SNF Chromatin Remodeling Complexes in Health and

Subscriber access provided by ORTA DOGU TEKNIK UNIVERSITESI KUTUPHANESI

Current Topic/Perspective

PRC2 and SWI/SNF Chromatin Remodeling Complexes in Health and Disease Cigall Kadoch, Robert A. Copeland, and Heike Keilhack Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.5b01191 • Publication Date (Web): 02 Feb 2016 Downloaded from http://pubs.acs.org on February 13, 2016

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Biochemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

PRC2 and SWI/SNF Chromatin Remodeling Complexes in Health and Disease Cigall Kadoch1, Robert A. Copeland2 and Heike Keilhack2# 1

Dana-Farber Cancer Institute and Harvard Medical School; 450 Brookline Avenue, Boston, MA 02215,

USA. 2

Epizyme Inc., 400 Technology Square, 4th floor, Cambridge MA 02139, USA.

#

Current address: Ribon Therapeutics, 485 Massachusetts Ave, Cambridge MA 02139, USA. Email

address: [email protected]

Abstract The dynamic structure of histones and DNA, also known as chromatin, is regulated by two classes of enzymes: those that place covalent modifications on either histone proteins or DNA, and those that use the energy generated by ATP hydrolysis to mechanically alter chromatic structure. Both classes of enzymes are often found in large protein complexes. In this review we describe two such complexes: polycomb repressive complex 2 (PRC2), with the protein methyltransferase EZH2 as its catalytic subunit, and the ATP-dependent chromatin remodeler SWItch/Sucrose Non-Fermentable (SWI/SNF). EZH2 catalyzes the methylation of lysine 27 on histone H3, a covalent chromatin modification that is associated with repressed heterochromatin. The catalytic activity of SWI/SNF, in contrast, leads to a state of open chromatin associated with active transcription. In this review, we discuss the biochemical properties of both complexes, outline the principles of their regulation and describe their opposing roles in normal development, which can be perturbed in disease settings such as cancer.

ACS Paragon Plus Environment

Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Introduction Eukaryotic cells require mechanisms to compact their ~ 1.8 meters of DNA, carrying the full genetic code for life, into the very small volume of a cell nucleus (typically ca. 2-6 µm3). This is achieved by complexing chromosomal DNA together with core histone proteins to form chromatin; chromatin is composed of basic units known as nucleosomes, which consist of 146 base pairs of double stranded DNA wrapped around an octameric core of histone proteins, much like thread wrapped around a spool. Nucleosomes along a stretch of chromosomal DNA can pack tightly against one another, thus providing a structural mechanism for compacting the large content of DNA into the nucleus. Ironically, this necessary mechanism of compaction creates a physical conundrum for eukaryotic cells as it imposes a steric barrier preventing transcription, replication, recombination and DNA repair machineries from binding to DNA. Hence, the highly compacted structure of chromatin must be relaxed in a gene-specific manner to allow for timely and appropriate gene transcription and other critical cellular processes to ensue. DNA accessibility is facilitated by two classes of enzymes, ATP-dependent nucleosome remodelers and histone modifying enzymes, two of which will be discussed thoroughly in this review: the mammalian SWItch/Sucrose Non-Fermentable (SWI/SNF) complexes and Polycomb Repressor Complex 2 (PRC2). Histone modifying enzymes such as PRC2 post-translationally modify the N-terminal tails of histone proteins to alter the structure of chromatin and provide binding sites for regulatory proteins. Many chromatin-associated proteins contain protein domains that bind these moieties such as the bromodomain that recognizes acetylated residues. Through direct interactions with histone tails, these proteins are targeted to specific sites on chromatin, such as transcriptionally active regions abundant in trimethylated lysine 4 of histone H3 (H3K4me3), or repressed regions marked with


Page 2 of 40

Page 3 of 40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

trimethylated lysine 27 of histone H3 (H3K27me3). By contrast, chromatin remodeling complexes (CRCs) utilize the energy of ATP hydrolysis to disrupt DNA-nucleosome contacts, transition nucleosomes along DNA, and eject or exchange nucleosomes as means to enable DNA accessibility. Both histone modifying complexes such as PRC2 and CRCs such as the SWI/SNF complex are required for several aspects of development and more recently have been strongly implicated in human disease.

PRC2 Complexes and the H3K27me3 Repressive Mark Chromatin remodeling requires highly coordinated activation and recognition elements to ensure the kinetic and genomic targeting specificity of these enzymatic reactions and the downstream gene-specific transcriptional activation or silencing that they elicit. Hence, the majority of chromatin remodeling enzymes reside within cells as components of multi-protein complexes. Different protein subunits of these complexes are responsible for different aspects of chromatin recognition and activation, such as binding to specific DNA sequences, transcription factors or histone proteins; recognition and binding of specific post-translational modifications to histone (e.g., recognition of acetyl-lysine by bromodomains and recognition of methyl-lysine by Chromo, Tudor and MBT, PHD and PWWP domains); and catalysis, per se. Composition of the PRC2 Complex PRC2 as isolated from mammalian cells consists of at least five protein subunits: EZH1 or EZH2, EED, SUZ12, RBBP4/7 and AEBP2.1 Despite their occurrence within multi-protein complexes, many chromatin remodeling enzymes can be expressed and purified as isolated proteins with retention of their catalytic activity.2 PRC2 is an exception to the generality. Isolated EZH1/2 is devoid of enzymatic activity and requires a minimum of two additional PRC2 subunits for robust methylation activity: EED


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

and SUZ12.3 Depending on the cellular context, various other PRC2 associated factors (both proteins and RNA entities) have been described to regulate the activity, targeting and specificity of PRC2.4 EZH1 or EZH2 act as the business end of the PRC2 complex, catalyzing the addition of methyl groups onto lysine residues of proteins (see below and Figure 1), while the other subunits have diverse, non-catalytic functions. The WD domain within the core EED subunit is configured to bind repressive chromatin marks, including H3K27me3, H3K9me3 and H4K20me3; hence EED has a role of recruiting PRC2 to sites within chromatin that require further repression.5-7 It has been suggested that binding of EED to H3K27me3 allosterically stimulates EZH2, providing a feed-forward mechanism for enhancing the H3K27 methylation reaction. Interestingly, EZH2 also methylates JARID2, a common PRC2 associated factor; methylated JARID2 can also bind to EED and thereby activates EZH2 in the absence of H3K27me3.8 This process is, for instance, used to regulate H3K27me3 deposition during ES cell differentiation. The complex stabilizing core unit SUZ12,9, 10 in contrast, has been suggested to sense activating marks such as H3K4me3, binding of which leads to allosteric inhibition of EZH2.11 A similar function in integrating activating histone marks has been suggested for RBBP4/7, a subunit that can bind histone tails, but with diminished affinity when they are methylated on H3K4.11 AEBP2 is required for the optimal enzymatic activity of the complex,1 likely due to its suggested central role in stabilizing the complex and as an allosteric cofactor bridging DNA-binding and enzymatically active components of the complex.9 Finally, PHF9, binds to pre-installed H3K36me3 and localizes the PRC2 complex to active chromatin regions facilitating H3K27me3 deposition and silencing of transcription (see references from Table 1). PRC2-mediated Protein Methylation With a few exceptions, including the enzyme DOT1L, all protein lysine methyltransferases (PKMTs) rely on a canonical, catalytic domain referred to as the SET (Su(var)3-9/Enhancer of zeste/Trithorax) domain.12 The SET domain contains within it a contiguous active site cavity that is


Page 4 of 40

Page 5 of 40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

structurally composed of a S-adenosylmethionine (SAM) binding site, a methyl-acceptor lysine binding site and an intervening channel for methyl group migration during the SN2 reaction mechanism;13 the SET domain-containing catalytic subunit of PRC2 consists of one of two highly homologous proteins, EZH1 or EZH2.14 PRC2, containing either EZH1 or EZH2, demonstrates a remarkable level of substrate specificity, catalyzing significant methylation of only one lysine (lysine 27 of histone 3; H3K27) among all the amino acids of the four core histone proteins (H2A, H2B, H3 and H4); this specificity for H3K27 methylation is retained among substrates of varying sizes and structural complexity from assembled nucleosomes to isolated histones to small peptide substrates of PRC2.15, 16 While H3K27 appears to be the dominant substrate of EZH2, various in vitro and cell culture studies have indicated additional substrates such as JARID2,8 H2BK120,17 talin18 and transcription factors such as STAT3,19 suggesting alternative roles of EZH2 beyond chromatin remodeling. Most of the biochemical characterization of PRC2 has been performed with the EZH2 catalytic subunit, in part because of the relevance of this subunit to human disease (vide infra). Hence, for the remainder of this section, we will focus on what is known of the catalytic mechanism of PRC2 containing EZH2. Where it has been studied, the biochemical properties of PRC2 containing either EZH2 or EZH1 are largely identical, although it has been suggested that EZH1 has overall lower methyltransferase activity and distinct chromatin binding properties.14 As for all PKMTs, PRC2 is thought to catalyze methyl transfer from SAM to lysine via a bi bi SN2 reaction mechanism, with SAM and H3K27 as the two reaction substrates and S-adenosylhomocysteine (SAH) and methylated H3K27 as the two reaction products.13 This mechanism implies direct transfer of the methyl group from SAM to the substrate lysine in the context of a ternary complex with the enzyme, rather than a double-displacement mechanism involving initial transfer of the methyl group from SAM to a site on the enzyme, followed by subsequent transfer of the methyl group from the enzyme to the substrate lysine (Figure 1). Consistent with this mechanism, steady state kinetic analysis of PRC2


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

catalyzed methylation of a peptide substrate (bracketing the sequence of the H3K27 site) demonstrated patterns of intersecting lines when velocity was plotted as a function of substrate concentrations in the form of double reciprocal plots. This pattern of intersecting lines is consistent with a ternary complex mechanism in which both substrates are simultaneously bound to the enzyme prior to group transfer. No evidence of a preferred order of substrate addition to the enzyme has been demonstrated,16 suggesting a random order ternary complex mechanism, but this could not be definitively determined on the basis of product inhibition patterns because the data were confounded by significant enzyme activation by the product peptide H3K27me3 (likely mediated through EED binding, see above). PRC2 is the only mammalian enzyme known to catalyze the methylation of H3K27. As such, this enzymatic complex must be responsible for all three methylation reactions at this lysine residue: mono-, di- and tri-methylation of H3K27 (H3K27me, H3K27me2 and H3K27me3).1, 3, 20 However, there is some controversy within the literature on the details of how the initial mono-methyl product, H3K27me, is produced. For example, it was observed that H3K27me is still detected in cells with non-functional PRC2.21,

22

Steady state kinetic studies of PRC2 containing wild-type (WT) EZH2 by Sneeringer et al.,

however, demonstrated that this enzyme preferentially performs the first methylation reaction (converting H3K27 to H3K27me) and that its catalytic efficiency wanes with further methylation reactions; the kcat/KM values for the three methylation reactions were 45.7 ± 3.0, 15.0 ± 2.0 and 3.4 ± 0.3 x 104 h-1nM-1 for the mono-, di- and tri-methylation reactions of PRC2 (WT EZH2), respectively.15 Interestingly the PRC2 associated factor PHF1 has been implicated in boosting the efficiency of the H3K27me2 to H3K27me3 conversion by WT EZH2,23 suggesting a cell or cell state specific modulatory function of this factor during deposition of the repressive H3K27me3 mark. Enzymes that perform multiple rounds of catalysis on macromolecular substrates can do so by either of two limiting mechanisms: distributive or processive catalysis. A distributive mechanism is one in which the enzyme binds substrate (here, H3K27), performs a single catalytic reaction and then


Page 6 of 40

Page 7 of 40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

releases the first reaction product (H3K27me). Subsequent rounds of catalysis on this first product require rebinding to an enzyme molecule. At the other end of the spectrum, a fully processive reaction is one in which the enzyme binds the substrate (H3K27) and performs all catalytic reactions on that substrate prior to release of the final product (H3K27me3). Thus, under steady state condition, where substrate is present in large molar excess over enzyme, only the substrate (H3K27) and the final product (H3K27me3) can accumulate during a fully processive enzyme reaction; any intermediate species (e.g., H3K27me and H3K27me2) would not accumulate to levels greater than the concentration of enzyme in the reaction mixture (as these intermediate species would only exist in the context of a binary complex with enzyme). Early studies of SET domain PKMTs suggested that these enzymes likely worked through a processive mechanism of catalysis.24 However, two independent lines of research now suggest that PRC2 functions as a distributive enzyme (illustrated in Figure 1). The first line of evidence comes from studies of recombinant PRC2 complex containing WT EZH2 as the catalytic subunit. Swalm et al. measured formation of H3K27, H3K27me, H3K27me2 and H3K27me3 as a function of reaction time using mass spectrometry detection for reactions initiated by addition of 500 nM H3K27 peptide to 10 nM PRC2.16 The data clearly demonstrated transient accumulation of > 300 nM H3K27me intermediate prior to accumulation of ca. 500 nM H3K27me2, with little detectable H3K27me3 formed throughout the time course of the experiment. These data are consistent with a distributive mechanism of catalysis and inconsistent with processive catalysis by PRC2. The second line of evidence for a distributive catalytic mechanism for PRC2 comes from studies of nonHodgkin lymphoma (NHL) cells that are heterozygous for mutations at Y646 within the SET domain of EZH2. Mutations of EZH2 Y646 to F, N, H, S or C were identified in subsets of patients with germinal center B-cell lymphomas (GCB; diffuse large B-cell lymphoma [DLBCL] and follicular lymphoma [FL]).25 Patients presenting with these lymphomagenic mutations were always found to be heterozygous, expressing approximately equal amounts of mutant and WT EZH2 at the message and protein levels.


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Subsequent biochemical studies conducted initially by Sneeringer et al. and later confirmed by Yap and colleagues suggested that lymphomagenesis was dependent on requisite enzymatic coupling of the activity of the WT and mutant enzymes.15, 26 The WT and mutant EZH2s displayed quite distinct patterns of substrate utilization. WT EZH2 is most effective at catalyzing the first methylation reaction (H3K27 to H3K27me), weaker at catalyzing the second reaction (H3K27me to H3K27me2) and very weak at catalyzing the third reaction (H3K27me2 to H3K27me3), as described above. In striking contrast, all of the GCB-lymphoma associated EZH2 Y646 mutant enzymes were much more effective than WT EZH2 in catalyzing the tri-methylation reaction (H3K27me2 to H3K27me3), weaker at catalyzing di-methylation and essentially unable (or in the case of the Y646F mutant very weak) to catalyze the initial monomethylation reaction. Thus, Sneeringer et al. proposed that the oncogenic hyper-trimethylation of H3K27 seen in heterozygous, mutant-bearing GCB-lymphoma cells required coupling of the activity of WT EZH2, to catalyze the mono-methylation reaction, and mutant EZH2 to drive hyper-trimethylation. Requisite coupling of enzymatic activity, as proposed by Sneeringer et al., can only occur if the WT and mutant EZH2 forms function as distributive enzymes; this is because coupling would require the release of the mono-methylated product from the WT enzyme, rebinding of this product to either WT or mutant enzyme to produce the di-methylated product, release of this second product and subsequent binding to and catalysis by the mutant enzyme. To test this hypothesis, Swalm et al. performed studies with recombinant PRC2 containing either WT or lymphomagenic mutants of EZH2.16 If coupling occurs between WT and mutant EZH2 then an equimolar mixture of the two EZH2 forms (5 nM of each enzyme for a total of 10 nM) should produce more H3K27 methylation than an equal concentration (10 nM) of WT enzyme alone, and much more H3K27 methylation than an equal concentration (10 nM) of mutant enzyme alone. This is exactly the pattern that was observed by these researchers for all of the Y646 mutants of EZH2, consistent with a distributive catalytic mechanism and coupled activity of WT and mutant EZH2 within heterozygous, mutant-bearing GCB-lymphoma cells. This disease mechanism is fully


Page 8 of 40

Page 9 of 40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

consistent with genetic studied in drosophila from over 20 years ago.27 The mutant allele that first identified E(z),28 the drosophila homolog of EZH2, is a missense mutation that alters the same active site tyrosine as Y646,29 and the gain of function behavior of the fly mutant required WT E(z) activity in trans.27 Regulatory Inputs into PRC2 Given the involvement of PRC2 in many biological processes, its activity needs to be tightly controlled in a gene locus and tissue specific manner. Three categories of control mechanisms (i.e., inputs) have been studied in detail (summarized in Table 1): 1) modulation of PRC2 binding and activity through pre-installed histone modifications; 2) EZH2 phosphorylation on specific threonine or serine residues; and 3) the binding of PRC2 associated factors. A number of studies have revealed a molecular crosstalk whereby PRC2 senses either repressive or activating histone marks in local chromatin environments via direct biochemical contact, and its activity is consequently either enhanced or inhibited, respectively.30-33 Studies in Drosophila revealed early on that enzymes involved in methylating H3K4 and H3K36 residues (TRX and ASH) were genetic suppressors of activating polycomb mutations, long before their enzyme functions were understood.34, 35 Intriguingly, activating chromatin marks rarely exist on the same histone tail with H3K27me3,11, 36, 37 but occur in the context of the bivalent domain on the 2 different tails of a histone octamer in the case of H3K4me3.38 The antagonism of H3K27 acetylation (H3K27ac, another activating mark) and H3K27 methylation39, 40 is further enforced by MSK1mediated phosphorylation of the neighboring serine 28 (S28), which enhances H3K27ac through displacement of the PRC2 complex.41,

42

Of note, while EZH1-containing PRC2 binds similarly well to

H3K27me3 peptides in the absence or presence of S28 phosphorylation, EZH2-containing PRC2 binding was significantly weakened by S28 phosphorylation; this observation suggests phosphorylation state specific differences between EZH1 and EZH2 mediated chromatin remodeling.43 Finally, the plethora of


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 10 of 40

PRC2 associated factors and their variants (both protein and RNA entities) enable multiple layers of tissue and cell state specific regulation of PRC2 activity and targeting.4

SWI/SNF Complex- Mediated ATP-Dependent Chromatin Remodeling Mammalian SWI/SNF (also called BAF) complexes are large multimeric assemblies of 10-15 subunits which utilize the energy derived from ATP hydrolysis to achieve the coordinate mobilization of nucleosomes and the modulation of chromatin accessibility. Through now decades of study, from the original discovery in yeast, to subsequent characterization and mechanistic studies in Drosophila and mammals, several key themes have emerged, which will be discussed in this review. These include the mechanisms of nucleosome remodeling, the importance of combinatorial assembly and paralog subunit specificity, and the opposition with PRC family members. In recent years, these complexes have become the subject of increasing attention owing to the striking frequency of mutation in a wide range of human cancers as well as neurodevelopmental disorders.44, 45 The Catalytic Activity of the SMARCA4 (BRG1) and SMARCA2 (BRM) ATPase Subunits. The mammalian SWI/SNF complexes are powered by two closely related paralog ATPase subunits, SMARCA4 and SMARCA2, which assemble into complexes in a mutually exclusive manner. SMARCA4 and SMARCA2 are co-expressed in most tissue types with the exception of ES cells in which SMARCA4 is the only ATPase expressed.46 The genes encoding these 190 kDa subunits were first identified in yeast in screens for mate type switching and sucrose fermentation,47, 48 and the realization that these genes were involved in chromatin regulation emerged with studies showing that genetic reversion of these mutations was achieved with second mutations in genes encoding histones.49 Subsequent biochemical characterizations later indicated that these ATPase proteins did not work in


Page 11 of 40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

isolation but rather with several other components, yielding the name ‘SWI/SNF complex’ for the multisubunit entity, which will be referred to throughout this review. The early days: a recap of in vitro studies. In vitro studies have long suggested that, the complex is able to break DNA-histone interactions, slide histone core octamers on DNA and dissociate the histone core from DNA.50-53 Genetic studies aimed to determine the functional role of complexes have demonstrated that SWI/SNF complex subunits are important for instructive functions such as controlling cell fate, lineage specification, and cell proliferation in vivo, likely via their roles in maintaining the full complex as a context-specific master transcriptional regulator.54-58 To achieve these important in vivo biological outputs, it is clear that SWI/SNF complexes likely do much more than simply moving and ejecting nucleosomes. For example, despite the definitive requirement for complex subunits in vivo, the majority of subunits are not required for the remodeling activity of the ATPase in vitro, further cautioning that in vitro mechanistic studies may have been only be partially informative. Initially this result was thought to be due to genome-wide complex targeting. Later studies, however, determined this to be a less likely explanation given that mutations in the non-required subunits (such as ARID1A, etc.) had phenotypes almost identical to rapid conditional deletion of the Brg1 and/or Brm ATPase.46, 59, 60

These data underscore a loss of function mechanism, indicating that individual subunit function was

central to the fundamental mechanism of the complexes. As such, a critical and unmet goal in the field is to develop more faithful systems to study the complexes, in vivo, using real chromatin templates containing both the modifications and complexity of the very genomic sites at which they carry out their functions. Moreover, due to a near complete lack of structural information for the complex as a whole or any subunit in isolation, mechanisms regarding subunit-specific function have been particularly difficult to assign. With these caveats in mind, we review the in vitro studies to date which have characterized the mechanisms of SWI/SNF (and SWI/SNF-related) complexes.


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 12 of 40

Several early structural studies focused on understanding the mechanisms by which ATP hydrolysis of CRC ATPase subunits catalyzes the movement of DNA-bound nucleosomes (Figure 2). DNAnucleosome interactions are driven by electrostatic interactions, rendering the unwrapping process energetically unfavorable. Despite this barrier, some of the first studies characterizing CRCs indicated that they have the ability to disrupt these contacts in order to promote nucleosome sliding, ejection, and nucleosome exchange on nucleosomal templates in vitro. One early mechanism hypothesized was that of ‘twist diffusion’, in which DNA twists around the nucleosome, thereby accommodating the gain of a base pair from the linker DNA. This twist then continues and propagates through the remaining length of the DNA-histone contact, unidirectionally advancing the nucleosome along DNA. However, this model was rejected when it was shown that instating large barriers to DNA twisting, including structural impediments such as DNA hairpins or biotin crosslinks, produced no defect in nucleosome sliding.61, 62 A more favored model became the ‘loop recapture’ model, which proposed that a loop of DNA is created by newly gained histone contacts with neighboring linker DNA.62 As in the twist diffusion model, this loop is then propagated around the nucleosome to forward the nucleosome along DNA. This ‘loop recapture’ model explains how DNA might move around the nucleosome without changes to rotational phasing, even though calculations revealed that this was energetically costly. DNA loops may be generated as a result of the translocase activity of the SWI/SNF, ISWI, and ACF ATPases.63-66 Specifically for SWI/SNF, Saha et al. postulated that the ATPase protein binds to a specific site on the nucleosome, and subsequently employs 3’ translocase activity to draw DNA from one entry/exit location and advance it to the other in a directional wave.67 DNA footprinting and crosslinking experiments in cells have suggested that the ATPases localize sites at which DNA-histone binding is weak, such that the torsional strain generated may be tolerated for the propagation of the loop.65, 67-69 In addition, imaging of RSC and SWI/SNF complexes using electron microscopy indicate that these complexes form multi-lobed C-shaped structures that surround the nucleosome in a central cavity with the DNA entry and exit sites exposed.69-


Page 13 of 40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

71

Binding of CRCs to their nucleosome attachment site generates significant repositioning of DNA

relative to the histone octamer, even in the absence of ATP hydrolysis, which is thought to facilitate the creation of a DNA loop required for ATP-dependent translocation.72 Comparative protein sequence analyses of chromatin remodelers, across species, suggested that the core ATPase domains of SMARCA4 and SMARCA2 are remarkably similar to those of the DEAD/H helicases, indicating that they most likely utilize (and require) ATP to facilitate movement of DNA by similar mechanisms. Indeed, these enzymes and the related RAD54 protein share an ability to translocate DNA similar to the DEAD/H helicases, despite no evidence for helicase activity of SWI/SNF proteins.73 Despite these similarities, these proteins are genetically non-redundant, demonstrating their functional specificity.74 More intricate analyses of the SWI/SNF and ISWI complexes have demonstrated differences in the reaction product, substrate selection, enzymatic activity, and targeting of these complexes, perhaps, in part explaining the non-redundancy. However, discussed above, our understanding of the relative ability of CRCs to remodel chromatinized templates may be only partial, due the inability to produce physiologically relevant templates in vitro. As such, nucleosome movement is likely only one aspect of CRC function; future studies will be important to identify additional specific mechanisms. Combinatorial Assembly Drives Biologic Specificity The SWI/SNF family of ATP-dependent CRCs in particular has undergone substantial changes in subunit composition as well as subunit assembly-specific mechanisms coincident with the evolutiondriven advent of multicellularity, appearance of linking histones, polycomb-mediated repression, and a greater genome size. The differentially employed diversity of mammalian BAF complex subunit compositions is essential for the development of specific cell fates, including the progression from pluripotent embryonic stem cells to multipotent progenitor cells, to committed, post-mitotic neurons.


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 14 of 40

Overview of Subunit Evolution. Yeast SWI2/SNF2 is incorporated into a 1.14 MDa multi-subunit complex of 8-11 subunits. Over the course of evolution from yeast and Drosophila complexes to mammalian SWI/SNF complexes, subunits have been gained, lost, and shuffled, likely to accommodate the epigenetic changes required in multicellular organisms with larger genomes. Five subunits of the mammalian SWI/SNF complex are conserved all the way back to yeast; these ySWI/ SNF orthologues are BRG1/hBRM, BAF155/170, BAF60, BAF53a/b, and BAF47. Several additional unique subunits (BAF250a/BAF250b, BAF200, BAF45a/b/c/ d, Brd9, and Brd7), and two subunits, BAF57 and beta-actin, which are related to Nhp10 and actin, are found in the yeast INO80 and SWR1 complexes. Five yeast subunits have been eliminated through evolution and are not present in mammalian subunits. Mammalian complexes are about 2 MDa, which is larger than the calculated molecular weight of the known subunits, indicating that several subunits have yet to be identified. In addition to the Brg1 and Brm ATPase domains (DEXDc and HELICc), as well as bromodomains, additional domains present in the accessory subunits most likely facilitate interactions with proteins (LXXLL, BAH, SANT, SWIRM, SWIB), DNA (ARID, HMG, Zn finger, Leucine zipper), and modified histones (bromodomain, chromodomain, and PHD domains), although this has yet to be confirmed. The bromodomain of Brg1 is not required for its in vivo function, as a mutant lacking the bromodomain fully rescued the knockout phenotype in both flies and mice.75, 76 However, additional bromodomains in Brd7 and Brd9 may function, either redundantly or specifically, in recruiting the complexes to specific genetic loci. Finally, while ySWI/SNF lacks the actin subunit, the mammalian BAF complex has approximately one actin molecule per complex, which may enhance the ATPase activity of BRG1/hBRM, as in the case of yeast SWR1.77 In addition to gains and losses of subunits over the course of evolution, the SWI/SNF family has exhibited an expansion of gene families that encode homologous BAF subunits. Whereas ySWI/SNF is monomorphic, in mammals, BAF complexes are arrayed from several possible options in each of the following gene families: Brg1/Brm, BAF250a/b, BAF155/BAF170, BAF60a/b/c, BAF45a/b/c/d, BAF57,


Page 15 of 40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

BAF53a/b, BAF47, and actin. Genetic studies indicate that subunit exchange helps drive the transition from pluripotency to multipotency to committed post- mitotic neurons in nervous system development. Pluripotent embryonic stem (ES) cells express only the Brg1 ATPase subunit (not Brm), express BAF155 but not BAF170, and BAF53a but not BAF53b,46, 78-80 consistent with a requirement for this particular assembly for pluripotency. Indeed, deletion of Brg1 results in early embryonic lethality during implantation,81 while Brm-deficient mice are viable, though larger than littermates.82 However, this has been recently disputed owing to questions regarding the construction of the original Brm knockout mouse.83 Differentiation of ES cells into neural progenitors leads to the activation of Brm and BAF170 as well as the repression of BAF60b.84 One of the most striking illustrations of the role of combinatorial assembly in the determination of cell fate comes from studies of the vertebrate nervous system, in which the two yeast SWI/SNF Arp4 homologues, BAF53a and BAF53b, are used sequentially in the progression from neural progenitors to mature, post-mitotic neurons. Crabtree and colleagues demonstrated that BAF53a is present in neural stem cells lining the ventricles and is rapidly replaced by BAF53b (via stark changes in subunit gene expression) precisely at cell cycle exit.84 BAF53b was shown to be a dedicated subunit of the neuronspecific nBAF complex; moreover, its deletion leads to death shortly after birth owing to a failure of dendritic morphogenesis.85 Similarly, loss of the Drosophila homologue of the BAF53a/b subunit, BAP55, during embryogenesis in results in aberrantly oriented class I dendrites and reduced arborization. In olfactory projection neurons, deletion of BAP55 leads to a highly specific neuronal mistargeting.57, 86 Neither BAF53a nor BAF53b possess ATPase activity, nor are they required for the enzymatic activity of Brg or Brm in in vitro nucleosome-based assays;77, 85 as such, these subunits must instead function within the complex by other means, perhaps in directing locus- or histone- specific complex targeting. Accompanying these changes, several other positions within the complex were demonstrated to exhibit subunit switching during this neural progenitor to neuron switch. Most notably, BAF45A, D switch for


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 16 of 40

BAF45B,C, and SS18 switches for CREST (SS18L1). The expression of BAF45B and C, as well as CREST is unique to the nervous system.55, 87 Yoo and colleagues determined that the mechanism underlying the switch in subunits during the development of the vertebrate nervous system is a triple-negative genetic circuit in which REST represses miR-9/9* and miR-124, leading to a repression of BAF53a and cell cycle exit, the activation of the alternative BAF53b subunit, and neural differentiation.55 This npBAF to nBAF switch is essential for the development of the vertebrate nervous system, since mutations affecting either state are lethal. Another example of selective assembly occurs in the developing heart, where BAF60c designates the region of the embryo with cardiogenic potential.54 Complexes containing BAF60c are uniquely required for heart development, and can directly facilitate the formation of heart tissue from mesoderm in the presence of tissue-specific factors.54, 88 In each of these cases, the expression of homologous subunits does not compensate for the loss of the correct subunit, arguing for the exquisite specificity and instructive role of each composite complex during cell fate decisions. While changes in subunit composition have been documented most extensively in specific developmental transitions such as those of the nervous system and the heart, mSWI/SNF subunit changes in human cancers driven by mutations to subunit genes largely have yet to be uncovered. Changes to complex composition may be directed either by changes in subunit gene expression or by protein stability of subunits. We have demonstrated that in specific cancers driven by mutation to a mSWI/SNF component, such as human synovial sarcoma driven by the SS18-SSX translocation which fuses SSX to the SS18 subunit, BAF complex composition is altered as BAF47 is unable to assemble.89 Additional proteomic mass spectrometry analyses as well as gene expression studies will continue to reveal cancer-specific BAF complex assemblies.


Page 17 of 40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

SWI/SNF – PRC2 Antagonism and Its Importance in Cancer Both polycomb and SWI/SNF complexes are highly conserved, however, the SWI/SNF subunit composition has changed substantially with evolution, likely to adapt to new requirements such as multicellularity, organogenesis and the presence of complex organ systems including the nervous system.90 Antagonism between polycomb and SWI/SNF complexes was first observed in Drosophila, in which polycomb and trithorax (of which SWI/SNF is a member) were found to oppose each other to regulate gene expression and development.34 This opposition is likely regulated by a combination of diverse mechanisms, such as variations in the compositions of both complexes, pre-existing chromatin modification, allosteric modulations of their respective enzymatic activities (for instance through posttranslational modifications or natural small molecule binding, such as phosphatidylinositol 4,5bisphosphate [PIP2] binding to SWI/SNF), and the presence and absence of tissue-specific transcription factors.90 One can imagine that slight changes to this fine-tuned balance can be deleterious to normal cell homeostasis, and indeed both members of the PRC2 and SWI/SNF complexes are heavily implicated in disease, including both cancer and developmental diseases (summarized in Figures 3 and 4).90, 91 It is known that mutation and/or overexpression of EZH2, the catalytic subunit of PRC2, for instance, can lead to aberrant H3K27me3 deposition, a repressed epigenetic state that has been observed in several cancer types. Furthermore, once can hypothesize that other genetic lesions than those affecting PRC2 complex members themselves result in aberrant PRC2 recruitment and/or H3K27me3 levels, such as loss of function mutations in the H3K27 demethylases (KDM6A) or H3K27 acetylases (CREBBP, EP300), or mutations in proteins impacting opposing activating methyl marks such as H3K4 (MLL family). In contrast, subsets of cancers such as T-cell acute lymphoblastic leukemia (T-ALL) or myeloproliferative disorders show genetic loss of EZH2 or other PRC2 components. Intriguingly, point mutations of the


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

H3K27 residue itself are driver lesions in cancer.92,

93

Page 18 of 40

H3K27M mutant histones act as competitive

inhibitors of EZH2,94 and cancers bearing such mutations show a global loss, but also local areas of gain of the H3K27me3 mark, suggesting altered targeting of the PRC2 complex in the H3K27M mutant setting.95 Together this suggests that perturbing the correct balance of PRC2 placed H3K27me3 in a given cellular background in either direction can drive changes leading to oncogenesis. Regarding SWI/SNF complexes, greater than 20% of human cancers bear mutations in one or more of the subunit genes; mutations can be either heterozygous and homozygous, somatic or germline, and often result in the loss of expression of a given complex member (summarized in Figure 4). SWI/SNF gene mutations occur in a broad spectrum of tumors: early stem cell-like, mesenchymal, epithelial and blood-borne cancer, as well as later stage adult cancers such as lung cancers, which has prompted questions as to whether they serve as driver or passenger mutations in a given cancer background. One strong line of evidence for their roles as true drivers in cancer comes from studies on early childhood cancers and cancers driven by well-defined genomic aberrations such as translocations. The most well-studied example of perturbed SWI/SNF – PRC2 antagonism in cancer (illustrated in Figure 5) is in malignant rhabdoid tumors (MRT), a rare, highly aggressive tumor type mainly found in children.96 MRT is characterized by bi-allelic loss of the SWI/SNF core component SMARCB1 (most cases) or the ATPase component SMARCA4 (rare cases).97, 98 Genetic loss of SMARCB1 has also been described in other human malignancies, e.g. epitheliod sarcoma, epithelioid malignant peripheral nerve sheath tumors, extraskeletal myxiod chondrosarcoma, myoepithelial carcinoma, renal medullary carcinoma and atypical chordoma.99 Other than the loss of either SMARCB1 or SMARCA4, MRTs display very few deleterious mutations, suggesting an oncogenic driver role for those lesions.100 Studies by Wilson and colleagues showed that MRTs display altered patterns of H3K27me3 due to inefficient PRC2 opposition by a SMARCB1-deleted SWI/SNF complex, generating an oncogenic gene expression pattern.101 Most notably, the INK4b-ARF-INK4a tumor suppressor locus is stably silenced in MRT cells.102 Genetic


Page 19 of 40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

knockout of EZH2 can prevent oncogenesis driven by loss of SMARCB1 in mice,102 and pharmacological inhibition of the EZH2 HMTase activity with small molecule inhibitors leads to context-specific killing of SMARCB1-negative MRT cells and durable tumor regressions in a MRT xenograft model.103 Intriguingly, presence of the SMARCA4 ATPase is necessary for tumorigenesis driven by SMARCB1 loss,104 while the alternative ATPase SMARCA2 is not expressed (without gene mutations) in the majority of MRT cell lines and 65% of MRT tumors.105 Non-mutational SMARCA2 silencing is also observed in 2 other rare tumor types that have recently been described to be SMARCA4-deleted: small cell cancer of the ovary, hypercalcemic type (SCCOHT) and undifferentiated thoracic sarcomas.106, 107 The observation that both ATPases are mutated or silenced yet cells remain viable seems paradoxical, as loss of both SWI/SNF ATPases generally results in rapid attenuation of proliferation of most cell lines in culture. Nonetheless, there are prior examples of cell lines lacking both SMARCA4 and SMARCA2 ATPases, such as the SW13 line.108,

109

Further studies will be needed to reveal the mechanisms underscoring the oncogenic

functions of these complexes. Intriguingly, transcriptome analyses showed that MRT, SCCOHT and SMARCA4-deleted thoracic sarcomas cluster together and up-regulate a group of developmental genes, pointing to the undifferentiated stem-like nature of such tumors and pointing toward a common disease mechanism and potentially a common vulnerability to EZH2 inhibition.107 Indeed, Epizyme recently reported preliminary evidence of antitumor activity in adult patients with both SMARCB1-negative MRT and SMARCA4-negative SCCOHT in the context of their phase 1 study of the EZH2 inhibitor tazemetostat (EPZ-6438).110 Based on gene expression and other similarities to MRTs (including a near pristine genome, sheet like arrangement of round cells, poorly differentiated phenotype, and high proliferative index), it has been suggested that these may be similar cancers that likely share some similar features in their progenitor cells.111 In contrast, the same transcriptome analysis mentioned above indicated that SMARCA4-deleted lung cancers cluster away from SMARCA4-deleted thoracic sarcomas, MRTs and SCCOHTs, suggesting a


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 20 of 40

divergent disease mechanism despite perturbation to the same SWI/SNF subunit.107 Of note, the majority of SMARCA4-deleted lung cancers still express SMARCA2;112 moreover, loss or inactivation of SMARCA2 is a unique synthetic lethality in such cancers.112-114 The finding that SMARCA4-deleted lung cancer cells are not sensitive to single agent EZH2 inhibitor115 but do respond to SMARCA2 inactivation fits with a hypothesis of a “switch of addiction”, dependent on when during cellular differentiation SMARCA4 (or SMARCB1) deletions are introduced. In more undifferentiated cancer cells of origin, PRC2 activity is generally high owing to its well-known role in regulating self-renewal. If PRC2 activity is unopposed due to a defective SWI/SNF complex, genes involved in differentiation remain silenced, including SMARCA2, and such cancers are vulnerable to EZH2 inhibition. This hypothesis is strengthened by the fact that in ES cells, SMARCA2 is not expressed and the only ATPase subunit available to incorporate into SWI/SNF complexes is SMARCA4.46 In more differentiated cancer cells of origin (such as those that give rise to epithelial tumors), EZH2 activity has been down-regulated, and now cancers harbor a SMARCA2-containing SWI/SNF complex that is oncogenic and can be targeted by SMARCA2 inhibition. In 2010, Young and colleagues performed a set of experiments that may shed further light on the different consequences of an aberrant PRC2 – SWI/SNF balance during oncogenesis in epithelial vs. mesenchymal tumors.116 Through studies in mice they discovered a dramatic tissue specificity of oncogene-stress induced expression of cell cycle inhibitors. While KRAS expression in the lung rarely induces p19Arf and lung tumors can exist in the presence of functional p19Arf, sarcomas always show robust activation, and for full-blown sarcomagenesis p19Arf expression needs to be lost (for instance through genetic mutation). As discussed above, the INK4b-ARF-INK4a locus is heavily silenced (without mutation) in MRTs due to ineffective PRC2 opposition by a SMARCB1-deleted SWI/SNF complex,102 and treatment of MRT cells with an EZH2 inhibitor increases mRNA expression of CDKN2A.103 It is hence plausible that sarcomas or other mesenchymal tumors can take different routes to CDKN2A inactivation,


Page 21 of 40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

either through genetic mutation or epigenetic silencing through oncogenic PRC2 activity. Lung cancers, however, have evolved to not require inactivation of CDKN2A, and as such, display other vulnerabilities that are not PRC2-mediated. While aberrant SWI/SNF-polycomb opposition in cancers has most often been demonstrated in SWI/SNF loss of function settings such as those discussed above, studies have indicated that in one particular cancer type, the directionality of this opposition appears to be opposite. Human synovial sarcoma is driven by and uniformly characterized by the t(X;18) chromosomal translocation, resulting in the SS18-SSX fusion protein.117 We demonstrated that this fusion protein replaces the WT SS18 subunit in BAF complexes, and results in dramatic changes to BAF complex structure and function. Most notably, complex subunit structure is altered, the BAF47 subunit is unable to assemble and hence destabilized at the protein level, and these altered complexes are mistargeted genome-wide to polycomb target sites.89 Importantly, proliferation of synovial sarcoma cells is dependent on BAF complexes containing the SS18SSX fusion, and both SWI/SNF complex localization and gene expression is reversed upon knock down of the fusion protein and reassembly of WT complexes. Further studies will be required to determine the specific mechanisms governing this gain of function, and response of these tumors to PRC2 complex inhibition. Additional investigation will be required to determine if SWI/SNF lesions besides SMARCA4 and SMARCB1 loss can direct the imbalance of SWI/SNF-PRC2 opposition and the heightened dependency on PRC2 in cancers. Recently, a synthetic lethal relationship with EZH2 inhibition has been suggested for ARID1A-mutant ovarian clear cell carcinoma,118 a very common mutation (~50%) in such cancers. Similar to the SMARCA4-SMARCA2 axis of the complex, other recent studies have demonstrated that in ARID1Adeficient cancer lines, ARID1B, the mutually exclusive ARID1A homolog, ranks as the top synthetic lethality.119 Thus, subunits within these two axes within the complex, the ATPase subunits (SMARCA4 and SMARCA2) as well as the ARID-containing 250kDa subunits ARID1A and ARID1B share the common


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 22 of 40

feature of context-specific disruption to the balance between SWI/SNF and polycomb. Further experiments will be needed to decipher the mechanisms underlying the oncogenic addiction of ARID1Amutant cancers and other SWI/SNF mutated tumor types.


Page 23 of 40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

Figure Legends and Tables Table 1: Regulatory Inputs into the Activity and Targeting of the PRC2 Complex Category Pre-existing chromatin modifications

EZH2 phosphorylation

PRC2 associated factors

Input Dense chromatin

Effect +

H3K27me3

+

H3K9me3

+

H2AK119Ub

+

H3K4me3 H3K36me3

-

H1K26me3

-

H3K27ac H3S28ph

-

T355 (CDK)

+

T377 (p38)

+

T497 (CDK)

-

S26 (AKT)

-

JARID2

+

PHF19

+

PHF1

+/-

MTF2

+/-

C17orf96

+

Proposed mechanism • Activation through neighboring nucleosomes with a fragment of their H3 histones (A31 to R42) • Mediated by SUZ12 • Activation through binding to EED • On the same or the other histone tail of a nucleosome octamer • Through EED without allosteric activation • Increased targeting to facilitate heterochromatin formation • Only on the opposite histone tail • Increased targeting of a JARID2/AEBP containing PRC2 complex • Allosteric inhibition, likely mediated through SUZ12 binding • Occurs on opposite histone tail compared to H3K27me3 • Competes with H3K27me3 and H3K9me3 binding at EED • Allosteric inhibition • Prevents H3K27 methylation • Displacement of EZH2-PRC2 • Enables H3K27ac formation • Increased PRC2 binding • Allosteric activation • Promotes interaction with YY1 targeting protein, which recruits PRC2 to repressed targets • Disrupts binding of EZH2, SUZ12 and EED Diminishes PRC2 activity • Fosters ubiquitination and degradation of EZH2 • Reduction in PRC2 histone methylation • Proposed to enable solo function of EZH2 (without other PRC2 subunits) as gene activator • PRC2 targeting to chromatin • PRC2 activation in the absence of pre-existing H3K27me3 through K116 methylation • Interaction with ncRNAs • Recruits H3K36me3 demethylases and PRC2 (through H3K36me3 binding) to chromatin • Enables silencing of regions of active transcription • Boosts catalytic efficiency in converting H3K27me2 to H3K27me3 • Binds H3K36me3 which inhibits PRC2 activity • Depending on the gene locus either promotes repression or activation • Stimulates PRC2 activity


References 120

5-7

7, 38, 121

122-124

11, 36-38

7

39, 40 41, 42

125, 126

127, 128

129, 130

131, 132

8, 133, 134

135-138

23, 139

140-142

143

Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ncRNAs

+

Nascent RNA (low Mw)

-

• Examples: XIST, HOTAIR • Enable locus specific targeting of PRC2, for instance during X chromosome silencing (XSIST) or silencing of HOX genes (HOTAIR) • Mechanism to sense areas of active transcription

Page 24 of 40

144-147

148-151

Figure 1: PRC2 Complexes and Their Catalytic Mode of Action. PRC2 complexes catalyze the placement of methyl groups to lysine 27 of histone H3 (H3K27), generating a repressive chromatin mark. Briefly, PRC2 catalyzes methyl group transfers in a distributed fashion, with S-adenosylmethionine (SAM) and H3K27 as the two reaction substrates (present in excess compared to enzyme at steady state) and S-adenosylhomocysteine (SAH) and methylated H3K27 (mono, di- and tri-methylated) as the two reaction products. Wild-type EZH2 cannot efficiently catalyze the dito tri-methylation step.

Figure 2: The Spectrum of PRC2 Subunit Perturbation in Human Cancers. (Left) Whole-exome sequencing efforts have revealed both gain- and loss- of function mutations in PRC2 subunits, EZH2, EED, and SUZ12. Mutations as well as amplifications/deletions in the genes encoding PRC2 subunits span a wide range of human cancers, from the specific gain-of-function, heterozygous point mutation in EZH2, to loss of expression in MDS, CMML, and other settings. (Right) Additional PRC2 subunits and associated factors and their cancer-specific perturbation.

Figure 3: Subunit Composition and Function of Mammalian SWI/SNF (BAF) Complexes. SWI/SNF complexes are combinatorically assembled into 10-15 subunit multimers from the products of 29 genes. The mutually exclusive ATPase subunits of these complexes, SMARCA4 and SMARCA2, catalyze ATP hydrolysis to enable the sliding, ejecting, and exchanging of nucleosomes on chromatinized templates.

Figure 4: SWI/SNF Complexes Are the Most Frequently Mutated Chromatin Regulators in Human cancer. Exon sequencing studies have revealed that the genes encoding SWI/SNF complex subunits are among the most frequently and broadly mutated in human malignancy, totaling over 20% of cancers. Specific subunits are mutated in specific malignancies, such as SMARCB1, SS18, and SMARCE1, however others are mutated or deleted in several cancer types, such as ARID1A (clear cell ovarian carcinoma, neuroblastoma, etc.), and SMARCA4 (NSCLC, etc.).

Figure 5: SWI/SNF-PRC2 Opposition: Models for Mechanism of Action, Subunits Implicated, and Relevant Therapeutic Approaches.


Page 25 of 40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

Loss of function mutations in SWI/SNF subunits result in a loss of its ability to oppose PRC2, as evidenced by increased H3K27me3 levels and gene repression.

References: [1] Cao, R., and Zhang, Y. (2004) SUZ12 is required for both the histone methyltransferase activity and the silencing function of the EED-EZH2 complex, Mol Cell 15, 57-67. [2] Chan-Penebre, E., Kuplast, K. G., Majer, C. R., Boriack-Sjodin, P. A., Wigle, T. J., Johnston, L. D., Rioux, N., Munchhof, M. J., Jin, L., Jacques, S. L., West, K. A., Lingaraj, T., Stickland, K., Ribich, S. A., Raimondi, A., Scott, M. P., Waters, N. J., Pollock, R. M., Smith, J. J., Barbash, O., Pappalardi, M., Ho, T. F., Nurse, K., Oza, K. P., Gallagher, K. T., Kruger, R., Moyer, M. P., Copeland, R. A., Chesworth, R., and Duncan, K. W. (2015) A selective inhibitor of PRMT5 with in vivo and in vitro potency in MCL models, Nat Chem Biol 11, 432-437. [3] Pasini, D., Bracken, A. P., Jensen, M. R., Lazzerini Denchi, E., and Helin, K. (2004) Suz12 is essential for mouse development and for EZH2 histone methyltransferase activity, EMBO J 23, 4061-4071. [4] Vizan, P., Beringer, M., Ballare, C., and Di Croce, L. (2015) Role of PRC2-associated factors in stem cells and disease, FEBS J 282, 1723-1735. [5] Hansen, K. H., Bracken, A. P., Pasini, D., Dietrich, N., Gehani, S. S., Monrad, A., Rappsilber, J., Lerdrup, M., and Helin, K. (2008) A model for transmission of the H3K27me3 epigenetic mark, Nat Cell Biol 10, 1291-1300. [6] Margueron, R., Justin, N., Ohno, K., Sharpe, M. L., Son, J., Drury, W. J., 3rd, Voigt, P., Martin, S. R., Taylor, W. R., De Marco, V., Pirrotta, V., Reinberg, D., and Gamblin, S. J. (2009) Role of the polycomb protein EED in the propagation of repressive histone marks, Nature 461, 762-767. [7] Xu, C., Bian, C., Yang, W., Galka, M., Ouyang, H., Chen, C., Qiu, W., Liu, H., Jones, A. E., MacKenzie, F., Pan, P., Li, S. S., Wang, H., and Min, J. (2010) Binding of different histone marks differentially regulates the activity and specificity of polycomb repressive complex 2 (PRC2), Proc Natl Acad Sci U S A 107, 19266-19271. [8] Sanulli, S., Justin, N., Teissandier, A., Ancelin, K., Portoso, M., Caron, M., Michaud, A., Lombard, B., da Rocha, S. T., Offer, J., Loew, D., Servant, N., Wassef, M., Burlina, F., Gamblin, S. J., Heard, E., and Margueron, R. (2015) Jarid2 Methylation via the PRC2 Complex Regulates H3K27me3 Deposition during Cell Differentiation, Mol Cell 57, 769-783. [9] Ciferri, C., Lander, G. C., Maiolica, A., Herzog, F., Aebersold, R., and Nogales, E. (2012) Molecular architecture of human polycomb repressive complex 2, Elife 1, e00005. [10] Jiao, L., and Liu, X. (2015) Structural basis of histone H3K27 trimethylation by an active polycomb repressive complex 2, Science 350, aac4383. [11] Schmitges, F. W., Prusty, A. B., Faty, M., Stutzer, A., Lingaraju, G. M., Aiwazian, J., Sack, R., Hess, D., Li, L., Zhou, S., Bunker, R. D., Wirth, U., Bouwmeester, T., Bauer, A., Ly-Hartig, N., Zhao, K., Chan, H., Gu, J., Gut, H., Fischle, W., Muller, J., and Thoma, N. H. (2011) Histone methylation by PRC2 is inhibited by active chromatin marks, Mol Cell 42, 330-341. [12] Richon, V. M., Johnston, D., Sneeringer, C. J., Jin, L., Majer, C. R., Elliston, K., Jerva, L. F., Scott, M. P., and Copeland, R. A. (2011) Chemogenetic analysis of human protein methyltransferases, Chem Biol Drug Des 78, 199-210. [13] Copeland, R. A., Solomon, M. E., and Richon, V. M. (2009) Protein methyltransferases as a target class for drug discovery, Nat Rev Drug Discov 8, 724-732.


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 26 of 40

[14] Margueron, R., Li, G., Sarma, K., Blais, A., Zavadil, J., Woodcock, C. L., Dynlacht, B. D., and Reinberg, D. (2008) Ezh1 and Ezh2 maintain repressive chromatin through different mechanisms, Mol Cell 32, 503-518. [15] Sneeringer, C. J., Scott, M. P., Kuntz, K. W., Knutson, S. K., Pollock, R. M., Richon, V. M., and Copeland, R. A. (2010) Coordinated activities of wild-type plus mutant EZH2 drive tumorassociated hypertrimethylation of lysine 27 on histone H3 (H3K27) in human B-cell lymphomas, Proc Natl Acad Sci U S A 107, 20980-20985. [16] Swalm, B. M., Knutson, S. K., Warholic, N. M., Jin, L., Kuntz, K. W., Keilhack, H., Smith, J. J., Pollock, R. M., Moyer, M. P., Scott, M. P., Copeland, R. A., and Wigle, T. J. (2014) Reaction coupling between wild-type and disease-associated mutant EZH2, ACS Chem Biol 9, 2459-2464. [17] Kogure, M., Takawa, M., Saloura, V., Sone, K., Piao, L., Ueda, K., Ibrahim, R., Tsunoda, T., Sugiyama, M., Atomi, Y., Nakamura, Y., and Hamamoto, R. (2013) The oncogenic polycomb histone methyltransferase EZH2 methylates lysine 120 on histone H2B and competes ubiquitination, Neoplasia 15, 1251-1261. [18] Gunawan, M., Venkatesan, N., Loh, J. T., Wong, J. F., Berger, H., Neo, W. H., Li, L. Y., La Win, M. K., Yau, Y. H., Guo, T., See, P. C., Yamazaki, S., Chin, K. C., Gingras, A. R., Shochat, S. G., Ng, L. G., Sze, S. K., Ginhoux, F., and Su, I. H. (2015) The methyltransferase Ezh2 controls cell adhesion and migration through direct methylation of the extranuclear regulatory protein talin, Nat Immunol 16, 505-516. [19] Dasgupta, M., Dermawan, J. K., Willard, B., and Stark, G. R. (2015) STAT3-driven transcription depends upon the dimethylation of K49 by EZH2, Proc Natl Acad Sci U S A 112, 3985-3990. [20] Nekrasov, M., Wild, B., and Muller, J. (2005) Nucleosome binding and histone methyltransferase activity of Drosophila PRC2, EMBO Rep 6, 348-353. [21] Schoeftner, S., Sengupta, A. K., Kubicek, S., Mechtler, K., Spahn, L., Koseki, H., Jenuwein, T., and Wutz, A. (2006) Recruitment of PRC1 function at the initiation of X inactivation independent of PRC2 and silencing, EMBO J 25, 3110-3122. [22] Pasini, D., Bracken, A. P., Hansen, J. B., Capillo, M., and Helin, K. (2007) The polycomb group protein Suz12 is required for embryonic stem cell differentiation, Mol Cell Biol 27, 3769-3779. [23] Sarma, K., Margueron, R., Ivanov, A., Pirrotta, V., and Reinberg, D. (2008) Ezh2 requires PHF1 to efficiently catalyze H3 lysine 27 trimethylation in vivo, Mol Cell Biol 28, 2718-2731. [24] Dillon, S. C., Zhang, X., Trievel, R. C., and Cheng, X. (2005) The SET-domain protein superfamily: protein lysine methyltransferases, Genome Biol 6, 227. [25] Morin, R. D., Johnson, N. A., Severson, T. M., Mungall, A. J., An, J., Goya, R., Paul, J. E., Boyle, M., Woolcock, B. W., Kuchenbauer, F., Yap, D., Humphries, R. K., Griffith, O. L., Shah, S., Zhu, H., Kimbara, M., Shashkin, P., Charlot, J. F., Tcherpakov, M., Corbett, R., Tam, A., Varhol, R., Smailus, D., Moksa, M., Zhao, Y., Delaney, A., Qian, H., Birol, I., Schein, J., Moore, R., Holt, R., Horsman, D. E., Connors, J. M., Jones, S., Aparicio, S., Hirst, M., Gascoyne, R. D., and Marra, M. A. (2010) Somatic mutations altering EZH2 (Tyr641) in follicular and diffuse large B-cell lymphomas of germinal-center origin, Nat Genet 42, 181-185. [26] Yap, D. B., Chu, J., Berg, T., Schapira, M., Cheng, S. W., Moradian, A., Morin, R. D., Mungall, A. J., Meissner, B., Boyle, M., Marquez, V. E., Marra, M. A., Gascoyne, R. D., Humphries, R. K., Arrowsmith, C. H., Morin, G. B., and Aparicio, S. A. (2011) Somatic mutations at EZH2 Y641 act dominantly through a mechanism of selectively altered PRC2 catalytic activity, to increase H3K27 trimethylation, Blood 117, 2451-2459. [27] Jones, R. S., and Gelbart, W. M. (1990) Genetic analysis of the enhancer of zeste locus and its role in gene regulation in Drosophila melanogaster, Genetics 126, 185-199. [28] Wu, C. T., Jones, R. S., Lasko, P. F., and Gelbart, W. M. (1989) Homeosis and the interaction of zeste and white in Drosophila, Mol Gen Genet 218, 559-564.


Page 27 of 40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

[29] Joshi, P., Carrington, E. A., Wang, L., Ketel, C. S., Miller, E. L., Jones, R. S., and Simon, J. A. (2008) Dominant alleles identify SET domain residues required for histone methyltransferase of Polycomb repressive complex 2, J Biol Chem 283, 27757-27766. [30] Margueron, R., and Reinberg, D. (2011) The Polycomb complex PRC2 and its mark in life, Nature 469, 343-349. [31] O'Meara, M. M., and Simon, J. A. (2012) Inner workings and regulatory inputs that control Polycomb repressive complex 2, Chromosoma 121, 221-234. [32] Di Croce, L., and Helin, K. (2013) Transcriptional regulation by Polycomb group proteins, Nat Struct Mol Biol 20, 1147-1155. [33] van Kruijsbergen, I., Hontelez, S., and Veenstra, G. J. (2015) Recruiting polycomb to chromatin, Int J Biochem Cell Biol 67, 177-187. [34] Kennison, J. A., and Tamkun, J. W. (1988) Dosage-dependent modifiers of polycomb and antennapedia mutations in Drosophila, Proc Natl Acad Sci U S A 85, 8136-8140. [35] Shearn, A. (1989) The ash-1, ash-2 and trithorax genes of Drosophila melanogaster are functionally related, Genetics 121, 517-525. [36] Young, N. L., DiMaggio, P. A., Plazas-Mayorca, M. D., Baliban, R. C., Floudas, C. A., and Garcia, B. A. (2009) High throughput characterization of combinatorial histone codes, Mol Cell Proteomics 8, 2266-2284. [37] Yuan, W., Xu, M., Huang, C., Liu, N., Chen, S., and Zhu, B. (2011) H3K36 methylation antagonizes PRC2-mediated H3K27 methylation, J Biol Chem 286, 7983-7989. [38] Voigt, P., LeRoy, G., Drury, W. J., 3rd, Zee, B. M., Son, J., Beck, D. B., Young, N. L., Garcia, B. A., and Reinberg, D. (2012) Asymmetrically modified nucleosomes, Cell 151, 181-193. [39] Tie, F., Banerjee, R., Stratton, C. A., Prasad-Sinha, J., Stepanik, V., Zlobin, A., Diaz, M. O., Scacheri, P. C., and Harte, P. J. (2009) CBP-mediated acetylation of histone H3 lysine 27 antagonizes Drosophila Polycomb silencing, Development 136, 3131-3141. [40] Pasini, D., Malatesta, M., Jung, H. R., Walfridsson, J., Willer, A., Olsson, L., Skotte, J., Wutz, A., Porse, B., Jensen, O. N., and Helin, K. (2010) Characterization of an antagonistic switch between histone H3 lysine 27 methylation and acetylation in the transcriptional regulation of Polycomb group target genes, Nucleic Acids Res 38, 4958-4969. [41] Gehani, S. S., Agrawal-Singh, S., Dietrich, N., Christophersen, N. S., Helin, K., and Hansen, K. (2010) Polycomb group protein displacement and gene activation through MSK-dependent H3K27me3S28 phosphorylation, Mol Cell 39, 886-900. [42] Lau, P. N., and Cheung, P. (2011) Histone code pathway involving H3 S28 phosphorylation and K27 acetylation activates transcription and antagonizes polycomb silencing, Proc Natl Acad Sci U S A 108, 2801-2806. [43] Stojic, L., Jasencakova, Z., Prezioso, C., Stutzer, A., Bodega, B., Pasini, D., Klingberg, R., Mozzetta, C., Margueron, R., Puri, P. L., Schwarzer, D., Helin, K., Fischle, W., and Orlando, V. (2011) Chromatin regulated interchange between polycomb repressive complex 2 (PRC2)-Ezh2 and PRC2-Ezh1 complexes controls myogenin activation in skeletal muscle cells, Epigenetics Chromatin 4, 16. [44] Kadoch, C., Hargreaves, D. C., Hodges, C., Elias, L., Ho, L., Ranish, J., and Crabtree, G. R. (2013) Proteomic and bioinformatic analysis of mammalian SWI/SNF complexes identifies extensive roles in human malignancy, Nat Genet 45, 592-601. [45] Lopez, A. J., and Wood, M. A. (2015) Role of nucleosome remodeling in neurodevelopmental and intellectual disability disorders, Front Behav Neurosci 9, 100. [46] Ho, L., Ronan, J. L., Wu, J., Staahl, B. T., Chen, L., Kuo, A., Lessard, J., Nesvizhskii, A. I., Ranish, J., and Crabtree, G. R. (2009) An embryonic stem cell chromatin remodeling complex, esBAF, is essential for embryonic stem cell self-renewal and pluripotency, Proc Natl Acad Sci U S A 106, 5181-5186.


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 28 of 40

[47] Neigeborn, L., and Carlson, M. (1984) Genes affecting the regulation of SUC2 gene expression by glucose repression in Saccharomyces cerevisiae, Genetics 108, 845-858. [48] Stern, M., Jensen, R., and Herskowitz, I. (1984) Five SWI genes are required for expression of the HO gene in yeast, J Mol Biol 178, 853-868. [49] Peterson, C. L., and Herskowitz, I. (1992) Characterization of the yeast SWI1, SWI2, and SWI3 genes, which encode a global activator of transcription, Cell 68, 573-583. [50] Imbalzano, A. N., Kwon, H., Green, M. R., and Kingston, R. E. (1994) Facilitated binding of TATAbinding protein to nucleosomal DNA, Nature 370, 481-485. [51] Kwon, H., Imbalzano, A. N., Khavari, P. A., Kingston, R. E., and Green, M. R. (1994) Nucleosome disruption and enhancement of activator binding by a human SW1/SNF complex, Nature 370, 477-481. [52] Clapier, C. R., and Cairns, B. R. (2009) The biology of chromatin remodeling complexes, Annu Rev Biochem 78, 273-304. [53] Narlikar, G. J., Sundaramoorthy, R., and Owen-Hughes, T. (2013) Mechanisms and functions of ATPdependent chromatin-remodeling enzymes, Cell 154, 490-503. [54] Lickert, H., Takeuchi, J. K., Von Both, I., Walls, J. R., McAuliffe, F., Adamson, S. L., Henkelman, R. M., Wrana, J. L., Rossant, J., and Bruneau, B. G. (2004) Baf60c is essential for function of BAF chromatin remodelling complexes in heart development, Nature 432, 107-112. [55] Yoo, A. S., Staahl, B. T., Chen, L., and Crabtree, G. R. (2009) MicroRNA-mediated switching of chromatin-remodelling complexes in neural development, Nature 460, 642-646. [56] Singhal, N., Graumann, J., Wu, G., Arauzo-Bravo, M. J., Han, D. W., Greber, B., Gentile, L., Mann, M., and Scholer, H. R. (2010) Chromatin-Remodeling Components of the BAF Complex Facilitate Reprogramming, Cell 141, 943-955. [57] Tea, J. S., and Luo, L. (2011) The chromatin remodeling factor Bap55 functions through the TIP60 complex to regulate olfactory projection neuron dendrite targeting, Neural Dev 6, 5. [58] Yoo, A. S., Sun, A. X., Li, L., Shcheglovitov, A., Portmann, T., Li, Y., Lee-Messer, C., Dolmetsch, R. E., Tsien, R. W., and Crabtree, G. R. (2011) MicroRNA-mediated conversion of human fibroblasts to neurons, Nature 476, 228-231. [59] de Bruijn, D. R., Peters, W. J., Chuva de Sousa Lopes, S. M., van Dijk, A. H., Willemse, M. P., Pfundt, R., de Boer, P., and Geurts van Kessel, A. (2006) Targeted disruption of the synovial sarcomaassociated SS18 gene causes early embryonic lethality and affects PPARBP expression, Hum Mol Genet 15, 2936-2944. [60] Krasteva, V., Buscarlet, M., Diaz-Tellez, A., Bernard, M. A., Crabtree, G. R., and Lessard, J. A. (2012) The BAF53a subunit of SWI/SNF-like BAF complexes is essential for hemopoietic stem cell function, Blood 120, 4720-4732. [61] Aoyagi, S., and Hayes, J. J. (2002) hSWI/SNF-catalyzed nucleosome sliding does not occur solely via a twist-diffusion mechanism, Mol Cell Biol 22, 7484-7490. [62] Strohner, R., Wachsmuth, M., Dachauer, K., Mazurkiewicz, J., Hochstatter, J., Rippe, K., and Langst, G. (2005) A 'loop recapture' mechanism for ACF-dependent nucleosome remodeling, Nat Struct Mol Biol 12, 683-690. [63] Saha, A., Wittmeyer, J., and Cairns, B. R. (2002) Chromatin remodeling by RSC involves ATPdependent DNA translocation, Genes Dev 16, 2120-2134. [64] Whitehouse, I., Stockdale, C., Flaus, A., Szczelkun, M. D., and Owen-Hughes, T. (2003) Evidence for DNA translocation by the ISWI chromatin-remodeling enzyme, Mol Cell Biol 23, 1935-1945. [65] Zofall, M., Persinger, J., Kassabov, S. R., and Bartholomew, B. (2006) Chromatin remodeling by ISW2 and SWI/SNF requires DNA translocation inside the nucleosome, Nat Struct Mol Biol 13, 339346.


Page 29 of 40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

[66] Blosser, T. R., Yang, J. G., Stone, M. D., Narlikar, G. J., and Zhuang, X. (2009) Dynamics of nucleosome remodelling by individual ACF complexes, Nature 462, 1022-1027. [67] Saha, A., Wittmeyer, J., and Cairns, B. R. (2005) Chromatin remodeling through directional DNA translocation from an internal nucleosomal site, Nat Struct Mol Biol 12, 747-755. [68] Schwanbeck, R., Xiao, H., and Wu, C. (2004) Spatial contacts and nucleosome step movements induced by the NURF chromatin remodeling complex, J Biol Chem 279, 39933-39941. [69] Dechassa, M. L., Zhang, B., Horowitz-Scherer, R., Persinger, J., Woodcock, C. L., Peterson, C. L., and Bartholomew, B. (2008) Architecture of the SWI/SNF-nucleosome complex, Mol Cell Biol 28, 6010-6021. [70] Leschziner, A. E., Lemon, B., Tjian, R., and Nogales, E. (2005) Structural studies of the human PBAF chromatin-remodeling complex, Structure 13, 267-275. [71] Chaban, Y., Ezeokonkwo, C., Chung, W. H., Zhang, F., Kornberg, R. D., Maier-Davis, B., Lorch, Y., and Asturias, F. J. (2008) Structure of a RSC-nucleosome complex and insights into chromatin remodeling, Nat Struct Mol Biol 15, 1272-1277. [72] Lorch, Y., Maier-Davis, B., and Kornberg, R. D. (2010) Mechanism of chromatin remodeling, Proc Natl Acad Sci U S A 107, 3458-3462. [73] Thoma, N. H., Czyzewski, B. K., Alexeev, A. A., Mazin, A. V., Kowalczykowski, S. C., and Pavletich, N. P. (2005) Structure of the SWI2/SNF2 chromatin-remodeling domain of eukaryotic Rad54, Nat Struct Mol Biol 12, 350-356. [74] Ho, L., and Crabtree, G. R. (2010) Chromatin remodelling during development, Nature 463, 474-484. [75] Laurent, B. C., Treich, I., and Carlson, M. (1993) The yeast SNF2/SWI2 protein has DNA-stimulated ATPase activity required for transcriptional activation, Genes Dev 7, 583-591. [76] Elfring, L. K., Daniel, C., Papoulas, O., Deuring, R., Sarte, M., Moseley, S., Beek, S. J., Waldrip, W. R., Daubresse, G., DePace, A., Kennison, J. A., and Tamkun, J. W. (1998) Genetic analysis of brahma: the Drosophila homolog of the yeast chromatin remodeling factor SWI2/SNF2, Genetics 148, 251-265. [77] Zhao, K., Wang, W., Rando, O. J., Xue, Y., Swiderek, K., Kuo, A., and Crabtree, G. R. (1998) Rapid and phosphoinositol-dependent binding of the SWI/SNF-like BAF complex to chromatin after T lymphocyte receptor signaling, Cell 95, 625-636. [78] Gao, X., Tate, P., Hu, P., Tjian, R., Skarnes, W. C., and Wang, Z. (2008) ES cell pluripotency and germlayer formation require the SWI/SNF chromatin remodeling component BAF250a, Proc Natl Acad Sci U S A 105, 6656-6661. [79] Kaeser, M. D., Aslanian, A., Dong, M. Q., Yates, J. R., 3rd, and Emerson, B. M. (2008) BRD7, a novel PBAF-specific SWI/SNF subunit, is required for target gene activation and repression in embryonic stem cells, J Biol Chem 283, 32254-32263. [80] Kidder, B. L., Palmer, S., and Knott, J. G. (2009) SWI/SNF-Brg1 regulates self-renewal and occupies core pluripotency-related genes in embryonic stem cells, Stem Cells 27, 317-328. [81] Bultman, S., Gebuhr, T., Yee, D., La Mantia, C., Nicholson, J., Gilliam, A., Randazzo, F., Metzger, D., Chambon, P., Crabtree, G., and Magnuson, T. (2000) A Brg1 null mutation in the mouse reveals functional differences among mammalian SWI/SNF complexes, Mol Cell 6, 1287-1295. [82] Reyes, J. C., Barra, J., Muchardt, C., Camus, A., Babinet, C., and Yaniv, M. (1998) Altered control of cellular proliferation in the absence of mammalian brahma (SNF2alpha), EMBO J 17, 6979-6991. [83] Thompson, K. W., Marquez, S. B., Lu, L., and Reisman, D. (2015) Induction of functional Brm protein from Brm knockout mice, Oncoscience 2, 349-361. [84] Lessard, J., Wu, J. I., Ranish, J. A., Wan, M., Winslow, M. M., Staahl, B. T., Wu, H., Aebersold, R., Graef, I. A., and Crabtree, G. R. (2007) An essential switch in subunit composition of a chromatin remodeling complex during neural development, Neuron 55, 201-215.


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 30 of 40

[85] Wu, J. I., Lessard, J., Olave, I. A., Qiu, Z., Ghosh, A., Graef, I. A., and Crabtree, G. R. (2007) Regulation of dendritic development by neuron-specific chromatin remodeling complexes, Neuron 56, 94108. [86] Parrish, J. Z., Kim, M. D., Jan, L. Y., and Jan, Y. N. (2006) Genome-wide analyses identify transcription factors required for proper morphogenesis of Drosophila sensory neuron dendrites, Genes Dev 20, 820-835. [87] Staahl, B. T., Tang, J., Wu, W., Sun, A., Gitler, A. D., Yoo, A. S., and Crabtree, G. R. (2013) Kinetic analysis of npBAF to nBAF switching reveals exchange of SS18 with CREST and integration with neural developmental pathways, J Neurosci 33, 10348-10361. [88] Ieda, M., Fu, J. D., Delgado-Olguin, P., Vedantham, V., Hayashi, Y., Bruneau, B. G., and Srivastava, D. (2010) Direct reprogramming of fibroblasts into functional cardiomyocytes by defined factors, Cell 142, 375-386. [89] Kadoch, C., and Crabtree, G. R. (2013) Reversible disruption of mSWI/SNF (BAF) complexes by the SS18-SSX oncogenic fusion in synovial sarcoma, Cell 153, 71-85. [90] Kadoch, C., and Crabtree, G. R. (2015) Mammalian SWI/SNF chromatin remodeling complexes and cancer: Mechanistic insights gained from human genomics, Science Advances 1, 1-17. [91] Volkel, P., Dupret, B., Le Bourhis, X., and Angrand, P. O. (2015) Diverse involvement of EZH2 in cancer epigenetics, Am J Transl Res 7, 175-193. [92] Schwartzentruber, J., Korshunov, A., Liu, X. Y., Jones, D. T., Pfaff, E., Jacob, K., Sturm, D., Fontebasso, A. M., Quang, D. A., Tonjes, M., Hovestadt, V., Albrecht, S., Kool, M., Nantel, A., Konermann, C., Lindroth, A., Jager, N., Rausch, T., Ryzhova, M., Korbel, J. O., Hielscher, T., Hauser, P., Garami, M., Klekner, A., Bognar, L., Ebinger, M., Schuhmann, M. U., Scheurlen, W., Pekrun, A., Fruhwald, M. C., Roggendorf, W., Kramm, C., Durken, M., Atkinson, J., Lepage, P., Montpetit, A., Zakrzewska, M., Zakrzewski, K., Liberski, P. P., Dong, Z., Siegel, P., Kulozik, A. E., Zapatka, M., Guha, A., Malkin, D., Felsberg, J., Reifenberger, G., von Deimling, A., Ichimura, K., Collins, V. P., Witt, H., Milde, T., Witt, O., Zhang, C., Castelo-Branco, P., Lichter, P., Faury, D., Tabori, U., Plass, C., Majewski, J., Pfister, S. M., and Jabado, N. (2012) Driver mutations in histone H3.3 and chromatin remodelling genes in paediatric glioblastoma, Nature 482, 226-231. [93] Wu, G., Broniscer, A., McEachron, T. A., Lu, C., Paugh, B. S., Becksfort, J., Qu, C., Ding, L., Huether, R., Parker, M., Zhang, J., Gajjar, A., Dyer, M. A., Mullighan, C. G., Gilbertson, R. J., Mardis, E. R., Wilson, R. K., Downing, J. R., Ellison, D. W., Zhang, J., Baker, S. J., and St. Jude Children's Research Hospital-Washington University Pediatric Cancer Genome, P. (2012) Somatic histone H3 alterations in pediatric diffuse intrinsic pontine gliomas and non-brainstem glioblastomas, Nat Genet 44, 251-253. [94] Lewis, P. W., Muller, M. M., Koletsky, M. S., Cordero, F., Lin, S., Banaszynski, L. A., Garcia, B. A., Muir, T. W., Becher, O. J., and Allis, C. D. (2013) Inhibition of PRC2 activity by a gain-of-function H3 mutation found in pediatric glioblastoma, Science 340, 857-861. [95] Chan, K. M., Fang, D., Gan, H., Hashizume, R., Yu, C., Schroeder, M., Gupta, N., Mueller, S., James, C. D., Jenkins, R., Sarkaria, J., and Zhang, Z. (2013) The histone H3.3K27M mutation in pediatric glioma reprograms H3K27 methylation and gene expression, Genes Dev 27, 985-990. [96] Bourdeaut, F., Chi, S. N., and Fruhwald, M. C. (2014) Rhabdoid tumors: integrating biological insights with clinical success: a report from the SMARCB1 and Rhabdoid Tumor Symposium, Paris, December 12-14, 2013, Cancer Genet 207, 346-351. [97] Versteege, I., Sevenet, N., Lange, J., Rousseau-Merck, M. F., Ambros, P., Handgretinger, R., Aurias, A., and Delattre, O. (1998) Truncating mutations of hSNF5/INI1 in aggressive paediatric cancer, Nature 394, 203-206. [98] Hasselblatt, M., Gesk, S., Oyen, F., Rossi, S., Viscardi, E., Giangaspero, F., Giannini, C., Judkins, A. R., Fruhwald, M. C., Obser, T., Schneppenheim, R., Siebert, R., and Paulus, W. (2011) Nonsense


Page 31 of 40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

mutation and inactivation of SMARCA4 (BRG1) in an atypical teratoid/rhabdoid tumor showing retained SMARCB1 (INI1) expression, Am J Surg Pathol 35, 933-935. [99] Margol, A. S., and Judkins, A. R. (2014) Pathology and diagnosis of SMARCB1-deficient tumors, Cancer Genet 207, 358-364. [100] Lee, R. S., Stewart, C., Carter, S. L., Ambrogio, L., Cibulskis, K., Sougnez, C., Lawrence, M. S., Auclair, D., Mora, J., Golub, T. R., Biegel, J. A., Getz, G., and Roberts, C. W. (2012) A remarkably simple genome underlies highly malignant pediatric rhabdoid cancers, J Clin Invest 122, 2983-2988. [101] Wilson, B. G., Wang, X., Shen, X., McKenna, E. S., Lemieux, M. E., Cho, Y. J., Koellhoffer, E. C., Pomeroy, S. L., Orkin, S. H., and Roberts, C. W. (2010) Epigenetic antagonism between polycomb and SWI/SNF complexes during oncogenic transformation, Cancer Cell 18, 316-328. [102] Kia, S. K., Gorski, M. M., Giannakopoulos, S., and Verrijzer, C. P. (2008) SWI/SNF mediates polycomb eviction and epigenetic reprogramming of the INK4b-ARF-INK4a locus, Mol Cell Biol 28, 3457-3464. [103] Knutson, S. K., Warholic, N. M., Wigle, T. J., Klaus, C. R., Allain, C. J., Raimondi, A., Porter Scott, M., Chesworth, R., Moyer, M. P., Copeland, R. A., Richon, V. M., Pollock, R. M., Kuntz, K. W., and Keilhack, H. (2013) Durable tumor regression in genetically altered malignant rhabdoid tumors by inhibition of methyltransferase EZH2, Proc Natl Acad Sci U S A 110, 7922-7927. [104] Wang, X., Sansam, C. G., Thom, C. S., Metzger, D., Evans, J. A., Nguyen, P. T., and Roberts, C. W. (2009) Oncogenesis caused by loss of the SNF5 tumor suppressor is dependent on activity of BRG1, the ATPase of the SWI/SNF chromatin remodeling complex, Cancer Res 69, 8094-8101. [105] Kahali, B., Yu, J., Marquez, S. B., Thompson, K. W., Liang, S. Y., Lu, L., and Reisman, D. (2014) The silencing of the SWI/SNF subunit and anticancer gene BRM in Rhabdoid tumors, Oncotarget 5, 3316-3332. [106] Karnezis, A. N., Wang, Y., Ramos, P., Hendricks, W. P., Oliva, E., D'Angelo, E., Prat, J., Nucci, M. R., Nielsen, T. O., Chow, C., Leung, S., Kommoss, F., Kommoss, S., Silva, A., Ronnett, B. M., Rabban, J. T., Bowtell, D. D., Weissman, B. E., Trent, J. M., Gilks, C. B., and Huntsman, D. G. (2015) Dual loss of the SWI/SNF complex ATPases SMARCA4/BRG1 and SMARCA2/BRM is highly sensitive and specific for small cell carcinoma of the ovary, hypercalcemic type, J Pathol. [107] Le Loarer, F., Watson, S., Pierron, G., de Montpreville, V. T., Ballet, S., Firmin, N., Auguste, A., Pissaloux, D., Boyault, S., Paindavoine, S., Dechelotte, P. J., Besse, B., Vignaud, J. M., Brevet, M., Fadel, E., Richer, W., Treilleux, I., Masliah-Planchon, J., Devouassoux-Shisheboran, M., Zalcman, G., Allory, Y., Bourdeaut, F., Thivolet-Bejui, F., Ranchere-Vince, D., Girard, N., Lantuejoul, S., Galateau-Salle, F., Coindre, J. M., Leary, A., Delattre, O., Blay, J. Y., and Tirode, F. (2015) SMARCA4 inactivation defines a group of undifferentiated thoracic malignancies transcriptionally related to BAF-deficient sarcomas, Nat Genet 47, 1200-1205. [108] Wong, A. K., Shanahan, F., Chen, Y., Lian, L., Ha, P., Hendricks, K., Ghaffari, S., Iliev, D., Penn, B., Woodland, A. M., Smith, R., Salada, G., Carillo, A., Laity, K., Gupte, J., Swedlund, B., Tavtigian, S. V., Teng, D. H., and Lees, E. (2000) BRG1, a component of the SWI-SNF complex, is mutated in multiple human tumor cell lines, Cancer Res 60, 6171-6177. [109] Reisman, D. N., Strobeck, M. W., Betz, B. L., Sciariotta, J., Funkhouser, W., Jr., Murchardt, C., Yaniv, M., Sherman, L. S., Knudsen, E. S., and Weissman, B. E. (2002) Concomitant down-regulation of BRM and BRG1 in human tumor cell lines: differential effects on RB-mediated growth arrest vs CD44 expression, Oncogene 21, 1196-1207. [110] Italiano, A., Keilhack, H., Toulmonde, M., Coindre, J. M., Michot, J. M., Massard, C., Ottesen, L., Reyderman, L., Blakemore, S. J., Kraljevic, S., Thomson, B., McDonald, A., Ho, P. T., and Ribrag, V. (2015) A phase 1 study of EPZ6438 (E7438), an Enhancer of ZesteHomolog 2 (EZH2) inhibitor: Preliminary activity in INI1-negative tumors, Annals of Oncology, ESMO in press.


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 32 of 40

[111] Foulkes, W. D., Clarke, B. A., Hasselblatt, M., Majewski, J., Albrecht, S., and McCluggage, W. G. (2014) No small surprise - small cell carcinoma of the ovary, hypercalcaemic type, is a malignant rhabdoid tumour, J Pathol 233, 209-214. [112] Wilson, B. G., Helming, K. C., Wang, X., Kim, Y., Vazquez, F., Jagani, Z., Hahn, W. C., and Roberts, C. W. (2014) Residual complexes containing SMARCA2 (BRM) underlie the oncogenic drive of SMARCA4 (BRG1) mutation, Mol Cell Biol 34, 1136-1144. [113] Hoffman, G. R., Rahal, R., Buxton, F., Xiang, K., McAllister, G., Frias, E., Bagdasarian, L., Huber, J., Lindeman, A., Chen, D., Romero, R., Ramadan, N., Phadke, T., Haas, K., Jaskelioff, M., Wilson, B. G., Meyer, M. J., Saenz-Vash, V., Zhai, H., Myer, V. E., Porter, J. A., Keen, N., McLaughlin, M. E., Mickanin, C., Roberts, C. W., Stegmeier, F., and Jagani, Z. (2014) Functional epigenetics approach identifies BRM/SMARCA2 as a critical synthetic lethal target in BRG1-deficient cancers, Proc Natl Acad Sci U S A 111, 3128-3133. [114] Vangamudi, B., Paul, T. A., Shah, P. K., Kost-Alimova, M., Nottebaum, L., Shi, X., Zhan, Y., Leo, E., Mahadeshwar, H. S., Protopopov, A., Futreal, A., Tieu, T. N., Peoples, M., Heffernan, T. P., Marszalek, J. R., Toniatti, C., Petrocchi, A., Verhelle, D., Owen, D. R., Draetta, G., Jones, P., Palmer, W. S., Sharma, S., and Andersen, J. N. (2015) The SMARCA2/4 ATPase Domain Surpasses the Bromodomain as a Drug Target in SWI/SNF-Mutant Cancers: Insights from cDNA Rescue and PFI-3 Inhibitor Studies, Cancer Res 75, 3865-3878. [115] Fillmore, C. M., Xu, C., Desai, P. T., Berry, J. M., Rowbotham, S. P., Lin, Y. J., Zhang, H., Marquez, V. E., Hammerman, P. S., Wong, K. K., and Kim, C. F. (2015) EZH2 inhibition sensitizes BRG1 and EGFR mutant lung tumours to TopoII inhibitors, Nature 520, 239-242. [116] Young, N. P., and Jacks, T. (2010) Tissue-specific p19Arf regulation dictates the response to oncogenic K-ras, Proc Natl Acad Sci U S A 107, 10184-10189. [117] Clark, J., Rocques, P. J., Crew, A. J., Gill, S., Shipley, J., Chan, A. M., Gusterson, B. A., and Cooper, C. S. (1994) Identification of novel genes, SYT and SSX, involved in the t(X;18)(p11.2;q11.2) translocation found in human synovial sarcoma, Nat Genet 7, 502-508. [118] Bitler, B. G., Aird, K. M., Garipov, A., Li, H., Amatangelo, M., Kossenkov, A. V., Schultz, D. C., Liu, Q., Shih Ie, M., Conejo-Garcia, J. R., Speicher, D. W., and Zhang, R. (2015) Synthetic lethality by targeting EZH2 methyltransferase activity in ARID1A-mutated cancers, Nat Med 21, 231-238. [119] Helming, K. C., Wang, X., Wilson, B. G., Vazquez, F., Haswell, J. R., Manchester, H. E., Kim, Y., Kryukov, G. V., Ghandi, M., Aguirre, A. J., Jagani, Z., Wang, Z., Garraway, L. A., Hahn, W. C., and Roberts, C. W. (2014) ARID1B is a specific vulnerability in ARID1A-mutant cancers, Nat Med 20, 251-254. [120] Yuan, W., Wu, T., Fu, H., Dai, C., Wu, H., Liu, N., Li, X., Xu, M., Zhang, Z., Niu, T., Han, Z., Chai, J., Zhou, X. J., Gao, S., and Zhu, B. (2012) Dense chromatin activates Polycomb repressive complex 2 to regulate H3 lysine 27 methylation, Science 337, 971-975. [121] Sidoli, S., Schwammle, V., Ruminowicz, C., Hansen, T. A., Wu, X., Helin, K., and Jensen, O. N. (2014) Middle-down hybrid chromatography/tandem mass spectrometry workflow for characterization of combinatorial post-translational modifications in histones, Proteomics 14, 2200-2211. [122] Blackledge, N. P., Farcas, A. M., Kondo, T., King, H. W., McGouran, J. F., Hanssen, L. L., Ito, S., Cooper, S., Kondo, K., Koseki, Y., Ishikura, T., Long, H. K., Sheahan, T. W., Brockdorff, N., Kessler, B. M., Koseki, H., and Klose, R. J. (2014) Variant PRC1 complex-dependent H2A ubiquitylation drives PRC2 recruitment and polycomb domain formation, Cell 157, 1445-1459. [123] Cooper, S., Dienstbier, M., Hassan, R., Schermelleh, L., Sharif, J., Blackledge, N. P., De Marco, V., Elderkin, S., Koseki, H., Klose, R., Heger, A., and Brockdorff, N. (2014) Targeting polycomb to pericentric heterochromatin in embryonic stem cells reveals a role for H2AK119u1 in PRC2 recruitment, Cell Rep 7, 1456-1470.


Page 33 of 40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

[124] Kalb, R., Latwiel, S., Baymaz, H. I., Jansen, P. W., Muller, C. W., Vermeulen, M., and Muller, J. (2014) Histone H2A monoubiquitination promotes histone H3 methylation in Polycomb repression, Nat Struct Mol Biol 21, 569-571. [125] Chen, S., Bohrer, L. R., Rai, A. N., Pan, Y., Gan, L., Zhou, X., Bagchi, A., Simon, J. A., and Huang, H. (2010) Cyclin-dependent kinases regulate epigenetic gene silencing through phosphorylation of EZH2, Nat Cell Biol 12, 1108-1114. [126] Kaneko, S., Li, G., Son, J., Xu, C. F., Margueron, R., Neubert, T. A., and Reinberg, D. (2010) Phosphorylation of the PRC2 component Ezh2 is cell cycle-regulated and up-regulates its binding to ncRNA, Genes Dev 24, 2615-2620. [127] Caretti, G., Di Padova, M., Micales, B., Lyons, G. E., and Sartorelli, V. (2004) The Polycomb Ezh2 methyltransferase regulates muscle gene expression and skeletal muscle differentiation, Genes Dev 18, 2627-2638. [128] Palacios, D., Mozzetta, C., Consalvi, S., Caretti, G., Saccone, V., Proserpio, V., Marquez, V. E., Valente, S., Mai, A., Forcales, S. V., Sartorelli, V., and Puri, P. L. (2010) TNF/p38alpha/polycomb signaling to Pax7 locus in satellite cells links inflammation to the epigenetic control of muscle regeneration, Cell Stem Cell 7, 455-469. [129] Wei, Y., Chen, Y. H., Li, L. Y., Lang, J., Yeh, S. P., Shi, B., Yang, C. C., Yang, J. Y., Lin, C. Y., Lai, C. C., and Hung, M. C. (2011) CDK1-dependent phosphorylation of EZH2 suppresses methylation of H3K27 and promotes osteogenic differentiation of human mesenchymal stem cells, Nat Cell Biol 13, 87-94. [130] Wu, S. C., and Zhang, Y. (2011) Cyclin-dependent kinase 1 (CDK1)-mediated phosphorylation of enhancer of zeste 2 (Ezh2) regulates its stability, J Biol Chem 286, 28511-28519. [131] Cha, T. L., Zhou, B. P., Xia, W., Wu, Y., Yang, C. C., Chen, C. T., Ping, B., Otte, A. P., and Hung, M. C. (2005) Akt-mediated phosphorylation of EZH2 suppresses methylation of lysine 27 in histone H3, Science 310, 306-310. [132] Xu, K., Wu, Z. J., Groner, A. C., He, H. H., Cai, C., Lis, R. T., Wu, X., Stack, E. C., Loda, M., Liu, T., Xu, H., Cato, L., Thornton, J. E., Gregory, R. I., Morrissey, C., Vessella, R. L., Montironi, R., MagiGalluzzi, C., Kantoff, P. W., Balk, S. P., Liu, X. S., and Brown, M. (2012) EZH2 oncogenic activity in castration-resistant prostate cancer cells is Polycomb-independent, Science 338, 1465-1469. [133] Landeira, D., and Fisher, A. G. (2011) Inactive yet indispensable: the tale of Jarid2, Trends Cell Biol 21, 74-80. [134] Kaneko, S., Bonasio, R., Saldana-Meyer, R., Yoshida, T., Son, J., Nishino, K., Umezawa, A., and Reinberg, D. (2014) Interactions between JARID2 and noncoding RNAs regulate PRC2 recruitment to chromatin, Mol Cell 53, 290-300. [135] Ballare, C., Lange, M., Lapinaite, A., Martin, G. M., Morey, L., Pascual, G., Liefke, R., Simon, B., Shi, Y., Gozani, O., Carlomagno, T., Benitah, S. A., and Di Croce, L. (2012) Phf19 links methylated Lys36 of histone H3 to regulation of Polycomb activity, Nat Struct Mol Biol 19, 1257-1265. [136] Brien, G. L., Gambero, G., O'Connell, D. J., Jerman, E., Turner, S. A., Egan, C. M., Dunne, E. J., Jurgens, M. C., Wynne, K., Piao, L., Lohan, A. J., Ferguson, N., Shi, X., Sinha, K. M., Loftus, B. J., Cagney, G., and Bracken, A. P. (2012) Polycomb PHF19 binds H3K36me3 and recruits PRC2 and demethylase NO66 to embryonic stem cell genes during differentiation, Nat Struct Mol Biol 19, 1273-1281. [137] Cai, L., Rothbart, S. B., Lu, R., Xu, B., Chen, W. Y., Tripathy, A., Rockowitz, S., Zheng, D., Patel, D. J., Allis, C. D., Strahl, B. D., Song, J., and Wang, G. G. (2013) An H3K36 methylation-engaging Tudor motif of polycomb-like proteins mediates PRC2 complex targeting, Mol Cell 49, 571-582. [138] Qin, S., Guo, Y., Xu, C., Bian, C., Fu, M., Gong, S., and Min, J. (2013) Tudor domains of the PRC2 components PHF1 and PHF19 selectively bind to histone H3K36me3, Biochem Biophys Res Commun 430, 547-553.


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 34 of 40

[139] Cao, R., Wang, H., He, J., Erdjument-Bromage, H., Tempst, P., and Zhang, Y. (2008) Role of hPHF1 in H3K27 methylation and Hox gene silencing, Mol Cell Biol 28, 1862-1872. [140] Walker, E., Chang, W. Y., Hunkapiller, J., Cagney, G., Garcha, K., Torchia, J., Krogan, N. J., Reiter, J. F., and Stanford, W. L. (2010) Polycomb-like 2 associates with PRC2 and regulates transcriptional networks during mouse embryonic stem cell self-renewal and differentiation, Cell Stem Cell 6, 153-166. [141] Casanova, M., Preissner, T., Cerase, A., Poot, R., Yamada, D., Li, X., Appanah, R., Bezstarosti, K., Demmers, J., Koseki, H., and Brockdorff, N. (2011) Polycomblike 2 facilitates the recruitment of PRC2 Polycomb group complexes to the inactive X chromosome and to target loci in embryonic stem cells, Development 138, 1471-1482. [142] Li, X., Isono, K., Yamada, D., Endo, T. A., Endoh, M., Shinga, J., Mizutani-Koseki, Y., Otte, A. P., Casanova, M., Kitamura, H., Kamijo, T., Sharif, J., Ohara, O., Toyada, T., Bernstein, B. E., Brockdorff, N., and Koseki, H. (2011) Mammalian polycomb-like Pcl2/Mtf2 is a novel regulatory component of PRC2 that can differentially modulate polycomb activity both at the Hox gene cluster and at Cdkn2a genes, Mol Cell Biol 31, 351-364. [143] Zhang, Z., Jones, A., Sun, C. W., Li, C., Chang, C. W., Joo, H. Y., Dai, Q., Mysliwiec, M. R., Wu, L. C., Guo, Y., Yang, W., Liu, K., Pawlik, K. M., Erdjument-Bromage, H., Tempst, P., Lee, Y., Min, J., Townes, T. M., and Wang, H. (2011) PRC2 complexes with JARID2, MTF2, and esPRC2p48 in ES cells to modulate ES cell pluripotency and somatic cell reprogramming, Stem Cells 29, 229-240. [144] Zhao, J., Sun, B. K., Erwin, J. A., Song, J. J., and Lee, J. T. (2008) Polycomb proteins targeted by a short repeat RNA to the mouse X chromosome, Science 322, 750-756. [145] da Rocha, S. T., Boeva, V., Escamilla-Del-Arenal, M., Ancelin, K., Granier, C., Matias, N. R., Sanulli, S., Chow, J., Schulz, E., Picard, C., Kaneko, S., Helin, K., Reinberg, D., Stewart, A. F., Wutz, A., Margueron, R., and Heard, E. (2014) Jarid2 Is Implicated in the Initial Xist-Induced Targeting of PRC2 to the Inactive X Chromosome, Mol Cell 53, 301-316. [146] Rinn, J. L., Kertesz, M., Wang, J. K., Squazzo, S. L., Xu, X., Brugmann, S. A., Goodnough, L. H., Helms, J. A., Farnham, P. J., Segal, E., and Chang, H. Y. (2007) Functional demarcation of active and silent chromatin domains in human HOX loci by noncoding RNAs, Cell 129, 1311-1323. [147] Tsai, M. C., Manor, O., Wan, Y., Mosammaparast, N., Wang, J. K., Lan, F., Shi, Y., Segal, E., and Chang, H. Y. (2010) Long noncoding RNA as modular scaffold of histone modification complexes, Science 329, 689-693. [148] Zhao, J., Ohsumi, T. K., Kung, J. T., Ogawa, Y., Grau, D. J., Sarma, K., Song, J. J., Kingston, R. E., Borowsky, M., and Lee, J. T. (2010) Genome-wide identification of polycomb-associated RNAs by RIP-seq, Mol Cell 40, 939-953. [149] Davidovich, C., Zheng, L., Goodrich, K. J., and Cech, T. R. (2013) Promiscuous RNA binding by Polycomb repressive complex 2, Nat Struct Mol Biol 20, 1250-1257. [150] Kaneko, S., Son, J., Shen, S. S., Reinberg, D., and Bonasio, R. (2013) PRC2 binds active promoters and contacts nascent RNAs in embryonic stem cells, Nat Struct Mol Biol 20, 1258-1264. [151] Cifuentes-Rojas, C., Hernandez, A. J., Sarma, K., and Lee, J. T. (2014) Regulatory interactions between RNA and polycomb repressive complex 2, Mol Cell 55, 171-185.


Page 35 of 40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42

Biochemistry

SAM

H3K27me

SAH

H3K27me2

H3K27

H3K27me3


Core PRC2 Complex 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 Loss 36 37 MPNST 38 39 40 41 42

Gain Breast (Ampl.)

Loss T-ALL MPNST

Biochemistry

Other EST (translocations)

Page 36 of 40

Additional PRC2 Subunits

Cancer Relevance

RBBP7 (RBAP46)

Gain: Prostate (Ampl.)

RBBP4 (RBAP48)

Gain: Breast, Ovarian (Ampl.) Loss: MPNST, pancreatic

AEBP2

Gain (Ampl.): Breast, Ovarian, Prostate

PRC2 Associated Factors PCLs • PHF19 • PHF1 • MTF2

Cancer Relevance PHF19: Gain: over-expressed in colon, skin, lung, rectal, cervical, uterus, and liver cancers PHF1: Loss: reduced expression in breast cancer Other: translocations in EST MTF2: unclear

Gain (Ampl.) Breast Ovarian

JARID2

Gain: breast (Ampl.), overexpressed in RMS Loss: CML (at transformation)

C17orf96

unclear

Gain (Mutation) Loss B-NHL MDS ncRNAs, for Melanoma MF example HOTAIR Ewing sarcoma CMML Gain (Ampl.) JMML T-ALL ACS Breast Paragon Plus Environment Ovarian Prostate

Gain: Overexpressed in many cancers, including gastric, breast and colorectal

Page 37 of 40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42

Biochemistry


Biochemistry

Page 38 of 40

1 2 3 4 Ovarian Clear Cell Carcinoma 5 6 Endometrioid Carcinoma 7 Bladder cancer 8 Clear Cell Renal Cell Neuroblastoma 9 Carcinoma (ccRCC) Hepatocellular Carcinoma 10 Leukemia/Lymphoma 11 Head and Neck Cancer 12 Myeloma 13 BCL7 14 Spinal/cranial (familial) 15 A,B,C BCL11 PBRM1 Synovial clear cell meningioma A,B 16 PHF10 BRD9 DPF2 BRD7 Sarcoma BCL7 17 18 A,B,C 19 ARID1A SS18 SS18 20 ARID2 ARID1B 21SMARCD1 ACTL6A ACTL6A SMARCD1 22 ACTB SMARCE1 SMARCE1 2 2 23 3 3 SMARCB1 24 SMARCB1 25 Malignant Rhabdoid SMARCC1 SMARCC1 SMARCA4 26 SMARCA4 Tumor SMARCA2 SMARCC2 SMARCC2 27 Epithelioid Sarcoma 28 29 30 Undifferentiated Thoracic Sarcoma (subset) 31 Breast Cancer 32 Lung Cancer 33 34 35 36 37 38 39 ACS Paragon Plus Environment 40 41 42

mSWI/SNF (BAF) Complex

ACTB

Polybromo-associated BAF Complex (PBAF)

Page 39 of 40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42

Biochemistry

mSWI/SNF Complexes

PRC2 Complex SMARCB1- and SMARCA4-deficient cancers: • Malignant rhabdoid tumors (MRT) • Atypical teratoid rhabdoid tumors(AT/RT) • Epithelioid sarcoma • Small cell carcinoma of the ovary, hypercalcemic type SCCOHT


EZH2i

Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42

Page 40 of 40

Striking the Right Balance

SWI/SNF

PRC2


SNF Chromatin Remodeling Complexes in Health and

Recommend Documents