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. Author manuscript; available in PMC: 2015 Feb 6.
Published in final edited form as: Mol Cell. 2014 Feb 6;53(3):498–505. doi: 10.1016/j.molcel.2014.01.010

The catalytic subunit of the SWR1 remodeler is a histone chaperone for the H2A.Z-H2B dimer

Jingjun Hong 1, Hanqiao Feng 1, Feng Wang 1, Anand Ranjan 1, Jianhong Chen 1, Jiansheng Jiang 2, Rodolfo Ghirlando 3, T Sam Xiao 2, Carl Wu 1, Yawen Bai 1,*
PMCID: PMC3940207  NIHMSID: NIHMS557564  PMID: 24507717

SUMMARY

Histone variant H2A.Z-containing nucleosomes exist at most eukaryotic promoters and play important roles in gene transcription and genome stability. The multi-subunit nucleosome-remodeling enzyme complex SWR1, conserved from yeast to mammals, catalyzes the ATP-dependent replacement of histone H2A in canonical nucleosomes with H2A.Z. How SWR1 catalyzes the replacement reaction is largely unknown. Here we determined the crystal structure of the N-terminal region (599–627) of the catalytic subunit Swr1, termed Swr1-Z domain, in complex with the H2A.Z-H2B dimer at 1.78 Å resolution. The Swr1-Z domain forms a 310 helix and an irregular chain. A conserved LxxLF motif in the Swr1-Z 310 helix specifically recognizes the αC helix of H2A.Z. Our results show that the Swr1-Z domain can deliver the H2A.Z-H2B dimer to the DNA-(H3–H4)2 tetrasome to form the nucleosome by a histone chaperone mechanism.

INTRODUCTION

Eukaryotic genomic DNA is packaged into chromatin through association with histones to form the nucleosome, the structural unit of chromatin (Kornberg, 1974; Kornberg and Thomas, 1974). The canonical nucleosome core consists of an octamer of histones with two copies of H2A, H2B, H3 and H4, around which ~146 bp of DNA winds in ~1.65 left-handed super-helical turns, which occludes about half of the DNA surface (Luger et al., 1997), making it poorly accessible to the transcriptional machinery. In cells, two major classes of enzymes participate in counteracting the constraints imposed on DNA by the nucleosome structure. The first class covalently modifies the nucleosome core histones by attaching them to chemical groups, which can alter the dynamics of the nucleosome (Fischle et al., 2003). The second class catalyzes mobility or reorganization of the nucleosome in an ATP-dependent fashion (Eberharter and Becker, 2004).

There are also minor nucleosomes containing variants of histones H2A and H3 in the chromatin (Ausio, 2006). For example, in budding yeast, the vast majority of promoters are characterized by two well-positioned nucleosomes containing histone variant H2A.Z, flanking a DNA region that is relatively depleted for histones (Cairns, 2009; Jiang and Pugh, 2009; Weiner et al., 2010). The H2A.Z nucleosome acts as a barrier that occludes the transcription start sites at the edge of the nucleosome free region and helps position downstream nucleosomes in the coding region (Jiang and Pugh, 2009). Budding yeast cells in which the H2A.Z gene is deleted exhibit slow growth (Carr et al., 1994; Santisteban et al., 2000), chromosome instability (Carr et al., 1994; Krogan et al., 2004), gene silencing defects (Meneghini et al., 2003), and sensitivity to genotoxic and environmental stress (Jackson and Gorovsky, 2000; Kobor et al., 2004; Mizuguchi et al., 2004). H2A.Z has also been implicated in DNA repair (Morrison and Shen, 2009) and in suppression of spurious noncoding transcription (Zofall et al., 2009). In metazoans, H2A.Z is localized to nucleosomes proximal to promoters of active genes (Rando and Chang, 2009).

The S. cerevisiae SWR1 complex (SWR1) is required for the incorporation of H2A.Z (Venters and Pugh, 2009; Kobor et al., 2004; Mizuguchi et al., 2004). The human homologues of SWR1, p400 and SRCAP, have also been identified (Gevry et al., 2007; Ruhl et al., 2006). SWR1-catalyzed H2A.Z replacement in vitro occurs in a stepwise manner, producing heterotypic nucleosomes containing both H2A and H2A.Z as intermediates and homotypic nucleosomes with only H2A.Z as end products (Luk et al., 2010). Two regions in the SWR1 subunits Swc2 and Swr1 have been identified to associate with H2A.Z-H2B dimer directly in vitro (Wu et al., 2005; Wu et al., 2009). In addition, an H2A.Z-specific chaperone, Chz1, is found to facilitate the H2A.Z/H2A exchange reaction (Luk et al., 2007; Zhou et al., 2008), suggesting that Chz1 delivers the H2A.Z-H2B dimer to the Swc2 and/or Swr1 subunits of SWR1.

Although many genetic and biochemical studies on SWR1 have been reported, little is known about how H2A.Z is incorporated into the nucleosome and how the replacement reaction is regulated. Here, we investigated the structural mechanism for the recognition of the H2A.Z-H2B dimer by the N-terminal region of the catalytic subunit Swr1. Our results suggest that the Swr1 subunit has a chaperone function for the H2A.Z-H2B dimer.

RESULTS

Structure of an Swr1 domain in complex with a single chain H2A.Z-H2B

To investigate the structural basis of H2A.Z recognition by the Swr1 subunit, we used nuclear magnetic resonance (NMR) spectroscopy to determine the precise Swr1 region that interacts with the H2A.Z-H2B dimer based on the earlier observation that the Swr1370-681 region, which is N-terminal to the ATPase domains, binds to the H2A.Z-H2B dimer (Fig. 1A, B and Fig. S1A–C) (Wu et al., 2009). We found that the Swr1599-627 region binds to the single chain H2B36-130 and H2A.Z22-118 chimera (scH2B-H2A.Z) used previously to determine the structure of the Chz1-scH2B-H2A.Z complex. Here we termed this H2A.Z-H2B binding region the Swr1-Z domain. Sedimentation experiments indicate that Swr1589-650 and scH2B-H2A.Z formed a 1:1 complex (Fig. 1C). We solved the structure of the Swr1589-638-scH2B-H2A.Z complex at 1.78 Å resolution (Fig. 1D and Table 1). The crystal structure is consistent with the secondary structures and dynamic behavior of the complex characterized by solution NMR (Fig. S1D).

Figure 1. Structure of the Swr1589-650-scH2B-H2A.Z complex.

Figure 1

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(A) Illustration of the location of the ATPase domains and the amino acid sequence in the region 599–627 of the Swr1 subunit. The letters in bold indicate the amino acids that are folded in the complex, whereas the regular letters indicate the disordered loop.

(B) Deviation of Cα chemical shifts of Swr1589-650 from random coil values: Swr1589-650 in the free form (upper panel) and in complex with scH2B-H2A.Z (lower panel). The regions in red are not observable because they form folded structures in the complex and the complex is larger than 25 kDa.

(C) Velocity sedimentation result of the Swr1589-650-scH2B-H2A.Z complex, showing a S20,w of 2.54S complex with a molecular weight of 27.7 kDa.

(D) Overall structure of the Swr1589-638-scH2B-H2A.Z complex. Only regions 599–605 and 614–627 in the Swr1-Z domain are folded in the complex. See also Figure S1.

Table 1.

Data collection and refinement statistics.

Data collection
Space group P21212
Cell dimensions
 a, b, c (Å) 65.61, 69.85, 101.60
 α, β, γ (°) 90.00, 90.00, 90.00
Molecules/asymmetric unit 2
Resolution (Å)* 50-1.78 (1.84-1.78)
Rmergea 0.05 (0.58)
I/σI 23.6 (2.5)
Completeness (%) 96.8 (76.7)
Redundancy 6.9 (4.8)
Unique reflections 43986
Refinement
Non-hydrogen atoms 3211
Water molecules 187
Rwork (%)b 21.1
Rfree (%)c 22.0
Average B-factor (Ǻ2)
Protein main chain 28.3
Protein side-chain 32.0
Water 35.0
RMSD from ideality
Bond lengths (Ǻ) 0.013
Bond angles (deg.) 1.6
Ramachandran analysis
Most-favored regions (%) 98.1
Additionally allowed regions (%) 1.9
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Values in parentheses are for highest-resolution shell.

a

Rmerge = Σh Σi |Ii(h) − <I(h)> |/ΣhΣi Ii(h), where Ii(h) and <I(h)> are the ith and mean measurement of the intensity of reflection h.

b

Rwork = Σh||Fobs (h)| − |Fcalc (h)||/Σh|Fobs (h)|, where Fobs (h) and F calc (h) are the observed and calculated structure factors, respectively.

c

Rfree is the R value obtained for a test set of reflections consisting of a randomly selected 5% subset of the dataset excluded from refinement.

In the crystal structure, the Swr1-Z domain forms a η(eta)-shaped structure and binds to one region of the scH2B-H2A.Z (Fig. 1D). The binding sites are distant from the connection point of H2A.Z and H2B in scH2B-H2A.Z (Fig. S2A), further validating the use of the single chain chimera. The Swr1-Z domain residues 599–605, which include a 310 helix, interact with the residues in the α3 and αC helices of H2A.Z and the N-terminal region of the α2 helix of H2B through hydrophobic interactions. Residues Leu601, Leu604 and Phe605 of the Swr1-Z domain form a hydrophobic cluster with the residues Leu91 and Ile95 in the α3 helix and Ile105 and Ile109 in the αC helix of H2A.Z (Fig. 2A and Fig. S2B). The Swr1-Z domain residues 606–613, which include 3 Gly residues and 3 acidic residues, formed a disordered loop (Fig. 1A, D). Following the disordered loop, the Swr1-Z domain residues 614–622 bind to the L2 loop and α3 helix of H2A.Z, and the L1 loop and α2 helix of H2B through electrostatic interactions (Fig. 1D and Fig. S2C). Negatively charged Swr1-Z domain residues Asp614, Asp616, Asp618, Asp619, Glu621 and Asp622 are surrounded by the positively charged residues Arg85 and Arg89 of H2A.Z and Lys60 of H2B (Fig. 2B). Finally, the Swr1-Z domain residues 623–627 interact with the α1 helix and the N-terminal region of the α2 helix of H2B through hydrophobic interactions (Fig. 2C). The Swr1-Z domain residues Phe623 and Val625 make hydrophobic interactions with Tyr45 in the α1 helix and Met62 in the N-terminal region of the α2 helix of H2B.

Figure 2. Specific interactions between the Swr1-Z domain and scH2B-H2A.Z.

Figure 2

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(A–C) Hydrophobic and electrostatic interactions between the Swr1-Z domain and scH2B-H2A.Z.

(D–F) Effects of mutations on the binding affinity between the Swr1-Z domain and scH2B-H2A.Z.

(G) Comparison of the H2A.Z αC helix in the Swr1-Z domain-scH2B-H2A.Z complex and the corresponding region in the H2A model.

(H) Effects of replacement of the αC residues in H2A.Z by those of H2A or vice versa on the binding affinity. Here, Kd(WT) is the dissociation equilibrium constant between the Swr1-Z domain and scH2B-H2A.Z. See also Figure S2 and Table S1.

Residues important for interactions between the Swr1-Z domain and scH2B-H2A.Z

To identify the residues important for interactions between the Swr1-Z domain and H2A.Z-H2B, we used isothermal titration calorimetry (ITC) to measure the binding affinity between Swr1504-650 and scH2B-H2A.Z. We found that Swr1504-650 binds to scH2B-H2A.Z tightly, with a dissociation equilibrium constant (Kd) of ~7 nM, whereas it binds to scH2B-H2A ~20 times weaker, with a Kd of ~151 nM (Fig. S2D, E and Table S1). Mutation of each of the Swr1-Z domain residues Leu601, Leu604, and Phe605 to Ala reduces the binding affinity by a factor of ~6 (Fig. 2A, D). A double mutation Leu601Gly/Phe605Gly reduces the binding affinity by more than a factor of 10. For the electrostatic interactions (Fig. 2B, E), a double mutation of Asp616Ala/Asp618Ala or Asp618Ala/Asp622Ala also reduces the binding affinity by more than a factor of 10. For the second hydrophobic cluster (Fig. 2C, F), mutation of each of the Swr1-Z domain residues Phe623 and Val625 to Ala decreases the binding affinity by a factor of ~6 and ~3, respectively. Double Ala mutations of Phe623Ala/Val625Ala and Phe623Ala/Asp622Ala reduce the binding affinity by factors of ~9 and ~29, respectively. These results are fully consistent with the crystal structure.

Residues responsible for specific recognition of H2A.Z-H2B by the Swr1-Z domain

In the structure of the Swr1-Z domain-scH2B-H2A.Z complex, the Swr1-Z domain residues Leu601 and Phe605 directly interact with H2A.Z-specific region (Ile105 — Arg-Ala-Thr-Ile109) in the αC helix of H2A.Z, whose corresponding H2A residues are Leu98-Gly-Asn-Val-Thr-Ile103 (Fig. 2G). The insertion of a residue in H2A in this region would disrupt the register of the hydrophobic residue Ile103 in the αC helix and prevent it from interacting with Leu601 in the Swr1-Z domain. We found that insertion of a Gly residue into the corresponding position in H2A.Z decreased the binding affinity by a factor of ~4 (Fig. 2H). Replacement of H2A.Z residues Ile105 — Arg-Ala107 by the corresponding residues Leu98-Gly-Asn-Val101 of H2A decreased binding affinity by a factor of ~14. Replacement of a longer region, Asp102-Ser-Leu-Ile — Arg-Ala107, of H2A.Z with the corresponding region, Asn95-Lys-Leu-Leu-Gly-Asn-Val101, in H2A had less effect on the binding affinity (~ a factor of 6), indicating that there are compensatory effects for the binding affinity by the two additional residues. We also made two mutants of H2A with corresponding inverse mutations (Fig. 2H), which bind to the Swr1-Z domain with binding affinities that were only a factor of ~2 or ~3 lower than that between the Swr1-Z domain and scH2B-H2A.Z. We conclude that the hydrophobic interactions between the residues Leu601 and Phe605 of the Swr1-Z domain and residues Ile105 and Ile109 of H2A.Z, which are facilitated by the deletion of one residue in the corresponding region of H2A, are largely responsible for specific recognition of H2A.Z by the Swr1-Z domain.

Chaperone properties of the Swr1-Z domain

In comparison with the structure of the H2A.Z-H2B histones in the nucleosome, we found that the αC helix of H2A.Z in the Swr1-Z domain-scH2B-H2A.Z complex was extended by two extra helical turns (Fig. 3A). The region after the αC helix did not show electron density and is not observable by NMR, presumably due to dynamic motions on the millisecond time scale. Overlay of the Swr1-Z domain-scH2B-H2A.Z and Chz1-scH2B-H2A.Z structures on the histones shows that the Swr1-Z domain and Chz1 bind to different locations on the H2A.Z-H2B (Fig. 3B and Fig. S3A). Sedimentation experiments showed that Swr1589-650, Chz163-124 and scH2B-H2A.Z form a complex with 1:1:1 ratio (Fig. 3C), indicating that Swr1589-650 and Chz1 can indeed bind to H2A.Z-H2B simultaneously. In contrast, in the Swr1-scH2B-H2A.Z complex, the Swr1-Z domain occupied the DNA- and H3-binding sites of the H2A.Z-H2B dimer in the nucleosome (Fig. 3D), which are the typical structural features of histone chaperones (Chz1 (Zhou et al., 2008), Scm3 (Zhou et al., 2011), Daxx (Elsasser et al., 2012; Liu et al., 2012), HJURP (Hu et al., 2011), and FACT (Hondele et al., 2013). Following a previous assay for chaperone function of FACT on H2A-H2B (Hondele et al., 2013)), we performed gel shift experiments to examine the chaperone function of the Swr1-Z domain. Indeed, we found that the Swr1-Z domain can prevent the H2A.Z-H2B dimer from aggregating on DNA (Fig. S3B, C). Moreover, we found that the Swr1-Z domain facilitated the incorporation of the H2A.Z-H2B dimer into the pre-reconstituted DNA-(H3–H4)2 tetrasome to form the nucleosome with a yield of ~50% (Fig. 3E, F and Fig. S3D), whereas the H2A.Z-H2B dimer in the absence of the Swr1-Z domain largely failed to form the nucleosome with the tetrasome (Fig. 3E).

Figure 3. Chaperone function of the Swr1-Z domain.

Figure 3

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(A) The structural difference between the C-terminal region of H2A.Z in the crystal structure of the Swr1-Z domain-scH2B-H2A.Z complex and that of H2A in the nucleosome (pdb ID: 1ID3).

(B) The Swr1-Z domain and Chz1 bind to opposite surfaces on H2A.Z/H2B.

(C) Velocity sedimentation of an equal mole mixture of Swr1589-650, scH2B-H2AZ, and Chz163-124, showing a S20,w of 2.92 S (36.0 kDa), indicating that Chz1, H2A.Z-H2B, and Swr1 form a heterotetrameric complex.

(D) The Swr1-Z domain blocks sites on H2A.Z-H2B for DNA and H3 binding in the nucleosome.

(E) Salt elution of the mixture of DNA-(H3–H4)2 tetrasome and H2A.Z-H2B with (solid line) and without (dashed line) the presence of the Swr1-Z domain.

(F) Comparison of the amount of histones H3 and H4 in the tetrasome input with those in the nucleosome. Upper panel shows SDS gel of the input tetrasome and the reconstituted nucleosome produced in the presence of the Swr1-Z domain. Lower panel shows the cross section of the H4 bands in the upper panel. See also Figure S3.

The Swr1-Z domain binding is important for H2A.Z incorporation

To examine the functional role of the Swr1-Z domain, we performed an in vitro H2A.Z/H2A-exchange assay using reconstituted mono-nucleosomes with a 40 bp linker DNA on one side of the nucleosome (Ranjan et al. 2013). We found that SWR1 containing the Leu601Gly/Phe605Gly double mutation (Dmut) or the Leu601Gly/Phe605Gly/D622A/F623A quadruple mutation (Qmut) or the Swr1-Z deletion in Swr1, did not disrupt the integrity of SWR1 (Fig. S4A) but impaired its ability to catalyze the incorporation of H2A.Z into the H2A nucleosome, in particular, for the formation of the final homotypic nucleosome with double H2A.Z (Fig. 4A and Fig. S4B). In addition, we found that the yeast strain with the above Swr1 mutations grew more slowly than the wild-type strain in the presence of caffeine, as did the Swr1 ATPase inactive strain (Fig. 4B and Fig. S4C) (Mizuguchi et al., 2004). H2A.Z ChIP assay also indicated that incorporation of H2A.Z was decreased in some promoter regions in the yeast strain that harbors the Swr1 mutations, when compared with that containing the WT Swr1 (Fig. 4C and Fig. S4D). These results indicate that the Swr1-Z domain has an important functional role in the replacement of H2A in the nucleosome by H2A.Z in vivo.

Figure 4. The Swr1-Z domain is important for SWR1 function and conserved.

Figure 4

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(A) Mutations in the Swr1-Z domain impair H2A.Z-H2B incorporation into the nucleosome in vitro. In this assay, the mono-nucleosome was reconstituted using the Widom ‘601’ DNA plus a 40-bp linker DNA at one end (Ranjan et al., 2013). The octameric nucleosome with two H2A or one H2A and one H2A.Z or two H2A.Z is represented with AA or ZA or ZZ, respectively.

(B) Mutations in the Swr1-Z domain led to slower cell growth in the presence of caffeine. K727G mutation in Swr1 completely inactivates the ATPase activity of SWR1. The standard deviation of three experiments for each point is close to the size of the symbol.

(C) Mutations in the Swr1-Z domain reduce the incorporation of H2A.Z at promoter regions of some genes. ARS610 is the control. The error bars are standard deviation of three ChIP experiments.

(D, E) NMR spectra of p400945-1017 and SRCAP501-607 in complex with H2A.Z/H2B. Peaks from free p400945-1017 and SRCAP501-607 are shown in red. Peaks from p400 and SRCAP in 1:1 ratio with H2A.Z-H2B are shown in blue. The disappeared p400945-1017 and SRCAP501-607 peaks upon addition of H2A.Z-H2B indicate they are associated with the histones, becomes parts of the folded region of the complex.

(F, G) ITC results of titrations of p400945-1017, SRCAP501-607, and their mutants to the H2A.Z-H2B dimer and the H2A-H2B dimer. See also Figure S4 and Table S2.

Corresponding regions of the Swr1-Z domain in human p400 and SRCAP

To answer whether p400 or SRCAP also uses the similar region to recognize the H2A.Z-H2B dimer, we examined the amino acid sequence of p400 and SRCAP. The overall amino acid sequence of the Swr1-Z domain is not conserved in the two proteins. However, we found that there are ΦxxΦΦ motifs (Φ: large hydrophobic residues) N-terminal to the ATPases in p400 and SRCAP, similar to the Swr1-Z domain LxxLF motif that is important for specific recognition of H2A.Z and for stabilization of the Swr1-scH2B-H2A.Z complex (Fig. 2G and Table S2). Using NMR, we tested the binding of the H2A.Z-H2B dimer by the p400945-1017 and SRCAP501-607 regions that include the ΦxxΦΦ motifs. We found that both p400945-1017 and SRCAP501-607 regions form stable complexes with the H2A.Z-H2B dimer (Fig. 4D, E). We further measured their binding affinities using ITC. p400945-1017 and SRCAP501-607 bound to the H2A.Z-H2B dimer with Kd values of ~5 nM and ~200 nM, respectively (Fig. 4F, G and Table S2). In contrast, they bound to the H2A/H2B dimer with Kd values of ~60 nM and undetectable, respectively. Moreover, mutation of the hydrophobic residues in the ΦxxΦΦ motifs of both p400945-1017 and SRCAP501-607 to Ala led to weaker binding (Fig. 4F, G and Table S2), suggesting that the conserved motif also play an important role for specific recognition of human H2A.Z-H2B dimer.

DISCUSSION

In this study, we determined the high-resolution crystal structure of the Swr1-Z-domain-scH2B-H2A.Z complex, which provides the first glimpse of how the Swr1 subunit of SWR1 recognizes the H2A.Z-H2B dimer at atomic resolution. We observed that the Swr1 subunit uses the intrinsically disordered Swr1-Z domain to specifically recognize H2A.Z-H2B through interactions between the LxxLF motif of the Swr1-Z domain and the hydrophobic residues in the αC helix of H2A.Z. Mutation of the hydrophobic residues in the LxxLF motif or deletion of the Swr1-Z domain leads to slow cell growth phenotype in the presence of caffeine. The same mutation also causes inefficient incorporation of H2A.Z into the H2A nucleosome in vitro and in vivo at some gene promoter regions. These results are in excellent agreement with the earlier conclusion that a crucial part of the histone signal by which SWR1 recognizes histone H2A.Z over H2A is attributed to the αC helix of H2A.Z and its replacement by the corresponding region in H2A results in a H2A.Z phenotype and reduced binding of SWR1 (Wu et al., 2005; Wu et al., 2009).

We further found that the Swr1-Z domain binds to the histone sites that interact with DNA in the nucleosome and has a histone chaperone function for delivering the H2A.Z-H2B dimer to the DNA-(H3–H4)2 tetrasome. In the Swr1 subunit, the Swr1-Z domain is very close to the ATPase domain, suggesting that the Swr1-Z domain could deliver H2A.Z-H2B to the nucleosome by coupling ATP hydrolysis and H2A-H2B eviction. These features of Swr1 are similar to those of Mot1, a SWI2/SNF2 remodeler, whose chaperone domain near the ATPase domain inhibits the transcription factor TBP from forming a dimer and interacting with DNA (Wollmann et al., 2011). It is possible that the ATPase domain plays a role in loosening the H2A-H2B dimer in the nucleosome by ATP-hydrolysis (Fig. S4E). The tighter binding of H2A.Z-H2B by the Swr1-Z domain could increase the local concentration of the H2A.Z-H2B dimer near the nucleosome, which allows the exchange reaction to occur through a mass-action-facilitated chaperone mechanism.

The Swc2 subunit also binds to the H2A.Z-H2B dimer preferentially over the H2A-H2B dimer, which could also enhance the concentration of H2A.Z-H2B near the nucleosome upon association of the DNA-binding domain of Swc2 with linker DNA (Ranjan et al. 2013). The Swr1 and Swc2 subunits may participate in the exchange reaction independently or in a cooperative manner. The observation that mutation or deletion of the Swr1-Z domain or deletion of the Swc2 subunit can impair the function of SWR1 in catalyzing the exchange reaction suggests that the two subunits work cooperatively to complete the exchange reaction.

It is reported that acetylation of H3 K56 in the nucleosome inhibits the incorporation of H2A.Z by SWR1 (Watanabe et al., 2013). It is hypothesized that the Swc2 subunit functions at the end of the reaction cycle to “lock” H2A.Z and that the acetylation of H3 K56 within the nucleosome disrupts Swc2 binding to the nucleosome (Watanabe et al., 2013). However, there is no direct structural information about how Swc2 binds to the H2A.Z-H2B dimer or the nucleosome containing H2A.Z. By overlaying the histone structures in the Swr1-Z domain-scH2B-H2A.Z complex with those in the nucleosome, we found that H3 K56 is very close to the negatively charged regions in the Swr1-Z domain (Fig. S4F, G). Thus, it is possible that the acetylation of H3 K56 inhibits the delivery function of the Swr1-Z domain chaperone.

Finally, we have identified the regions corresponding to the Swr1-Z domain in human p400 and SRCAP. Over-expression of H2A.Z and its incorporation by p400 in promoter regions of several cell-proliferation-promoting genes have been associated with breast cancer (Hua et al., 2008; Svotelis et al., 2010). It was proposed that one strategy for breast cancer therapy is to use small molecules to disrupt the recognition between H2A.Z-H2B and p400 (Rangasamy, 2010). Assuming that p400945-1017 also plays an important chaperone role as the Swr1-Z domain, the p400945-1017-H2A.Z-H2B complex could provide a target for screening of small molecules to disrupt the formation of the complex.

EXPERIMENTAL PROCEDURES

Cloning, expression, and purification of recombinant proteins

Various regions of Swr1 DNA were amplified from the plasmid harboring the coding sequence of Swr1 and cloned into pET vectors. Mutations were made using the QuikChange kit. E. Coli BL21 was used for protein expression. Proteins were purified using reverse phase HPLC and lyophilized. Isotope-labeled proteins for NMR studies were expressed by growing E. coli cells in M9 media with 15NH4Cl, U-13C6-Glucose, and with or without D2O as the sole source for nitrogen, carbon, and deuterium, respectively.

Demonstration of the chaperone function of the Swr1-Z domain

DNA was mixed with pre-incubated H2A.Z-H2B with or without the Swr1-Z domain and precipitates were removed by centrifugation. The soluble complexes were run on agarose gel and stained with ethidium bromide. Tetrasomes were reconstituted using salt dialysis and purified with DEAE-5PW ion-exchange column. The pre-reconstituted tetrasome was mixed with the H2A.Z-H2B dimer with or without the Swr1-Z domain. The solution was centrifuged to remove aggregates and then loaded to a DEAE-5PW ion-exchange column.

ITC and analytical ultracentrifugation experiments

The ITC experiments were performed on a VP-ITC microcalorimeter. The data was fitted to one set of binding sites model using Origin 7.0 software. Sedimentation velocity experiments were conducted for the protein complex on a Beckman Optima XL-A or Beckman Coulter ProteomeLab XL-I analytical ultracentrifuge. Data were collected using the absorbance optical system in continuous mode at 280 nm, which was analyzed using SEDFIT software.

Crystallization, diffraction data collection, and structure determination

Crystals were grown at room temperature from hanging drops. The crystals were flash frozen in liquid nitrogen. X-ray diffraction data was collected at GM/CA-CAT (beam line 23ID-D) in the Advanced Photon Source. The structure was solved using molecular replacement method.

Cell growth analysis

The mutations of the Swr1-Z domain for phenotypic analysis were made in the pSwr1-2Flag plasmid, which was introduced into an Swr1Δ strain (Research Genetics). The overnight cultures were separately seeded into 50 ml fresh medium with or without caffeine. The cell density (OD600) of each culture was measured every two hours.

H2A.Z incorporation assay and Chip-qPCR

Nucleosomes were reconstituted using recombinant histones and standard methods of salt gradient dialysis. Reaction mix had nucleosome, SWR1-2F, H2A.Z/H2B-3F dimer and ATP in exchange buffer. Histone-exchange reaction was stopped by adding excess Lambda DNA and nucleosomes were resolved in native PAGE. ChIP assays were carried out with growing of yeast cells to mid-log phase. Cells were cross-linked by 1% formaldehyde. The reaction was quenched with glycine. Purified DNA from cells was analyzed by running real-time PCR reactions on iCycler real-time PCR detection system.

Supplementary Material

01

Highlights.

  • We determined the structure of the Swr1-Z domain-H2A.Z-H2B complex

  • The conserved Swr1-Z LxxLF motif recognizes the αC helix of H2A.Z

  • The Swr1-Z domain is a histone chaperone important for SWR1 function

  • Human p400 and SRCAP include domains similar to Swr1-Z

Acknowledgments

We thank the staff at the Advanced Photon Source (APS) (23-ID beamline) for technical support; S. Li for protein purification and D. Wei for purification of SWR1; Dr. B. Zhou for assistance in ITC data collection and analysis; Dr. J. Barrowman for editing the manuscript. This research was supported by the Intramural Research Programs of the National Cancer Institute, National Institute of Allergy and Infectious Diseases, and the National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, and National Cancer Institute grant Y1-CO-1020, National Institute of General Medical Sciences grant Y1-GM-1104, and U.S. Department of Energy grant DE-AC02-06CH11357 (APS).

Footnotes

ACCESSION CODES Coordinates and structural factors have been deposited in the Protein Data Bank, with ID 4M6B.

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