Dangerous liaisons: interplay between SWI/SNF, NuRD, and Polycomb in chromatin regulation and cancer

1Chromatin is fundamental to all processes involving the eukaryotic genome. The nucleosome—147 bp of DNA wrapped tightly in ∼1.7 left-handed superhelical turns around an octamer of histones H2A, H2B, H3, and H4—is the fundamental repeating unit of chromatin. The need to compact genomic DNA (∼2 m for the human genome) to fit into the cellular nucleus (with a diameter of only ∼10 µm) is often presented as the rationale for nucleosomes. However, the packing fraction of DNA within the nucleus of a somatic cell is typically only about 1%, leaving ample unoccupied space. Therefore, rather than solving a physical packaging problem, nucleosomes instead provide a functional organization of the genome, enabling regulation of its replication, repair, and transcription. In fact, the most pertinent consequence of packaging genomic DNA into chromatin is that nucleosomes can impede access of DNA-binding proteins, such as transcription factors. Consequently, chromatin remodeling constitutes a fundamental level of gene expression control.

2Central to chromatin organization, ATP-dependent chromatin remodeling enzymes (remodelers) are molecular motors dedicated to the assembly, positioning, or disruption of nucleosomes. By modulating the presentation of DNA, chromatin remodelers provide a fundamental level of gene expression control. In addition, chromatin state is regulated through a plethora of posttranslational modifications, in particular of the unstructured N-terminal histone tails that protrude from the nucleosome. These modifications, when present at specific residues on the histone N-terminal tails, can promote or antagonize the recruitment of regulatory proteins and may directly affect the compaction of the chromatin fiber. The local pattern of histone modifications is closely correlated with the transcriptional state of the associated gene or regulatory DNA element. For example, histone acetylation is generally associated with active chromatin irrespective of which residue is modified. In contrast, for histone methylation, the specific residue that is modified determines whether it is an active or a repressive mark. For example, while methylation of histone H3 at Lys4 (H3K4) by the MLL/COMPASS methyltransferases is associated with active transcription, trimethylation at Lys27 (H3K27me3) is central to gene silencing by the Polycomb system. Although remodelers and histone-modifying enzymes, such as members of the Polycomb group, catalyze fundamentally different biochemical reactions, they function in an integrated manner to determine chromatin state. Here, we review how remodelers and Polycombs modulate the chromatin template to regulate gene expression. We also examine the interplay between the SWI/SNF (switch/sucrose nonfermenting) and NuRD (nucleosome remodeling and deacetylase) remodelers with Polycombs in human cancer and how our expanding understanding of their biology is guiding the development of new cancer treatments.

3To understand how chromatin remodelers are powerful regulators of gene transcription, it is first necessary to understand their mechanisms of action. A chromatin remodeling reaction can have a variety of different outcomes. Through a sliding mechanism, a remodeler can move a nucleosome along the DNA template (Fig. 1A). Remodelers can generate a poorly understood remodeled state, in which the DNA becomes more accessible, but the histone octamer does not translocate to a new position. The action of remodelers can also result in a partial disruption of the nucleosome structure (e.g., through the eviction of a histone H2A/H2B dimer), while some remodelers mediate the exchange between histone variants. Finally, remodeling can lead to the complete eviction of the histone octamer. Whereas there are compelling examples of each of these mechanisms, their relative importance in vivo remains unclear. There are four major families of remodelers named after their central ATPase: SWI/SNF, INO80, ISWI, and CHD (Fig. 1B;; ). Remodelers are further defined by unique sets of associated proteins that can modulate their activity or recruitment to chromatin. The various remodelers perform a wide-range of mostly nonredundant functions in the maintenance, transmission, and expression of eukaryotic genomes. For example, CHD1 and the ISWI class ACF remodelers mediate the formation of regular nucleosomal arrays, whereas SWI/SNF mediates their local disruption. INO80 class remodelers catalyze the exchange between the canonical histone H2A and the variant H2A.Z in nucleosomes.

4The basic action of the ATPase in different remodeling complexes appears to be largely similar. Here, we highlight the salient aspects of our current understanding of remodeler function, in particular those relevant for SWI/SNF and NuRD. All remodelers contain a single motor subunit that belongs to the superfamily of ATP-dependent DNA and RNA translocases and helicases. The ATPase domain within the catalytic subunit is split into two domains with homology to the ATPase domain of the Escherichia coli RecA DNA-binding protein, referred to as lobes 1 and 2. The catalytic subunits of remodelers contain class-specific domains that can modulate their activity or mediate binding to DNA or histones (Fig. 1B). The noncatalytic subunits of remodeler complexes provide a plethora of additional functionalities, including regulation of the ATPase, providing contacts with DNA, histones, histone chaperones or sequence-specific transcription factors. A large body of studies on the mechanisms and structures of remodelers engaged with nucleosomes suggest a common mode of action. Fundamental to chromatin remodeling is the ATP-dependent translocation of DNA along the histone core of the nucleosome. Studies on classic translocases revealed that they move along one of the DNA strands, named the tracking strand, while the other strand is referred to as the guide strand (Fig. 2A). The ATPases of SWI/SNF, ISWI, and CHD1 all bind to the nucleosomal DNA at superhelical position 2 (SHL + 2), which is located two helical turns away from the nucleosomal dyad (Fig. 2B;;;;; ). In the resting state, the two lobes have an open conformation, separating the Walker A and B motifs in lobe 1 from a crucial arginine in lobe 2. ATP binding is accompanied by a conformational change (referred to as “closed state”) that creates a binding pocket comprising the Walker A and B motifs in lobe 1 and the catalytic arginine in lobe 2. Following ATP hydrolysis, the two lobes open up again, creating a cycle of ATP-binding and hydrolysis that drives movement of the translocase along the DNA tracking strand using an inchworm mechanism with a 1-bp step per ATP hydrolysis. However, nucleosome remodeling involves additional contacts between the remodeler and the nucleosome (Fig. 2C). These include binding of the ATPase to the opposite DNA gyre and the histone core. In particular, an acidic patch formed by histones H2A and H2B is frequently in physical contact with remodelers. Moreover, some remodelers interact with the N-terminal tail of histone H4 or contain additional DNA-binding domains that bind the linker DNA. Binding to these additional sites fixes the position of the translocase, preventing it from walking along the nucleosomal DNA. Instead, the remodeler will now pull the DNA toward the octamer dyad. This creates a ratcheting cycle in which the DNA is locally distorted, and through a combination of translational and rotational displacement peeled off the histone core. Throughout this remodeling process the histone core does not appear to undergo a major deformation. In summary, nucleosome remodeling depends on ATP-dependent DNA translocation driven by a motor domain that is fixed onto the nucleosome through additional DNA and histone contacts. Protein domains outside the RecA lobes play crucial roles in remodeler functionality. Finally, in most remodelers, the ATPase activity is modulated by accessory subunits that determine remodeler function and targeting to specific genomic loci.

5At its most basic level, remodelers control gene transcription by mobilizing nucleosomes to make gene regulatory elements more or less accessible to the transcription machinery. Nucleosomes present a barrier for RNA polymerase II (RNAPII), and consequently there is no basal transcription on chromatin templates. Rather, gene transcription on chromatin requires sequence-specific transcription factors, which use coregulators, including remodelers, histone-modifying enzymes, and chaperones. Different remodelers perform diverse, nonredundant functions in the transcription cycle. Several ISWI class remodelers function in the assembly and generation of regularly spaced nucleosomal arrays (Fig. 3A). This plays a crucial role in the packaging of newly synthesized DNA following replication. Moreover, studies in yeast revealed that the generation of evenly spaced nucleosomes in gene bodies by ISWI and CHD1 remodelers helps to repress cryptic initiation of transcription. SWI/SNF remodelers have been implicated in generating an open chromatin conformation at gene promoters and enhancers (Fig. 3B). Induction of the unfolded protein response transcription program in Drosophila cells caused extensive changes in nucleosomal DNA accessibility, without accompanying changes in nucleosome occupancy. Several studies suggested the presence of “fragile” nucleosomes, with DNA that is highly accessible, at regulated promoters. A recent study showed that these fragile nucleosomes are partially unwrapped RSC remodeling intermediates, which result from cooperation between RSC and general regulatory transcription factors. In addition to changing DNA accessibility through remodeling, remodelers can also affect the composition of the histone core. For example, the INO80 class remodelers mediate the replacement of canonical histone H2A by the H2A.Z variant. H2A.Z containing nucleosomes are enriched around transcription start sites and increase accessibility of nucleosomal DNA.

6A key mechanism of gene selectivity is the cooperation between remodelers and sequence-specific DNA-binding transcription factors. Remodeler recruitment can be re-enforced by local chromatin changes. Histone acetylation can promote the recruitment of SWI/SNF to specific loci through recognition of acetylated histones by a bromo domain. As discussed below, SWI/SNF and the Polycomb repressors function antagonistically on many regulatory DNA elements. The NuRD complexes can oppose SWI/SNF at shared regulatory elements (Fig. 3C). NuRD can generate repressive chromatin through nucleosome placement at regulatory DNA elements and histone deacetylation. The Polycomb system might further advance the formation of a repressive chromatin environment through H3K27 methylation and chromatin compaction (discussed below). It must be stressed that the behavior of a substantial proportion of promoters does not conform to generalizations derived from averaging results from genome-wide studies, which should be considered more as rules of thumb than as dogmas that apply to all genes.

7The large multisubunit SWI/SNF complex was the first remodeler described and remains the best studied. SWI/SNF was originally identified genetically in Saccharomyces cerevisiae through screens for genes that were involved in expression of the HO nuclease, required for mating type switching (SWI), and expression of the SUC2 invertase, required for sucrose fermentation (SNF). Several of the encoded proteins, including the Snf2 ATPase, turned out to reside in a common complex, named SWI/SNF. The observation that mutations in histones alleviated the requirement for SWI/SNF and changes in chromatin structure in snf2 mutants, suggested that SWI/SNF functions through targeting chromatin. Indeed, in vitro biochemical analysis of yeast SWI/SNF revealed ATP-dependent chromatin remodeling and increased DNA accessibility. These results supported a scenario in which SWI/SNF activity opens-up promoter chromatin to promote transcriptional activation. Following the discovery of yeast SWI/SNF, related complexes were identified in mammalian cells that performed similar chromatin remodeling and gene regulatory functions.

8There are two main subtypes of SWI/SNF complexes that are broadly conserved among eukaryotes (Fig. 4A; Table 1). The first includes yeast SWI/SNF, Drosophila BAP, and mammalian BAF, while the second class includes yeast RSC, fly PBAP, and mammalian PBAF. The corresponding complexes contain a variable number of identical subunits and paralogs that form a common core, associated with a set of signature subunits that are unique to either SWI/SNF-BAF or RSC-PBAF. Sth1, the ATPase of RSC, is a paralog of Snf2, the motor subunit of SWI/SNF. RSC and SWI/SNF contain four additional paralogs and share three subunits. The remaining subunits are unique for each complex. Both complexes are involved in activation of RNAPII transcription of largely nonoverlapping sets of genes and have been implicated in different aspects of DNA repair. RSC is also involved in RNA polymerase III transcription and several nontranscriptional chromosomal functions throughout the cell cycle. Human cells contain two distinct SWI/SNF ATPases, named SMARCA4/BRG1 and SMARCA2/hBRM, which are both equally related to yeast Swi2/Snf2 and Sth1. Either ATPase associates with about eight additional subunits to form a core complex shared by both BAF and PBAF. In addition, there are two sets of mutually exclusive signature subunits that associate with the common core to form either BAF or PBAF. Polybromo (PBRM1), BRD7, ARID2, and PHF10 are specific for PBAF, whereas ARID1A/B and DPF1/2/3 define BAF. The differential incorporation of an array of paralogous subunits further increases the functional diversity of (P)BAF complexes. The isolated SMARCA2/4 ATPases are capable of remodeling in vitro, albeit at a lower level than the whole SWI/SNF complex. The association of the core subunits SMARCB1, SMARCC1, and SMARCC2 with SMARCA4 suffices to restore untargeted remodeling activity to the level of the full SWI/SNF complex. It is instructive to compare the roles of Drosophila BAP and PBAP, because they share an identical remodeling core (Table 1). Functional dissection of fly SWI/SNF revealed that the subunits comprising the core play key architectural or enzymatic roles, whereas the BAP- and PBAP-specific subunits determine most of the genomic targeting and functional selectivity. BAP and PBAP have shared functions but also unique effects on gene expression, development, and cell cycle progression. Recent comprehensive analyses of yeast and human SWI/SNF complexes provided detailed insights into their assembly, architecture, and functional organization. Collectively, structure–function dissection of the SWI/SNF remodelers suggests that they could be considered holoenzymes, in which different modules provide different functionalities that direct the remodeling activity.

9Recently, a third type of mammalian SWI/SNF complex was identified, named GBAF (glioma tumor suppressor candidate region gene 1 [GLTSCR1] BAF) or ncBAF (noncanonical BAF). GBAF/ncBAF comprises BRD9, GLTSCR1/1L, SMARCA2/4, ACTL6A/B, Actin, SMARCC1, SMARCD1, BCL7, and SS18/L1 (Fig. 4A; Table 1). Surprisingly GBAF lacks the conserved core subunit SMARCB1, which stimulates chromatin remodeling activity and genomic targeting of the canonical SWI/SNF complexes. The presence of genes with homology to the GBAF-specific subunits in Drosophila raise the possibility that GBAF might be evolutionarily conserved. BRD9 plays a key role in directing GBAF to a specific set of genomic loci, in part through binding to BRD4. GBAF targets includes CTCF- and promoter-proximal sites, and in embryonic stem cells (ESCs), loci associated with naive pluripotency. In summary, the SWI/SNF remodelers comprise a wide range of complexes that perform specialized, rather than generic functions.

10NuRD complexes bring together ATP-dependent chromatin remodeling and HDAC activities (Fig. 4B;; ). Unlike SWI/SNF, which is present in all eukaryotes examined, NuRD remodelers, are restricted to metazoans. Mammalian NuRD complexes harbor one of three chromodomain ATP-dependent helicases (CHD3-5) and one of the histone deacetylases HDAC1 or HDAC2. CHD3-5 are unique amongst the CHD family in that they possess double PHD fingers in their N terminus (Fig. 1B). In addition, NuRD complexes contain one of two scaffolding proteins (GATAD2A/B), histone chaperones (RBBP4/7), one histone tail- and DNA-binding protein (MTA1–3), and one of the CpG-binding proteins (MBD2/3). Notably, MBD2, but not MBD3, has been proposed to preferentially bind methylated CpG residues. Alternatively, DNA methylation has been suggested to be required for the binding of both MBD2 and MBD3. Finally, the small DOC1 (deleted in oral cancer 1) protein is an integral subunit of all NuRD complexes and plays a role in its recruitment to target loci. NuRD performs pivotal functions during development and stem cell differentiation.

11NuRDs can act as transcriptional corepressors, which are recruited through sequence-specific transcription factors to induce robust gene silencing. However, a global role for NuRDs in fine-tuning transcription has also been observed, in particular in ESCs. Genome-wide studies revealed that CHD4 associates with the majority of promoters and enhancers in the mammalian genome, where it dampens the levels of cognate gene transcription. Binding to the histone H3.3 variant might help to recruit NuRD to regions of active chromatin, where it then fine-tunes the level of transcription. Temporal analysis of gene repression induced by the transcription factor Ikaros revealed that NuRD drives nucleosome invasion, RNAPII eviction, and reduced activator binding at target loci. This required chromatin remodeling by CHD4 but was independent of HDAC activity. Histone deacetylation occurs later and contributes to the maintenance of gene silencing. The use of a MBD3-inducible system in ESCs revealed wide association with active chromatin, and a role for MBD3-NuRD in transcriptional dampening that mainly involves nucleosome invasion. Again, deacetylation of H3K27 followed attenuation of transcription, rather than preceding it. Thus, a model is emerging in which NuRD acts broadly at many enhancers and promoters to dampen and fine-tune active gene expression. In summary, NuRD acts as a global modulator of transcription but can also function as a transcription factor recruited corepressor. These different modes of NuRD action might reflect a difference in the level of local NuRD recruitment by transcription factors versus a more general affinity of NuRD for open chromatin.

12NuRD refers to a multitude of different protein assemblages. Consequently, studies based on a single subunit may only reflect the function of a particular subset of NuRD complexes. For example, MBD2-NuRD, rather than MBD3-NuRD, appears to form repressive chromatin. Whereas Mbd3 KO mice are early embryonic lethal, Mbd2 KO mice are viable. Therefore, caution must be taken not to generalize observations based on a particular subunit to all NuRD family complexes. The NuRDs appear to be more loosely assembled than SWI/SNF, and separation of remodeling- and HDAC modules has been reported. These submodules may associate with selective partners that recruit them to distinct genomic loci. For example, a complex comprising the Drosophila Mi2 ATPase and the zinc-finger homeobox protein MEP1 has been identified. A NuRD-related HDAC module, lacking CHD3-5 and MBD2/3, associates with PWWP2A/B, which recognizes the active chromatin features H2A.Z and H3K36me3 (Fig. 4B). Recruitment of this module by PWWP2A/B to active genes reduces the level of histone acetylation to decrease transcriptional elongation. Thus, some NuRD subunits are also part of alternate complexes that lack either remodeling or HDAC activity.

13Polycomb group (PcG) proteins are a large family of conserved chromatin regulators that are essential for maintaining cellular identity in higher eukaryotes. PcG genes were discovered in Drosophila as repressors of homeotic (Hox) genes. The maintenance of established patterns of Hox and other developmental gene expression requires the antagonistic activities of the PcG and Trithorax group (TrxG) proteins. While the PcG proteins maintain repression, the TrxG proteins contribute to sustaining active gene transcription. The names of many of the PcG genes were inspired by the phenotype of extra sex combs appearing on the second and third pair of legs of male flies, when normally these bristles only form on the first pair of legs. This distinctive phenotype is caused by the derepression of the sex combs reduced Hox gene and provided a powerful diagnostic for subsequent genetic screens to identify additional PcG- and TrxG genes.

14In Drosophila, most PcG proteins function as part of two broad classes of multiprotein complexes, named PRC1 and PRC2. The core of Drosophila PRC2 is formed by E(z), Su(z)12, CAF1-p55, and Esc (Fig. 5A). E(z) is a SET domain containing histone methyltransferase that, as part of PRC2, trimethylates histone H3K27 (H3K27me3), which is essential for PcG repression. There are two main forms of PRC2 in flies, defined by whether they contain the alternative subunits Pcl (Polycomb-like) or Jarid2. The PRC1 class is further subdivided into canonical (cPRC1) and noncanonical PRC1 (ncPRC1) (Fig. 5B). Drosophila cPRC1 comprises Pc, Psc [or Su(z)2], Ph, and Sce (also known as dRing), and while Scm associates with cPRC1, it is considered a substoichometric subunit. cPRC1 binds PRC2-mediated H3K27me3 via the chromodomain of Pc and is thought to mediate repression through chromatin compaction and loop formation. Fly ncPRC1, originally named dRAF (dRing-associated factors), is defined by a core complex composed of Sce, Psc, and Kdm2 but lacks Pc and Ph and can include Rybp. ncPRC1 couples the removal of the active H3K36me2 mark with the deposition of H2AK118ub, whereas cPRC1 lacks appreciable H2A ubiquitylation activity. While the H2AK118ub mediated by ncPRC1 is required for viability and helps to promote PRC2 mediated H3K27me3, it is not essential for Hox gene repression.

15In mammals, PcG proteins perform essential functions during development, cell differentiation and disease. As in Drosophila, genome-wide binding studies in mammalian cells confirmed that PcG proteins directly bind to the gene loci of Hox and other key developmental regulators. Although the key PcG proteins, PRC organization, and histone modifications are all conserved, the Polycomb system has expanded in mammals, compared with flies. In mammals, both cPRC1 and ncPRC1 are defined by a heterodimeric RING-PCGF (PcG ring finger) core, which can function as an E3 ubiquitin ligase to monoubiquitinate H2AK119 (Lys118 in Drosophila). There are two variants of the RING subunit in mammals, RING1A and RING1B, each of which can form a heterodimer with one of six variants of the PCGF subunit PCGF1–6 (Fig. 5B; ). cPRC1 contains either PCGF2 (MEL18) or PCGF4 (BMI1) in addition to one chromobox (CBX2, CBX4, CBX6, CBX7, or CBX8), one sex combs midleg (SCMH1, SCML1, and SCML2), and one polyhomeotic (PHC1–3) subunit.

16As in flies, the PRC2 complex is responsible for mediating all H3K27me1/2/3 on chromatin. The core PRC2 complex is composed of one of the two histone H3K27 methyltransferases, Ezh1 or Ezh2, together with Suz12 and Eed, which are required for histone methytransferase activity. In addition, several accessory proteins associate with core PRC2, which are thought to modulate the recruitment and enzymatic activity of the complex (Fig. 5A; ). Drosophila Pcl has three mammalian homologs (Phf1, Mtf2, and Phf19), while Jarid2 and Aebp2 (Jing in flies) are conserved as well. Additional interacting proteins include Epop, Pali1, and Pali2. Comprehensive proteomic analyses showed that PRC2 primarily assembles into two mutually exclusive combinations, termed PRC2.1 and PRC2.2. PRC2.1 is defined as containing one of the three Pcl proteins, while PRC2.2 is defined as containing Aebp2 and Jarid2. There is additional variation within the PRC2.1 subtype, such that one Pcl protein is a constant and defining feature, while the presence of EPOP and PALI1 are mutually exclusive. Therefore, while PRC2.1 and PRC2.2 are similar to Drosophila Pcl-PRC2 and Jarid2-PRC2, respectively, additional accessory proteins of PRC2.1 have emerged during evolution, providing additional opportunities for regulation (Fig. 5A).

17The recruitment of PcG proteins has been well-studied in flies. It is mediated by specific cis-regulatory DNA sequences named Polycomb response elements (PREs). PREs are essential for the establishment and propagation of Polycomb-repressed chromatin. The sequence-specific transcription factor Pho plays a central role in the recruitment of PRC1 and PRC2 to PREs. Pho and PRC1 bind to PREs cooperatively, generating a nucleosome free region. Pho associates with Sfmbt to form PhoRC. Next, Scm, through binding to Sfmbt, PRC1, and PRC2, provides a functional link between these three complexes. SAM-domain mediated polymerization of Scm and Ph might further contribute to the generation of PcG silenced domains or long-range interactions, possibly directed by H3K27me3. While PHO-binding sites are necessary, they are not sufficient for PRC recruitment, consistent with the essential contributions of additional transcription factors. In summary, PcG recruitment in flies is achieved through a network of protein–protein interactions and DNA-binding that tether PRCs to regulatory DNA elements. Rather than a universal hierarchical process, PcG repression involves a mix of cooperative and redundant mechanisms that differ in different contexts.

18In mammals, the role of PRC2-mediated H3K27me3 in directing cPRC1 to target genes via chromodomains within its CBX subunits (the mammalian Pc homologs) is well-defined. Indeed, the majority of H3K27me3 and PRC2-associated genes are cobound by cPRC1 complexes in multiple mammalian cell types. After binding, cPRC1 is thought to confer gene repression by chromatin compaction through its CBX and PHC1–3 subunits. While the role of H3K27me3 in directing cPRC1 is well-defined in mammalian cells, much less is known about the role of PRC2-mediated H3K27me2. H3K27me2 is present on almost all intergenic euchromatin regions in both mammals and Drosophila and has been proposed to form a repressive “genomic blanket” to prevent the misfiring of cell type-specific enhancers of alternative lineages. Thus, the PRC2 complex is believed to contribute to the maintenance of cellular identity via both H3K27me3 and H3K27me2.

19As in Drosophila, ncPRC1 is responsible for the majority of H2A monoubiquitination in mammalian cells. The ncPRC1 assemblages all lack the CBX (Pc in flies) and PHC (Ph in flies) proteins. Instead, they contain one of the paralogs RYBP or YAF2 that interact directly with RING1A or RING1B. Another distinctive feature of mammalian ncPRC1 assemblages is that they contain any of PCGF1/3/5/6, each of which associates with specific additional subunits (Fig. 5B). For example, PCGF6-containing ncPRC1 harbors several transcription factors, including E2F6, MAX, and MGA, whereas the PCGF1-containing ncPRC1 includes the lysine demethylase KDM2B together with BCOR, SKP1, and the deubiquitylating enzyme USP7 (Fig. 5B). KDM2B can bind to unmethylated CpG islands via its CxxC motif and recruits PCGF1-ncPRC1, which can then deposit H2AK119ub (Fig. 5C;; ). This H2AK119ub modification has been suggested to contribute to the recruitment of PRC2.2 via its Jarid2 subunit, which contains a ubiquitin-binding motif. Supporting this model, reduced H2AK119ub in ESCs lacking either RING1 proteins or combinations of ncPRC1-specific PCGFs leads to a partial reduction in PRC2 and H3K27me3 levels on Polycomb target genes. However, while the loss of RING1A/B in both mouse colon and skin cells in vivo leads to complete loss of H2AK119ub, global levels of H3K27me3 are maintained. Therefore, it appears that while recognition of H2AK119ub by JARID2-PRC2.2 contributes to local accumulation of H3K27me3 in some contexts, it cannot be the sole mechanism. It is likely that even in the absence of H2A ubiquitylation, Polycomb-like proteins can still direct PRC2.1 to mediate H3K27me3. Supporting this, Drosophila embryos engineered to lack all H2AK118ub maintain the repression of Hox genes and do not exhibit a Polycomb phenotype. Furthermore, the loss of Pcl, but not Jarid2, leads to Hox gene derepression in Drosophila. Although the mechanisms still need to be worked out in mammalian cells, Polycomb-like proteins may provide alternate means of engagement with chromatin through binding to H3K36me2/3 and H3K27me3 via their conserved Tudor domain or interaction with GC-rich DNA via their winged helix (WH) domain.

20In summary, it is clear that there are multiple interdependent interactions between the various PRCs that mediate their association with chromatin in mammalian cells (Fig. 5C). The ncPRC1 complexes can direct H2AK119ub to chromatin via either KDM2B binding to CpG islands or DNA-binding transcription factors. The H2AK119ub modification is in turn recognized by PRC2.2, which then deposits H3K27me3. PRC2.1 is targeted to chromatin via its Polycomb-like proteins. The combined action of both PRC2 subtypes results in a genomic profile of H3K27me3, which is then recognized by cPRC1. The activities of cPRC1 mediate long-range interactions and compact the chromatin via SAM-directed oligomerization, generating Polycomb-repressed domains (Fig. 5D). In conclusion, despite the general conservation of the Polycomb system in multicellular eukaryotes, expansion and subfunctionalization in vertebrates enables diverse mechanisms of recruitment and fine-tuning of gene expression.

21The SWI/SNF, NuRD, and PcG complexes do not function in isolation but are part of a regulatory network that also involves other chromatin regulators and transcription factors. This was first highlighted by genetic studies in Drosophila that identified suppressors of PcG mutations, forming the TrxG genes. The TrxG genes encode a diverse set of proteins involved in various aspects of chromatin regulation and transcription. They include the MLL/COMPASS histone H3K4 methyltransferases (that includes Trx), subunits of the mediator complex, the cohesin subunit Rad21, the Brd4-related fs(1)h, the remodelers Brm and Kismet, and the sequence-specific DNA-binding proteins Gaga and Zeste. TrxG genes were identified in two different ways. Some, including the founding member trx, were identified as activators of Hox gene expression. Others, such as Brm, were discovered in screens for suppressors of Pc mutations. Brm encodes the fly homolog of yeast Swi2/Snf2 (Table 1; ). The SWI/SNF core subunit Moira (Mor) and BAP-selective subunit Osa were also identified as suppressors of Pc. Direct tests revealed that the PBAP signature subunits Bap170, Polybromo, and Sayp are also suppressors of Pc. The transcription factor Zeste belongs to the TrxG and has many binding sites within PREs. A combination of biochemical and in vivo observations showed that Zeste mediates the maintenance of an activated state through recruitment of (P)BAP. Zeste binds to its DNA sites in a chromatin template by itself, but (P)BAP is required for transcription activation. Zeste tethers (P)BAP via direct binding to the TrxG subunits Mor (core), Osa (BAP) and Bap170 (PBAP). In conclusion, genetic studies in Drosophila established that SWI/SNF and Polycomb act antagonistically in a dosage-dependent manner. Analysis of the role of SWI/SNF in human cancer revealed the conservation of this mechanism of gene control. In rhabdoid tumor cells, the loss of SMARCB1 compromises SWI/SNF opposition of Polycomb repression. Genome-wide analysis revealed that SWI/SNF counteracts Polycomb repression of a multitude of genes, in particular at bivalent promoters. Artificial recruitment of SWI/SNF leads to a rapid (within minutes), ATP-dependent eviction of PRCs, which is independent of RNAPII transcription. These studies suggest that SWI/SNF can remove PRCs from the chromatin through a direct mechanism (Fig. 3B). Conversely, in vitro studies showed that PRC1 can inhibit chromatin remodeling by SWI/SNF. Collectively, these studies suggest that SWI/SNF and Polycomb compete in a dosage-dependend manner to generate opposing chromatin states.

22NuRD and SWI/SNF also act antagonistically on common regulatory elements. In ESCs, SWI/SNF, and NuRD have opposite effects on the nucleosome organization of shared targets. Likewise, in oral squamous carcinoma cells NuRD and SWI/SNF compete for binding to genes that encode master regulators of the epithelial-to-mesenchymal transition (EMT). NuRD mediates the formation of repressive chromatin through nucleosome invasion, histone deacetylation, and subsequent Polycomb recruitment. In contrast, SWI/SNF generates open chromatin and counteracts Polycomb. These results suggest that transcriptional control involves a dynamic equilibrium between opposing chromatin modulating enzymes rather than a static chromatin state. In agreement with this notion, the induction of repressive chromatin in pre-B cells by the transcription factor Ikaros is accompanied by the replacement of SWI/SNF by NuRD. The Drosophila Ikaros-related transcription factor Hunchback represses the Hox genes early in development through NuRD recruitment. After expression of Hunchback ceases later in development, gene repression is maintained by the Polycomb system. These early observations in Drosophila first suggested that NuRD might create a chromatin environment that facilitates subsequent Polycomb repression (Fig. 3C). This notion has been expanded by studies in mice, ESCs, and human cancer. The loss of NuRD-mediated histone deacetylation activity in Mbd3 null ESCs correlates with a loss of PRC2 association on a subset of CHD4 target genes. Thus, deacetylation of H3K27 might be a prerequisite for methylation and subsequent stabilization of PRC2 binding (Fig. 3C). Likewise, the depletion of CHD5 leads to a loss of H3K27me3 on CHD5 and PRC2 cobound genes. The CHD3–5 proteins all contain two PHD and two chromodomains (Fig. 2A), which have been proposed to act synergistically to determine their histone-binding specificity. The double PHD domains bind to unmodified H3K4, while the double chromodomains bind to H3K27me3 in vitro. Thus, PRC2 might also help NuRD recruitment, creating a positive feedback loop for NuRD and PRC2 targeting to chromatin. Alternatively, through an indirect process, transcriptional repression by NuRD might allow the default binding of PRC2 to CpG islands of silenced genes. In conclusion, at least on a subset of regulatory sites, SWI/SNF and NuRD compete for access to chromatin and generate opposite chromatin states.

23Cancer genome sequencing studies revealed that chromatin regulatory proteins are among the most highly mutated in human cancer. This indicates that disruptions in the biochemical mechanisms that control chromatin dynamics may play a significant role in the development of many cancers. Approximately 30% of newly identified potential cancer “driver” mutations occur in genes that encode chromatin regulators. Within this functional class, genes encoding selective SWI/SNF subunits are remarkably prone to mutations. More than 20% of all cancers have mutations in SWI/SNF members, making these complexes the most frequently mutated remodelers in cancer (Fig. 6). Genes encoding different SWI/SNF subunits are mutated at high frequencies in specific, nonoverlapping malignancies, indicating that they might have nonredundant tumor-suppressive functions. In addition to clear truncating, loss-of-function or loss-of-expression mutations, a substantial portion of mutations in SWI/SNF genes are substitution mutations that are distributed rather evenly across the coding sequence at relatively low frequency. Although these substitutions might affect protein stability, their potential role as drivers of cancer remains to be determined. Genes encoding members of the PRC2 complex are also mutated at elevated frequencies in particular cancers (Fig. 6). Intriguingly, these mutations can lead to either increased or decreased levels of H3K27me3, which varies depending on tissue or disease subtype. Below, we discuss instructive examples of how altered remodeler or Polycomb function promotes the development of cancer, highlighting our burgeoning understanding of the underlying molecular mechanisms. We start by reviewing rhabdoid tumors and synovial sarcoma in detail, because they are the best-understood cancers that are caused by aberant SWI/SNF function. Next, we review examples of cancers with additional SWI/SNF, NuRD, or PRC2 alterations. Finally, we discuss how perturbing the balance of SWI/SNF, NURD, and Polycomb activities may affect transcriptional programs and cell identity to promote oncogenesis.

24The first evidence for a causative role for mSWI/SNF defects in oncogenesis came from studies on rhabdoid tumors (RTs). RTs are deadly pediatric cancers of the central nervous system (CNS), kidney and soft tissues, which typically occur in children <2 yr of age. These cancers are also referred to as atypical teratoid RTs (ATRT) when located in the CNS or extracranial malignant RTs (ecMRT) when located elsewhere in the body. The vast majority of RT cases (∼99%) have biallelic loss of the SMARCB1 gene (Fig. 6). A small minority of RTs are associated with mutations in SMARCA4 rather than SMARCB1. Families harboring germline mutations in SMARCB1 or SMARCA4 are predisposed to the development of RT, referred to as RT predisposition syndrome (RTPS). The genomes of RTs display no substantial genomic instability and have very low mutational burden, with loss of SMARCB1 as the sole recurring event. Thus, inactivation of SMARCB1 appears to be sufficient to drive oncogenesis in the absence of collaborating genetic abnormalities. While biallelic loss of Smarcb1 leads to early lethality during mouse development, haplo-insuffient mice are prone to develop tumors that resemble human RTs, showing loss of heterozygosity that is typical for a tumor suppressor. Reversible conditional inactivation of Smarcb1 in mice revealed that, while it is essential for the survival of most normal cells, it also causes highly penetrant and aggressive cancers. The vast majority of these tumors were T-cell lymphoma with only rare cancers that resembled RTs. Nonetheless, these mouse experiments established that loss of Smarcb1 causes cancer in mouse models. Inactivation of Smarcb1 at different stages of mouse development leads to dramatically different outcomes. While full Smarcb1 deletion during early development is lethal, its partial inactivation at embryonic day 6–10 (E6–E10) results in highly penetrant rapid onset CNS tumors that closely resemble human RT. In agreement with earlier observations, loss of Smarcb1 in adult mice causes lymphomas instead of RT. The predisposition to the development of childhood RTs is not fully penetrant among RTPS families with germline mutations in SMARCB1, and its development in adults from these families is extremely rare. However, these adults frequently develop multiple schwannomas and benign tumors involving cranial and peripheral nerves. Thus, both patient data and mouse studies provide strong evidence for developmental stage as the major factor determining the consequences of SMARCB1 inactivation.

25While several studies have addressed the alterations in gene expression in RT; interpretations have been complicated by the substantial heterogeneity among tumors. An embryonic gene expression signature distinguishes RT from other cancers that are SMARCB1-deficient. Consistent with SWI/SNF's role as a member of the TrxG, dysregulation of Hox genes is frequently observed in RTs. Although more research is required to determine the significance of specific transcriptional perturbations in RT, the retention of an embryonic signature is likely a key aspect in the development of these tumors. Furthermore, loss of SMARCB1 can affect cell cycle control in multiple ways. For example, inactivation of SMARCB1 has been implicated in silencing of CDKN2A, encoding the pivotal tumor suppressor p16INK4a, in cell lines, mouse models, and human tumors. CDKN2A is a well-characterized target of PcG proteins, linked to their ability to suppress cellular senescence. Therefore, a key consequence of the loss of SMARCB1 in RTs is that it compromises the ability of the SWI/SNF complex to counteract Polycomb-mediated repression of CDKN2A.

26The loss of SMARCB1 does not substantially debilitate the structural integrity of BAF or PBAF but does compromise their recruitment to key target sites. In addition, SMARCB1/Snf5 binds the SMARCA4/Snf2 catalytic subunit and stimulates nucleosome remodeling by mammalian and yeast SWI/SNF. Recent genome-wide studies demonstrated that SMARCB1 is important for recruitment of SWI/SNF to developmental enhancers. Loss of SWI/SNF binding at these sites is associated with PRC2 binding and repression of cognate developmental gene promoters, indicating that the SWI/SNF–Polycomb antagonism observed at the CDKN2A locus occurs genome wide. Importantly, EZH2 is essential for tumor formation after conditional loss of SMARCB1 in mice, and treatment with small molecule EZH2 inhibitors led to regression of RT in a xenograft model. Thus, an important aspect of RT development is the loss of SWI/SNF antagonism to PcG repression. While the loss of SMARCB1 compromises both BAF and PBAF, it does not affect GBAF that lacks this subunit. Therefore, it is of interest that chemical probes that target the GBAF subunit BRD9 inhibit proliferation of RT cell lines. Collectively, these observations suggest that RTs might be especially vulnerable to combined targeting of BRD9 and EZH2. In addition, due to their stable genomes, the canonical tumor suppressor pathways remained intact in RT, and might be exploited as well. In conclusion, the detailed dissection of gene control in RT starts to provide leads for potential therapeutic intervention.

27Synovial sarcoma is an aggressive, poorly differentiated malignancy that can occur in patients of all ages but is particularly common in children and young adults with a peak incidence between 20 and 30 yr of age. The hallmark genetic abnormality is a chromosomal translocation involving chromosomes X and 18, t(X;18). This rearrangement fuses the N terminus of the BAF and GBAF subunit SS18 (also known as SYT) to the C terminus of one of three related proteins, SSX1, SSX2, or SSX4. Like RTs [other than t(X:18)], synovial sarcoma tumors contain few, if any, genetic abnormalities. This suggests that the SS18-SSX fusion protein is the primary driver of disease development. Indeed, conditional expression of SS18-SSX leads to the development of a synovial sarcoma-like disease in mice. Similar to results observed in Smarcb1-deficient mouse models, both cellular context and developmental timing are critical for the ability of SS18-SSX to induce tumors. For example, while the expression of SS18-SSX in mature muscle lineages causes myopathy without tumorigenesis, when expressed in immature muscle progenitor cells, adult mice develop aggressive tumors with 100% penetrance.

28SS18-SSX dominantly assembles into SWI/SNF, causing the eviction and proteasomal degradation of both the wild-type SS18 and SMARCB1. Therefore, in both RT and synovial sarcoma, SMARCB1 is lacking from BAF (but not from PBAF). However, the assembly of SS18-SSX into BAF, and not SMARCB1 loss, appears to be the primary pathogenic alteration. SS18-SSX does not associate with PBAF, but it assembles into both BAF and GBAF. Given their compositional differences, it is likely that incorporation of SS18-SSX has unique functional effects as part of BAF versus GBAF. These differences need to be explored given the recent demonstration that synovial sarcoma cells are exquisitely dependent on GBAF function. Depletion of SS18-SSX correlates with increased PRC2 mediated H3K27me3 and repression of many SS18-SSX target genes. SS18-SSX containing BAF and GBAF appear to be more potent antagonists of Polycomb function than their wild-type counterparts. This suggests that SS18-SSX containing complexes may act dominantly to aberrantly activate Polycomb target genes. Consistent with this notion, SS18-SSX activates the expression of many developmental regulators. Moreover, cells expressing SS18-SSX appear to be blocked in their capacity to differentiate. Depletion of SS18-SSX triggers downregulation of developmental gene expression signatures and differentiation toward a mesenchymal cell fate. It has also been suggested that SS18-SSX associates with KDM2B, which may lead to its mis-direction to KDM2B-bound loci. Collectively, these results suggest that SS18-SSX containing BAF and GBAF oppose Polycomb mediated repression of a primitive development transcriptional program, causing a block to cellular differentiation.

29Studies on RT and synovial sarcomas established that defects in SWI/SNF subunits SMARCB1 and SS18 cause cancer. Sequencing studies revealed that the genes encoding additional SWI/SNF subunits are frequently mutated in a wide array of other cancer types (Fig. 6). Here, we restrict our discussion to a selection of SWI/SNF subunits for which functional studies are beginning to provide mechanistic insights. The BAF-specific ARID1A gene is the most commonly mutated SWI/SNF component in cancer (Fig. 6). Its function is primarily lost due to truncating mutations as has been reported in multiple cancers, including bladder cancer, uterine endometrial carcinoma, and neuroblastoma. Moreover, recent pan-cancer analyses of almost 10,000 adult and 1600 pediatric cancers further demonstrate the significance of ARID1A mutations in multiple malignancies. While our mechanistic insights on the effects of ARID1A loss are still limited, recent studies suggested that impaired enhancer function might play a central role. In a mouse model of colon cancer, based on the conditional deletion of Arid1a, loss of ARID1A correlated with reduced binding of SWI/SNF at enhancers and reduced expression of cognate genes. Another study established that ARID1A loss leads to reduced chromatin accessibility at enhancers, impeding transcription factor binding. Consistent with the observed reductions in gene expression and chromatin accessibility, many of these enhancers had reduced levels of H3K27ac. Furthermore, the deletion of Arid1a in a mouse model of hepatocellular cancer also leads to a reduction in chromatin accessibility and associated gene expression. Truncating mutations of ARID1A abrogate its ability to associate with the core of the SWI/SNF complex. Thus, like its Drosophila homolog Osa, loss of ARID1a impairs BAF recruitment to chromatin. Given the reductions in both chromatin accessibility and H3K27Ac at enhancers following loss of ARID1a, it is tempting to speculate that the absence of BAF allows NuRD to promote repressive chromatin at these loci. This could contribute to an impaired ability of these cancer cells to activate key genes required for differentiation. Finally, oncogene-induced senescence and activation of p53 in hepatocellular carcinoma cells depends on ARID1B/BAF.

30PBRM1 loss-of-function genetic alterations are present in ∼40% of kidney renal clear cell carcinoma (KIRC), making it second to the VHL tumor suppressor in terms of frequency (Fig. 6; ). Gene expression analysis revealed substantial changes due to loss of PBAF function in these cells, which correlates with responsiveness to immune checkpoint therapy. In an independent screen to identify barriers to killing of cancer cells by cytotoxic T cells, inactivation of Pbrm1, Arid2 and Brd7 was found to mediate resistance. Both studies suggest that PBAF control of interferon-stimulated gene expression promotes immune resistance. Loss of PBAF function increased tumor cell secretion of chemokines that recruit effector T cells. Thus, targeting PBAF might sensitize cancer cells to immunotherapy. Paradoxically, in clear cell renal cell carcinoma, loss of PBAF function might promote both tumorigenicity and susceptibility to antitumor immunity.

31SMARCA4 is the most commonly mutated Snf2-like ATPase in cancer, including several adult and pediatric malignancies (Fig. 6;;;; ). Similar to ARID1A, many SMARCA4 mutations are predicted to inactivate, suggesting that it functions as a tumor suppressor. A portion of the cancer-associated SMARCA4 mutations occur within the ATPase domain. Introduction of these SMARCA4 ATPase mutations into mouse ESCs leads to a loss of chromatin accessibility and H3K27Ac at enhancers, accompanied by transcriptional down-regulation, and accumulation of Polycomb and H3K27me3 at the promoters of associated genes. However, as observed for SMARCB1-deficient RT cells, increased Polycomb activity occurs specifically at gene promoters and not at enhancers. These observations indicate that loss of SWI/SNF function in cancer leads to alterations in enhancer landscapes. We speculate that this might be due to increased NuRD activity at these sites. The reduced activity of SWI/SNF-dependent enhancers correlates with an increased activity of Polycomb at the associated gene promoters, and attenuated transcription. These effects appear to alter the developmental potential of SWI/SNF mutant cells, making them linger in an undifferentiated (or incompletely differentiated) state. A recent analysis of somatic histone mutations in cancer revealed a substantial number of alterations in the globular domain that have been implicated in remodeler function. These include mutations homologous to yeast mutants that alleviate the need for SWI/SNF, and alterations in the acidic patch (Fig. 3C) that are predicted to impair remodeling by ISWI.

32In addition to its well-established roles in cell differentiation programs, disruption of NuRD function has also been implicated in oncogenesis. However, cancer-associated mutations in NuRD subunits occur less frequently than in SWI/SNF components (;; http://www.cbioportal.org). The gene encoding the CHD5 member of NuRD is a tumor suppressor that is frequently deleted in high-risk neuroblastoma and glioma. It is expressed in normal neuronal tissues and in low-risk neuroblastomas, but its levels are reduced in tumors from high-risk neuroblastoma patients. CHD5 is required for terminal neuronal differentiation and has a dual role in facilitating the activation of neuronal genes, as well as the repression of a cohort of Polycomb target genes. Therefore, its loss in neuroblastoma has been proposed to impede the ability of neural cells to undergo terminal differentiation. The role of CHD4 in human cancer is more complicated. In some cancers, CHD4 is mutated with increased frequency, but compelling evidence that these are driver mutations remains lacking. Moroever, CHD4 has also been linked with pro-oncogenic functions. The recruitment of CHD4-NuRD by the DNA-binding transcription factor ZFHX4 is crucial for the maintenance of therapy-resistant tumor-initiating cells in glioblastoma. CHD4 is required for the maintenance of MLL-AF9 rearranged acute myeloid leukemia cells but not for growth of normal hematopoietic cells. Conversely, MBD3-CHD4-NuRD prevents tumorigenesis by constraining a B-cell transcriptional program through restriction of lineage-specific enhancers and promoters. This process involves opposing activities of NuRD and SWI/SNF, again emphasizing the importance of chromatin balance in developmental transcription control. The DOC1 subunit of NuRD is lost in the majority of oral cancers and correlates with tumor invasion and adverse outcomes. The loss of DOC1 affects recruitment of NuRD to a selection of genes that control proliferation and EMT. At these loci, NuRD and SWI/SNF compete for binding and generate opposite chromatin states. SWI/SNF drives formation of open chromatin and counters Polycomb binding. In contrast, NuRD mediates nucleosome invasion, histone deacetylation, recruitment of Polycomb and KDM1A, and transcriptional repression. Although not as well explored as the connection between SWI/SNF and cancer, these studies indicate that alterations in NuRD integrity can contribute to oncogenesis. Mutations in or deregulation of NuRD can disturb the balance with SWI/SNF, leading to the corruption of developmental programs and proliferation control. As for SWI/SNF, subunit-dependent gene selectivity is likely to explain the association with specific types of cancer.

33Initial links between deregulation of Polycomb function and cancer were related to their overexpression rather than driver mutations. However, there is little evidence of driver mutations in genes encoding subunits of cPRC1 in human cancer. So, while overexpression of PRC1 components such as PCGF4/BMI1 might promote oncogenesis, it might also be a consequence of the proportion proliferating or stem like cells in the tumor, rather than a cause. This limited evidence of cancer driver mutations in genes encoding cPRC1 subunits might be related to the high level of redundancy among cPRC1 subunits, which would limit the impact of mutations. However, somatic mutations in the ncPRC1 subunit BCOR have been identified in AML and myelodysplastic syndrome, and mouse studies support a tumor suppressor function in leukemia. Bcor has a key function in the regulation of the hematopoietic stem cell transcription network and its inactivation drives expansion of myeloid progenitor cells and cooperates with oncogenic Kras to drive the development of leukemia. Thus, while the roles of some PRC1 components in cancer seems mostly related to their requirement for stem cell survival, BCOR appears to be a bona fide tumor suppressor.

34Multiple cancer genome sequencing studies have revealed that the function of PRC2 is disrupted on a genetic level in a wide array of cancers, leading to both loss and gain of activity. Prior to these studies, EZH2 was reported to be highly expressed in multiple cancers as a result of being an E2F-regulated gene, downstream from the pRB pathway. However, elevated EZH2 expression is not considered to lead to deregulation of H3K27 methylations, because the stoichiometry of the core PRC2 complex would require correspondingly high levels of the EED and SUZ12 subunits. In 2010, recurrent heterozygous mutations of the EZH2 catalytic SET domain in 22% of Diffuse large B-cell lymphomas (DLBCLs) and 10% of follicular lymphomas (FLs) were reported for the first time (Fig. 6; ). These mutations were originally thought to induce loss-of-function effects on EZH2 enzymatic activity; however, additional work demonstrated that the mutant EZH2 has altered substrate preferences. The preferred activity of wild-type EZH2 is the conversion of H3K27me0 to me1 and H3K27me1 to H3K27me2, while the enzyme is relatively inefficient at the final conversion step to H3K27me3. Owing to changes in substrate-binding modality, lymphoma associated EZH2 SET domain mutants have an enhanced ability to convert H3K27me2 to me3. As a result, these “change-of-function” EZH2 mutants cooperate with the wild-type enzyme, pushing the kinetics of PRC2 activity toward increased H3K27me3 production. This leads to aberrantly high global levels of H3K27me3, with concomitant reductions of H3K27me2 levels in EZH2 mutant lymphoma cells. Importantly, in a mouse model, conditional expression of this “change-of-function” EZH2 mutant in B-cell lineages replicates these H3K27 methylation changes and, while augmented by overexpression of BCL2 or loss of p53, is sufficient to cause malignancy.

35Our understanding of the molecular mechanisms underlying oncogenesis in these EZH2 mutant contexts is still limited. ChIP-seq (chromatin immunoprecipitation [ChIP] combined with high-throughput sequencing) experiments have suggested that increased levels of H3K27me3 mark the promoter regions of genes required for cell cycle exit and terminal B-cell differentiation. This correlates with reduced expression of many of these genes, suggesting that mutant EZH2 impedes the differentiation potential of immature B-cells. Moreover, cPRC1 appears to maintain the repression of these genes, which may be linked to recruitment of the CBX8 containing cPRC1 via binding to H3K27me3 at these sites. To date, the consequences of reduced H3K27me2 levels in these cells and its functional relationship with H3K27me3 have yet to be examined. This might be important given that H3K27me2 is known to mark up to 70% of all histone H3 in normal cells. Moreover, H3K27me2 has been suggested to perform repressive functions at intergenic sites that are likely altered in EZH2 mutant lymphoma cells.

36In contrast to lymphomas, the genes encoding core PRC2 subunits are tumor suppressors in other cancer types. For example, in pediatric T-cell acute lymphoblastic leukemias (T-ALLs) and malignant peripheral nerve sheath tumors (MPNSTs) the EZH2, EED, and SUZ12 genes are subject to deletions or inactivating mutations. These loss-of-function alleles are associated with reductions in the global levels of both H3K27me2 and H3K27me3. Functional studies showed that the loss of PRC2 activity in T-ALL and MPNST leads to aberrant activation of some PRC2 target genes. It is likely that the loss of PRC2-mediated H3K27me3 at these genes leads to increased activity of SWI/SNF chromatin remodellers. The function of PRC2 is also deregulated in pediatric diffuse intrinsic pontine gliomas (DIPGs), a highly aggressive pediatric brainstem tumor. The discovery that up to 80% of DIPG have mutations in two genes encoding histone H3F3A (H3.3) or HIST3H1B (H3.1) led to the finding of global reductions in H3K27me2 and H3K27me3 in these cells. Subsequent work has suggested that H3K27M can act as a dominant-negative mutation that, at least partially, inhibits PRC2.

37Taken together, it is clear that the function of PRC2 is deregulated in several types of cancer, accompanied by changes in the global levels and genomic distributions of H3K27me2 and H3K27me3. While it remains unclear how these epigenomic changes contribute to the development of cancer, it is likely that they confer context-dependent blocks to cellular differentiation and increased vulnerability to aberrant cancer signaling pathways.

38Normal gene expression control depends on the equilibrium between SWI/SNF opposed by NuRD and Polycomb (Fig. 7A). As discussed in this review, many cancers have a disturbance of this chromatin balance. This common feature might be exploited therapeutically. For example, in the context of loss of SWI/SNF function, unrestrained Polycomb activity appears to mediate aberrant repression of transcriptional programs related to cellular differentiation and tumor suppression. Consequently, targeting PRC2 function in SWI/SNF mutant cancers may reverse the oncogenic chromatin changes that underlie disease development. Consistent with this, codeletion of Ezh2 and Snf5 is not compatible with cancer development in mice. The recent development of small-molecule EZH2 methyltransferase inhibitors has provided an opportunity to examine the clinical utility of PRC2 inhibition in multiple cancers, including those carrying inactivating mutations in genes encoding SWI/SNF subunits. As a proof of concept, SMARCB1-deficient RT cells are more sensitive to treatment with EZH2 inhibitors than wild-type cells. Moreover, EZH2 inhibition blocks the progression of SMARCB1 mutant cancers in vivo, which has motivated the establishment of ongoing clinical trials with EZH2 inhibitors in RTs. Early results have reported clinical responses in a subset of SMARCB1 negative patients. EZH2 inhibitor treatment of non-small cell lung cancer cells bearing SMARCA4 mutations sensitizes these cells to chemotherapy, an effect that is not apparent in SMARCA4 wild-type cells. Furthermore, Arid1a mutant, but not wild-type, ovarian cancer cell lines are also sensitive to reduction of EZH2. These findings indicate that targeting PRC2 activity in SWI/SNF mutant cancers may provide an effective means to treat this large cohort of patients. Finally, targeting HDAC activity, particularly HDAC2, blocks the progression of Arid1a mutant ovarian cancer cells. Thus, NuRD complexes, harboring HDAC1/2, may also be relevant in SWI/SNF mutant cancers. However, the general efficacy of HDAC inhibitors remains to be systematically examined in SWI/SNF mutant contexts.

39Accumulating evidence indicates that the SWI/SNF complexes themselves may be an effective therapeutic target in SWI/SNF mutant cancers. For example, GBAF is essential for the continued growth and survival of synovial sarcoma cells. The selective targeting of the GBAF subunit BRD9 in a mouse model of synovial sarcoma effectively blocked tumor progression, supporting the therapeutic promise of this approach. Synthetic lethality is another potential strategy for cancers with loss-of-function mutations in genes encoding SWI/SNF subunits. Typically, the residual SWI/SNF complexes are essential in these cancer cells, which may be exploited therapeutically (Fig. 7B). For example, SMARCB1 deficient cells require residual SWI/SNF function, because concomitant loss of SMARCB1 and SMARCA4 blocks tumor development. Moreover, SMARCB1 mutant RT cells also depend on BRD9 function. ARID1A mutant cancer cells often require the function of its paralog ARID1B. Likewise, SMARCA4 mutant lung cancer cells depend on SMARCA2 for their continued growth and survival. Thus, targeting paralog function in these settings may provide an effective therapeutic option (Fig. 7B). Recent drug development efforts are beginning to uncover molecules that specifically target SWI/SNF subunits such as ARID1A, BRD7, and BRD9, which will allow this idea to be tested.

40The mutation of EZH2 in B-cell lymphomas motivated the development of small-molecule inhibitors that act as SAM-competitive binders of the SET domain and exhibit high selectivity for EZH1/2. These compounds are capable of reducing global levels of H3K27me1/2/3 in cells, regardless of their EZH2 mutational status. Nevertheless, EZH2 “change-of-function” mutant cells are particularly sensitive to these drugs. This motivated the establishment of clinical trials to examine their effects in lymphoma patients. Recent reporting on these ongoing clinical studies indicated significant responses in EZH2 mutant patients, with as many as 92% of EZH2-mutant follicular lymphoma patients responding to treatment. Functional studies indicated that EZH2 inhibition leads to reductions in global H3K27me3 levels, which is associated with up-regulation of PRC2 target genes. A complicating factor is the identification of secondary mutations in both wild-type and mutant EZH2 alleles in B-Cell lymphoma cells lines, which can cooperate to confer resistance to EZH2 inhibitors. This emphasizes the probability that acquired resistance to EZH2 inhibitors will also occur in patients. Thus, alternative targets to disrupt both wild-type and mutant EZH2 in cancer will be needed. The development of potent EED inhibitors, which block its ability to allosterically activate PRC2 via its engagement with H3K27me3, might help to bypass this complication. Importantly, cell lines that had developed resistance to EZH2 SET domain inhibitors remained sensitive to this EED inhibitor, indicating that they may help to circumvent primary acquired resistance. However, an alternative strategy may be needed to treat patients with T-ALL and MPNST that harbor loss-of-function mutations in PRC2. In the context of the chromatin balance paradigm (Fig. 7A), it is tempting to speculate that specific inhibitors of SWI/SNF might open up new opportunities for treatment of these cancer types. In conclusion, the detailed knowledge of the molecular mechanisms and biology of chromatin remodelers and Polycombs, is now enabling the development of promising new therapeutic approaches to treat many human cancer types.