INO80 and SWR complexes: relating structure to function in chromatin remodeling

Review

Special Issue: Chromatin Dynamics

INO80 and SWR complexes: relating structure to function in chromatin remodeling Christian B. Gerhold1 and Susan M. Gasser1,2 1 2

Friedrich Miescher Institute for Biomedical Research, Maulbeerstrasse 66, CH-4058 Basel, Switzerland Faculty of Natural Sciences, University of Basel, Basel, Switzerland

Virtually all DNA-dependent processes require selective and controlled access to the DNA sequence. Governing this access are sophisticated molecular machines, nucleosome remodelers, which regulate the composition and structure of chromatin, allowing conversion from open to closed states. In most cases these multisubunit remodelers operate in concert to organize chromatin structure by depositing, moving, evicting, or selectively altering nucleosomes in an ATP-dependent manner. Despite sharing a conserved domain architecture, chromatin remodelers differ significantly in how they bind to their nucleosomal substrates. Recent structural studies link specific interactions between nucleosomes and remodelers to the diverse tasks they carry out. We review here insights into the modular organization of the INO80 family of nucleosome remodelers. Understanding their structural diversity will help to shed light on how these related ATPases modify their nucleosomal substrates. Dynamic chromatin structures Given their large size, eukaryotic genomes face several challenges that prokaryotic genomes do not. The first is the massive compaction required to fit the genome into the nuclear compartment. The second is the selective modulation of this compact structure that is needed to regulate gene expression. Third, the alteration of transcriptional profiles that occurs during cell differentiation and development. Cell type identity depends at least in part on the interplay of primary sequence with factors that interfere with or promote higher levels of chromatin compaction. This epigenetic regulation involves DNA methylation as well as histone modification, nucleosome packing, and precise nucleosome positioning. Together these enable the multiplicity of gene responses and expression patterns that are observed in different cell types. To form a nucleosome, DNA is wrapped around a histone octamer almost twice [1]. Higher-order folding of the nucleosomal fiber is likely to involve a range of fiber Corresponding author: Gasser, S.M. ([email protected]). Keywords: chromatin remodeling; nucleosome binding; SWR-C; INO80-C; Arp subcomplexes. 0962-8924/ ß 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tcb.2014.06.004

conformations (reviewed in [2]) which collectively solve the challenge of fitting DNA into the nucleus. Accessibility to sequence requires the reversal of a compacted chromatin structure and its re-establishment in a temporally and spatially controlled manner. We summarize here recent findings on two important regulators of nucleosome and DNA accessibility – the yeast chromatin remodeling complexes SWR-C (see Glossary) and INO80-C (SRCAP and hINO80 complexes in human) – to gain insight on how their structures and nucleosome-binding modes influence their ultimate enzymatic function. Glossary AAA+: ATPase associated with diverse cellular activities. ACF: ATP-utilizing chromatin assembly and remodeling factor (ISWI-type remodeler). Arp: actin-related protein. Protein with structural similarity to actin. BTAF1: B-TFIID transcription factor-associated (Mot1 homolog in humans). CHD: chromodomain helicase DNA-binding. ATP-dependent remodeler with SANT and SLIDE domains. ChIP-exo: chromatin immunoprecipitation coupled with exonuclease to determine protein–DNA binding sites with high resolution. Gcn5: general control nonderepressible. Functions as a HAT. HAT: histone acetyltransferase. HDACs: histone deacetylases. HAND: the HAND domain resembles an open hand and, together with SANT and SLIDE domains, functions as a nucleosome-spacing domain. HSA: helicase-SANT-associated domain (recruits Arps and actin to the remodeler). Ies: Ino eighty subunit. Ies1–6 are specific subunits of budding yeast INO80. Ies2 and Ies6 are conserved in man. INO80: inositol-requiring 80. ATP-dependent multisubunit chromatin remodeler. Mot1: modifier of transcription 1. ATP-dependent chromatin remodeler. Does not remodel nucleosomes but the interaction between DNA and TATA box binding proteins (TBPs) and TBPs themselves. NCP: nucleosome core particle. Histone octamer with at least 146 bp of DNA. PBAF: Polybromo, Brg1-associated factors (RSC homolog in humans). PTH: post HSA domain (domain between HSA and Snf2 ATPase, putative regulatory role). QAOS: quantitative amplification of ssDNA. RSC: remodels the structure of chromatin (SWI/SNF-type remodeler). RvB: RuvB-like proteins. AAA+ ATPases with an oligonucleotide-binding fold. SANT: SWI3, ADA2, N-COR, TFIIB. Involved in nucleosome spacing. SLIDE: SANT-like ISWI domain. Involved in nucleosome spacing. Snf2: sucrose non-fermenting-type ATPase. Similar to ATPase sequences of chromatin remodelers. SRCAP: SNF2-related CBP activator protein (SWR-C in humans). Sth1: Snf two homolog 1. Snf2 ATPase subunit of the RSC remodeling complex. SWI/SNF: yeast mating-type switching and sucrose non-fermenting. SWR-C: sick with RSC/Rat1 complex. ATP-dependent multisubunit chromatin remodeler.

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which specifically displaces the TATA box binding protein (TBP) from promoter DNA [7]. One subclass of the Snf2-like ATPases includes both Ino80 and Swr1, and each serves as a scaffold for an array of different subunits that form semi-stable subcomplexes of their own. The Ino80- and Swr1-containing holocomplexes (INO80-C and SWR-C, respectively) share several subunits, although the majority of subunits are distinct. Their accessory components are essential for remodeler function and contribute to the modular architecture of the Ino80/Swr1 family (Figure 1C). Common to the Ino80 and Swr1 ATPases is a long insertion within their Snf2-ATPase domain, which is responsible for the recruitment of the Rvb1/2 helicase, a subcomplex found in both remodelers [8–10]. Rvb1/2 proteins form a hexameric AAA+ ATPase, related to the microbial RuvB, which facilitates the migration of strandexchange structures during recombination in bacteria [11]. A further prominent feature shared by Ino80 and Swr1 is the helicase SANT-associated (HSA) domain which is required for the recruitment of actin and actin-related proteins (Arp) [12]. Both, INO80-C and SWR-C contain actin and Arp4 as subunits, as well as other complex-specific Arp

ATP-dependent chromatin-remodeling enzymes: the Ino80 and Swr1 subfamily ATP-dependent chromatin remodelers act in concert to regulate fluctuations in chromatin organization, often in response to post-translational modifications (PTMs) on histones or other acute signaling pathways that trigger DNA repair, changes in gene expression, or other DNAbased events that are necessary for progression through the cell cycle. Remodelers are thus vital housekeepers as well as specific effectors of epigenetic regulation [3,4]. All remodelers act through a Snf2 (sucrose non-fermenting)type ATPase which forms a large family of ATPases subordinate to the SF2 helicase superfamily (Figure 1A,B) [5]. This catalytic domain serves as a motor that functionally alters DNA–protein interactions, and accessory modules are often required for regulation of its functionality (Box 1). Indeed, most nucleosome remodelers function as large, multisubunit molecular machines whose subunit complexity helps them to deliver a broad range of remodeling activities (reviewed in [4,6]). Whereas the substrate of such remodelers is usually the nucleosome, there are some notable exceptions, such as Mot1 (BTAF1 in humans),

Helicases

(A)

(B) INO80/SWR1

Superfamily SF1

SF2

SF3

SWI/SNF Family DEADbox helicases

RecG helicases

DEAH/DExH helicases

TypeI/III restriconenzymes

Snf2

Snf2 ATPases CHD

Subfamily Snf2-like

Ino80-like

Mot1-like

Rad54-like

Rad5/16-like

SMARCAL/distant

ISWI Group EP400

Ino80

Swr1

Etl1/Fun30

(C)

RvB1/RvB2 heterododecamer

(D)

Head Nhp10 module (body) Ies5

Ies3

Nhp10

INO80-C

Arp8 module(foot) Arp5 module (neck)

Taf14 Arp8

Arp5

Ies4

Arp5.com Ies6

Nhp10 module

Ies2

Ies1

Ino80

HSA

PTH

Dexx /RecA1

x6 acn

Arp4

HELICc /RecA2

Insert

Arp8 module

Foot

RvB2

RvB1/RvB2 heterohexamer

RvB1 x3

Swr1

HSA

H2A.Zbinding

Dexx /RecA1

Insert

Neck

Body

HELICc /RecA2

C-module Swc5

Bdf1 Swc4

N-module Swc3

Swc7

Yaf9

Swc2 Arp6

SWR-C

Swr1

Swc6 C-module

TRENDS in Cell Biology

Figure 1. Diversity among ATP-dependent chromatin remodelers. There are two main classifications for Snf2 type ATPase remodelers in the literature. These are either based on sequence conservation or on subunit composition and functionality. INO80-type remodelers constitute their own group in both classifications based on the insertion sequence in their ATPase domain. (A) Snf2 ATPases belong to the superfamily 2 of helicases and can be further subclassified according to the sequence of their ATPase domain. The remodelers ISWI, CHD, and SWI/SNF, that constitute separate groups in an alternative classification, belong to the group of Snf2-like remodelers. (B) An alternative classification of the remodelers according to subunit composition and functionality is shown. Snf2-like remodelers are split into separate groups of SWI/SNF, CHD, and ISWI. (C) Similar domain architectures and shared subunits of INO80 (upper) and SWR-C (lower) would suggest similar topology of these chromatin remodelers. (D) SWR-C and INO80-C show very diverse topologies based on electron microscopy (EM) structures.

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Review Box 1. Regulation of the Ino80 Snf2 ATPase  Snf2-like ATPases share translocase activity that is essential for the remodeling reaction [94].  INO80-type remodeler family members contain a sequence insertion in their Snf2 ATPase domains [95].  This insertion recruits the RvB1/2 hetero-oligomer and either Arp5–Ies6 (INO80-C) or the C-module (SWR-C) to the remodeler [15].  INO80-C has two ARP modules: Arp5–Ies6 and Arp8–Arp4–actin [9,14].  Both ARP modules and the Nhp10 module contribute to nucleosome binding [14,27].  Ino80 ATPase activity is stimulated by nucleosomes in an Arp5 module-dependent manner, whereas the Arp8 module is required for DNA-dependent stimulation [14].  We suggest that Arp5–Ies6 couples the nucleosome to the remodeler whereas Arp8–Arp4–actin accentuates the force of ATP hydrolysis for remodeling activity.  Remodeling activity is absent in INO80-C lacking the Arp5 module and is strongly impaired in INO80-C lacking Arp8–Arp4–actin. Loss of the Nhp10 module does not significantly reduce nucleosome remodeling [14].

proteins. Given the number of shared subunits and their similarity in primary sequence, one might predict that INO80-C and SWR-C would have highly analogous topologies. Surprisingly, recent work suggests that this is not the case. Structural insights into the INO80-C and SWR-C complexes: related, but different Recent electron microscopy (EM) structures of INO80-C and SWR-C revealed that the topologies of the holocomplexes are strikingly different [13,14]. Whereas SWR-C appeared to be very compact, INO80-C formed an elongated embryo-like shape with a head–neck–body–foot topology (Figure 1D). The most striking and perhaps most unanticipated difference concerns the RvB module: INO80-C harbors a double-ring heterododecamer, whereas SWR-C contains a single-ring heterohexamer. Moreover, two INO80-C-specific subunits, Arp5 and Ies6 (Ino eighty subunit 6), form a module (Sebastian Fenn, PhD thesis, Ludwig-Maximilians-Universita¨t Mu¨nchen, 2011) that physically associates with the RvB1/2 proteins in the INO80-C complex [14,15]. By contrast, deletion of the insertion in the ATPase subunit of Swr1 leads to loss of RvB1/2 and the C-module (Arp6, Swc2, Swc3, and Swc6) [8] (Figure 1C). The fact that INO80-C harbors a RvB subcomplex that is twice the size of that bound to SWR-C argues for context-specific assembly of the RvB helicase, even though its oligomeric composition depends on intrinsic structural folds (oligonucleotide-binding folds) [16]. These differences suggest that RvB proteins serve different roles within these remodeling complexes. In addition to the divergence in RvB stoichiometry, the overall contours of INO80-C and SWR-C differ significantly. The EM reconstructions of the remodeler complexes are supported by lysine-specific crosslinking and mass spectrometry of linked peptides [13,14]. Crosslinking techniques provide distance constraints and argue that, in INO80-C, Arp4 must be located near the Arp8 module in the foot structure, distant from RvB1 which is in the head (Figure 1C,D). By contrast, in SWR-C, RvB1 formed

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crosslinks to Arp4 and the HSA domain of Swr1. This underscores major differences in the topologies of INO80-C and SWR-C. It should be noted that the flexibility of these multiple-subunit molecular machines is a major challenge to structural studies, and requires that complexes are chemically fixed before structural analysis. Therefore, the currently available structures of SWR-C and INO80-C most likely represent only one of several possible conformations and compositions. Depending on the substrate bound or the stage of the reaction, SWR-C and INO80-C may therefore resemble each other more closely in some conformations. Clinging, grasping, or engulfing the nucleosomal substrate Interactions between INO80-C and the nucleosome core particle (NCP) were deduced from a combination of crosslinking data and class averages of negatively stained EM images of the INO80–nucleosome complex [14]. In parallel, the 3D SWR-C structure bound to a NCP was itself reconstructed from EM images [13]. From these structures it was obvious that their modes of nucleosome binding were very distinct. The way SWR-C and INO80-C interact with nucleosomes also differed from that shown for other remodelers (Figure 2). We summarize these differences below. The structures of the SWI/SNF subfamily remodelers (RSC, PBAF, and SWI/SNF) reveal a NCP binding cavity which at least partially engulfs the nucleosomal substrate [17–19] (Figure 2A). In contrast to SWI/SNF remodelers,

(A)

(B)

RSC Nucleosome engulfing

ISWI Nucleosome ruler ATPase domain

ISWI

SNF2h (ACF)

Sth1

(C)

SWR-C

(D)

Nucleosome clinging

INO80-C Nucleosome grasping

Swr1 RvB1/2 module

N-module C-module

Arp8 module -

RvB1/2 module

Nhp10 module

Arp5–Ies6 module

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Figure 2. Different nucleosome-binding modes of chromatin remodelers. (A) SWI/ SNF-type remodelers engulf the NCP with a central cavity that accommodates the nucleosome. (B) ISWI-type remodelers bind adjacent to nucleosomes to function as molecular rulers for DNA linker length. They space nucleosomes with respect to one another. Two different models have been proposed: ISWI uses a dinucleosome as a substrate to alter linker length and ACF functions as dimeric motor to move a central nucleosome. Both models can be merged into one hypothesis in which two ISWI-type remodelers work on a trinucleosome substrate to regulate DNA linker length. (C) SWR-C clings to chromatin. The structure of the remodeler opens up upon binding to the nucleosome. One side of the NCP remains exposed and most of the contacts are mediated by the Swr1 scaffold protein. (D) INO80-C grasps the nucleosome. The foot/Arp8 module of INO80-C undergoes a major structural rearrangement to bind to one side of the nucleosome while the other side faces the RvB1/2 module. The Nhp10 module holds the nucleosome in place.

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Review ISWI-type remodelers have been shown to interact with nucleosomes in two distinct ways. In one model – based on the human ISWI remodeler ACF, which contains the Snf2h ATPase bound to Acf1 – two remodeler complexes are thought to bind to a single nucleosome, forming a bidirectional motor that allows proper nucleosomal spacing through coordinated ATPase interplay. This is supported by the 3D EM reconstruction in the presence of an activated ATP mimic (Figure 2B) [20]. In apparent contradiction to this, X-ray crystallography and EM of the nucleosomespacing module of the related yeast ISW1a suggested that one yeast ISWI remodeler simultaneously interacts with two adjoining nucleosomes to regulate their spacing [21]. This may indicate that ISWI remodelers function differently in the two species. Alternatively, one can try to merge the two sets of evidence into a common mechanism by suggesting that two ISWI-type remodelers work in concert as a dimeric ruler to regulate DNA linker length on either side of a central nucleosome. In either model, the DNAbinding HAND–SANT–SLIDE domain of the ATPase appears to be crucial for mediating nucleosome spacing [21,22]. A similar mode of action may hold true for CHD-type remodelers, which also contain SANT and SLIDE domains within the catalytic Chd1 subunit. Interestingly, these substructures were only revealed within Chd1 once it was crystallized, and not by sequence alignment [23]. Although the mode of action for nucleosome sliding on isolated templates is fairly well established (summarized in [24]), it remains a challenge to determine how these remodelers function at the molecular level on the higher-order chromatin substrates that they encounter in living cells. In contrast to the above mechanisms, SWR-C and INO80-C bind to their nucleosomal substrates in distinct ways. Rather than engulfing the nucleosome, SWR-C appears to cling to its substrate through the Swr1 ATPase, which subtly opens the compact structure of the remodeler. Swr1 itself provides most of the molecular contacts between the remodeler and nucleosome, which has one face of the octamer exposed and one in contact with SWR-C (Figure 2C) [13]. To accommodate this mode of binding, SWR-C may need to hang on to nucleosomes through accessible linker DNA [25]. Although the C-module of SWR-C is important for NCP binding and dimer exchange [8], it contributes only marginally to the interface between the remodeler and its substrate [13]. It is proposed that a structural rearrangement of SWR-C, mediated through its C-module, may be necessary for nucleosome binding during dimer exchange [13]. Thus, it is likely that the holoSWR-C complex is able to bind to more than one nucleosome, or else binds simultaneously to a nucleosome and a histone dimer by making use of different domains. INO80-C appears to bind to the NCP through a distinct mechanism. This remodeler neither engulfs the nucleosome, nor clings peripherally to a chromatin array, but instead appears to grasp its substrate ‘like a hand’, employing a subdomain of the holocomplex (Figure 2D). In contrast to SWR-C, which expands its structure upon nucleosome binding, the INO80-C–NCP complex appears more highly compacted. Crosslinking studies place the nucleosome squarely between the INO80-C head and 622

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flexible foot modules [14]. The Nhp10 module positions the NCP towards the RvB1/2 dodecamer while the Arp8 module encases the other side of the nucleosome (Figure 2D). We note that, even though the structural data on INO80-C and SWR-C assign one nucleosome-binding site in each remodeler, it is possible that both SWR-C and INO80-C bind to two or more nucleosomes under physiological conditions. Moreover, as discussed below, there could be changes in conformation that occur as the remodelers act on their substrates. Consistent with this reflection, the chromatin-remodeler SWI/SNF is known to act preferentially on dinucleosomes, although there is no clear model describing how it would bind to two NCPs [26]. Multiple chromatin-binding modules allow flexible relationships between nucleosome and remodeler Both INO80-C and SWR-C contain more than one module that can bind to nucleosomes [8,14,25,27]. Tables 1 and 2 list the proteins that have significant affinity for DNA or histones, individually or in subcomplexes within INO80-C or SWR-C. These modules can be expressed and purified as discrete subcomplexes, and most likely act in concert to recruit the holo-remodeler to its chromatin substrate. Two general principles explain how these multiple chromatinbinding domains might work. The first principle is that of concerted chromatin binding, in which several subunits concomitantly contribute to chromatin association of the remodelers. The second is conditional or transitory chromatin-binding modes. Concerted binding by multiple subunits Both SWR-C and INO80-C influence the genomic distribution of the histone variant H2A.Z (Htz1 in yeast) [28–34]. Notably, SWR-C deposits H2A.Z in a stepwise manner [32], whereas INO80-C is thought to help to evict this histone variant, particularly at non-promoter sites [33]. Within SWR-C, Swc2 and Swr1 are largely responsible for H2A.Z affinity [8,35,36]. Insights into the biochemistry of the SWR-C-catalyzed exchange reaction suggest that Swc2 acts as a molecular lock, preventing H2A.Z removal from the nucleosome [37]. In other words, the H2A.Z binding of Swr1 most likely incorporates H2A.Z into nucleosomes, whereas Swc2 binding of H2A.Z appears to prevent the reverse reaction. Evidence that the concerted action of Swr1 and Swc2 is important stems from the fact that H2A.Z deposition in vivo is lost not only in swr1D but also in swc2D mutants [38]. PTMs of histones in nucleosomes containing H2A.Z also seem to be important for the action of either SWR-C or INO80-C [33,37]. Acetylation of histone H3 at K56 has a major impact on the variant exchange reaction in vitro. This specific modification appears to open the Swc2 lock, allowing reversal of the enzymatic activity, and leading to H2A.Z eviction and H2A incorporation by SWR-C [37]. H3K56ac also stimulates the eviction of H2A.Z by INO80-C [33,37], and thus both may eventually lead to the depletion of H2A.Z-containing nucleosomes from newly replicated chromatin. The contributions of other remodeler subunits in reading histone PTMs remain largely unexplored, but two

Review

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Table 1. Composition and chromatin binding by INO80-C S. cerevisiae INO80-C Subunits organized in Subunit characteristics subcomplexes Arp8 Binds weakly to dsDNA, H2A–H2B dimers, H3–H4 tetramers, Arp8 or whole nucleosomes, possibly as an Arp8 homodimer. foot module [52,96]. Mammalian Arp8 helps to recruit INO80-C to DNA damage [42]. Also a subunit of SWR-C and NuA4 complexes. Displays Arp4 higher affinity for H3–H4 tetramers than for nucleosomes [52]. Helps to recruit complexes to DNA damage in yeast [41]. Actin Integral subunit of complexes SWR-C, INO80-C, and NuA4. Involved in chromatin affinity and remodeling efficiency in INO80-C [97]. Taf14 YEATS-domain protein and integral subunit of several chromatin associated complexes. May bind to acetylated histones [39]. Ies4 No chromatin-binding role, but is a target of Mec1. Phosphorylated upon DNA damage [92]. Nhp10 High mobility group protein that binds to Isw2 and Nhp10 recognizes distorted DNA ends [98]. Implicated in stable body module INO80-C to gH2AX binding [83]. Enigmatic protein that accumulates in the cytosol upon Ies1 hypoxia [99]. Has affinity for several ribosome-associated proteins [100] but no chromatin interactions have been reported to date. Ies3 Two-hybrid interaction of Ies3 and the telomerase protein Est1 [101]. Affinity capture MS suggests an interaction with Isw2 [102]. Ies5 No chromatin-binding properties reported, but the mutant has synergistic growth defects with Rad50, Rad51, and Rad54 (DNA repair) [83]. Arp5 At high resolution, Arp5 has a slightly different chromatin Arp5 occupancy than INO80-C [78]. neck module May recruit Arp5 to the INO80-C complex [14]. No clear Ies6 chromatin-binding reported. RvB1 AAA+ ATPase, binds to ss and dsDNA and RNA. Has 30 to 50 RvB1/RvB2 helicase activity and an insertion into the AAA+ domain head module resembles the OB-fold of RPA [103]. RvB2 Closely related to RvB1. Potential structural role within the INO80-C complex [14]. Scaffold ATPase Ies2 Mammalian Ies2 regulates the Ino80 ATPase [10]. Ino80 A scaffold protein with Snf2 ATPase domain that is required for chromatin remodeling [45,95].

candidates for this activity are the YEATS domain proteins Yaf9 (in SWR-C) and Taf14 (in INO80-C). Both may serve as epigenetic readers that recognize H3K56ac or other PTMs in the context of the remodeler [39]. If so, then the N-module of SWR-C (containing Arp4, actin, Bdf1, Swc4, and Yaf9), and the Arp8 subcomplex of INO80-C (Arp8, Arp4, actin, Taf14, and Ies4) may target their respective complexes to nucleosomes bearing specific chromatin modifications, even though neither the N-module nor the Arp8 subcomplex contributes significantly to NCP affinity within the holocomplex [8,14,25]. These Arp4-containing modules do contribute to remodeler recruitment at specific loci. Specifically, the N-module of SWR-C has been termed the DNA repair/telomere boundary module [40] and, in INO80-C, SWR-C, and NuA4, Arp4 is implicated in recruitment of the complex to sites of DNA damage [41]. In mammalian INO80-C, Arp8 may replace Arp4 for remodeler recruitment to DNA damage [42] but, given that Arp8, Arp4, and actin are in the same subcomplex, the overall mechanism may be the same.

Module characteristics The Arp8 module is a nucleosome-binding module and is located in the foot of the INO80-C complex. It helps to recruit INO80-C to damaged chromatin. There is potential cooperative binding of Arp8–Arp4– actin–HSA to nucleosomes [52]. Therefore, binding of the Arp8 module to nucleosome may facilitate binding of a second nucleosome or second Arp8. Upon NCP–INO80-C binding, the Arp8 module undergoes a major conformational shift to grasp the nucleosome [14]. Nucleosome remodeling is strongly impaired but not abolished in Arp8 module-deficient INO80-C [14]. The pointed end of actin is accessible within the module. Actin mutations affect INO80-C–nucleosome affinity [97]. The Nhp10 module is located within the body of INO80 and forms a platform for NCP binding. INO80-C lacking the Nhp10 module has reduced affinity to nucleosomes but is only marginally deficient for remodeling [14]. The least-conserved part of INO80-C complex.

Arp5–Ies6 recruitment to the neck of INO80-C requires RvB proteins [15]. This subcomplex is essential for nucleosome remodeling [14,27]. Arp5-Ies6 may bind to chromatin independently of INO80-C [78]. Present as heterododecamer in INO80-C. Located in head of INO80-C [14]. Has preferential 50 to 30 helicase activity as a heterohexamer, although human RvB1/ RvB2 helicase activity is unclear [104].

The HSA domain of the Ino80 protein is likely involved in DNA binding [52,67].

In conclusion, both INO80-C and SWR-C harbor at least one subcomplex that is primarily responsible for NCP binding within the complex (the Nhp10 subcomplex and the C-module, respectively) and a second module that contributes to the selective recruitment of the remodeler, possibly sensing changes in chromatin modification or structure (Arp8- and the N-module, respectively). This concept of concerted binding through differential affinities could allow the remodelers both to fulfill general housekeeping roles and to accumulate at specific sites for specialized functions. Conditional or dynamic chromatin binding Several lines of evidence indicate that some chromatinbinding sites may be masked in the canonical remodeler structure but be revealed and functional under specific conditions, contributing at least transiently to remodeler function. Such ‘conditional’ chromatin-binding sites within the remodeler allow further regulation of nucleosome remodeling. 623

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Table 2. Composition and chromatin binding by SWR-C S. cerevisiae SWR-C Subunits organized in subcomplexes N-module

Subunit characteristics

Module characteristics

Bdf1

An integral subunit of TFIID [105]. Contains two bromodomains that bind to acetylated lysines in H3 and H4. Prevents SIR protein spreading into euchromatin likely by outcompeting Sir2 deacetylase for acetylated H4 [106].

Arp4

Integral subunit of SWR-C, INO80-C, and NuA4 complexes. Higher affinity for H3–H4 tetramers than for nucleosomes [52]. Helps to recruit complexes to DSBs in yeast [41]. Exists as cytoplasmic network in filamentous form and in globular form with Arp4 in SWR-C, INO80-C, and NuA4 complexes. Contributes to INO80 chromatin binding and remodeling [95]. Copurifies with the N terminus of Swr1 in a yaf9D background. No obvious contribution to nucleosome or H2A.Z binding [8]. A YEATS domain protein that binds to H3 and H4 in vitro. Important for H2A.Z incorporation in vivo [107]. SWR-C from the yaf9D strain only slightly drops in nucleosome affinity. Mediates Swc4, actin, and Arp4 recruitment to SWR-C (possibly also to NuA4) [8]. May antagonize Sir3 occupancy near telomeres. The N-terminal SANT domain is essential for yeast survival. Integral subunit also of the NuA4 complex (Eaf2) [40]. Recruits SWR-C to heterochromatic regions possibly for activation [108]. Part of a Swc4/5 subcomplex that can be detected on nucleosomes independently of Swr1 [34]. Located close to the N-module in SWR-C. Not required for subunit recruitment to complex, but may mask an additional chromatin binding site [8]. Swc5 relocates to the cytosol upon hypoxia, perhaps after being displaced from SWR-C under stress [99]. Binds to nucleosomes with Swc4 and possibly Swc7 independently of Swr1 [34]. Directly binds to H2A.Z and acts as a molecular lock to prevent H2A.Z eviction [8,37]. Targets SWR-C to NFRs [25]. Swc3 association with SWR-C requires Swc2 [8]. Not involved in H2A.Z incorporation in vitro [35] but H2A.Z deposition is reduced in vivo in swc3D strains [30]. Integral subunit for the C-module [8]. Important for H2A.Z deposition in vivo [30,38] possibly by Swc2 recruitment to Swr1. Integral subunit for the C-module [8]. Important for H2A.Z deposition in vivo possibly by Swc2 recruitment to Swr1. Can bind to nuclear pores independently of Swr1 [82]. AAA+ ATPase, binds to ss and dsDNA and RNA. An insertion into the AAA+ domain resembles the OB-fold of RPA [103]. Closely related to RvB1. Snf2 ATPase domain required for H2A.Z deposition [31]. Binds to H2A.Z with its Swr1-Z domain (amino acids 599–627) [36]. Serves as a scaffold protein: the N terminus recruits the N-module whereas the C terminus recruits the C-module and the RvB1/2 heterohexamer [8,25].

The N-module is the DNA repair/telomere boundary module of SWR-C and participates in NuA4. The Nmodule bound to the N terminus of Swr1 does not significantly bind to nucleosomes although some independent subunits bind to H3 or H4 (Arp4 or Yaf9) [25]. These individual chromatin-binding sites may be buried within the complex or else bind to masked surfaces of histones H3 and H4. The YEATS domain of Yaf9 and bromodomain of Bdf1 should prefer acetylated histones, although histone acetylation does not significantly alter N-SWR1 affinity for dinucleosomes in vitro [25]. DNA linker length between dinucleosomes does not affect N-module binding to chromatin. The Nmodule does not contact the nucleosome in the EM structure [13]. Nonetheless, the histone variant exchange reaction is significantly hampered if SWRC lacks the N-module [8]. The N-module may stabilize intermediate states of the remodeling/ variant exchange reaction.

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Swc4 and Swc5 are evenly distributed over the first four nucleosomes of regulated genes (+1!+4), whereas most of the other SWR-C subunits bind to the +1 nucleosome. Swc7 also found at (+1!+4), with decreasing occupancy [34]. The roles of Swc4/5/ (7) are unclear. They may be deposited by SWR-C to facilitate later recruitment of the complex. The C-module binds to the ATPase insertion of Swr1 and mediates nucleosome affinity of SWR-C. SWR-C purified from a swc6D or arp6D background lacks the module and nucleosome affinity decreases 10-fold [8]. The C-module (Swc6 and Arp6) may mask a DNAbinding site in SWR-C [25]. Upon nucleosome binding, the C-module undergoes a positional shift, possibly revealing this DNA-binding site [13]. The shift may also reveal the nucleosome-binding site on SWR-C. The EM structure of the NCP–SWR-C complex does not show significant contacts between the C-module and the nucleosome [13]. Assembled as heterohexamer in SWR-C by the Swr1 ATPase insertion [8,13]. Both 50 to 30 and 30 to 50 DNA helicase activity are attributed to yeast RvB1/RvB2, but the activity of human RvB1/RvB2 is unclear [104]. Swr1 appears to contribute to most of the contacts between SWR-C and the nucleosome in an EM structure [13].

Review One example of this may be the C-module of SWR-C (Swc2, Swc3, Swc6, and Arp6). The association of this module with Swr1 depends on Arp6 and Swc6, and the SWR-C complex purified from either swc6D or arp6D backgrounds lacks Swc2 and Swc3, as well as Swc6 and/or Arp6 [8]. The remodeler lacking this C-module has a 10-fold reduced affinity for nucleosomes in vitro [8], even though the EM structure of the SWR-C–NCP complex showed only marginal contact between the C-module and the nucleosome [13]. We propose that this subcomplex undergoes a major positional change upon SWR-C binding to the NCP, revealing alternative binding sites. The evidence is as follows: using swc2D and arp6D backgrounds, different SWR-C complexes were isolated: one lacking Swc2/3, one lacking Swc2/3 with reduced Swc6/ Arp6, and one lacking the entire C-module. None of the complexes showed significant affinity for dinucleosomes with short linker lengths, but binding was restored when linker length reached 140 bp. Surprisingly, the SWR-C variant that lacked the entire C-module recovered dinucleosome binding, whereas the complex lacking only Swc2 and Swc3 did not [25]. This suggested that Swc6/Arp6 of the C-module may mask additional chromatin-binding sites within the complex that mediate long linker DNA recognition. Long linker DNA is, of course, reminiscent of nucleosome-free regions (NFR), which are particularly relevant at promoters and at sites of DNA damage. Finally, a SWR-C complex lacking only Swc5 appeared to have a stronger affinity for nucleosomes than did the intact SWRC complex, suggesting that Swc5 may also suppress Swr1NCP binding, even though Swc5 is needed for the H2A.Z exchange reaction [8]. A further player in context-specific chromatin binding may be Arp4, which in yeast is an essential protein [43] that binds to Swr1, Ino80, and Eaf2 of NuA4 [12,31,44,45]. Arp4 forms a heterodimer with actin [46], as does its human homolog, Baf53a (ACTL6a), a component of human SWI/SNF-like BAF and PBAF complexes [47,48]. In yeast SWI/SNF and RSC, Arp4/ actin is replaced by Arp7/9, forming a very similar dimer [49,50]. Intriguingly, Arp4 binds to core histones [51] and intact nucleosomes in vitro, and has even higher affinity for (H3–H4)2 tetramers [52]. This suggests that Arp4 may interact with intermediates in the remodeling reaction. The fact that Arp4 alone has a higher affinity for the (H3–H4)2 histone tetramer than in the context of the INO80-C complex suggests that Arp4–histone binding sites are masked within the complex [52]. However, when Arp8 is absent (for instance in SWR-C or NuA4 complexes) the Arp4 high-affinity site may be revealed. Given that the Arp8–Arp4–actin module together with the HSA subdomain of Ino80 binds to nucleosomes with a high Hill coefficient, this subdomain may allow INO80-C to bind cooperatively to nucleosomes [52] by interacting with a second nucleosome through the Arp8 module. At present, it is unknown if cooperative binding is physiologically relevant or if PTMs alter the relative importance of these histone-binding modules, but the ‘hidden’ affinities of Arp homologs are implicated in both.

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Versatile remodeling activities of INO80-C and SWR-C Despite the relatedness of the SNF2-family of ATPases, their roles in vivo are distinct. INO80-C has a wide range of activities and has been implicated in nucleosome remodeling [45], nucleosome eviction [53,54], replacement of the H2A.Z histone variant-containing nucleosomes [33], and the establishment of nucleosomal spacing [55], whereas SWR-C is specialized for deposition of the H2A.Z histone variant [30,31]. Both contribute to gene activation, particularly given that H2A.Z is preferentially enriched at the +1 nucleosome of open reading frames (reviewed in [56]). In addition, INO80-C and SWR-C confer distinct but reproducible defects in DNA repair. We discuss here how these remodelers affect gene expression. Histone eviction The budding yeast PHO5 promoter is a well-studied example in which nucleosome eviction is required to expose an activator binding site to mediate transcriptional induction on low phosphate medium [57]. Full activation of the PHO5 promoter requires SWI/SNF, INO80-C, and the histone acetyltransferase of the SAGA complex, Gcn5 [58,59], as well as RSC, an abundant Sth1-dependent remodeler [60]. INO80-C and SWI/SNF remodelers act in concert, each contributing in a partially redundant manner to nucleosome eviction [59]. Loss of either SWI/ SNF or INO80-C activity still allows gene activation, but induction in the deletion mutants (snf2D or ino80D) is markedly delayed, and double mutants show additive delays and reduced overall levels of expression [58,59]. Finally, a temperature-sensitive allele of Sth1 also compromises expression from the PHO5 promoter under particular conditions in vivo [60]. Each remodeler, however, may preferentially handle nucleosomes in a distinct state or context. Supporting the involvement of INO80-C in vivo, it was shown that the subdiffusive movement of the lacO-tagged PHO5 promoter increased in an INO80-C-dependent manner upon PHO5 induction [61]. The authors suggested that this increase in mobility stems from nucleosome loss. In fission yeast, deletion of the INO80-C subunit Arp8 led to an overall increase of nucleosomes in the genes whose expression is controlled by INO80-C [62], consistent with a role for the S. pombe homolog in histone eviction, at least at active genes. Whereas RSC was found to evict promoter nucleosomes in vitro [63], this has not been shown for INO80-C [32,33]. Loss of histones H3, H2A.Z, and H2B was also observed after the introduction of a specific DNA double-strand break (DSB), in a manner that was not only dependent on INO80-C [53] and RSC (reviewed in [64]) but also on enzymes implicated in end-resection, such as the MRX (Mre11–Rad50–Xrs2) complex and the ATP-dependent remodeler Fun30 [54,65,66]. In yeast arp8D mutants histone loss and Rad51 binding at DSBs are reduced but not eliminated [54]. End-resection and RPA loading were delayed in arp8D strains, as scored by the accumulation of single-stranded (ss)DNA overhangs and ChIP assays [53], as well as in fun30D strains [65]. Intriguingly, in the case of arp8, impaired resection was not observed by Southern blot [54], even though it could be scored by 625

Review QAOS, a quantitative method that monitors ssDNA near an induced break [53]. Unlike loss of INO80-C, loss of SWR-C did not lead to impaired resection, at least at a GAL-HO-induced cut at the MAT locus [53,65]. Nucleosome sliding The spacing of nucleosomes along genomic sequence is achieved by several Snf2-type remodelers which generally function by sliding histone octamers along DNA. This activity not only ensures a regular spacing of nucleosomes but also mediates nucleosomal phasing with respect to a nucleosome-depleted promoter and regulates DNA sequence accessibility by the positioning of individual nucleosomes (reviewed in [24]). The INO80-C complex may also be involved in nucleosome spacing, given that purified INO80-C remodeling complex can shift nucleosomes with long linker DNA closer together to achieve a spacing of 30 bp in vitro [55]. In contrast to the ISWI family, for which nucleosome spacing is well characterized, INO80-C does not rely on the histone H4 tail and may instead be downregulated by H2A tails [55]. HAND–SANT–SLIDE domains of ISWI-type remodelers are crucial for nucleosome spacing [22], and SANT and SLIDE domains have been found to be structurally conserved in CHD1, despite a lack of sequence similarity [23]. It is therefore possible that Ino80 or an accessory subunit may harbor a structural equivalent to the SANT and SLIDE motifs, allowing nucleosome spacing through a related mechanism. Alternatively, the binding of a nucleosome by the flexible Arp8 module could help INO80-C to space nucleosomes: in other words, a dinucleosome substrate could bind to both sides of the Arp8 module, such that the DNA-binding HSA domain [52,67] would space the octamers. Dimer exchange SWR-C has no reported enzymatic activity in nucleosome sliding or eviction, but instead mediates histone dimer exchange by replacing canonical H2A–H2B dimers with H2A.Z–H2B dimers in a two-step reaction. SWR-C can discriminate between canonical and H2A.Z-containing nucleosomes, and the Swr1 ATPase is specifically stimulated by free H2A.Z–H2B dimers and H2A-containing nucleosomes. For this reason, free H2A.Z–H2B dimer does not exchange with nucleosomal H2A.Z–H2B, nor is there efficient exchange of a H2A–H2B dimer with H2A–H2B nucleosomes. In addition, the ATPase activity of SWR-C is low compared to INO80-C or SWI/SNF [31,32]. The deposition of H2A.Z influences transcription, particularly in subtelomeric regions of budding yeast (reviewed in [56,68,69]). Although not subject to SIR repression, subtelomeric domains in yeast contain extended regions of deacetylated histones [70]. In this context, the anti-silencing activity of H2A.Z at the +1 nucleosome of a promoter is particularly crucial [71]. Given that H2A.Z is often present at inactive but inducible genes, it is likely that this variant primarily keeps promoters open for other factors which subsequently trigger transcriptional initiation [72]. INO80-C ensures the depletion of H2A.Z–H2B from unacetylated nucleosomes, in a reaction that may follow 626

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the replication fork [33]. It should be noted, however, that another group was unable to detect INO80-C-driven dimer exchange [32], leading us to query whether depletion of H2A.Z–H2B does not reflect whole-nucleosome exchange or loss in vivo. Interestingly, the replacement reaction may be coupled to or modulated by PTMs on histones, given that H2A.Z acetylation by NuA4 appears to regulate its dynamics. In this way SWR-C, INO80-C, and NuA4 could collectively govern H2A.Z localization (reviewed in [28]). The acetylation of histone H3 on K56, which accompanies the deposition of nucleosomes on newly replicated DNA, may also influence H2A.Z–H2B dimer replacement. Its presence appears to reverse the enzymatic activity of SWR-C in vitro, resulting in H2A.Z–H2B dimer replacement by H2A–H2B [37]. H3K56ac also accelerates the eviction reaction by INO80-C [37]. We note that histone H3K56 acetylation increases DNA breathing at nucleosomes but shows only a modest positive effect on RSC or SWI/SNF remodeling activity [73]. Whether altered access to the nucleosome or a specific interaction between H3K56ac and the remodeler renders the INO80-C replacement reaction more efficient remains to be shown. It is also not fully clear if H3K56ac switches SWR-C directionality in vivo. Nonetheless, constitutive H3K56 acetylation in vivo did lead to a decreased steady-state level of promoterproximal H2A.Z [37]. That post-translational histone modification might reverse the enzymatic reaction of a chromatin remodeler is a new concept, although histone PTMs are known to influence the remodeling reaction. One example is the requirement for phosphorylation and subsequent acetylation of H2A.v in D. melanogaster for exchange by dTIP60 and Domino/p400 [74]. Precise distribution of INO80-C and SWR-C on genomic nucleosomes Nucleosome positions are clearly organized with respect to NFR boundaries at the promoters and terminators of genes. At the 50 end, and to a lesser extent at the 30 end, nucleosomes are organized in uniformly spaced arrays (reviewed in [75]). This is in part determined passively by the underlying DNA sequence which can favor nucleosome localization thermodynamically (reviewed in [76]). However, ATP-dependent chromatin remodelers are able to override this preference for nucleosome positioning, and this in turn helps to regulate gene expression [77]. INO80C binds with considerable enrichment at nucleosomes around the 50 NFR (-1, +1, and +2 nucleosomes), and at the terminal nucleosome immediately upstream of the 30 NFR [78], as well as at origins of replication and tRNA genes [79]. The majority of promoters at which INO80-C can be detected by chromatin immunoprecipitation (ChIP) are either up- or downregulated in ino80 or arp8 mutants [79,80]. Very high resolution ChIP coupled with exonuclease digestion (ChIP-exo) has allowed precise mapping of INO80-C and SWR-C with respect to the +1 nucleosome [34]. The exact location for each TAP-tagged remodeler subunit around the +1 nucleosome showed a similar pattern across many yeast genes, indicating one preferred binding mode for each remodeler at this particular location

Review

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fact that Arp4 and Taf14 are members of multiple complexes implicated in transcription. Their enrichment may actually reflect the colocalization of the other complexes at the +1 nucleosome. Ironically, however, Taf14 is highly enriched at a smaller subset of genes than are other INO80 subunits [34]. According to the ChIP-exo data, the binding modes of INO80-C and SWR-C at the +1 nucleosome appear to be much more similar than the structural models of NCPbound remodelers suggest. For example, ChIP-exo indicates that both remodelers bind asymmetrically to the NFR-proximal part of the +1 nucleosome. Hereby, the respective RvB module as well as the scaffold Snf2 ATPase subunit (Ino80 or Swr1) seem to play major roles in mediating association with the nucleosome (Figure 3A) [34]. To resolve the discrepancy between EM data and ChIPexo models, we will speculate on the existence of semiindependent subcomplexes of the INO80-C and SWR-C remodelers, even though our arguments are based largely on the differential recovery of subunits with the +1 nucleosome (Figure 3B) [13,14,34]. Given that the ChIP-exo data

[34] (Figure 3A). As mentioned above, Swc2 is the only subunit of SWR-C that bound to the NFR, and it may be responsible for targeting the remodeler to open regions [25,34]. By contrast, Swc2 occupancy appears to be much weaker than Swr1 occupancy around the +1 nucleosome in most of the 4972 genes assessed. This could mean that Swc2 association to the NFR and the +1 nucleosome is unstable in some contexts, allowing Swc2 to dissociate from SWR-C. This might have profound consequences on H2A.Z deposition, given that SWR-C purified from a swc2D strain showed a reversed enzymatic activity in vitro [37]. It cannot, however, be fully ruled out that TAP tagging of complex subunits subtly influences their expression, association within the complex, or substrate binding. In contrast to this, the INO80-C subunit Arp8 was found to bind broadly over the NFR, the +1 nucleosome, and to some extent other nucleosomes, whereas the occupancy of other subunits of the Arp8 subcomplex (Arp4, Ies4, and Taf14) peaked exclusively at the +1 nucleosome. These differences could stem from the elongated structure of the Arp8 module within INO80-C [14,81] or might reflect the (A)

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Figure 3. Contribution of INO80-C and SWR-C modules to nucleosome binding. (A) Discrepancy in nucleosome binding based on distinct modes of analysis. Here we compare the structural models of nucleosome-bound INO80 and SWR-C with the high-resolution data of ChIP-exo derived from occupancy of the respective subunits at +1 nucleosomes. The ChIP-exo models differ significantly from the models based on EM and/or crosslinking. (B) Potential stable subcomplexes of INO80-C and SWR-C. Subcomplexes of INO80-C and SWR-C can be expressed as stable modules independent of the Snf2 ATPase scaffold protein such as Arp5–Ies6, Rvb1/2, or Nhp10–Ies3–Ies5 may also function outside the remodeler. Some subcomplexes are implicated in chromatin binding without the rest of the remodeler (Swc4–Swc5 possibly together with Swc7) and other subunits have been shown to have functions distinct from the holo-complex (e.g., Arp6). Differential and context specific subunit compositions of remodelers may help to explain the discrepancy between the nucleosome-binding modes derived from EM/crosslinking and ChIP-exo.

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represent occupancy averaged over a population of cells, it is possible that the subunit composition of the remodeler varies in different cells, or responds differentially to formaldehyde fixation in different contexts. It is even possible that subcomplexes of remodelers dissociate, or have independent binding patterns, as well as secondary functions (Figure 3B). One example of this is the Arp6 subunit of SWR-C. Arp6 and Swr1 do not map identically across the genome, and Arp6 has functions that are independent of Swr1 – such as the recruitment of ribosomal protein genes to nuclear pores [82]. Another example of an independent subcomplex may involve Swc4 and Swc5. The chromatin occupancy of these two proteins suggests that they constitute a subcomplex that localizes to nucleosomes independently of holo-SWR-C [34]. It will be interesting to assess how this subcomplex is deposited or retained at nucleosomes, given that neither Swc4 nor Swc5 participate in the nucleosome-binding affinity of SWR-C [8]. Their interaction may instead be mediated by Swc7 which appears to distribute in a manner intermediate between the majority of SWR-C subunits and Swc4/Swc5 [34]. Again, however, Swc7 has little affinity on its own for nucleosomes and may instead be localized by interaction with another DNA-binding factor. Similarly, Ies5 and Nhp10 of INO80-C colocalize with the general transcription factor Reb1, whereas Ies3 and Ies1, part of the same Nhp10 module, clearly bind to nucleosomes [34]. The Nhp10 subcomplex can bind to both DNA and nucleosomes, and contributes to the overall

SW R

INO80-C and SWR-C maintain genomic integrity In addition to gene regulation, both INO80-C and SWR-C are implicated in maintenance and repair of the genome (Figure 4) given that mutants deficient in either complex render cells hypersensitive to DNA-damaging agents. ino80 and swr1 mutants do not, however, show identical sensitivities; for example, mutations compromising INO80-C render cells sensitive to replication fork arrest on hydroxyurea, which is not the case for swr1D mutants [31,80]. Consistently, both remodelers are actively recruited to DNA DSBs [53,80,83], whereas only INO80C was recovered at stalled replication forks [79]. It was initially thought that phosphorylated H2A.X (gH2A in yeast) was responsible for INO80-C recruitment to DSBs [80,83], but more recent work has shown that remodeler recruitment is strongly reduced in G1-phase cells, and that loss of the H2A phosphoacceptor site leads to an accumulation of cells in G1 [84]. Thus, the reduction in remodeler recruitment to DSB in histone H2A phosphoacceptor site mutants is most likely an indirect effect of impaired cell cycle progression [84]. INO80-C remodeler recruitment indeed occurs at breaks preferentially in S/G2-phase, and may precede the resection needed for homologous recombination (HR). Both INO80-C and RSC remodelers participate in nucleosome eviction at DSBs and facilitate Rad51 binding

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Figure 4. INO80-C and SWR-C in DSB repair. INO80-C is implicated in histone variant exchange, nucleosome eviction, and nucleosome remodeling, and is also able to space nucleosomes. SWR-C replaces H2A–H2B with H2A.Z–H2B in nucleosomes. Both remodeling complexes play roles in the DNA double strand break (DSB) repair pathways. Inter alia, they are responsible for increased chromatin mobility at the site of damage and INO80-C, at least, also confers augmented chromatin movement genome-wide. Moreover, SWR-C contributes to the relocation of persistent DSBs to the nuclear periphery irrespective of the cell cycle, whereas INO80-C contributes only to DSB break binding to Mps3 and only in S-phase cells [87].

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Review [54,64]. In addition, INO80, RSC, and Fun30 remodelers all appear to contribute to end-resection. Finally, it was recently shown that both INO80-C and SWR-C contribute to the subdiffusive mobility of chromatin, which increases after induction of a DSB [61,85–87]. Chromatin movement augments after damage both at the site of cleavage and genome-wide, albeit to a lesser degree [85,88]. This increased local and global chromatin movement depends on intact INO80-C activity [88] and may render the homology search more efficient for repair by HR. A final role for SWR-C and INO80-C in yeast may be to regulate the sequestration of DSBs at the nuclear periphery, which appears to regulate promiscuous recombination [87,89–91]. Interestingly, INO80-C and SWR-C contribute to DSB relocalization to the nuclear envelope in distinct ways: SWR-C is needed for the relocalization to either nuclear pores or Mps3 in either G1- or S-phase, whereas INO80-C is only required for the association with Mps3 in S- or G2-phase cells [87]. INO80-C and in particular SWRC thus may be part of a contingency plan that involves the spatial segregation of DNA break(s) to enable either protection from recombination or the promotion of alternative pathways of repair. Whether these pathways require histone eviction remains to be examined. Concluding remarks and future directions In summary, we propose that the multifunctional remodelers, INO80-C and SWR-C, have more than one structure and subunit composition during their chromatin-binding and -remodeling reactions. This, and the conditional association of holo-complexes with chromatin, may explain the diversity of phenotypes observed when distinct subunits within a multisubunit complex are deleted. This is particularly evident for subunits of INO80-C or SWR-C in assays that monitor sensitivity to DNA damage. In addition, dissecting the role of each INO80-C and SWR-C subunit in chromatin binding will help us to understand the molecular toolbox that is at the disposal of these remodeling complexes. (Box 2). We suspect that the plasticity of remodeler structure will be controlled by signals from kinases and phosphatases, as well as HATs and HDACs, which all respond to acute signals. Indeed, INO80-C is a validated target of the Mec1/ATR kinase [92,93] and may also be differentially regulated through the cell cycle. It remains to be seen to what extent the plasticity of nucleosome remodelers helps them to carry out their regulation of epigenetic states and gene expression.

Box 2. Outstanding questions  What is the role of the RvB oligomers within the remodelers?  What recruits INO80-C and SWR-C differentially to DSBs through the cell cycle?  What is the role of histone modification in regulating remodeler activity?  What are the roles of the remodeler subcomplexes that bind to chromatin independently of the ATPase subunit?  How do multisubunit remodelers associate with higher-order chromatin structures?  How can the discrepancy between nucleosome-binding models derived from EM/crosslinking or ChIP-exo be resolved?

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Acknowledgments We thank Dr Kenji Shimada, Andrew Seeber, and Dr Kristina Lakomek for critical reading of the manuscript. We apologize to all those colleagues whose important work is not cited owing to space limitations. The authors have been supported by research grants from the Human Frontiers Science Program, and the Swiss National Science Foundation. C.B.G. has a Framework Program (FP7) Marie Curie Intra European Fellowship. The laboratory of S.M.G gratefully acknowledges the continued support of the Novartis Research Foundation.

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