechassa M.L. Persinger J. Brahma S. Paul S. Ranish J. Bartholomew B. Loss of Snf5 Induces Formation of an Aberrant SWI/SNF Complex.Cell Rep. 2017; 18: 2135-2147Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar), we found similar clustering of the majority of both core and ATPase subunits, with the SNF2-centered ATPase module containing ARP7, ARP9 (potential orthologs of ACTL6A), and RTT102. SWI3 (SMARCC ortholog) and SNF12 (SMARCD ortholog), along with yeast-specific SNF6 and SWP82, form the core module, and SWI1 (ARID1 ortholog) and SNF5 (SMARCB1 ortholog) subunits cluster and bridge the core and ATPase modules (Figures 2D and S2I–S2L). Using correlation analyses of crosslinks within individual subunit regions and domains across mammalian, fly, and yeast complexes, we found that the most highly conserved interactions were between regions of the BAF core, OSA or ARID1, and ATPase modules (Figures 2E, 2F, S2M, and S2N). Taken together, SWI/SNF complexes retain specific modular organization across evolutionarily distant branches of life, indicating functional conservation of subunit architecture. Complex purifications (Figures 1B and 1D) coupled with these cross-linking mass spectrometry analyses suggested the presence of an early subcomplex containing SMARCD and SMARCC followed by SMARCE1 and SMARCB1 subunits (Figure 3A). Indeed, SMARCC1 purifications showed enrichment of the same subcomplex module (Figure 3B; Table S2). Similar results were obtained from SMARCB1, SMARCE1, and SMARCD2 purifications (Figures S3A–S3I; Table S2) using both mass spectrometry and fluorometric approaches and demonstrated SMARCB1 association with the BAF core module of cBAF and PBAF (Figures S3A–S3G). Of note, ncBAF-specific BRD9 and GLTSCR1 and GLTSCR1L components were completely absent in these three purifications, further suggesting that these subunits mark complexes of unique composition and lack several ubiquitously expressed, highly conserved subunits.Figure S3Purification and Mass Spectrometry Analyses of the BAF Core Module, Related to Figure 3Show full caption(A) Native HA-SMARCB1 BAF complexes purified from WT HEK293T cells and subjected to glycerol gradient centrifugation. Collected fractions were SDS-PAGE separated and silver stained.(B) Graphical representation of peptide relative abundance in each density gradient fraction identified by mass spectrometry analysis. Total spectral counts from fraction of highest peptide abundance for each subunit are indicated.(C) Clustering heatmap of HA-SMARCB1 density gradient mass spec fractions displayed as Z-scores.(D) Native HA-SMARCB1 BAF complexes were prepared as in (A) but each fraction was labeled using IRDye 680RD NHS ester.(E) IRDye 680RD detection performed on Fractions 9 and 12 from (A). Identified proteins are labeled.(F) Clustering heatmap of HA-SMARCB1 density gradient IRDye 680RD quantification displayed as a Z-score.(G) Graphical representation of IRDye 680RD quantification and peptide relative abundance in each density gradient fraction from two independent biological replicates of data displayed in (A) and (D).(H) Native HA-SMARCE1 BAF complexes purified from WT HEK293T cells and subjected to glycerol gradient centrifugation; collected fractions were SDS-PAGE separated and silver stained (left). Clustering heatmap and spectral counts of HA-SMARCE1 density gradient mass spec fractions (right).(I) Native HA-SMARCD2 BAF complexes purified from WT HEK293T cells and subjected to glycerol gradient centrifugation; collected fractions were SDS-PAGE separated and silver stained (left). Clustering heatmap and spectral counts of HA-SMARCD2 density gradient mass spec fractions (right).(J) HEK293T nuclear extracts were immunodepleted using indicated antibodies. Input, IP and flow through fractions were loaded on to SDS-PAGE and analyzed using WB with indicated antibodies.(K) Representative colloidal blue near infra-red detection of fractions 12-15 from DPF2-purified BAF complexes. Identified proteins are labeled and their approximated stoichiometry relative to DPF2 bait are indicated in parentheses.(L) Evolutionary conservation of the SMARCC subunits. Conserved domains and regions are indicated.(M) Co-IP/immunoblot analysis of BAF core module WT and subunit KO cells. Antibodies used for detection are indicated.(N) Native HA-SMARCB1 BAF complexes were purified from ΔSMARCD 293T cells and subjected to glycerol gradient centrifugation, collected fractions were SDS-PAGE separated and silver stained (left).(O) Silver stain analysis of Fraction 8 of the HA-SMARCB1 gradient in WT HEK293T cells. Subunits are labeled.(P) Native HA-SMARCD1 BAF complexes were purified from ΔSMARCB1 cells and were subjected to glycerol gradient centrifugation. Collected fractions were SDS-PAGE separated and silver stained (left). Clustered heatmap and spectral counts of the mass spec analysis performed on selected pulled fractions (right).(Q) Samples from HA-SMARCD1 gradient in Figure 3G were PAGE-separated and silver stained (short development time).View Large Image Figure ViewerDownload Hi-res image Download (PPT) (A) Native HA-SMARCB1 BAF complexes purified from WT HEK293T cells and subjected to glycerol gradient centrifugation. Collected fractions were SDS-PAGE separated and silver stained. (B) Graphical representation of peptide relative abundance in each density gradient fraction identified by mass spectrometry analysis. Total spectral counts from fraction of highest peptide abundance for each subunit are indicated. (C) Clustering heatmap of HA-SMARCB1 density gradient mass spec fractions displayed as Z-scores. (D) Native HA-SMARCB1 BAF complexes were prepared as in (A) but each fraction was labeled us" @default.
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• W2895850607 title "Modular Organization and Assembly of SWI/SNF Family Chromatin Remodeling Complexes" @default.
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