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Mol Cell Biol. 2010 May; 30(10): 2391–2400.
Published online 2010 March 22. doi:  10.1128/MCB.01106-09
PMCID: PMC2863710

SWR1 Complex Poises Heterochromatin Boundaries for Antisilencing Activity Propagation [down-pointing small open triangle]

Abstract

In eukaryotes, chromosomal processes are usually modulated through chromatin-modifying complexes that are dynamically targeted to specific regions of chromatin. In this study, we show that the chromatin-remodeling complex SWR1 (SWR1-C) uses a distinct strategy to regulate heterochromatin spreading. Swr1 binds in a stable manner near heterochromatin to prepare specific chromosomal regions for H2A.Z deposition, which can be triggered by NuA4-mediated acetylation of histone H4. We also demonstrate through experiments with Swc4, a module shared by NuA4 and SWR1-C, that the coupled actions of NuA4 and SWR1-C lead to the efficient incorporation of H2A.Z into chromatin and thereby synergize heterochromatin boundary activity. Our results support a model where SWR1-C resides at the heterochromatin boundary to maintain and amplify antisilencing activity of histone H4 acetylation through incorporating H2A.Z into chromatin.

Both histone modification and chromatin-remodeling enzymes affect nucleosome dynamics in Saccharomyces cerevisiae. Histone modification enzymes covalently modify various histones through chemical means, such as lysine acetylation, serine phosphorylation, lysine and arginine methylation, ubiquitylation, deimination, ADP ribosylation, and proline isomerization (18). Chromatin-remodeling enzymes use the energy of ATP hydrolysis to induce nucleosome mobility or disrupt histone-DNA interactions (38). NuA4 is a histone acetyltransferase complex responsible for global histone H4 acetylation (1, 12, 39). The catalytic subunit of NuA4 is Esa1, which associates with Yng2p and Epl1p to form a smaller complex in vivo named Piccolo NuA4 (8). Piccolo NuA4 has a nontargeted histone acetyltransferase activity (8), while the full NuA4 complex, united by the Eaf1 protein, carries out site-specific and targeted acetylation reactions (2). The SWR1 complex (SWR1-C) is an ATP-dependent chromatin-remodeling complex that catalyzes the exchange of H2A-H2B with H2A.Z-H2B (H2A.Z is also known as Htz1) (17, 31). Notably, the platform proteins Eaf1 and Swr1 in NuA4 and SWR1-C, respectively, not only exhibit considerable sequence similarity to each other but also display strong and distinct homologies to human p400/Domino (2), suggesting an evolutionary merging of functions in higher eukaryotes.

Heterochromatin was originally defined cytologically as genome blocks where the structure of chromatin is highly condensed throughout the cell cycle and hence remains transcriptionally silenced (36). The main structural proteins of silent chromatin in S. cerevisiae are known as silent information regulator (Sir) proteins and include Sir2, Sir3, and Sir4. The Sir complex propagates along chromatin to form an ordered and compact structure that is usually restrictive to transcription (35).

Heterochromatin spreading is limited by boundary elements between silenced and active chromatin (35). These barriers appear to fall into two classes (6). The first class of barrier confers a structural characteristic to the boundary region, such as gaps in nucleosomes or association with nuclear pores. The second class of barrier is more dynamic and involves modification of heterochromatin-proximal regions in a wide area. Local chromatin dynamics can counteract the repressive Sir activity and prevent further spread of silencing.

Both SWR1-C and NuA4 have been linked to antisilencing functions at heterochromatin boundaries (11, 24, 27), however, their functional interplay in antisilencing is not yet clear. In this study, we dissected the individual and coupled actions of SWR1-C and NuA4 at antisilencing regions. By studying the functions of Swc4, we discovered an intrinsic relationship between NuA4 and SWR1-C in the establishment of a chromatin boundary.

MATERIALS AND METHODS

Yeast strains and reagents.

Antibodies, yeast strains, and primers used in this study are listed in Tables S1, S2 and S3 in the supplemental material, respectively.

ChIP assay.

Most chromatin immunoprecipitation (ChIP) assays were performed as described previously (47). For Esa1 ChIP, we followed an optimized protocol (20). ChIP products were directly analyzed by real-time PCR using SYBR green as a label (Toyobo). Enrichment values were the average immunoprecipitation (IP)/whole-cell extract (WCE) ratios from triplicate samples with standard errors of the means (SEM). Relative occupancy of protein/modification at indicated loci was the enrichment value at indicated loci relative to the control loci (PRP8 for hemagglutinin [HA]-tagged Swr1 [Swr1-HA], Htz1-HA, and tandem affinity purification (TAP)-tagged Swc4 [Swc4-TAP], TELVIR for H4 and AcH4K8, region R for Myc-tagged Esa1 [Esa1-Myc], and ACT1 for Sir2-Myc and Sir3-Myc).

Coimmunoprecipitation assay.

Yeast whole-cell extract was prepared from ~150 ml yeast culture (optical density at 600 nm [OD600] = 1.0) and then incubated with 25-μl of IgG Sepharose beads (Amersham Biosciences) for 90 min at 4°C. Beads were then pelleted and washed three times with 1 ml of phosphate-buffered saline (PBS). After washing, the beads were resuspended in SDS sample buffer and subjected to SDS-PAGE electrophoresis and Western blotting.

Immunostaining assay.

Immunostaining experiments were performed as previously described (47). Confocal microscopy was performed on a Leica TCS SP2 microscope with a 63× lambda blue objective (oil). Similar filtration and threshold levels were standardized for all images.

RESULTS

SWR1-C is abundant at antisilencing regions as well as silenced HMR loci.

To understand the antisilencing mechanisms for SWR1-C-mediated H2A.Z deposition and NuA4-mediated histone H4 acetylation, we first examined their relative abundances at regions near heterochromatin. Using chromatin immunoprecipitation (ChIP) combined with real-time PCR, we detected the relative abundance of HA-tagged Swr1, HA-tagged H2A.Z, and acetylated histone H4 at regions near HMR and within the subtelomeric regions of chromosomes III and XIV (Fig. (Fig.1A).1A). To distinguish between NuA4-mediated histone H4 acetylation and histone H4K16 acetylation mediated by SAS complex (26), we used anti-acetyl H4 lysine 8 (AcH4K8) antibody instead of anti-tetra-acetylated H4 antibody. The specificity of anti-AcH4K8 antibody was validated by Western blotting and ChIP (Fig. 1B and C). As expected, the occupancy of Swr1 was high at most of the euchromatic regions we tested. The occupancy of Swr1 was undetectable at distal telomeres but was surprisingly high at HMR loci (Fig. (Fig.1D,1D, regions D, E, F and G), where the nucleosomes are largely hypoacetylated. This result was unexpected, because a previous study had shown that SWR1-C was recruited to hyperacetylated chromatin domains through its Bdf1 subunit (25, 46). It is possible that a subunit(s) other than Bdf1 also functions to stabilize the association of SWR1-C complex with chromatin.

FIG. 1.
SWR1-C is abundant at antisilencing regions as well as silenced HMR loci. (A) Schematic diagram of four major antisilencing chromosomal domains. Shown are the HMR silent mating-type cassette and its proximal regions and regions near the ends of chromosome ...

We next sought to determine if changes in heterochromatin structure affected chromatin association of Swr1 and H2A.Z. Deletion of SIR2, which encodes one of the structural components of heterochromatin, disrupted heterochromatin silencing of both HM loci and telomeres (35). Interestingly, in the absence of Sir2, the occupation pattern of Swr1 at most regions, including distal telomeres, was barely altered (Fig. (Fig.1D).1D). Based on these data, we conclude that SWR1-C binds to chromatin in a manner that is independent of the chromatin silencing status.

Preassociation of SWR1-C with chromatin is essential but not sufficient for H2A.Z incorporation.

Although SIR2 deletion caused a dramatic elevation of H2A.Z levels at HMR loci where Swr1 was present (Fig. 1D and E, regions E, F, G, and H), it did not lead to an increase in H2A.Z levels at distal telomeres where Swr1 was absent (Fig. 1D and E, regions R, S, T, X, Y, and Z), suggesting that the chromatin binding of SWR1-C contributed to the replacement of H2A-H2B with H2A.Z-H2B. In addition, we found that Swr1 occupied many more chromatin regions than H2A.Z. For example, Swr1 was highly enriched at HMR, HML, and regions I and J, while H2A.Z was not enriched over background levels at those sites (Fig. 1D and E). Thus, the preassociation of SWR1-C with chromatin is not the sole driving force for H2A.Z deposition.

NuA4-mediated H4 acetylation is required for H2A.Z incorporation at Swr1-enriched regions.

Next, we considered if NuA4-dependent H4 acetylation contributed to H2A.Z deposition. Interestingly, the relative abundance of H4K8 acetylation and H2A.Z binding correlated nicely at most of the chromosomal loci we examined (Fig. 1E and F), implying a potential requirement of histone H4 acetylation for H2A.Z incorporation into nucleosomes. Additionally, EAF1 deletion, which eliminated H4K8 acetylation (Fig. (Fig.1F),1F), greatly reduced H2A.Z levels in the corresponding boundary regions (Fig. (Fig.1E).1E). Moreover, when a set of randomly selected intergenic regions was examined for H2A.Z occupation, we surprisingly found that H2A.Z was specifically incorporated into regions where we observed significantly high levels of Swr1 and acetylated H4K8 (Fig. (Fig.1G,1G, regions CLB6 and DYN1). Therefore, NuA4-dependent H4 acetylation is likely required for SWR1-C-mediated H2A.Z replacement of H2A at heterochromatin boundaries.

Targeted H4 acetylation triggers H2A.Z deposition at Swr1-enriched regions.

To investigate further the interplay between NuA4-mediated H4 acetylation and SWR1-C-mediated H2A.Z deposition, we adopted an artificial recruitment system (34) where an array of lac operator sites (lacO) was integrated into a locus ~4 kb from HMR or a locus ~4 kb from the end of chromosome XIV-R (Fig. (Fig.1A).1A). Esa1 or Swr1 was artificially targeted to the lacO loci by being fused to the LacI repressor. Western blot analysis with anti-LacI antibody showed that the expression levels of fusion proteins were comparable to each other (Fig. (Fig.2A).2A). As expected, tethered Esa1 acetylated chromatin near lacO (Fig. 2B and E). In contrast, tethered Swr1 increased H2A.Z incorporation into chromatin in an inefficient way (Fig. 2C and F). Consistently, tethered Esa1 but not Swr1 attenuated Sir2 binding at regions near lacO (Fig. 2D and G). This result coincided with the above statement that chromatin recruitment of SWR1-C per se was insufficient for H2A.Z deposition. Notably, recruited Esa1 also gave rise to a marked increase in H2A.Z levels at HMR-proximal regions (Fig. (Fig.2F).2F). The increase was apparently Swr1 dependent, because targeted Esa1 failed to boost H2A.Z levels at telomere XIV-R, where Swr1 was absent (Fig. (Fig.1D1D and and2C).2C). Hence, Esa1 targeting was able to directly induce H2A.Z incorporation at Swr1-enriched regions.

FIG. 2.
NuA4-mediated H4 acetylation triggers SWR1-C-mediated H2A.Z deposition. (A) Western blotting was performed with whole-cell extracts from strains with exogenous LacI fusion genes indicated on the top. Antibodies used are indicated on the right. (B to G) ...

Since both histone H4 and H2A.Z were substrates for NuA4 (1, 3), we wondered which event, either H4 acetylation or H2A.Z acetylation, was the signal that led to SWR1-C-dependent H2A.Z deposition. To this end, we mutated specific lysines on H4 or H2A.Z to arginines (H4K5,8,12R and HTZ1K14R). A lysine-to-arginine mutation was unable to be acetylated and thereby mimicked an unacetylated state. Interestingly, in H4K5,8,12R cells, tethered Esa1 reduced H2A.Z incorporation to a level much lower than that observed in wild-type cells (Fig. (Fig.2F).2F). In contrast, the HTZ1K14R mutation had a modest influence on H2A.Z incorporation induced by Esa1 tethering (Fig. (Fig.2F).2F). These data support the idea that H2A.Z K14 acetylation is not required for its deposition at boundaries (3), and NuA4-mediated H4K5,8,12 acetylation triggers H2A.Z deposition at heterochromatin boundaries.

Swc4 is required for both NuA4-mediated histone acetylation and SWR1-C-mediated H2A.Z incorporation at antisilencing regions.

We next considered the genetic interaction between NuA4 and SWR1-C in antisilencing. Unfortunately, double mutation of the key components of NuA4 and SWR1-C (e.g., eaf1Δ swr1Δ) caused synthetic lethality (17), thereby introducing challenges to the direct assessment of the genetic interaction between these two complexes. In a large-scale screening for mother-selfless genes in the BY4743 strain background (28), we coincidentally discovered that cells carrying a deletion of the SWC4 gene, which encodes a shared subunit of NuA4 and SWR1-C, were viable, although exhibiting a slow-growth phenotype (Fig. 3A to D). The Swc4 protein contains an N-terminal SANT domain and a C-terminal Yaf9-interacting domain (YID) (7) (Fig. (Fig.3E).3E). A SANT domain deletion mutant (swc4santΔ) phenocopied swc4Δ cells in yeast growth (Fig. (Fig.3F),3F), suggesting an essential role for the SANT domain in mediating Swc4's function. In addition, comparison of the transcriptional profiles of swc4yidΔ cells to that of htz1Δ cells revealed a marked overlap (Fig. (Fig.3G),3G), suggesting that the YID domain mediates Swc4's functions in SWR1-C-dependent H2A.Z deposition. Using analytical-scale affinity purification, we observed that Swc4 stably associated with Esa1 and Swr1 (Fig. 3H and I). Deletion of the SANT domain or the YID domain did not dissociate Swc4 from either of the main complexes (Fig. 3H and I).

FIG. 3.
Swc4 is a nonessential subunit shared by NuA4 and SWR1-C. (A and B) Shown is the tetrad dissection analysis of heterozygous SWC4/swc4Δ diploid yeast strains in a BY4743 background. Specific genetic backgrounds are shown on the top. Spores were ...

Proteins containing SANT domains had central roles in many chromatin-modifying complexes (9, 10). SWC4 deletion did not detectably alter global acetylation level of histone H4 (Fig. (Fig.3J),3J), suggesting that Swc4, as is the case for Eaf1 (2), regulates the site-specific roles of the NuA4 complex. We therefore asked whether Swc4 contributed to the actions of NuA4 and SWR1-C specifically at boundaries. ChIP experiments were performed to monitor the chromatin changes in swc4Δ cells. Interestingly, deletion of SWC4 resulted in hypoacetylation of histone H4K8 and dramatic reduction of H2A.Z levels at all the boundary regions we tested (Fig. 4A and B), most likely because deletion of SWC4 reduced binding of Swr1 and Esa1 with chromatin (Fig. 4C and D). Additionally, Swc4 itself was associated with regions (Fig. (Fig.4E)4E) that were also bound by Esa1 or Swr1 (Fig. (Fig.4E).4E). These data indicate that Swc4 directly regulates the chromatin association of both NuA4 and SWR1-C and thereby is indispensable for histone H4 acetylation and H2A.Z incorporation at heterochromatin boundaries.

FIG. 4.
Swc4 couples the actions of both NuA4 and SWR1-C on H2A.Z incorporation at boundaries. (A to E) Relative enrichments, as assayed for Fig. Fig.1F,1F, of AcH4K8 (A), Htz1-HA (B), Esa1-Myc (C), Swr1-HA (D), or Swc4-TAP (E) in cells with the indicated ...

Increased H2A.Z incorporation at subtelomeric regions reinforces the antisilencing activity of histone H4 acetylation.

We have shown above that H2A.Z deposition at chromosomal regions adjacent to heterochromatin required the coordinated action of SWR1-C and NuA4. Since Swc4 stabilized both SWR1-C and NuA4 at boundaries, tethered Swc4 might confer an even higher activity for H2A.Z incorporation at these sites. To test this idea, we utilized the lacO-LacI system again to target LacI-fused Swc4 to the subtelomeric region of chromosome XIV, where both SWR1-C and NuA4 were physiologically absent (Fig. 4C and D). As expected, targeted Swc4 increased subtelomeric H4K8 acetylation (Fig. (Fig.4F).4F). Strikingly, Swc4 targeting also boosted H2A.Z levels to greater than 10-fold above the basal level at the regions near lacO (Fig. (Fig.4G),4G), which was hardly seen when Esa1 or Swr1 was targeted (Fig. (Fig.2C).2C). Thus, Swc4 likely recruits both NuA4 and SWR1-C and couples the actions of SWR1-C and NuA4 toward H2A.Z deposition.

To examine whether Swc4 targeting conferred higher antisilencing activity than Esa1 or Swr1 targeting did, we examined Sir2 association at chromosomal regions near lacO and found that subtelomeric association of Sir2 was markedly reduced when Swc4-LacI was tethered to the lacO loci (Fig. (Fig.4H).4H). The abundance of Sir2 at region Z in the Swc4-tethering strain was even lower than that in the Esa1-tethering strain (Fig. (Fig.4H4H and and2D).2D). Additionally, the mRNA levels of PAU6, a gene located within the subtelomeric region of chromosome XIV, were highest in the Swc4-targeting strain (Fig. (Fig.4I).4I). Thus, tethered Swc4 conferred higher antisilencing activity than tethered Esa1 or tethered Swr1. The increased antisilencing activity was likely attributable to the increased H2A.Z deposition, because tethered Swc4 elevated histone H4 acetylation to a level similar to that observed in the Esa1-LacI strain (Fig. (Fig.4F4F and and2B).2B). Taken together, these data support the argument that incorporation of H2A.Z at the subtelomeric region enhances antisilencing activity normally attributed to histone H4 acetylation.

Synergistic action of NuA4 and SWR1-C confers broader antisilencing activity.

To further elucidate the functional relationship between NuA4 and SWR1-C in antisilencing, we carried out ChIP experiments to examine the distribution of Sir2 and Sir3 in eaf1Δ, swr1Δ, and swc4Δ cells. Under physiological conditions, chromatin-bound Sir proteins were primarily localized to silent chromatin regions, with limited spreading outside the boundaries (Fig. 5A and B, gray bars). In eaf1Δ cells, the binding of Sir proteins was increased in both heterochromatin and its nearby regions (Fig. 5A and B, pink bars), suggesting that NuA4 generally counteracts the association of Sir complexes with chromatin. In swr1Δ cells, the relative enrichment of both Sir2 and Sir3 was specifically increased at regions adjacent to heterochromatin, and this increase was accompanied by a modest reduction of Sir protein levels within heterochromatin (Fig. 5A and B, blue bars), a result that is typical of a Sir complex overspreading phenotype (27). Notably, in swc4Δ cells, Sir proteins were now enriched in the adjacent euchromatin, and the levels of Sir proteins in this euchromatin were much higher than we observed in swr1Δ or eaf1Δ cells (Fig. 5A and B). Since all three mutants we tested did not alter the expression of any of the major silencing-related genes, including RAP1, SIR1, SIR2, SIR3, SIR4, and HHF1 (data not shown), we concluded that these mutations had not likely affected Sir spreading in a nonspecific and indirect manner. Collectively, these data support the idea that Swc4 coordinates and integrates the effects of NuA4 and SWR1-C on the association of the Sir complex with chromatin.

FIG. 5.
Swc4 mediates a synergistic action of NuA4 and SWR1-C toward preventing Sir-dependent heterochromatin spreading. (A and B) Relative enrichments of Sir2-Myc (A, left panel) and Sir3-Myc (B, left panel) for wild-type (WT) and mutant cells. Average mutant/WT ...

To further examine heterochromatin spreading in swr1Δ, eaf1Δ, and swc4Δ cells, the subcellular localization of Myc-tagged Sir2 was examined by immunostaining using an anti-Myc antibody. In wild-type cells, we detected both a strong signal within a restricted nuclear subdomain and a weaker punctate pattern of staining (Fig. (Fig.5C).5C). The punctate Sir2 staining likely represented telomere-localized Sir2 (14). In either eaf1Δ or swr1Δ cells, there were no distinguishable changes to the Sir2-Myc staining pattern compared with that in wild-type cells (Fig. (Fig.5C).5C). However, in the swc4Δ mutant, the Sir2 staining pattern differed from that of wild-type cells by the presence of more nuclear foci (Fig. (Fig.5C).5C). Simultaneous immunostaining of Sir2 and Nop1, the yeast fibrillarin homolog, showed their colocalization and indicated an intact nucleolus in all the mutant strains (Fig. (Fig.5C).5C). Please note for Fig. Fig.5C5C that the weak Nop1 staining of one of the swc4Δ cells was not a result of dispersed or diminished nucleolar Nop1 but occurred because the nucleolus was slightly out of focus. These data, coupled with our ChIP data, have led us to postulate that the increase in punctate Sir2 staining in swc4Δ cells was most likely a result of the ectopic spread of the Sir2 protein into a wider range of euchromatic regions.

Previous work had shown that the ectopic spread of Sir proteins resulted in a transcriptional repression of genes adjacent to heterochromatin (27). We also examined the transcriptional changes in genes adjacent to heterochromatin in our mutant strains. The mRNA levels of representative genes proximal to or distant from heterochromatin (Fig. (Fig.1A)1A) were analyzed by real-time quantitative PCR. SIR2 deletion elevated the expression of the heterochromatin-proximal, but not the distal, genes (Fig. (Fig.5D),5D), revealing a limited ectopic spreading of Sir-mediated silencing proteins under normal physiological conditions. In swr1Δ, eaf1Δ, or swc4Δ mutant cells, the mRNA levels expressed by most genes we tested were decreased (Fig. (Fig.5D),5D), suggesting that both NuA4 and SWR1-C are required for normal expression of genes near heterochromatin. Notably, the SWC4 deletion strain showed the highest repressive effects on genes adjacent to heterochromatin. For example, the expression of the GTT1 gene, which was ~20 kb from the chromosome end, was slightly reduced in swr1Δ cells and eaf1Δ cells but was strongly repressed in swc4Δ cells (Fig. (Fig.5D),5D), suggesting that Swc4 has the greatest role in boundary activity of the three proteins we tested. Double mutations, eaf1Δ sir2Δ, swr1Δ sir2Δ, and swc4Δ sir2Δ, largely relieved the repressive effects on the transcription profile of the genes tested (Fig. (Fig.5D),5D), suggesting that the transcriptional repression in the mutant strains was Sir dependent. Since SWC4 deletion represented a synthetic defect of both NuA4 and SWR1-C, we concluded that NuA4 and SWR1-C synergistically work to antagonize Sir complex-mediated transcriptional silencing.

DISCUSSION

Chromatin-modifying complexes are usually targeted to specific regions in a dynamic manner to transiently modulate chromosomal processes like transcription and DNA damage repair (5, 41). In this study, we established that SWR1-C works at regions near heterochromatin to propagate an antisilencing signal that involves histone H4 acetylation and the promotion of H2A.Z incorporation. Genetic and functional assays examining the role of Swc4 in antisilencing have led us to propose that it is the synergistic actions of NuA4 and SWR1-C that lead to the efficient incorporation of H2A.Z at regions near heterochromatin and thereby contribute substantively to the levels of heterochromatin-euchromatin boundary activity (Fig. (Fig.66).

FIG. 6.
Model for the actions of NuA4 and SWR1-C at heterochromatin boundaries. Step 1: SWR1-C associates with chromatin through the shared module (Arp4, Act1, Yaf9, and Swc4). Without histone acetylation, Sir proteins tend to spread along the chromatin fiber. ...

SWC4 is a nonessential gene in the BY4743 background.

In this study, we unexpectedly identified SWC4 as a nonessential gene in the BY4743 background (Fig. 3A to D). The severe slow-growth phenotype of swc4Δ cells probably led others to conclude that SWC4 was an essential gene (2, 4, 23, 29). Our genetic and biochemical data have revealed that Swc4 is a coregulator of both NuA4 and SWR1-C (Fig. (Fig.4).4). Since Swc4 is a member of both NuA4 and SWR1-C (2, 17, 19, 30, 43), the cellular dysfunctions in swc4Δ cells are likely a result of a synthetic defect of both NuA4 and SWR1-C. Our findings indicate that subunits shared between NuA4 and SWR1-C are not coincident but instead underlie the cooperation between NuA4 and SWR1-C. It remains unclear how swc4Δ cells survive with the synthetic defect of inactivation of both NuA4 and SWR1-C. Since all swc4Δ spores were viable (Fig. 3A to D), it seems unlikely that a suppressor mutation has occurred in every isolate of swc4Δ cells. One possibility is that the Swc4 regulates the chromosomal functions of NuA4 and SWR1-C at limited regions. It will be intriguing to explore the potential roles of Swc4 in mediating these complexes' collaboration in other chromatin domains.

Shared subunits at the center of circuitry between SWR1-C and NuA4.

One attractive mechanism for the synergism between SWR1-C and NuA4 involves the differing association of unique subunits of the two complexes with the four shared subunits Act1, Arp4, Swc4, and Yaf9. Yaf9 contributes to the functions of both NuA4 and SWR1-C (45). Since there is physical interaction between Swc4 and Yaf9 (7, 44), it is likely that Swc4 links Yaf9 to NuA4 and SWR1-C and therefore mediates the function of Yaf9 in these two complexes. The actin protein Act1 and the actin-related protein Arp4 are two essential skeletal components that link the shared module with the main respective components of the complexes (40, 44). Interestingly, Arp4 is capable of direct interaction with both histones and nucleosomes (16). We propose that these four shared subunits cooperatively facilitate the general association of SWR1-C and NuA4 with chromatin.

The recruitment of SWR1-C to chromatin also involves binding of the Bdf1 subunit to acetylated histones through its bromodomain (21, 46). Likewise, the association of full NuA4 with promoters also involves the recognition of methylated histone H3 lysine 4 by the Yng2 subunit through its PHD domain (32). We propose that the shared subunits act as a docking platform for chromatin while other subunits of the complexes provide for the binding specificity.

Chromatin-modifying complexes synergize boundary activity.

The synergistic relationship between NuA4-dependent histone H4 acetylation and SWR1-C-dependent incorporation of H2A.Z has been previously described (13, 24, 33, 46). In this study, we have built on these pioneering studies and demonstrated that the coupled action of NuA4 and SWR1-C efficiently antagonizes Sir protein-dependent heterochromatin spreading. Taking advantage of Swc4, the versatile coregulator of NuA4 and SWR1-C, we found that heterochromatin became much more pervasive when both NuA4 and SWR1-C were deregulated (Fig. (Fig.5).5). Using the LacI-lacO system, we showed that corecruitment of NuA4 and SWR1-C conferred higher antisilencing activity than either module anchoring did alone (Fig. (Fig.2D2D and 4H and I).

Synergistic antisilencing actions of H2A.Z incorporation and other chromatin modifications, such as SET1-mediated histone H3K4 trimethylation and SAS-mediated histone H4K16 acetylation, have also been reported (37, 42). Since H3K4 trimethylation is proposed to regulate NuA4 recruitment (32), it will be interesting to know whether H3K4 trimethylation contributes to boundary function through recruitment of the NuA4 complex.

SWR1-C prepares boundary regions to propagate antisilencing activities.

The genome-wide chromatin association pattern of H2A.Z has been mapped (15, 22, 33, 46), but the blueprint for SWR1-C has not been well drawn. One speculation is that the binding profile of SWR1-C overlaps that of H2A.Z. Our results demonstrated that Swr1 covered a wider range of chromosomal domains than H2A.Z did. Swr1 was highly enriched at some heterochromatic regions, but H2A.Z was not (Fig. 1D, E, and G). However, targeted Swr1 did not boost H2A.Z levels in hypoacetylated regions (Fig. (Fig.2C).2C). We propose that recruitment of SWR1-C provides potential but by itself is not sufficient for specific chromatin regions to incorporate H2A.Z.

H2A.Z incorporation appeared to take place at histone H4K8-hyperacetylated regions (Fig. 1E, F, and and1G).1G). These results are in agreement with an earlier finding that the genome-wide occupancy of H2A.Z positively correlated with AcH4K8 and AcH4K12 (46). Esa1 tethering enhanced histone H4 acetylation and induced chromatin-bound SWR1-C to exchange H2A-H2B with H2A.Z-H2B. The incorporated H2A.Z then efficiently counteracted the encroachment of heterochromatin silencing (27). Since histone H4 acetylation per se was a natural antagonist of silencing, H2A.Z incorporation can further amplify its antisilencing activity (Fig. (Fig.66).

It remains unclear how NuA4-mediated histone acetylation induces SWR1-C-mediated H2A.Z deposition. One possibility is that histone acetylation facilitates the chromatin-remodeling process by neutralizing the positive charges on the histone surface and thereby leading to a looser configuration of the chromatin fiber. Interestingly, histone H4K16, catalyzed by the SAS complex, is also required for H2A.Z incorporation (37). It is possible that NuA4 and SAS cooperatively define the nucleosomal surface used for SWR1-C-mediated H2A.Z deposition.

Supplementary Material

[Supplemental material]

Acknowledgments

We thank Brian A. Lenzmeier (Buena Vista University, Storm Lake, IA) for his critical reading of our manuscript. We thank Susan M. Gasser, who generously provided the plasmids for the lacO targeting system.

This project was supported by grants from the Ministry of Science and Technology of China (2005CB522402) and the National Natural Science Foundation of China (90919027).

We declare that no competing interests exist.

Footnotes

[down-pointing small open triangle]Published ahead of print on 22 March 2010.

Supplemental material for this article may be found at http://mcb.asm.org/.

REFERENCES

1. Allard, S., R. T. Utley, J. Savard, A. Clarke, P. Grant, C. J. Brandl, L. Pillus, J. L. Workman, and J. Cote. 1999. NuA4, an essential transcription adaptor/histone H4 acetyltransferase complex containing Esa1p and the ATM-related cofactor Tra1p. EMBO J. 18:5108-5119. [PubMed]
2. Auger, A., L. Galarneau, M. Altaf, A. Nourani, Y. Doyon, R. T. Utley, D. Cronier, S. Allard, and J. Cote. 2008. Eaf1 is the platform for NuA4 molecular assembly that evolutionarily links chromatin acetylation to ATP-dependent exchange of histone H2A variants. Mol. Cell. Biol. 28:2257-2270. [PMC free article] [PubMed]
3. Babiarz, J. E., J. E. Halley, and J. Rine. 2006. Telomeric heterochromatin boundaries require NuA4-dependent acetylation of histone variant H2A.Z in Saccharomyces cerevisiae. Genes Dev. 20:700-710. [PubMed]
4. Ben-Aroya, S., C. Coombes, T. Kwok, K. A. O'Donnell, J. D. Boeke, and P. Hieter. 2008. Toward a comprehensive temperature-sensitive mutant repository of the essential genes of Saccharomyces cerevisiae. Mol. Cell 30:248-258. [PubMed]
5. Berger, S. L. 2007. The complex language of chromatin regulation during transcription. Nature 447:407-412. [PubMed]
6. Bi, X., and J. R. Broach. 2001. Chromosomal boundaries in S. cerevisiae. Curr. Opin. Genet. Dev. 11:199-204. [PubMed]
7. Bittner, C. B., D. T. Zeisig, B. B. Zeisig, and R. K. Slany. 2004. Direct physical and functional interaction of the NuA4 complex components Yaf9p and Swc4p. Eukaryot. Cell 3:976-983. [PMC free article] [PubMed]
8. Boudreault, A. A., D. Cronier, W. Selleck, N. Lacoste, R. T. Utley, S. Allard, J. Savard, W. S. Lane, S. Tan, and J. Cote. 2003. Yeast enhancer of polycomb defines global Esa1-dependent acetylation of chromatin. Genes Dev. 17:1415-1428. [PubMed]
9. Boyer, L. A., M. R. Langer, K. A. Crowley, S. Tan, J. M. Denu, and C. L. Peterson. 2002. Essential role for the SANT domain in the functioning of multiple chromatin remodeling enzymes. Mol. Cell 10:935-942. [PubMed]
10. Boyer, L. A., R. R. Latek, and C. L. Peterson. 2004. The SANT domain: a unique histone-tail-binding module? Nat. Rev. Mol. Cell Biol. 5:158-163. [PubMed]
11. Chiu, Y. H., Q. Yu, J. J. Sandmeier, and X. Bi. 2003. A targeted histone acetyltransferase can create a sizable region of hyperacetylated chromatin and counteract the propagation of transcriptionally silent chromatin. Genetics 165:115-125. [PubMed]
12. Clarke, A. S., J. E. Lowell, S. J. Jacobson, and L. Pillus. 1999. Esa1p is an essential histone acetyltransferase required for cell cycle progression. Mol. Cell. Biol. 19:2515-2526. [PMC free article] [PubMed]
13. Durant, M., and B. F. Pugh. 2007. NuA4-directed chromatin transactions throughout the Saccharomyces cerevisiae genome. Mol. Cell. Biol. 27:5327-5335. [PMC free article] [PubMed]
14. Gotta, M., S. Strahl-Bolsinger, H. Renauld, T. Laroche, B. K. Kennedy, M. Grunstein, and S. M. Gasser. 1997. Localization of Sir2p: the nucleolus as a compartment for silent information regulators. EMBO J. 16:3243-3255. [PubMed]
15. Guillemette, B., A. R. Bataille, N. Gevry, M. Adam, M. Blanchette, F. Robert, and L. Gaudreau. 2005. Variant histone H2A.Z is globally localized to the promoters of inactive yeast genes and regulates nucleosome positioning. PLoS Biol. 3:e384. [PMC free article] [PubMed]
16. Harata, M., Y. Oma, S. Mizuno, Y. W. Jiang, D. J. Stillman, and U. Wintersberger. 1999. The nuclear actin-related protein of Saccharomyces cerevisiae, Act3p/Arp4, interacts with core histones. Mol. Biol. Cell 10:2595-2605. [PMC free article] [PubMed]
17. Kobor, M. S., S. Venkatasubrahmanyam, M. D. Meneghini, J. W. Gin, J. L. Jennings, A. J. Link, H. D. Madhani, and J. Rine. 2004. A protein complex containing the conserved Swi2/Snf2-related ATPase Swr1p deposits histone variant H2A.Z into euchromatin. PLoS Biol. 2:E131. [PMC free article] [PubMed]
18. Kouzarides, T. 2007. Chromatin modifications and their function. Cell 128:693-705. [PubMed]
19. Krogan, N. J., K. Baetz, M. C. Keogh, N. Datta, C. Sawa, T. C. Kwok, N. J. Thompson, M. G. Davey, J. Pootoolal, T. R. Hughes, A. Emili, S. Buratowski, P. Hieter, and J. F. Greenblatt. 2004. Regulation of chromosome stability by the histone H2A variant Htz1, the Swr1 chromatin remodeling complex, and the histone acetyltransferase NuA4. Proc. Natl. Acad. Sci. U. S. A. 101:13513-13518. [PubMed]
20. Kurdistani, S. K., and M. Grunstein. 2003. In vivo protein-protein and protein-DNA crosslinking for genomewide binding microarray. Methods 31:90-95. [PubMed]
21. Ladurner, A. G., C. Inouye, R. Jain, and R. Tjian. 2003. Bromodomains mediate an acetyl-histone encoded antisilencing function at heterochromatin boundaries. Mol. Cell 11:365-376. [PubMed]
22. Li, B., S. G. Pattenden, D. Lee, J. Gutierrez, J. Chen, C. Seidel, J. Gerton, and J. L. Workman. 2005. Preferential occupancy of histone variant H2AZ at inactive promoters influences local histone modifications and chromatin remodeling. Proc. Natl. Acad. Sci. U. S. A. 102:18385-18390. [PubMed]
23. Lin, Y. Y., Y. Qi, J. Y. Lu, X. Pan, D. S. Yuan, Y. Zhao, J. S. Bader, and J. D. Boeke. 2008. A comprehensive synthetic genetic interaction network governing yeast histone acetylation and deacetylation. Genes Dev. 22:2062-2074. [PMC free article] [PubMed]
24. Lu, P. Y., N. Levesque, and M. S. Kobor. 2009. NuA4 and SWR1-C: two chromatin-modifying complexes with overlapping functions and components. Biochem. Cell Biol. 87:799-815. [PubMed]
25. Matangkasombut, O., and S. Buratowski. 2003. Different sensitivities of bromodomain factors 1 and 2 to histone H4 acetylation. Mol. Cell 11:353-363. [PubMed]
26. Meijsing, S. H., and A. E. Ehrenhofer-Murray. 2001. The silencing complex SAS-I links histone acetylation to the assembly of repressed chromatin by CAF-I and Asf1 in Saccharomyces cerevisiae. Genes Dev. 15:3169-3182. [PubMed]
27. Meneghini, M. D., M. Wu, and H. D. Madhani. 2003. Conserved histone variant H2A.Z protects euchromatin from the ectopic spread of silent heterochromatin. Cell 112:725-736. [PubMed]
28. Meng, F. L., Y. Hu, N. Shen, X. J. Tong, J. Wang, J. Ding, and J. Q. Zhou. 2009. Sua5p a single-stranded telomeric DNA-binding protein facilitates telomere replication. EMBO J. 28:1466-1478. [PubMed]
29. Micialkiewicz, A., and A. Chelstowska. 2008. The essential function of Swc4p—a protein shared by two chromatin-modifying complexes of the yeast Saccharomyces cerevisiae—resides within its N-terminal part. Acta Biochim. Pol. 55:603-612. [PubMed]
30. Mitchell, L., J. P. Lambert, M. Gerdes, A. S. Al-Madhoun, I. S. Skerjanc, D. Figeys, and K. Baetz. 2008. Functional dissection of the NuA4 histone acetyltransferase reveals its role as a genetic hub and that Eaf1 is essential for complex integrity. Mol. Cell. Biol. 28:2244-2256. [PMC free article] [PubMed]
31. Mizuguchi, G., X. Shen, J. Landry, W. H. Wu, S. Sen, and C. Wu. 2004. ATP-driven exchange of histone H2AZ variant catalyzed by SWR1 chromatin remodeling complex. Science 303:343-348. [PubMed]
32. Morillon, A., N. Karabetsou, A. Nair, and J. Mellor. 2005. Dynamic lysine methylation on histone H3 defines the regulatory phase of gene transcription. Mol. Cell 18:723-734. [PubMed]
33. Raisner, R. M., P. D. Hartley, M. D. Meneghini, M. Z. Bao, C. L. Liu, S. L. Schreiber, O. J. Rando, and H. D. Madhani. 2005. Histone variant H2A.Z marks the 5′ ends of both active and inactive genes in euchromatin. Cell 123:233-248. [PMC free article] [PubMed]
34. Rohner, S., S. M. Gasser, and P. Meister. 2008. Modules for cloning-free chromatin tagging in Saccharomyces cerevisiae. Yeast 25:235-239. [PubMed]
35. Rusche, L. N., A. L. Kirchmaier, and J. Rine. 2003. The establishment, inheritance, and function of silenced chromatin in Saccharomyces cerevisiae. Annu. Rev. Biochem. 72:481-516. [PubMed]
36. Schultz, J. 1936. Variegation in Drosophila and the inert chromosome regions. Proc. Natl. Acad. Sci. U. S. A. 22:27-33. [PubMed]
37. Shia, W. J., B. Li, and J. L. Workman. 2006. SAS-mediated acetylation of histone H4 Lys 16 is required for H2A.Z incorporation at subtelomeric regions in Saccharomyces cerevisiae. Genes Dev. 20:2507-2512. [PubMed]
38. Sif, S. 2004. ATP-dependent nucleosome remodeling complexes: enzymes tailored to deal with chromatin. J. Cell Biochem. 91:1087-1098. [PubMed]
39. Smith, E. R., A. Eisen, W. Gu, M. Sattah, A. Pannuti, J. Zhou, R. G. Cook, J. C. Lucchesi, and C. D. Allis. 1998. ESA1 is a histone acetyltransferase that is essential for growth in yeast. Proc. Natl. Acad. Sci. U. S. A. 95:3561-3565. [PubMed]
40. Szerlong, H., K. Hinata, R. Viswanathan, H. Erdjument-Bromage, P. Tempst, and B. R. Cairns. 2008. The HSA domain binds nuclear actin-related proteins to regulate chromatin-remodeling ATPases. Nat. Struct. Mol. Biol. 15:469-476. [PMC free article] [PubMed]
41. van Attikum, H., and S. M. Gasser. 2005. The histone code at DNA breaks: a guide to repair? Nat. Rev. Mol. Cell Biol. 6:757-765. [PubMed]
42. Venkatasubrahmanyam, S., W. W. Hwang, M. D. Meneghini, A. H. Tong, and H. D. Madhani. 2007. Genome-wide, as opposed to local, antisilencing is mediated redundantly by the euchromatic factors Set1 and H2A.Z. Proc. Natl. Acad. Sci. U. S. A. 104:16609-16614. [PubMed]
43. Wu, W. H., S. Alami, E. Luk, C. H. Wu, S. Sen, G. Mizuguchi, D. Wei, and C. Wu. 2005. Swc2 is a widely conserved H2AZ-binding module essential for ATP-dependent histone exchange. Nat. Struct. Mol. Biol. 12:1064-1071. [PubMed]
44. Wu, W. H., C. H. Wu, A. Ladurner, G. Mizuguchi, D. Wei, H. Xiao, E. Luk, A. Ranjan, and C. Wu. 2009. N terminus of Swr1 binds to histone H2AZ and provides a platform for subunit assembly in the chromatin remodeling complex. J. Biol. Chem. 284:6200-6207. [PubMed]
45. Zhang, H., D. O. Richardson, D. N. Roberts, R. Utley, H. Erdjument-Bromage, P. Tempst, J. Cote, and B. R. Cairns. 2004. The Yaf9 component of the SWR1 and NuA4 complexes is required for proper gene expression, histone H4 acetylation, and Htz1 replacement near telomeres. Mol. Cell. Biol. 24:9424-9436. [PMC free article] [PubMed]
46. Zhang, H., D. N. Roberts, and B. R. Cairns. 2005. Genome-wide dynamics of Htz1, a histone H2A variant that poises repressed/basal promoters for activation through histone loss. Cell 123:219-231. [PMC free article] [PubMed]
47. Zhou, J., B. O. Zhou, B. A. Lenzmeier, and J. Q. Zhou. 2009. Histone deacetylase Rpd3 antagonizes Sir2-dependent silent chromatin propagation. Nucleic Acids Res. 37:3699-3713. [PMC free article] [PubMed]

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