Diverse Roles and Interactions of the SWI/SNF Chromatin Remodeling Complex Revealed Using Global Approaches
[['Chromosomes undergo a wide variety of dynamic processes including transcription , replication , repair and packaging .', 'Each of these activities requires the recruitment and congregation of a particular set of factors and chromosomal elements .', 'For example visualization of nascent mRNA in HeLa cells has led to a model of transcription units being clustered into “factories” thereby facilitating optimal engagement of RNA Polymerase II ( Pol II ) and coordination with other crucial holoenzyme complexes [1]–[3] .', 'In addition to RNA Pol II and transcription factors , transcriptional assemblages include proteins critical to regulating chromatin .', 'The accessibility of nuclear proteins to DNA is often controlled by ATP-dependent chromatin remodeling complexes , which are thought to play a role in a number of different cellular transactions by reshaping the epigenetic landscape .', 'The SWI/SNF ( switch/sucrose nonfermentable ) chromatin remodeling proteins were first discovered in Saccharomyces cerevisiae as components of a 2 MDa complex that repositions nucleosomes for vital tasks such as transcriptional control , DNA repair , recombination and chromosome segregation [4] , [5] .', 'Mammalian SWI/SNF is comprised of approximately ten subunits and the combinations of these subunits , some of which have multiple isoforms , enable multiple varieties of SWI/SNF complexes to exist both within a given cell and across cell types [6] .', 'Among these subunits either of the two ATPases , Brg1 or Brm , is sufficient to remodel nucleosome arrays in vitro , however maximal nucleosome remodeling activity is achieved when the SWI/SNF subunits BAF155 , BAF170 and Ini1 are present in a 2∶1 stoichiometry relative to Brg1 [7] .', 'Whereas the ATPases have an obvious catalytic function , the roles of the other SWI/SNF subunits are largely obscure .', 'Several reports indicate that BAF155 and BAF170 provide scaffolding functions for other SWI/SNF subunits as well as regulating their protein levels [8] , [9] .', 'SWI/SNF also contains β-actin and the actin-related protein BAF53 , suggesting a possible bridge to nuclear organization or signal transduction , e . g . through phosphatidylinositol signaling [10] , [11] .', 'Phosphatidylinositol 4 , 5-bisphosphate has been shown to bind to Brg1 and promote binding to actin filaments [12] .', 'Mutations resulting in loss of Ini1 function are associated with rare but aggressive pediatric cancers [13] , [14] .', 'The SWI/SNF subunits Brg1 [15] and ARID1A [16]–[18] are likewise thought to have tumor suppressor roles based on mutations recovered from other tumor types .', 'Curiously , Ini1 alone has a unique and largely undefined role in HIV-1 infection that includes binding of Ini1 to HIV-1 integrase and the cytoplasmic export of Ini1 and its incorporation into HIV-1 particles [19]–[21] .', 'The role of SWI/SNF components in cancer and tumor suppression is poorly understood despite extensive study .', 'Detailed investigations of individual loci have implicated SWI/SNF in various transcriptional pathways including the cell cycle and p53 signaling [22] , insulin signaling [23] , and TGFβ signaling [24] , as well as signaling through several different nuclear hormone receptors [25] .', 'Although in vitro experiments and single-gene studies have been informative and have laid the foundation for understanding chromatin remodeling , a global analysis of targets of SWI/SNF is expected to yield a more extensive view into the biological roles of SWI/SNF components and their involvement in human disease .', 'In this study we present two complementary global analyses of SWI/SNF subunits to provide a more systematic view of SWI/SNF functions .', 'First we performed ChIP-Seq with the ubiquitous SWI/SNF components Ini1 , BAF155 , BAF170 as well as the Brg1 ATPase .', 'Second , in a parallel set of studies we performed mass spectrometry identification of proteins that co-immunoprecipitate with SWI/SNF components .', 'Using our ChIP-Seq results the resulting chromosomal locations were integrated with published annotations to yield a more complete understanding of SWI/SNF on a genome-wide scale .', 'We find SWI/SNF components frequently occupy transcription start sites ( TSSs ) , enhancers , CTCF regions and many regions occupied by Pol II .', 'Further analyses of the SWI/SNF regions we identified by ChIP-Seq reveals that SWI/SNF factors target genes and signaling pathways involved in cell proliferation and cancer .', 'Our investigation of SWI/SNF protein interactions detected not only the expected co-occurrences of individual SWI/SNF factors with each other but also with cellular components such as nuclear matrix proteins , key transcription factors and centromere proteins implying a ubiquitous role in gene regulation and nuclear function .', 'We find an overrepresentation of both SWI/SNF-associated chromosomal regions and proteins in cell cycle and chromosome organization .', 'Collectively our results suggest that SWI/SNF is at the nexus of multiple signal transduction pathways , essential chromosomal functions and nuclear organization .'], ['We identified the targets of four SWI/SNF components , Ini1 ( SMARCB1 ) , Brg1 ( SMARCA4 ) , BAF155 ( SMARCC1 ) and BAF170 ( SMARCC2 ) , using ChIP-Seq .', 'Chromatin complexes were isolated from HeLa S3 nuclei following independent immunoprecipitations with antibodies for each factor .', 'Each of these antibodies was characterized by both immunoblot and mass spectrometry analyses ( see Materials and Methods ) .', 'Reads that mapped uniquely to the genome were retained ( 29–33 million reads per data set; Table', '1 ) and significant binding regions were identified using the PeakSeq program with q-value<0 . 05 [26] .', 'The peaks were compared to a similarly-sized data set of uniquely mapped ChIP DNA reads obtained from control immunoprecipitation experiments using normal IgG ( i . e . a control serum that is not directed to any known antigens ) .', 'Using this approach we identified many Ini1- , Brg1- , BAF155- and BAF170-associated regions ( Table 1 ) .', 'The majority of SWI/SNF binding occurs near ( ±2 . 5 kb ) protein-coding genes , a distribution that is significant relative to a random target list ( p<1×10−16; Genome Structure Correction ( GSC ) test [27]; see Materials and Methods ) .', 'Several examples of SWI/SNF positioning relative to genic regions are shown in Figure 1 and Figure S1 .', 'In order to further examine SWI/SNF locations with respect to gene-rich and gene-poor regions we obtained a set of histone H3K27me3 domains that were identified in HeLa cells ( Table S1; [28] ) because this chromatin mark often occurs in gene-poor and repressed ( i . e . heterochromatin ) regions .', 'Although most SWI/SNF-binding occurs outside H3K27me3 domains , we observed that SWI/SNF is occasionally found in heterochromatin regions , as shown in Figure 2 .', 'In this example a 7 . 5 Mb heterochromatin region on Chr16 contains a single gene , the neuronal cadherin CDH8 , that is repressed and lacks RNA Pol II , however several SWI/SNF binding regions are found nearby .', 'We have performed considerable analyses of the targets for the individual SWI/SNF factors , particularly with respect to elements representing several major classes of genomic features including promoters ( Ensembl protein-coding genes ) , RNA Pol II sites [26] , CTCF sites [28] , and predicted enhancers [29] .', 'All of these features were identified in HeLa cells ( Table 1 , Tables S1 and S2; see Materials and Methods ) .', 'In comparisons between the individual target lists for Ini1 , Brg1 , BAF155 and BAF170 with promoters , RNA Pol II sites , CTCF sites and enhancers we found that each SWI/SNF factor is significantly overrepresented for each of these major classes of genomic elements ( p<1×10−16 , GSC test , see Materials and Methods ) .', 'To arrive at a single unified and more conservative list of SWI/SNF locations , we first took the union of all regions for Ini1 , BAF155 , BAF170 and Brg1 , resulting in 69 , 658 SWI/SNF regions .', 'We then trimmed this list to a high-confidence set of 49 , 555 sites by eliminating those regions where either only a single SWI/SNF subunit was present or that those regions that did not co-occur with either promoters , RNA Pol II sites , CTCF sites or predicted enhancers .', 'We used this list of 49 , 555 SWI/SNF regions for all subsequent analyses unless otherwise noted ( Table S3 ) .', 'The four major classes of genomic features mentioned above were overrepresented in both the 69 , 658 SWI/SNF regions as well as the more conservative list 49 , 555 SWI/SNF regions ( p<1×10−16 , GSC test ) .', 'We next examined the configurations of our 49 , 555 SWI/SNF regions ( Figure 3A and Table 2 ) .', 'Ini1 , BAF155 and BAF170 have been described as forming a ‘core’ based on their ability to stimulate remodeling activity of the Brg1 ATPase in reconstitution experiments [7] .', 'Among our data 30 , 310 regions ( 61% ) have two or more SWI/SNF components and 9 , 760 regions ( 20% ) contain the core of Ini1 , BAF155 and BAF170; for the purposes of this study we call this the ‘core set’ .', 'Among putative complexes comprised of two or more SWI/SNF subunits , we observed BAF155 was the subunit most common to each binding region .', 'Only 770 SWI/SNF subunit co-occurrences were recovered that lacked BAF155 as compared to 6 , 467 for BAF170 and 14 , 824 for Ini1 .', 'This finding is consistent with several previous studies showing that BAF155 is important for SWI/SNF complex stability [8] , [9] .', 'BAF155 may increase the stability of the complex during assembly , or BAF155 may be easier to detect by ChIP .', 'One of the primary functions of chromatin remodeling complexes is to assist in gene regulation .', 'Among the SWI/SNF regions in our high-confidence union set of 49 , 555 sites , 29% correspond to the 5′ ends of protein-coding Ensembl genes , 40% correspond to Pol II sites , 17% correspond to CTCF sites and 43% correspond to predicted enhancer regions ( Figure 3B; Table 3 ) .', 'The various combinations of these four elements account for a total of 90% of the SWI/SNF union regions; 4 , 800 ( 10% ) of the SWI/SNF regions are unclassified using the above elements .', 'Similar trends were observed for the 9 , 760 SWI/SNF “core” regions where Ini1 , BAF155 and BAF170 all co-occur ( Table 3 ) .', 'None of these four particular SWI/SNF subunits or any combinations thereof exhibited a differential preference for one type of element ( Table S4 ) .', 'There are some differences between the SWI/SNF core and union regions .', 'The SWI/SNF core regions are overrepresented for RNA Pol II ( p<9 . 9×10−16; hypergeometric test ) and 5′ ends ( p<6 . 5×10−211; hypergeometric test ) relative to all of the SWI/SNF high-confidence union regions; however the SWI/SNF high-confidence union regions are overrepresented for enhancer regions relative to the Ini1-BAF155-BAF170 core ( p<2 . 4×10−67; hypergeometric test ) .', 'Neither the SWI/SNF core nor the high-confidence union regions were over- or underrepresented for CTCF sites relative to each other ( p>0 . 05; hypergeometric test ) .', 'Enhancers are often characterized by long-range interactions .', 'We examined the locations of SWI/SNF binding regions in the 150 kb CIITA region where numerous chromosomal looping interactions have been mapped at high resolution in HeLa cells using 3C ( Chromosome Conformation Capture ) .', 'Brg1 has been previously mapped at several sites in this locus in these cells [30] .', 'Superimposition of these 3C data on our SWI/SNF ChIP-Seq data ( Figure', '4 ) reveals that all six of the 3C interacting regions in the CIITA locus ( −50 kb , −16 kb , −8 kb , pIV , +40 kb and +59 kb ) are bound by SWI/SNF components .', 'Moreover certain individual SWI/SNF component binding regions that appeared initially as orphans may now be seen as part of a complete complex when joined with a distal element .', 'For example Ini1 at pIV when joined with BAF155 and BAF170 regions at the −16 kb element forms a SWI/SNF core .', 'Thus in the CTIIA locus SWI/SNF regions are often associated with 3C regions and many of the regions bound by individual factors may in fact be part of entire SWI/SNF complexes inside the nucleus .', 'Overall our ChIP-Seq results are summarized in Table 1 , Table 2 , Table 3 , and Figure 3 and indicate that SWI/SNF likely contributes to gene regulation through many different avenues in light of its binding to promoters , enhancers and CTCF sites .', 'Furthermore SWI/SNF may facilitate looping interactions among these various elements as it has been shown in vitro that SWI/SNF can interact simultaneously with multiple DNA sites and generate loops between them [31] .', 'Interestingly we found a slightly higher presence of the SWI/SNF core at TSSs and with Pol II than the SWI/SNF union regions with these elements ( Table 3 ) .', 'Thus a complete core of Ini1 , BAF155 and BAF170 may be required for effective promoter function whereas only a subset of these factors may be required for enhancer function .', "Alternatively a full SWI/SNF core may be more difficult to recover from a single enhancer element as compared to a more compact promoter region due to the enhancer's presumed interaction with many different distal elements .", 'As detailed above SWI/SNF regions are enriched for Pol II .', 'To explore the prevalence of SWI/SNF with transcriptional machinery we asked whether the converse would also be true , namely if regions bound by RNA polymerases are enriched for SWI/SNF .', 'Indeed Pol II regions are enriched for SWI/SNF binding regions ( p<1×10−16 , GSC test ) .', 'Although Pol II overlaps extensively with SWI/SNF it differs from SWI/SNF in its concordance with CTCF and enhancer regions ( Table S5 ) .', 'Pol II regions lacking SWI/SNF show a five-fold decrease in CTCF sites and a two-fold decrease in enhancer regions as compared to those Pol II regions containing SWI/SNF .', 'We further compared our SWI/SNF regions with binding intervals identified for RNA polymerase III ( Pol III ) , which in addition to transcribing tRNA and other non-protein coding RNAs has an emerging role in the formation of boundary elements [32] , [33] .', 'Pol III localization data were obtained from published ChIP-Seq studies using HeLa cells ( [34] , [35]; Tables S1 and S6 ) and constitute 478 known and novel Pol III-associated regions .', 'Pol II is often associated with Pol III ( Table 4; reviewed in [32] .', 'Therefore we examined whether SWI/SNF was associated with Pol III binding regions independently of Pol II .', 'Of the 478 Pol III regions , 253 Pol III intervals lack Pol II and among these 39% ( 98/253 ) contain one or more SWI/SNF components .', 'These results suggest that SWI/SNF association with Pol III can occur independently of Pol II .', 'Overall 65% ( 309/478 ) of Pol III regions and 84% ( 19 , 541/23 , 320 ) of Pol II regions have at least one SWI/SNF factor associated with them .', 'The Ini1-BAF155-BAF170 core is found at 41% ( 195/478 ) of Pol III regions and 52% ( 12 , 079/23 , 320 ) of Pol II regions .', 'From the colocalizations of SWI/SNF , Pol II and Pol III we see that there is substantial overlap among these factors yet each of these factors also has distinct characteristics .', 'SWI/SNF is known to act as both an activator and repressor of transcription [36] .', 'We examined the locations of four SWI/SNF components relative to transcribed regions in HeLa S3 cells using the RNA-Seq data of Morin et al . [37] , Ini1 , Brg1 , BAF155 and/or BAF170 are present at or near the 5′ ends ( ±2 . 5 kb ) of 71 to 92% of active protein-coding genes .', 'As noted above , SWI/SNF occupancy in promoters is similar to that of Pol II and each of the factors is individually enriched in promoter regions ( p<1×10−16 , GSC test ) .', 'Although the majority of Ini1 , Brg1 , BAF155 and BAF170 target genes are expressed , an appreciable fraction of gene targets have little or no detectable mRNA in HeLa cells .', 'A closer examination of the union regions where a SWI/SNF component is located in the promoter of an inactive gene reveals that 58% ( 2 , 063/3 , 565 ) of these promoters are co-associated with Pol II suggesting transcriptional stalling ( reviewed in [38] , [39] .', 'Considering that SWI/SNF components bind near many expressed regions and that SWI/SNF factors occur in a multitude of configurations ( Figure 3 and Table S3 ) , we examined transcript expression levels for all possible combinations of Ini1 , Brg1 , BAF155 and BAF170 occurrences .', 'Using the RNA-Seq data of Morin et al . [37] , we examined transcript expression levels corresponding to each of these configurations ( Figure 5 ) .', 'We see that the highest levels of transcription are associated with the following four configurations:', '1 ) the complete core of Ini1 , BAF155 and BAF170;', '2 ) the complete core plus Brg1;', '3 ) Ini1 and BAF155 only and', '4 ) Brg1 , BAF155 and BAF170 .', 'Although BAF155 is the subunit that is common to all of the configurations associated with the highest levels of transcription , it does not appear to be the sole driver of transcriptional activity .', 'Compared against each other , all three components of the core complex taken individually have nearly indistinguishable profiles .', 'Despite the involvement of Brg1 in two of the four configurations with the highest expression levels , most other configurations involving Brg1 are restricted to profiles associated with the lowest expression levels .', 'One inference from these data is that certain combinations of SWI/SNF subunits are likely synergistic in promoting transcription whereas other combinations may be inhibitory or unstable .', 'We also examined SWI/SNF occurrences relative to 48 , 403 non-canonical small RNAs from HeLa cells ( ≤156 bp; Table S1 ) where most ( 83%; p<1×10−16 , GSC test ) of these small RNAs are near protein-coding genes [40] .', 'Approximately one third ( 30% ) of this entire small RNA set is within 1 kb of a target from our high-confidence union list of 49 , 555 SWI/SNF regions .', 'The incidence of small RNA-SWI/SNF co-associated regions was nearly equivalent in protein-coding genes and intergenic regions .', 'From this we surmise that SWI/SNF may contribute to gene regulation of a variety of transcripts , many of which are newly annotated and of unknown function .', 'Prior research has shown that a variety of signaling cascades are linked to SWI/SNF [25] .', 'To gain further insights into potential actions of SWI/SNF components we examined the underlying Gene Ontology ( GO ) and Kyoto Encyclopedia of Genes and Genomes ( KEGG ) designations of their gene targets to determine significantly overrepresented annotations and pathways ( Table 5 and Table S8 ) .', 'SWI/SNF gene targets were associated with ‘Pathways in cancer’ and several specific cancers types , e . g . chronic myeloid leukemia and pancreatic cancer .', 'A number of signaling pathways and cellular processes that are “hallmarks of cancer” [41] were also overrepresented among the gene targets of Ini1 , Brg1 , BAF155 and BAF170 .', 'These include the Wnt , ErbB , p53 , MAPK , and insulin signaling pathways , and processes endemic to oncogenesis and cancer progression such as DNA repair , the cell cycle and apoptosis .', 'From these analyses we surmise the recruitment of SWI/SNF components is likely to influence the molecular basis of cancer through several potential mechanisms .', 'The SWI/SNF-enriched pathways are highly interconnected .', 'Using the 49 , 555 SWI/SNF targets we identified a total of 24 KEGG signaling or biochemical pathways ( Figure 6 , yellow nodes ) .', 'Interestingly , these pathways partition into three groups ( Figure 6A–6C ) .', 'Two of the groups ( Figure 6A and 6B ) comprise sets of pathways exhibiting at most one degree of separation , e . g . ‘inositol phosphate metabolism’ and ‘amino sugar and nucleotide sugar metabolism’ .', 'The third group ( Figure 6C ) consists of three pathways that are unrelated to any other pathways in the KEGG database .', 'As displayed in Figure S2 directly related pathways such as ‘p53 signaling’ and the ‘cell cycle’ have shared components and many of the genes encoding these components are occupied by SWI/SNF factors .', 'Thus , our results demonstrate that SWI/SNF is involved in many closely related signaling pathways and cellular processes and may help serve to coordinate expression of genes involved in these processes .', 'The genomic binding data demonstrates that SWI/SNF localization is coupled with a broad range of functional elements , suggesting that SWI/SNF may also be found with a broad range of associated proteins .', "To further examine the scope of SWI/SNF's roles in the nucleus we analyzed proteins associated with SWI/SNF subunits using co-immunoprecipitation followed by mass spectrometry .", 'The SWI/SNF components Ini1 , BAF155 , BAF170 , Brg1 , Brm and ARID1A were immunoprecipitated from HeLa S3 nuclei , the resulting proteins were gel-separated and peptides were generated for analysis by mass spectrometry ( See Materials and Methods; Table S9 ) .', 'In addition to the factor-specific antibodies , parallel immunoprecipitations were performed using non-specific IgG antibodies .', 'Proteins identified in these “control IgG” immunoprecipitations were excluded as potential SWI/SNF co-purifying factors .', 'We identified a total of 101 proteins that were specifically associated with at least one of the SWI/SNF components assayed ( Figure 7 , turquoise edges; Table S10 ) .', 'Of the non-SWI/SNF subunits detected , 5 of these interactions were found previously in HeLa cells ( e . g . estrogen receptor alpha [42] , and 96 were new to this study .', 'Interestingly one of the novel interactions we observed in HeLa cells , BAF155 with NUF2 , has been previously observed in yeast between the yeast homolog of BAF155 ( SWI3 ) and NUF2 [15] .', 'Using the 101 nodes that we identified as proteins co-purifying with SWI/SNF in our undirected approach we ascertained overrepresented GO categories ( Table 6 ) .', 'Several of these designations such as ‘cell cycle’ and ‘chromosome organization’ coincide with the categories obtained from GO and pathway analyses of SWI/SNF ChIP-Seq targets , suggesting the possibility of highly-interactive network structures .', 'Many of the proteins that were novel to this study reinforce and expand upon other published reports of SWI/SNF characterizations .', 'For example SWI/SNF components have been localized by immunofluorescence to mitotic kinetochores and spindle poles [43] , and Brg1-deficient mice show dissolution of pericentromeric heterochromatin domains [44] .', 'From our immunoprecipitations BAF155 and BAF170 were associated with a number of kinetochore and centrosomal proteins ( e . g . BUB1B , CENPE and NUF2 , Figure 8 , green circles ) .', 'The role of SWI/SNF in the maintenance of kinetochore and spindle function is unknown .', 'We detected a variety of transcription factor activators and repressors ( e . g . NFκB1 , NFκB2 , RelA , PML and NFX1 ) as well as DNA repair ( ERCC5 and RAD50 ) and cell cycle ( e . g . CCNB3 and CDCA2 ) proteins ( Figure S3 ) .', 'Some of the SWI/SNF interacting proteins themselves interact with one another .', 'For example we detected several different proteins integral to estrogen and insulin signaling ( Figure 7; Table S10 ) .', 'We also identified proteins associated with only one SWI/SNF factor; these may either be interactions with a specific SWI/SNF component or an inability to detect the protein in the immunoprecipitations .', 'We developed an expanded network of SWI/SNF associations by including proteins that were found by others to co-purify with SWI/SNF subunits ( Figure 7 , black edges ) .', 'Only those factors that showed a one-degree separation with a SWI/SNF component in HeLa cells are displayed and all interactions are annotated in Table S10 .', 'SWI/SNF interacting proteins are associated with numerous UniProt keywords ( Figure 8; [45] ) .', 'Overall these results suggest a role for SWI/SNF components in a wide array of nuclear processes and diseases .', 'Some of these processes may take place in nuclear substructures .', 'Higher order chromatin structure is facilitated by the nuclear lamina and tethering of genes to the nuclear periphery is one epigenetic mechanism of gene regulation [46] , [47] .', 'Intriguingly we and others have detected SWI/SNF components with various nuclear envelope-associated proteins ( Figure 7 and Table S10 ) including lamin A , EMD ( emerin ) and BAF/BANF1 ( Barrier to Autointegration Factor , which although similar in name is not a SWI/SNF subunit ) .', 'Two of the nuclear membrane proteins , SYNE1 and C14orf49 , that we isolated in association with BAF155 are part of LINC complexes that link the nucleoskeleton and cytoskeleton [48] , [49] .', 'Numerous studies point to a high degree of functional organization in cell nuclei [46] .', 'Emerging nuclear organization models would benefit greatly from a catalogue of processes and chromatin characteristics mapped to particular genomic elements .', 'For example , the nuclear lamins are thought to influence chromatin organization , DNA replication and transcription [47] , [50] .', 'Our immunoprecipitation results demonstrating that SWI/SNF components are associated with lamin A/C ( Figure 7 and Table S10 ) along with immunoprecipitation , immunolocalization and cell fractionation experiments from others demonstrating an association between SWI/SNF and nuclear lamina ( e . g . emerin Figure 7; [51] ) prompted us to investigate whether SWI/SNF and the lamins can be located to the same genomic sequences .', 'We isolated lamin A/C and lamin B ChIP DNA from HeLa S3 nuclei and performed ChIP-chip on tiling arrays covering the ENCODE pilot regions ( see Materials and Methods and Table S1 ) .', 'Most of the 1 , 770 lamin A/C regions mapped to H3K27me3 domains ( 76%; 1 , 337/1 , 770 ) whereas the 1 , 270 lamin B regions were less commonly associated with H3K27me3 ( 29%; 372/1 , 270 ) .', 'Comparing regions where signal was detectable for the SWI/SNF , lamin A/C and lamin B experiments revealed that SWI/SNF has a much higher overlap with lamin B than lamin A/C .', 'We found that 38% ( 297/784 ) of SWI/SNF sites are within 100 bp of a lamin B region whereas only 5% ( 41/784 ) of SWI/SNF sites are within 100 bp of a lamin A/C region ( Table 7 ) .', 'For both lamin types the colocalization with SWI/SNF regions is significant relative to random target lists ( lamin B , p<1×10−16; lamin A/C , p<1×10−15; GSC tests ) .', 'SWI/SNF-lamin B intersecting regions contained approximately the same proportion of CTCF sites in the ENCODE regions as did all SWI/SNF sites in the ENCODE regions ( p>0 . 05 hypergeometric test; Table 7 ) .', 'Enhancers are underrepresented in the SWI/SNF-lamin B regions relative to all SWI/SNF locations in the ENCODE regions ( p<1 . 9×10−36; hypergeometric test ) .', 'The SWI/SNF-lamin B regions are overrepresented for Pol II ( p<2 . 9×10−39; hypergeometric test ) and 5′ ends ( p<7 . 3×10−37; hypergeometric test ) relative to all SWI/SNF locations in the ENCODE regions .', 'In crosslinked chromatin SWI/SNF is detected primarily with lamin B , but as noted from the above mass spectrometry experiments , in solubilized , non-cross-linked cells SWI/SNF is detected with lamin A/C and not lamin B ( Figure 1 and Figure 7 ) .', 'We interpret these results to indicate that SWI/SNF , lamin A/C and lamin B co-associate in different nuclear contexts but are all part of a broader interacting network with specific sub-associations .', 'SWI/SNF and the lamins have each been implicated in DNA replication ( see above; [52] , [53] ) .', 'One of the proteins we detected as associated with SWI/SNF is the replication protein RepA and another regulator of DNA replication , geminin , has been found to co-purify with SWI/SNF in HeLa cells ( Figure 7 , red circles; [54] ) .', 'We investigated whether there might be a relationship among SWI/SNF , lamins and DNA replication origins .', 'We obtained a set of 282 DNA replication origins identified in HeLa cells for the ENCODE regions ( [55]; Table S1 ) .', 'Of these 282 replication origins , 90 ( 32% ) occur within 100 bp of a SWI/SNF region ( p<1×10−16 , GSC test ) , 86 ( 31% ) occur at the 5′ ends of protein-coding genes and 151 ( 54% ) occur within 100 bp of a lamin B region .', 'In contrast to lamin B , only 17% ( 48/282 ) of the replication origins were near a lamin A/C region .', 'These results are consistent with nuclear staining patterns observed in mouse 3T3 cells showing colocalization between lamin B and sites of DNA replication whereas the same colocalization patterns were not observed for replication foci and lamin A [52] .', 'Of the 86 replication origins in promoter regions , 88% ( 76/86 ) intersected a lamin B region and most ( 78% or 67/86 ) were within a 100 bp of a SWI/SNF region .', 'These data indicate that SWI/SNF components are located near many DNA replication origins , particularly those located in promoter regions .', 'The coincidence of chromatin remodeling factors , promoters , lamins and replication origins at the same subset of genomic regions suggests that these loci may be particularly favorable for the formation of both DNA and RNA polymerase assembly and chromatin tethering .', 'As shown in Figure 1 the interplay among these elements as well as with Pol II , CTCF and heterochromatin regions is complex and interwoven , such that each may share many different supporting and counteracting roles .'], ['SWI/SNF performs a crucial function in gene regulation and chromosome organization by directly altering contacts between nucleosomes and DNA .', 'In the work presented here we undertook a two-pronged approach ( ChIP-Seq and IP-mass spectrometry ) to move towards a more thorough understanding of these functions .', 'Our ChIP-Seq analyses demonstrate that SWI/SNF components overlap extensively with important regions that require tight control of the dynamics of nucleosome occupancy such as promoters , enhancers and CTCF sites .', 'Not only does the SWI/SNF complex change the accessibility of DNA but it also acts in concert with an extensive host of cooperating factors , thereby facilitating combinatorial control among various genomic elements .', "In addition to our ChIP-Seq results , the diversity and number of proteins that co-purify with SWI/SNF as identified in our mass spectrometry experiments further supports SWI/SNF's involvement with a variety of functionally distinct complexes .", 'RNA polymerases II and III are extensively colocalized with SWI/SNF components .', 'Studies of transcription in HeLa cells have estimated that the number of active RNA II polymerases exceeds the number of transcriptionally active sites by at least one order of magnitude , leading to the proposal of “transcription factories” [1]–[3] .', 'The number of RNA Pol II transcription factories in HeLa cells has been estimated between 5 , 000 and 8 , 000 where each factory can be typified by several looped loci , their resulting transcripts and distal elements such as enhancers .', 'We infer that SWI/SNF regions are prevalent in transcriptional assemblages and their associated regulatory loops , given that >90% of our high-confidence union targets are associated with genic or regulatory regions and that 65% of Pol III and 84% of Pol II regions colocalize with at least one SWI/SNF factor ( Table 4 , Tables S5 and S6 ) .', 'Interestingly we observed that SWI/SNF components often occur independently of each other and in various configurations across the genome , and similarly our mass spectrometry data point to heterogeneity of SWI/SNF complexes .', 'We speculate that several mechanisms may underlie these various configurations and their associated genomic features , including', '1 ) synergism or antagonism of the individual SWI/SNF factors in influencing expression ( e . g . Figure 5 ) ;', '2 ) failure to detect individual subunits due to epitope masking as a consequence of variation with local environments;', '3 ) the capture of incomplete complexes that may in fact be completed upon superposition of genome-wide 3C data once such data become available ( e . g . Figure 4 ) ;', '4 ) the existence of SWI/SNF sub-complexes that deviate from the conventional composition of SWI/SNF assemblies ( e . g . [56] ) or', '5 ) the capture of intermediates in a multistep assembly or remodeling process .', 'This last view is consistent with a model of stochastic assembly that may 
['Introduction', 'Results', 'Discussion', 'Materials and Methods']
['A systems understanding of nuclear organization and events is critical for determining how cells divide , differentiate , and respond to stimuli and for identifying the causes of diseases .', 'Chromatin remodeling complexes such as SWI/SNF have been implicated in a wide variety of cellular processes including gene expression , nuclear organization , centromere function , and chromosomal stability , and mutations in SWI/SNF components have been linked to several types of cancer .', 'To better understand the biological processes in which chromatin remodeling proteins participate , we globally mapped binding regions for several components of the SWI/SNF complex throughout the human genome using ChIP-Seq .', 'SWI/SNF components were found to lie near regulatory elements integral to transcription ( e . g . 5′ ends , RNA Polymerases II and III , and enhancers ) as well as regions critical for chromosome organization ( e . g . CTCF , lamins , and DNA replication origins ) .', 'Interestingly we also find that certain configurations of SWI/SNF subunits are associated with transcripts that have higher levels of expression , whereas other configurations of SWI/SNF factors are associated with transcripts that have lower levels of expression .', 'To further elucidate the association of SWI/SNF subunits with each other as well as with other nuclear proteins , we also analyzed SWI/SNF immunoprecipitated complexes by mass spectrometry .', 'Individual SWI/SNF factors are associated with their own family members , as well as with cellular constituents such as nuclear matrix proteins , key transcription factors , and centromere components , implying a ubiquitous role in gene regulation and nuclear function .', 'We find an overrepresentation of both SWI/SNF-associated regions and proteins in cell cycle and chromosome organization .', 'Taken together the results from our ChIP and immunoprecipitation experiments suggest that SWI/SNF facilitates gene regulation and genome function more broadly and through a greater diversity of interactions than previously appreciated .']
['Genetic information and programming are not entirely contained in DNA sequence but are also governed by chromatin structure .', 'Gaining a greater understanding of chromatin remodeling complexes can bridge gaps between processes in the genome and the epigenome and can offer insights into diseases such as cancer .', 'We identified targets of the chromatin remodeling complex , SWI/SNF , on a genome-wide scale using ChIP-Seq .', 'We also identify proteins that co-purify with its various components via immunoprecipitation combined with mass spectrometry .', 'By integrating these newly-identified regions with a combination of novel and published data sources , we identify pathways and cellular compartments in which SWI/SNF plays a major role as well as discern general characteristics of SWI/SNF target sites .', 'Our parallel evaluations of multiple SWI/SNF factors indicate that these subunits are found in highly dynamic and combinatorial assemblies .', 'Our study presents the first genome-wide and unified view of multiple SWI/SNF components and also provides a valuable resource to the scientific community as an important data source to be integrated with future genomic and epigenomic studies .']
['cellular structures', 'chromosome structure and function', 'genome sequencing', 'epigenetics', 'cell nucleus', 'chromatin', 'chromosome biology', 'biology', 'systems biology', 'cell biology', 'genetics', 'genomics', 'molecular cell biology', 'genetics and genomics']