Assembly of the Arp5 (Actin-related Protein) Subunit Involved in Distinct INO80 Chromatin Remodeling Activities*

Background: DNA is packaged into nucleosomes, which are positioned by ATP-dependent chromatin remodelers. Results: The Arp5 subunit of the INO80 complex that lacks unique insertion domains stimulates ATP hydrolysis without facilitating nucleosome sliding. Conclusion: The Arp5-Ies6 subunit module is required for productive chromatin remodeling in vitro. Significance: Subunit modules contribute to different chromatin remodeling functions and nucleosome organization that regulates DNA-templated processes. ATP-dependent chromatin remodeling, which repositions and restructures nucleosomes, is essential to all DNA-templated processes. The INO80 chromatin remodeling complex is an evolutionarily conserved complex involved in diverse cellular processes, including transcription, DNA repair, and replication. The functional diversity of the INO80 complex can, in part, be attributed to specialized activities of distinct subunits that compose the complex. Furthermore, structural analyses have identified biochemically discrete subunit modules that assemble along the Ino80 ATPase scaffold. Of particular interest is the Saccharomyces cerevisiae Arp5-Ies6 module located proximal to the Ino80 ATPase and the Rvb1-Rvb2 helicase module needed for INO80-mediated in vitro activity. In this study we demonstrate that the previously uncharacterized Ies2 subunit is required for Arp5-Ies6 association with the catalytic components of the INO80 complex. In addition, Arp5-Ies6 module assembly with the INO80 complex is dependent on distinct conserved domains within Arp5, Ies6, and Ino80, including the spacer region within the Ino80 ATPase domain. Arp5-Ies6 interacts with chromatin via assembly with the INO80 complex, as IES2 and INO80 deletion results in loss of Arp5-Ies6 chromatin association. Interestingly, ectopic addition of the wild-type Arp5-Ies6 module stimulates INO80-mediated ATP hydrolysis and nucleosome sliding in vitro. However, the addition of mutant Arp5 lacking unique insertion domains facilitates ATP hydrolysis in the absence of nucleosome sliding. Collectively, these results define the requirements of Arp5-Ies6 assembly, which are needed to couple ATP hydrolysis to productive nucleosome movement.

Eukaryotic genomes are organized within chromatin, making chromatin manipulation essential to DNA-templated processes. Included among chromatin modifiers are enzymes that post-translationally modify histones and chromatin remodelers that alter the position and composition of nucleosomes. ATP-dependent chromatin remodeling is a conserved essential process, and deficiencies in these enzymes result in viability defects, developmental abnormalities, and disease initiation in different species (1,2). The form and structure of nucleosomes influence many chromatin-associated functions including, but not limited to, the recruitment of factors involved in transcription, replication, and DNA repair.
The INO80 chromatin remodeling complex is highly conserved with roles in transcription, genome stability, embryonic stem cell identity, and disease pathogenesis (1,(3)(4)(5)(6)(7). In vitro, the INO80 complex moves nucleosomes into regularly spaced arrays (8), which is reflected in the deregulation of nucleosome abundance and positioning in vivo (9,10). Previous research has demonstrated that the large multisubunit INO80 complex influences diverse DNA-templated processes through subunit specification of distinct chromatin remodeling activities (3,11). Indeed, each chromatin remodeling event is a complex multistep process involving chromatin recruitment and substrate selection followed by ATP hydrolysis and nucleosome positioning (2). All chromatin remodelers contain SNF2-type ATPases that "couple" ATP hydrolysis to nucleosome sliding activity (12)(13)(14), with "coupling efficiency" indicative of the amount of ATP hydrolyzed per sliding event (15). Each chromatin remodeling step needs to be tightly controlled for optimal enzyme activity and subsequent chromatin in diverse DNA-templated processes.
Recently, the structure of the 1.3-MDa Saccharomyces cerevisiae INO80 complex was described (16,17), providing clues to the functional organization of this remodeler. The complex was found to be modular with biochemically separable subcomplexes consisting of distinct subunits assembling onto the Ino80 ATPase scaffold. Thus, it is likely that the INO80 complex elicits diverse functions that are attributed to unique modules within the larger complex. Of particular interest are the actin-related proteins (Arps) 5 that are critical for chromatin remodeling function (18). Of the 10 Arps in S. cerevisiae, four are primarily cytoplasmic with cytoskeleton functions, whereas the remaining six are in chromatin remodeling complexes. The INO80 complex contains Arp4, Arp8, and Arp5, all of which are required for in vitro chromatin remodeling (19). Arp4 and Arp8 assemble as a separate module in the helicase-SANT-associated domain of Ino80 (16,20) and are proposed to facilitate interactions between the nucleosome and remodeler (19,21). The N-terminal domain of Ino80 ATPase assembles subunits that are less conserved among species (16), yet some of these subunits have directed functions in DNA damage recognition (22) and telomere stability (23) in S. cerevisiae.
The INO80 subfamily of chromatin remodelers is uniquely distinguished by an insertion region that splits the ATPase domain (3,24). The insertion region is evolutionarily conserved and necessary for the recruitment of mammalian RuvBL1-RuvBL2 (Rvb1-Rvb2 in yeast) (25), which are AAAϩ helicases with homology to the bacterial RuvB helicase involved in Holliday Junction migration (26), yet their roles in chromatin remodeling remain unclear. In addition, the insertion region of mammalian Ino80 is needed for ACTR5-INO80C module (Arp5-Ies6 in yeast) assembly within the INO80 complex (25), although its requirement for the yeast complex has not previously been reported. However, Rvb1-Rvb2 module is necessary for Arp5-Ies6 association with the INO80 complex (27). Struc-tural analyses of the S. cerevisiae INO80 complex also demonstrate that the Arp5-Ies6 module is within close proximity of the Ino80 ATPase domain and Rvb1-Rvb2 subunits (16), thus linking Arp5-Ies6 to critical enzymatic components of the INO80 complex. In vivo, Arp5-Ies6 are important for nucleosome positioning (9), DNA damage responses (28 -31), replication (32), transcriptional regulation (33,34), and mitotic stability (35). In vitro, this subunit module is critical for INO80-mediated ATP hydrolysis, nucleosome sliding, and histone exchange that reconstructs nucleosomes by removing the Htz1 variant (H2A.Z in mammals) (16,17,19).
In this study we delineate the assembly of the Arp5-Ies6 module within the S. cerevisiae INO80 complex and its resulting influence on INO80 function. Through biochemical analysis, individual INO80 subunits and domains are identified as necessary for Arp5-Ies6 association with the INO80 complex. Furthermore, we identify critical domains within Arp5 that couple ATP hydrolysis to nucleosome sliding. Additionally, important differences between the requirements for Arp5-Ies6 assembly with the INO80 complex in different species are discussed.

Experimental Procedures
Yeast Strains-The strain list is shown in Table 1. Strain construction was in S288C background using standard techniques. All FLAG epitopes were chromosomally integrated to ensure endogenous expression of protein. Arp5 mutants were also integrated at chromosomal locus. Plasmids encoding IES6 consist of endogenous promoter (Ϫ500 bp) and terminator (ϩ500 bp) sequences. 5-Fluoro-orotic acid was used in synthetic media at 1 mg/ml. FLAG Affinity Purifications-Protein complexes were purified using anti-FLAG affinity beads as previously described (22,28,36). Briefly, cells were frozen in liquid nitrogen then broken Chromatin fractionations were performed as previously described (37) with some modification. 50 ml (A 660 ϳ0.5) of spheroplasted cells were resuspended in lysis buffer. For micrococcal nuclease (MNase) digestion, MgCl 2 was replaced by CaCl 2 . 150 units of MNase or 10 units of DNase I was added to 150 l of lysate for 30 min at 30°C before centrifugation at 13,500 rpm for 15 min to separate soluble and chromatin pellets. Percent solubilization was calculated by quantifying the Arp5 chemiluminescence signal relative to the H3 signal before and after digestion with the following calculation: Arp5 [DNase I-or MNase-treated (S2) fraction/insoluble chromatin (C) fraction]/H3 [DNase I-or MNase-treated (S2) fraction/insoluble chromatin (C) fraction]. Non-saturating chemiluminescence signal was obtained using Bio-Rad ChemiDoc imager.
ATPase reactions contained 2 nM INO80 d and Arp5-Ies6 complexes and 1 nM mononucleosomes in reaction buffer (25 mM HEPES, pH 7.4, 75 mM KCl, 0.37 mM EDTA, 0.35 mM EGTA, 0.02% Nonidet P-40, 1 mM DTT, 100 g/ml BSA, 10% glycerol). After incubation at 30°C for 30 min, reactions were initiated by the addition of 5 mM MgCl 2 and 100 mM ATP. All reactions were stopped by the addition of 85 mM EDTA after 60 min. Reactions were added to Malachite Green solution (0.375 mM Malachite Green oxalate, 8.5 mM ammonium molybdate, 1 mM HCl); color development was allowed to proceed for 3 min before the addition of sodium citrate to 5.6% and measurement at 620 nm. ATPase activity was calculated via conversion of free phosphates using a sodium phosphate standard curve.

Association of the Arp5-Ies6 Subunit Module with the INO80
Complex-Arp5 and Ies6 comprise a structurally distinct module of the INO80 complex important for INO80 catalytic activity and chromatin remodeling (16,19). Accordingly, deletion of either Arp5 or Ies6 resulted in a loss of both subunits with the purified INO80 complex, whereas the association of other subunits with the INO80 complex was unchanged (Fig. 1A). To identify other subunits involved in Arp5-Ies6 association with the INO80 complex, we performed additional Ino80 purifications in wild-type and INO80 subunit deletion strains. Interestingly, Ies2 was needed for Arp5-Ies6 association, as deletion resulted in the loss of the Arp5-Ies6 module within the INO80 complex (Fig. 1A). Importantly, the total protein levels of Ies2 and Arp5 were not decreased in these deletion strains. However, deletion of ARP5 resulted in an undetectable amount of Ies6 protein in whole cell extracts, whereas no substantial change in Ies6 was observed in ies2⌬ cells (Fig. 1B). Although the precise reason for decreased Ies6 protein is currently unknown, it may be that Arp5 influences Ies6 protein stability. Deletion of other subunits, such as Ies1, did not influence the association of other subunits or subunit modules with the INO80 complex.
Biochemically, Ies2 is a relatively uncharacterized component of the INO80 complex, although structural studies place it within 30 Å of the ATPase domain (16). As mentioned, the Ino80 ATPase domain is separated by a large insertion region, a characteristic that is distinct from other chromatin remodelers including Snf2 of the SWI/SNF complex (Fig. 1C). Deletion of this insertion region of Ino80 resulted in loss of Arp5, Ies6, Ies2, and Rvb1-Rvb2 association with the complex (Fig. 1D). Notably, Arp5 or Ies6 deletion did not alter Ies2 or Rvb1-Rvb2 association the INO80 complex (Fig. 1A). Likewise, Ies2 deletion did not dramatically alter association of Rvb1-Rvb2 with INO80 complex (Fig. 1A). These results demonstrate that Ies2 confers association of Arp5-Ies6 with a region of the INO80 complex critical for enzymatic activity.
As Ies6 is needed for Arp5 assembly with the INO80 complex ( Fig. 1A), we sought to further examine this interaction. Ies6 is a relatively uncharacterized subunit with one annotated domain, the evolutionarily conserved YL1 nuclear protein C-terminal (YL1-C) domain ( Fig. 2A). This domain is found in the YL1 family of proteins that exhibit DNA binding and is implicated in transcriptional regulation (39). In our protein purifications, deletion of the YL1-C domain resulted in loss of the Arp5-Ies6 module with INO80 complex (Fig. 2B). However, co-purification of Ies6 and Arp5 was maintained. In addition, deletion of the YL1-C domain resulted in fitness defects similar to that of complete IES6 deletion (Fig. 2C). Therefore, the YL1-C domain needed for Arp5-Ies6 association with the INO80 complex is critical for Ies6 cellular function.
Collectively, these results and those previously published (27) demonstrate that Ies2, Rvb1-Rvb2, the insertion region of Ino80, and the YL1-C domain of Ies6 are all involved in the association of the Arp5-Ies6 module with the Ino80 ATPase subunit and implicate the coordinated activity of these subunits in INO80 chromatin remodeling.

Distinct Domains of Arp5 Are Required for Association with
Ies6 and the INO80 Complex-As mentioned, the INO80 complex contains Arps 4, 5, and 8, of which Arps 5 and 8 are specific to the INO80 complex. All Arps have significant identity to actin within the actin fold domains. S. cerevisiae Arp4, Arp5, and Arp8 are 30% identical (53% similar), 26% identical (51% similar), and 21% identical (44% similar) to actin, respectively (18). Previous research demonstrates that Arp4 and Arp8 contain unique insertions regions that facilitate subunit specific interactions, namely actin binding and nucleosome association (21,40). To investigate Arp5-specific function, we biochemically characterized the conserved insertion regions unique to Arp5.
We identified four insertion regions within Arp5 that are distinct from actin and Arp4 (Fig. 3A) yet are shared with other species (Table 2) and thus may represent conserved Arp5-specific functional domains. Genomic deletions were made encompassing these insertion domains, referred to as arp5D1⌬ to D4⌬. Replacement of wild-type ARP5 with these arp5 insertions mutants resulted in reduced fitness (Fig. 3B). arp5D1⌬ and -D4⌬ exhibit fitness similar to that of complete ARP5 deletion, whereas arp5D2⌬ and -D3⌬ have intermediate fitness defects (Fig. 3B). These arp5 insertion mutants remain under the control of the endogenous promoter; however, Western analysis identifies reductions in protein abundance, which may be indicative of altered protein stability and contribute to decreased cellular fitness (Fig. 3C).
Purification of FLAG-tagged Ies6 from strains expressing these Arp5 insertion mutants confirmed that Arp5D2⌬ associates with Ies6 (Fig. 4A). In addition, in these purifications where Ies6 protein is enriched, co-purification of Arp5D3⌬ is detectable. However, in agreement with the purifications of the Arp5 insertion mutants (Fig. 3D), abundance of co-purifying subunits of the INO80 complex was greatly reduced. Furthermore, no detectable amount of Ies6 was found in purifications from arp5D1⌬ and -D4⌬ strains. Western blotting of whole cell extracts revealed a dramatic reduction in both Ies6-FLAG and Arp5D1⌬ and -D4⌬ protein (Fig. 4B). This suggests that the decrease of co-purifying Ies6 with Arp5D1⌬ and -D4⌬ may be a consequence of reduced Ies6 protein in the cell (Figs. 3D and 4A) rather than a loss of Arp5-Ies6 interaction. Ies6-FLAG was also reduced in the Arp5D3⌬ mutant (Fig.  4B); thus, the decrease in co-purifying Ies6 with Arp5D3⌬ ( Fig. 3D) was also likely a consequence of total protein amounts in the cell. However, when Ies6-FLAG is purified and subsequently enriched, approximately stoichiometric amounts of co-purifying Arp5D3⌬ can be observed (Fig. 4A).
As in Fig. 1B, these results also point to a mechanism by which Ies6 may be stabilized by the presence of Arp5. Collectively, these assays indicate that Arp5D2⌬ and -D3⌬ have reduced INO80 complex association while retaining Ies6 interaction at levels corresponding to abundance of Ies6 cellular protein.
Purification of FLAG-tagged Ino80 from cells expressing Arp5D1⌬ to -D4⌬ demonstrates that the mutant Arp5-Ies6 module has decreased association with the complex (Fig. 4C). Although Arp5 mutants and Ies6 was not detected in silverstained gels, ArpD2⌬ and D3⌬ was observed in Western analysis using an Arp5 specific antibody. Collectively, these results demonstrate that deletion of Arp5-specific insertion regions 2 and 3 result in disrupted association with the INO80 complex, whereas deletion of insertion domains 1 and 4 have greatly reduced Arp5 and Ies6 protein levels, thereby precluding examination of their interaction with the INO80 complex.
Association of the Arp5-Ies6 Subunit Module with Chromatin-Cell lysate fractionation was performed to identify the determinants of Arp5-Ies6 association with chromatin (Fig.  5A). In these assays, histone H3 and cytosolic hexokinase demarcate the chromatin and soluble fraction, respectively. Deletion of INO80, IES2, and IES6, but not IES1, resulted in a prominent reduction in Arp5 association with chromatin concomitant with an increase in the soluble fraction (Fig. 5B). These results suggest that the Arp5-Ies6 module is recruited to chromatin through association with the INO80 complex, as deletion of IES2 resulted in loss of the Arp5-Ies6 interaction with the complex (Fig. 1A). Interestingly, all Arp5 insertion region mutants retain significant association with chromatin (Fig. 5C). This result is unexpected, as Arp5D1⌬ and -D4⌬ have no measurable association with purified INO80 complex in our assays. To more directly assess the chromatin association of these Arp5 mutants, nucleases were added to the insoluble fraction to solubilize chromatin associated proteins while leaving non-chromatin associated proteins insoluble (Fig. 5A). As can be seen in Fig. 5C and D, MNase and DNase I efficiently solubilize histone H3 and wild-type Arp5. In addition, Arp5D2⌬ is solubilized at a level comparable with wild-type. However, Arp5D3⌬ is solubilized less relative to wild-type Arp5, and Arp5D1⌬ and -D4⌬ were not solubilized with either DNase I or MNase digestion (Fig. 5C and D). Thus, the deficiencies in Arp5D1⌬ and -D4⌬ to associate with other INO80 complex subunits and chromatin may be indicative of these mutants forming insoluble aggregates.
Influence of the Arp5-Ies6 Subunit Module in Chromatin Remodeling-In vitro biochemical assays were performed to determine the ability of Arp5D2⌬ and -D3⌬ to stimulate INO80-mediated ATP hydrolysis and nucleosome sliding. As Arp5D2⌬ and -D3⌬ had reduced association with the INO80 complex (Fig. 4C), we purified wild-type and mutant Arp5-Ies6 from ies2⌬ cells and normalized the amount of soluble Arp5-Ies6 module used in in vitro assays. The module was then added to the INO80 complex lacking Arp5-Ies6 (INO80 d ), which was purified via Ino80-FLAG from ies6⌬ cells (Fig. 1A). INO80 d had minimal ATPase activity (Fig.  6A) in agreement with other studies demonstrating reduced nucleosome-stimulated ATPase activity of the S. cerevisiae INO80 complex purified from arp5⌬ cells (16,19). The Arp5-Ies6 module alone, either mutant or wild type, also did not exhibit ATPase activity. However, the addition of the wild-type Arp5-Ies6 module to the INO80 d complex stimulated ATPase activity in vitro. Mutant Arp5D2⌬-Ies6 and D3⌬-Ies6 also significantly stimulate INO80-mediated ATPase activity, albeit to a reduced level from that of wildtype Arp5-Ies6. However, only the Arp5-Ies6 module from wild-type cells, and not from arp5D2⌬ or D3⌬ cells, was able to stimulate INO80-mediated nucleosome sliding in vitro (Fig. 6B).
The lack of nucleosome sliding activity of INO80 d complexes in the presence of Arp5-Ies6 modules lacking insertion regions 2 and 3 may be due to the inability of Arp5D2⌬-Ies6 and D3⌬- Ies6 to stimulate enough INO80-mediated ATP hydrolysis to facilitate nucleosome sliding or that insertion regions 2 and 3 are required for sliding activity but dispensable for minimal ATP hydrolysis. However, it should be noted that previous studies have detected a Ͼ50% reduction in ATPase activity mediated by the INO80 complex lacking specific subunits; yet nucleosome sliding could still be detected (19). As such, Arp5 insertion regions 2 and 3 may "uncouple" different INO80-mediated activities, such as ATP hydrolysis and nucleosome sliding.

Discussion
Results presented in this study delineate the assembly of the S. cerevisiae Arp5-Ies6 module, which is required for INO80 chromatin remodeling. Biochemical purifications reveal particular regions within the catalytic ATPase domain of the INO80 complex required for its association with Arp5-Ies6. Specifically, Arp5-Ies6, Ies2, and Rvb1-Rvb2 require the Ino80 insertion domain to associate with the INO80 complex. As previously reported, Rvb1-Rvb2 are required for Arp5 association with the INO80 complex (27), although Ies2 and Ies6 were not discussed. Additionally, our results demonstrate that Arp5-Ies6, but not Rvb1-Rvb2, is dependent on Ies2 for assembly within the INO80 complex. Thus, a hierarchy of subunit assembly is proposed on the Ino80 ATPase as follows: 1) the Ino80 insertion domain is required for Ies2 and Rvb1-Rvb2 assembly; 2) Ies2 is needed for Arp5-Ies6 association; 3) Rvb1-Rvb2 is needed for Arp5-Ies6 assembly; 4) Ies6 YL1-C domain is needed for Arp5-Ies6 association.
It is not yet known if Ies2 association with the INO80 complex is dependent on the Rvb1-Rvb2 module or if they indepen-    dently associate with the Ino80 insertion domain. However, data presented here demonstrate that Arp5-Ies6 is not dependent on both Ies2 and Rvb1-Rvb2 for assembly within the INO80 complex, as IES2 deletion resulted in the loss of Arp5-Ies6, yet Rvb1-Rvb2 remained associated. Future studies investigating the association of Ies2 in the absence of Rvb1-Rvb2 will clarify the relationship between these subunits. Our model of subunit arrangement is supported by chemical cross-linking between the Ino80 insertion domain, Rvb1-Rvb2, Ies2, Arp5, and Ies6 (16). Moreover, EM analyses identified distinct "head, neck, body, and foot" subunit modules that assemble along the Ino80 ATPase scaffold. Notably, Arp5-Ies6 and Ies2 are situated at the neck of the INO80 complex that contains the ATPase domain, located between the body module and Rvb1-Rvb2 head module. Indeed, Ies2 is within 30 Å of the ATPase domain and has been proposed to play a role in facilitating INO80 complex structural formation, as it can be chemically cross-linked to multiple subunits along the head, neck, body, and foot of the INO80 complex. Thus, Ies2 association may induce conformational changes conducive to Arp5-Ies6 association.
Importantly, Arp5-Ies6 are at the "catalytic center," within close proximity to both the RecA ATPase domain of Ino80 required for nucleosome sliding (24) and the large Rvb1-Rvb2 AAAϩ helicase module capable of RNA and DNA binding (26). Indeed, loss of Arp5-Ies6 resulted in a dramatic reduction of in vitro ATPase activity and nucleosome sliding of the S. cerevisiae INO80 complex (Refs. 16, 17, 19 and Fig. 6). Histones H2A and H2B preferentially cross-link to Arp5-Ies6, Ies2, the Ino80 ATPase domain, and Rvb1-Rvb2 (16). Notably, some of these cross-links are formed between regions of the histone that are not exposed on the surface of the nucleosome. The INO80 complex exchanges the Htz1 histone variant for canonical H2A, which has recently been found to dependent on Arp5-Ies6 (17,41). Collectively, these results suggest that the Arp5-Ies6 module, which bridges the head and neck modules of the INO80 complex, facilitates H2A-H2B dimer "remodeling." Characterization of domains within Arp5 that are distinct from other Arps, yet conserved among Arp5 from different species, demonstrates that insertion regions 2 and 3 is dispensable for Ies6 interaction and minimal INO80 complex association. Deletion of insertion domains 1 and 4 disrupt Arp5 solubility and may reflect the complexity of Arp5 folding into mature soluble protein. Interestingly, wild-type Arp5-Ies6 stimulates INO80-mediated chromatin remodeling in vitro and may be indicative of dynamic chromatin remodeling assemblies in vivo. Arp5 insertion regions 2 and 3 are dispensable for nucleosomestimulated ATPase activity yet are required for nucleosome sliding. Although uncoupling of ATP hydrolysis and nucleosome sliding has not previously been reported for the INO80 complex, it has been observed for the Chd1 and ISWI remodelers, which are functionally related (15,42). Thus, the Arp5-Ies6 subunit module bridges different enzymatic activities within the same complex (42). Indeed, Arp5 insertion domain 2 contains a site that can be cross-linked to H2B, suggesting that this domain is involved in H2A-H2B remodeling.
The SWR1 complex, another member of the INO80 chromatin remodeling subfamily, is also differentiated by distinctive insertion regions that split the RecA ATPase domain (24,43). The SWR1 complex exchanges canonical H2A in the nucleosome for the histone variant Htz1 (43), whereas INO80 has been implicated in the removal of Htz1 (41). INO80 histone exchange function is dependent on Ies6 (17), whereas SWR1 activity is dependent on Swc2 (44), both of which require the insertion region of the ATPase subunit for association with their respective complexes (Ref. 44 and Fig. 1). Similar to Ies6, Swc2 also contains a YL1-C domain. Results presented in this study demonstrate that the Ies6 YL1-C domain is needed for association of the Arp5-Ies6 module with the INO80 complex. Thus, it may be that Swc2 YL1-C facilitates Swc2 and, consequently, Htz1 association with the SWR1 complex. Again, these results bolster common modes of subunit assembly for INO80 and SWR1 as well as chromatin remodeling of H2A-H2B by YL1-C-containing subunits.
However, the results presented here reveal important distinctions between the functional assembly of the mammalian and yeast INO80 complex. It was recently reported that siRNA-mediated depletion of INO80B (yeast Ies2) in HEK293T cells results in minimal loss of ACTR5-INO80C (yeast Arp5-Ies6) with the INO80 complex (25). Furthermore, INO80B is required for nucleosome and DNA-stimulated ATPase activity, whereas ACTR5 and INO80C are not. These results are in contrast to others from S. cerevisiae demonstrating loss of ATPase activity in IN080 complexes purified from cells with ARP5 or IES6 deletion (16,17,19). Different methodologies of purifica- Arp5-Ies6 tions and depletion of subunits (siRNA knockdowns versus genomic deletions) may influence these conflicting results. However, it may reflect different mechanisms of INO80 complex function in different species. Indeed, the insertion domain of Ino80, which is required for Ies2 and Arp5-Ies6 assembly, is less conserved (14% identical; 24% similar) than the full protein (27% identical; 35% similar) or functional domains such as the helicase-SANT-associated domain (37% identical; 54% similar) and the ATPase domain (67% identical; 75% similar). In particular, the two regions of human INO80 that were reported as crucial for Arp5-Ies6 binding (25) are relatively less conserved (9 and 14% identical; 9 and 22% similar) and may provide a binding platform for Arp5-Ies6 assembly with human INO80 that is distinct from yeast. Likewise, S. cerevisiae Arp5 insertion regions 2 and 3 ( Table 2), which are critical for INO80-mediated nucleosome sliding, are relatively less conserved among mammals than other species and may also reflect divergent modes of chromatin remodeling that have evolved in higher eukaryotes.
It is also noteworthy that although both yeast Ies2 and mammalian INO80B have a conserved PAPA-1-like domain, they are not considered orthologs due to a lack of a common ancestor. Indeed, phylogenic analysis distinguishes these two proteins as being on different branches, indicative of distinct functional evolutionarily lineages (data not shown). In contrast Arp5, Ies6, and Ino80 have mammalian orthologs. Thus, Ies2 and INO80B may have evolved differently to regulate INO80 chromatin remodeling in disparate species. Interestingly, Ies2 function differs even within the fungal kingdom as Ies2 (but not Arp5, Ies6, or Ino80) is one of 20 genes essential for anaerobic growth in Saccharomyces yet is absent from other fungi that are unable to grow in anaerobic conditions (45). Thus, it may be that INO80-mediated chromatin remodeling is important for expression of transcriptional programs necessary for growth regulation in response to nutrient and oxygen availability. Ies2 may provide additional regulation for the association of the critical Arp5-Ies6 module that facilitates chromatin remodeling. Although these speculations require additional examination, it is evident that the regulated association of the Arp5-Ies6 module is important for the function of the INO80 complex in different species.