Histone Acetylation near the Nucleosome Dyad Axis Enhances Nucleosome Disassembly by RSC and SWI/SNF Nilanjana Chatterjee,a* Justin A. North,b Mekonnen Lemma Dechassa,d,e Mridula Manohar,c Rashmi Prasad,a Karolin Luger,d,e Jennifer J. Ottesen,c Michael G. Poirier,b,c Blaine Bartholomewa Department of Epigenetics and Molecular Carcinogenesis, UT M. D. Anderson Cancer Center, Smithville, Texas, USAa; Department of Physicsb and Department of Chemistry and Biochemistry,c Ohio State University, Columbus, Ohio, USA; Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, Colorado, USAd; Howard Hughes Medical Institute, Colorado State University, Fort Collins, Colorado, USAe

Signaling associated with transcription activation occurs through posttranslational modification of histones and is best exemplified by lysine acetylation. Lysines are acetylated in histone tails and the core domain/lateral surface of histone octamers. While acetylated lysines in histone tails are frequently recognized by other factors referred to as “readers,” which promote transcription, the mechanistic role of the modifications in the lateral surface of the histone octamer remains unclear. By using X-ray crystallography, we found that acetylated lysines 115 and 122 in histone H3 are solvent accessible, but in biochemical assays they appear not to interact with the bromodomains of SWI/SNF and RSC to enhance recruitment or nucleosome mobilization, as previously shown for acetylated lysines in H3 histone tails. Instead, we found that acetylation of lysines 115 and 122 increases the predisposition of nucleosomes for disassembly by SWI/SNF and RSC up to 7-fold, independent of bromodomains, and only in conjunction with contiguous nucleosomes. Thus, in combination with SWI/SNF and RSC, acetylation of lateral surface lysines in the histone octamer serves as a crucial regulator of nucleosomal dynamics distinct from the histone code readers and writers.

N

ucleosomes, the basic building blocks of eukaryotic chromatin, impose a physical barrier to regulatory proteins and repress various DNA-mediated transactions (1). Despite spontaneous partial unwrapping and rewrapping of DNA near the entry/ exit site (2, 3), nucleosomes are quite stable and show limited mobility. Fourteen major histone-DNA contacts at 10.5-bp intervals primarily contribute to this stability, and about 14 kCal/mol of energy is required to break these contacts (4, 5). ATP-dependent chromatin remodelers like RSC and SWI/SNF reposition (6) or evict (7, 8) nucleosomes by breaking these histone-DNA contacts during the course of remodeling. However, specific point mutations in the core histones can weaken key histone-DNA or histone-histone interactions in the nucleosome and partially alleviate the requirement for SWI/SNF (9). These mutations, termed SIN (SWI/SNF-independent) mutations, when present at the nucleosomal dyad or in the histone dimer-tetramer interface, decrease stability while increasing thermal mobility of nucleosomes and thereby may substitute for SWI/SNF function (10, 11). Incorporation of various posttranslational modifications (PTMs) into histones regulates nucleosome structure and dynamics. While the majority of PTMs reside in the unstructured N-terminal tail domain of histones, many have been identified in the ␣-helical histone fold motif that constrains the DNA superhelix to form the compact nucleosome core (12–14). Unlike the histone tail PTMs, those in the histone core are often buried and hence are less likely to be accessible for regulatory factor binding. Some of these nucleosome core PTMs are located in the histone-DNA interface (15, 16). Several of them colocalize with known SIN mutations (15) and reduce DNA binding affinity (15–17) or interfere with DNA wrapping about the lateral surface of the nucleosome (17, 18). Acetylation of histone H3 lysines 115 (H3K115ac) and 122 (H3K122ac) were first identified to occur individually and in combination (H3K115acK122ac) based on mass spectrometric analysis of bovine histones (12). Recently, H3K122ac was shown

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to be catalyzed by the histone acetyl transferases p300/CBP in response to estrogen receptor signaling (19). In the context of the nucleosome structure, lysine residues K115 and K122 are located in the L2 loop and helix ␣3, respectively, of the H3 histone fold. They project toward the backbone of the major groove of DNA and flank two SIN mutation sites at R116 (20) and T118 (5, 20) near the dyad symmetry axis, the region of the nucleosome with the highest density of histone-DNA interactions. Genomewide, H3K122ac marks are enriched at active gene promoters predominantly upstream of the transcription start site (TSS) (19). Biophysical studies with H3K115ac and H3K122ac nucleosomes showed that these modifications reduce the affinity of the histone octamer for DNA and mildly increase thermal repositioning of nucleosomes (16). When combined with mechanical unwrapping of DNA, these modifications facilitate release of histone octamer from DNA (17), and these modifications also enhance nucleosome disassembly rates by a DNA repair factor (21). In vivo, these PTMs are implicated in altering gene silencing at ribosomal DNA

Received 30 April 2015 Returned for modification 8 June 2015 Accepted 11 September 2015 Accepted manuscript posted online 28 September 2015 Citation Chatterjee N, North JA, Dechassa ML, Manohar M, Prasad R, Luger K, Ottesen JJ, Poirier MG, Bartholomew B. 2015. Histone acetylation near the nucleosome dyad axis enhances nucleosome disassembly by RSC and SWI/SNF. Mol Cell Biol 35:4083–4092. doi:10.1128/MCB.00441-15. Address correspondence to Blaine Bartholomew, [email protected]. * Present address: Nilanjana Chatterjee, Department of Biochemistry and Biophysics, University of California San Francisco, San Francisco, California, USA. Supplemental material for this article may be found at http://dx.doi.org/10.1128 /MCB.00441-15. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

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loci and telomeres (22), DNA repair (22, 23), and transcription activation (19, 23). The effects of H3 dyad acetylation on nucleosome structure and ATP-dependent chromatin remodeling are unknown. In this study, we incorporated these acetyl lysines into the folded histone H3 core via expressed protein ligation and determined their influence on nucleosome structure and remodeling. We found that the crystal structure of the nucleosome is minimally altered by these acetylations individually and in combination, indicating that they do not directly impact DNA wrapping of nucleosomes. We found that H3K115 or H3K122ac enhances dimer eviction as well as complete nucleosome disassembly by SWI/SNF and RSC in the context of dinucleosomes. We also establish a functional link between histone core acetylation and ATP-dependent chromatin remodeling that is independent of bromodomains, as the remodeling activity of a SWI/SNF mutant enzyme missing the Snf2 bromodomain (⌬Br SWI/SNF) was enhanced by H3 core acetylation, similar to the wild-type complex. MATERIALS AND METHODS Protein purification. SWI/SNF and ⌬Br SWI/SNF were immunoaffinity purified as described previously (24). RSC was similarly purified from the Saccharomyces cerevisiae strain YNC001 expressing C-terminally FLAGtagged Rsc2. Xenopus laevis core histones were overexpressed in Escherichia coli, purified, and refolded into octamers as described previously (25). Native peptide ligation and acetylated histone octamer refolding. Truncated histone H3 (residues 1 to 109) was overexpressed as a fusion protein with GyrA intein and a chitin binding domain in E. coli BL21(DE3), purified, and refolded as described previously (16). Thiolysis with 100 mM mercaptoethanesulfonate for 24 h at 4°C generated H3(1– 109) protein with a C-terminal thioester. An H3 peptide (residues 110 to 135) was chemically synthesized with acetylated lysine K115 or K122 or both and an N-terminal cysteine. Overnight peptide ligation at room temperature with a 10-fold molar excess of the acetylated H3 peptide to the H3 thioester, as described previously (26), generated the full-length acetylated H3 and was purified by ion exchange chromatography over a TSKgel SP-5PW column (Tosoh Bioscience). Purity and identity were assessed by SDS-PAGE and matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS). Acetylated H3 was refolded with other core Xenopus laevis histones to generate either a single-site or a dual-site acetylated H3 octamer, and the octamer was purified by Superdex 200 gel filtration chromatography. DNA synthesis and nucleosome reconstitution. Carrier DNA synthesis and mononucleosome [29N (601)59] and dinucleosome [40N (601)-31-N (603)6] assembly with acetylated H3 octamers were done as previously described (8, 24). Nucleosome crystallization. Acetylated H3 nucleosomes were reconstituted on 147-bp palindromic ␣-satellite DNA using salt dialysis, concentrated, and crystallized as described previously (5). Data were collected at the Advanced Light Source (Berkeley, CA). Structural data were processed using d*TREK (27). Molecular replacement was carried out using the Protein Databank sequence 1P3L. Refinement and model building were done using CNS (28) and Coot (29). The figures were prepared with PyMOL (30). Nucleosome binding assays with competitor DNA. 40N31N6 dinucleosomes or 29N59 mononucleosomes with nonacetylated H3, H3K115ac, H3K122ac, or H3K115acK122ac at a 10 nM final concentration were titrated with remodeler to determine conditions for full binding in the absence of competitor DNA. Binding reaction mixtures of 7 ␮l were incubated at 30°C for 30 min with saturating amounts of RSC, SWI/SNF, or ⌬Br SWI/SNF and 1.8 to 50 ng of sheared salmon sperm DNA competitor. The reaction mixtures also contained 20 mM HEPES-NaOH (pH 7.8), 3 mM MgCl2, 6% (vol/vol) glycerol, 70 mM NaCl, and 0.1 mg/ml

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bovine serum albumin. The binding reactions were analyzed on a native 4% (79:1 acrylamide to bisacrylamide) polyacrylamide gel. The relative amounts of remodeler bound to acetylated versus nonacetylated nucleosomes with increasing amounts of competitor DNA correlate to the ratio of the observed KD (equilibrium dissociation constant) of the remodeler for acetylated versus nonacetylated nucleosomes. The means and standard deviations were calculated from two independent experiments. Rate of ATP hydrolysis. The rate of ATP hydrolysis was determined under conditions of either saturating enzymes or nucleosomes as described previously (24). ATPase assay mixtures with saturating amounts of nucleosomes had 3 nM RSC, SWI/SNF, or ⌬Br SWI/SNF and 15 nM dinucleosomes containing either unmodified H3 or H3K115ac, H3K122ac, or H3K115K122ac. The means and standard deviations were calculated from three independent experiments. Nucleosome mobilization assays. Dinucleosomes (10 nM) containing either unmodified H3 or H3K115ac, H3K122ac, or H3K115K122ac were bound for 15 min at 30°C with saturating amounts of RSC, SWI/ SNF, or ⌬Br SWI/SNF as determined by gel shift assay. The reaction temperature was lowered to 18°C or 25°C, and dinucleosome remodeling was initiated by adding 4 or 55 ␮M ATP, respectively. Mononucleosome remodeling was done at 25°C with 55 ␮M ATP after binding the nucleosomes with saturating amounts of enzyme. At regular time intervals, samples were removed and stopped by adding 7 ␮l of sample to 2 ␮l of a 1:1 mixture of 10 mg/ml sonicated salmon sperm DNA and 10 mM ␥-thioATP. Samples were analyzed on 4% (35.36:1 acrylamide to bisacrylamide) native polyacrylamide gels in 0.5⫻ Tris-borate-EDTA. The percentage of species I or II or mobilized nucleosomes was determined by finding the band intensity of the remodeled product and dividing it by the total intensity of all nucleosomes within the lane. The percentage of remodeled product was plotted versus time using GraphPad Prism, and the graph was fitted nonlinearly to a hyperbolic curve of the form y ⫽ x/(n ⫹ x), with the Solver add-in function in Excel over 1,000 iterative cycles (31). The initial rate of remodeling was determined by differentiation of the curve and solving for t equals 0. The means and standard deviations were estimated from two independent repeats. Similar results were also obtained with linear or one-phase exponential fitting of the data.

RESULTS

H3 K115 and K122 acetylation does not alter the structure of the nucleosome. H3 lysines 115 and 122 are located near the nucleosome dyad and oriented toward the DNA phosphate backbone, such that acetylation might interfere with DNA binding and in turn destabilize nucleosomes (16). To investigate how these PTMs are accommodated within the nucleosome and which, if any, DNA-histone interactions are disrupted, we determined the crystal structure of nucleosomes containing H3K115ac, H3K122ac, and H3K115acK122ac to a resolution of 2.4, 2.5, and 3.0 Å, respectively (see Table S1 in the supplemental material). In data not shown, both SDS-PAGE and MALDI-MS analysis (16) demonstrate the purity of the single- or dual-site acetylated histone octamers and the absence of any unligated histone H3 (residues 1 to 109). Overall, the refined structures of the three acetylated nucleosomes were highly similar to that of the unmodified nucleosome, with all root mean square deviations below 0.4 Å. In particular, the protein backbone around acetylated K115 and K122 and the DNA conformation near the acetylation sites are both unchanged (Fig. 1A and B). In the unmodified nucleosome, the amine groups of K115 and K122 are located 5.1 and 4.8 Å from the nearest DNA phosphate group (Fig. 1C and D). Thus, they do not form direct interactions with the DNA, but rather form water-mediated hydrogen bonds with the DNA backbone (32). The side chains of acetylated K115 and K122 are not well defined in any of the crystal

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FIG 1 Acetylation of H3 K115 and K122 side chains has no apparent effect on nucleosome structure. (A) The structure of the H3-H4 tetramer inside nucleosomes is shown along with DNA. Histone H3 (␣2, L2, and ␣3 regions) is shown in blue; K115 and K122 residues are shown in red as a stick model. An arrow indicates the nucleosome dyad. (B) The structure of the H3 K115 K122 acetylated nucleosome, compared with unmodified nucleosome (PDB ID 1kx5 [32]) by aligning with PyMOL. The DNA and H3 in unmodified nucleosome are shown in wheat and blue, respectively; acetylated nucleosome DNA and H3 are shown in cyan and yellow, respectively; K115 and K122 residues are in red, and acetylated K115 and K122 are shown in green. (C and D). Close-up view for comparison of acetylated and unacetylated K115 and K122. The distance from unacetylated K115 and K122 to the closest phosphate DNA backbone of unmodified nucleosome (GUA 2, guanidine at position 2 [in blue] from the dyad) is shown.

structures and have poor electron density, which likely reflects their high flexibility. The conformation of the DNA around the acetylated K115 and K122 residues is unchanged (Fig. 1C and D). The flexible lysine side chain assumes more than one conformation upon acetylation to avoid steric clashes with the DNA. The modifications likely abrogate the water-mediated interactions of the lysine side chain amine group with DNA, thereby resulting in reduced DNA-histone binding free energy near the nucleosomal dyad, as reported earlier (16). Histone H3 acetylation at K115 and K122 does not affect RSC and SWI/SNF binding to nucleosomes. H3K122ac is significantly enriched near transcription start sites, where chromatin remodelers SWI/SNF and RSC are targeted to reposition and disassemble nucleosomes, thereby facilitating transcription (19, 33, 34). In addition, H3K115ac occurs with H3K122ac, suggesting they may function together. The crystal structures revealed that acetyllysines at 115 and 122 are solvent accessible but not necessarily available for protein binding. Therefore, we investigated whether these PTMs influence recruitment of the ATP-dependent chromatin remodelers RSC and SWI/SNF. H3K115ac, H3K122ac, or H3K115acK122ac did not significantly change the affinity of SWI/SNF or RSC for nucleo-

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somes, as measured by electrophoretic mobility shifts assays (EMSA) with dinucleosomes (see Fig. S1A to D in the supplemental material), suggesting that acetylation of these lysines does not influence SWI/SNF and RSC binding. We also deleted the SWI/SNF bromodomain (⌬Br SWI/SNF), since it binds acetylated histones, and we found that removal of this domain did not influence SWI/SNF binding to nucleosomes containing H3K115ac, H3K122ac, or H3K115acK122ac (see Fig. S1E and F). These experiments indicated no significant contribution of the bromodomain to interactions with nucleosomes containing these acetylations. In contrast, recruitment of SWI/SNF and RSC was enhanced by acetylation of histone H3 tails, as shown using free DNA to compete for binding to nucleosomes (24). For that reason, we carried out similar DNA competition experiments with dinucleosomes containing H3K115ac and/or H3K122ac and found that these modifications did not significantly change the ability of DNA to compete with binding of ⌬Br SWI/SNF, SWI/SNF, or RSC to nucleosomes (Fig. 2; see also Fig. S2 in the supplemental material). This confirmed our EMSA measurements, indicating that H3K115ac and/or H3K122ac do not change the affinities of SWI/SNF and RSC for nucleosomes.

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FIG 2 The presence of H3K115ac and H3K122ac separately or in combination does not affect the binding of RSC and SWI/SNF to dinucleosomes. (A) The affinity of the remodeler for modified and unmodified nucleosomes was determined by native gel shift assay and competition with free DNA, as shown for RSC. (Similar gel shift assays are shown for SWI/SNF and ⌬Br SWI/SNF in Fig. S2 in the supplemental material.) (B to D) The amount of RSC (B), SWI/SNF (C), or ⌬Br SWI/SNF (D) bound to dinucleosomes with H3K115ac, H3K122ac, both H3K115 and H3K122ac, or unacetylated H3 with different amounts of competitor DNA is plotted. The data are from two independent binding experiments.

H3K115ac and H3K122ac do not enhance the nucleosomestimulated ATPase activity of RSC and SWI/SNF. Acetylation of K115 and K122 enhances thermal sliding (16), release of histone octamers from mechanically unwrapped nucleosomes (17), and the rate of in vitro transcription (19). We therefore investigated the impact of these modifications on SWI/SNF and RSC ATPdependent chromatin remodeling. We first investigated the effect of H3K115ac and H3K122ac, individually and combined, on the ATPase activities of RSC, SWI/SNF, and ⌬Br SWI/SNF, and we found that they did not alter the rates of ATP hydrolysis in the presence of dinucleosomes (Fig. 3; see also Table S2 in the supplemental material). There were no significant differences in the rate of ATP hydrolysis with unacetylated and acetylated dinucleosomes when the ATPase assays were done with saturating amounts of enzyme, as determined by EMSA (Fig. 3; see also Fig. S1 and Table S2 in the supplemental material), or with saturating amounts of nucleosomes (see Fig. S3 and Table S3 in the supplemental material). H3K115ac and H3K122ac do not enhance nucleosome mobilization but stimulate dimer displacement. In order to determine the effects of H3K115ac and H3K122ac on the basic nucleosome mobilization properties of RSC and SWI/SNF independent of nucleosome disassembly and H2A-H2B dimer displacement, we next used mononucleosomes (Fig. 4; see also Fig. S4 in the supplemental material). There were no significant differences in the rates of nucleosome movement with RSC, SWI/SNF, and ⌬Br SWI/SNF when unmodified nucleosomes or those containing

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H3K115ac and H3122Kac were used (Fig. 4; see also Fig. S4 and Table S4 in the supplemental material). H3K115ac and H3K122ac combined did not change the rate by which RSC moved mononucleosomes; however, there was a modest increase in the rate with which SWI/SNF moved mononucleosomes (compare Fig. 4B with C and D, open versus closed squares; see also the top of Table S4). In addition, we observed a slight increase in nucleosome disruption upon remodeling with nucleosomes containing H3K115ac and H3K122ac, compared to that with unmodified nucleosomes (Fig. 4A; see also Fig. S4). We used the same dinucleosomal substrates as in the ATPase assays to determine the impact of acetylation near the dyad axis on dinucleosome mobilization and disassembly. The conditions were similar to those used for determining the rates of ATP hydrolysis with saturating amounts of enzyme, as shown in Fig. 3. We previously showed that two remodeled species are evident during dinucleosome remodeling by SWI/SNF and RSC (8, 24), which were separated by native gel electrophoresis and are referred to as species I and II (Fig. 5A and 6A; see also Fig. S5 and S6 in the supplemental material). Species I was previously shown to have one H2A-H2B dimer removed, while species II has an entire nucleosome displaced from the DNA template (8). Remodeling was done under low ATP concentrations and temperature to better examine the earlier steps of nucleosome mobilization, rather than the later step of nucleosome disassembly. H3K115ac and H3K122ac individually and together only slightly enhanced the rate of nucleosome movement versus unmodified

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FIG 3 The nucleosome-stimulated ATPase activities of SWI/SNF type remodelers is not altered by H3K115ac or H3K122ac. (A) The ATPase activities of RSC, SWI/SNF, and ⌬Br SWI/SNF with modified and unmodified dinucleosomes were determined by separating inorganic phosphate (Pi) from ATP by thin-layer chromatography on polyethylimine-cellulose. (B to D) Three independent experiments were each done with RSC (25 nM) (B), SWI/SNF (25 nM) (C), and ⌬Br SWI/SNF (14 nM) (C), and the amount of Pi was plotted relative to the reaction time. These experiments were performed with saturating amounts of enzyme relative to nucleosomes (10 nM) at 18°C with 4 ␮M ATP. (In Fig. S3 in the supplemental material, ATPase activity assays were done instead with saturating amounts of nucleosomes relative to remodeler at 18°C with 9.3 ␮M ATP, and the results were similar to those shown here.)

nucleosomes with RSC (1.3- to 1.5-fold), as seen via gel shift assay (Fig. 5A and B; see also Fig. S5 and Table S4 in the supplemental material). The effect was slightly more pronounced with SWI/SNF and ranged from 2.1- to 2.6-fold. No additional stimulation in the rate of nucleosome movement was observed when both lysines were acetylated (Fig. 5C; see also Fig. S5 and Table S4), and remodeling was not dependent on the presence of bromodomains (compare Fig. 5C and D, SWI/SNF versus ⌬Br SWI/SNF; see also Table S4). In general, there was not much of an effect of H3K115ac or H3K122ac on dinucleosome movement by RSC or SWI/SNF, consistent with the results obtained for mononucleosomes. H3K115ac or H3K122ac alone modestly increased the rate of dimer displacement by ⬃2.5-fold and, when combined, stimulated dimer displacement by as much as ⬃4- to 5-fold with SWI/ SNF and RSC (Fig. 5E to G; see also Table S5 in the supplemental material). These results suggest that changing the dynamics of the H3-H4 interface may impact the ability of remodelers to remove H2A-H2B dimers. The enhanced H2A-H2B displacement was independent of the presence or number of bromodomains present in the complex (⌬bromo SWI/SNF versus SWI/SNF versus RSC). Nucleosome disassembly of dinucleosomes by RSC and SWI/ SNF is stimulated by H3K115ac and H3K122ac. Our studies with low ATP concentrations indicated that H3K115ac and H3K122ac could be stimulating the nucleosome displacement activity of RSC and SWI/SNF (Fig. 5; see also Fig. S5 in the supplemental material), and it was important to revisit these findings under more optimal conditions for nucleosome disassembly, with a higher ATP concentration (Fig. 6; see also Fig. S6 in the supplemental

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material). H3K115ac and H3K122ac synergistically enhanced the disassembly of one of two nucleosomes by RSC and SWI/SNF (Fig. 6B and C; see also Table S6 in the supplemental material). Individually, H3K115ac or H3K122ac increased the rate of nucleosome disassembly by RSC or SWI/SNF by a factor of 2.5 to 3 (see Table S6 in the supplemental material). Similar assays done with H3K115ac and H3K122ac combined showed ⬃5- to 7-fold increases in the rate of nucleosome disassembly by SWI/SNF and RSC compared to nonacetylated dinucleosomes. In contrast to acetylation near the dyad axis, it was shown previously that acetylated H3 histone tails do not enhance nucleosome disassembly by SWI/SNF and RSC, although these modifications do stimulate nucleosome movement and binding of the complexes (24). Nucleosome disassembly by RSC and SWI/SNF was an order of magnitude slower than nucleosome mobilization or H2A/H2B displacement and was stimulated the most by H3 core acetylation, as seen in these experiments. Furthermore, the stimulation in the rate of nucleosome disassembly by H3 core acetylation was equal for RSC, SWI/SNF, and ⌬Br SWI/ SNF, again suggesting that the enhancement in nucleosome disassembly due to H3 core acetylation is not linked to bromodomain binding to histone core acetylation (see Table S6). DISCUSSION

Posttranslational modifications identified recently in the histone regions near the dyad axis are gaining recognition as regulators of chromatin structure and dynamics, particularly those on the nucleosome lateral surface that have the potential to influence

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determined by gel shift assay, and the reactions were performed at 25°C with 55 ␮M ATP. A typical gel shift assay is shown for RSC. (The gel shift assays for SWI/SNF and ⌬Br SWI/SNF are shown in Fig. S4 in the supplemental material.) Two independent experiments were done with 25 nM enzyme and 10 nM mononucleosomes. The time points are 5 s, 15 s, 45 s, 2 min, 6 min, 18 min, and 30 min after addition of ATP. (B to D) The amount of mononucleosomes moved was determined by tracking the disappearance of the original H3 acetylated or unacetylated nucleosomes and plotted versus time for RSC (B), SWI/SNF (C), and ⌬Br SWI/SNF (D).

histone-DNA interactions (18). Acetylation of H3K115 and K122 exemplifies such modifications. Acetylation of these residues is implicated in transcription activation and DNA repair in vivo, and acetylation of K122 has recently been shown to be enriched at transcription start sites and enhancers of actively transcribing genes and to stimulate transcription in vitro (19, 22, 23). The unresolved question has been how acetylation of core H3 sites such as K122 promotes histone eviction and is so closely connected with transcription activation and if there are synergistic effects when modification sites are combined. A clue to this question is the in vitro observations that acetylation of lysines at the nucleosomal dyad enhanced histone eviction upon mechanical stress (17) and in conjunction with histone chaperone Nap1 promoted histone eviction (19). These effects could be due to structural changes in the nucleosome. However, this does not seem to be the case, because incorporation of these modifications into nucleosomes, as shown here by X-ray crystallography, does not cause a noticeable change in DNA conformation or histone-DNA interactions beyond the observation that nucleosomes containing these acetylations are more mobile. Instead, these acetylations might reduce the nucleosome stability by disrupting water-mediated histone-DNA interactions, which are exploited by the SWI/SNF family of ATP-dependent chromatin remodelers to enhance nucleosome disassembly and turnover. Interestingly, mutations near the nucleosome dyad that relieve the requirement of SWI/SNF for mating type switching in budding yeast (9) did not significantly alter the nucleosome X-ray structure but did reduce nucleosome stability (10). We have found that acetylation of lysines 115 and 122 of his-

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tone H3 enhances the efficiency of ATP-dependent disassembly of nucleosomes mediated by RSC and SWI/SNF. These remodelers translocate DNA at an internal location two helical turns from the dyad axis to reposition nucleosomes (8, 35, 36). H3 tail acetylation enhances nucleosome repositioning by RSC and SWI/SNF, but it does not enhance the type of nucleosome disassembly that requires adjacent nucleosomes (8, 24). On the other hand, acetylation of the H3 core lysines at residues 115 and 122 enhanced nucleosome disassembly rates of RSC and SWI/SNF and was not coupled to changes in the rates of ATP hydrolysis or of mononucleosome movement. Individually, H3K115ac and H3K122ac were equally effective at stimulating about ⬃2- to 3-fold RSC- or SWI/SNF-mediated nucleosome disassembly. Combining both modifications increased the impact on disassembly more than any modification separately, enhancing disassembly ⬃5- to 7-fold over that with no modification. A similar effect was also seen on the ability of SWI/SNF and RSC to promote the eviction of an H2A-H2B dimer in conjunction with H3K115ac and H3K122ac. The additive effect of changing the dynamics of these two contacts of histones with DNA has a significant impact on the ability of RSC and SWI/SNF to mediate nucleosome disassembly in nucleosomal arrays. Acetylation of histone H3 near the nucleosome dyad could enhance remodeling either by altering the dynamics of the histone-DNA interface of nucleosomes or by interacting with RSC and SWI/SNF through their bromodomains to allosterically regulate their catalytic activity. Our observations are inconsistent with allosteric regulation. First, deletion of the

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FIG 5 H3K115ac and H3K122ac modestly enhance H2A-H2B dimer eviction by RSC and SWI/SNF. (A) A typical gel shift assay is shown and was used to determine the rate at which dinucleosomes initially moves, as evidenced by loss of the original dinucleosomes. The rate at which the first H2A/H2B dimer is displaced from dinucleosomes was determined by tracking the appearance of remodeled species I, and for displacement of one nucleosome it was based on the appearance of remodeled species II, consistent with previous data (8). Reactions were at 18°C with 4 ␮M ATP to enhance resolution of earlier stages in remodeling; in the example shown, RSC was used. (Examples for SWI/SNF and ⌬Br SWI/SNF are shown in Fig. S5 in the supplemental material.) (B to D) The amount of dinucleosome moved versus time was plotted as in Fig. 4; results are for RSC (B), SWI/SNF (C), and ⌬Br SWI/SNF (D). (E to G) H2A-H2B dimer displacement correlates with appearance of species I and was plotted versus time for all three enzymes with modified and unmodified dinucleosomes for RSC (E), SWI/SNF (F), and ⌬Br SWI/SNF (G). The data are from two independent experiments.

acetyl-lysine binding bromodomain in Snf2 did not affect enhancement of SWI/SNF remodeling by H3K115ac and H3K122ac. Second, H3 core acetylation enhanced remodeling rates equally for both RSC and SWI/SNF, irrespective of RSC having more bromodomains than SWI/SNF. Further evidence for the effects of H3K115ac and H3K122ac being independent of bromodomains is the observation that recruitment is also not affected by H3 core acetylation. Our binding data are consistent with recent findings from others, who failed to identify an H3K122 Acspecific binding protein using an unbiased SILAC (stable isotope labeling with amino acids in cell culture) approach (19). Thus, the increase in the rate of nucleosome disassembly or dimer eviction seen with H3K115ac and H3K122ac is not due to these modifications recruiting RSC and SWI/SNF or allosterically modulating their catalytic activities but rather by

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changing nucleosome dynamics in a way that facilitates nucleosome disassembly by these remodelers. Our previous (24) and current findings together provide a mechanistic model as to how the location of histone acetylation in the context of nucleosome structure can dictate the outcome of ATP-dependent chromatin remodeling (Fig. 7). The acetyl marks in histone H3 tails emanating out of the nucleosomes recruit SWI/SNF complexes and allosterically regulate their nucleosome-mobilizing activity by interacting through their bromodomains (24). On the other hand, the H3K115 and H3K122 acetyl marks near the dyad axis of nucleosomes reduce the affinity of histone octamer for DNA (16), likely disrupting water-mediated interactions between histones and DNA at the dyad, which helps poise nucleosomes for disassembly by SWI/ SNF and RSC. When combined with external factors like me-

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FIG 6 H3K115ac and H3K122ac enhance H2A-H2B nucleosome disassembly by RSC and SWI/SNF. (A) Similar remodeling assays were done as shown in Fig.

5, except that temperature and ATP concentration were increased to 25°C and 55 ␮M, respectively, so that nucleosome disassembly could be more readily tracked by gel shift assay. A representative gel shift is shown for SWI/SNF, and those for RSC and ⌬Br SWI/SNF are shown in Fig. S6 in the supplemental material. (B to D) The rate of nucleosome disassembly was determined by following the rate of appearance of nucleosome species I and II, as described for Fig. 5. Data are from two independent replicates.

chanical unwrapping of DNA (17), or histone chaperones like Nap1 (19), or as shown here, with ATP-dependent chromatin remodelers like SWI/SNF, these histone H3 PTMs near the nucleosomal dyad facilitate disassembly of nucleosomes and Acetylation of H3 N-terminal Tail

histone octamer eviction. This model also suggests that in chromatin regulation there are other factors, such as histone acetylation, that can influence the mode of action of SWI/SNFtype complexes to change from primarily moving nucleosomes Acetylation of H3 Near Dyad Axis

Recruitment

Increase H2A-H2B displacement from adjacent nucleosome

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FIG 7 Effects of H3 acetylation on the SWI/SNF family of chromatin remodelers The effect that H3 acetylation has on SWI/SNF remodeling depends on the location of the acetylated lysines in the nucleosome structure. Acetylation of lysines in the flexible histone tail region of H3 (shown in green) has been shown to recruit SWI/SNF and RSC (shown in gray) in a bromo domain-dependent manner and to stimulate their nucleosome-mobilizing activity (24). H3 tail acetylation, however, does not enhance nucleosome disassembly by either of these complexes (24). Acetylation of lysines in the more structured region near the dyad axis (also shown in green), in contrast, does not influence remodeler recruitment or nucleosome mobilization. Instead, they reduce histone-DNA binding free energy at the dyad without altering DNA wrapping in the nucleosome crystal structure, and they keep nucleosomes poised for disassembly. When coupled with ATP-dependent chromatin remodeling by SWI/SNF and RSC, these PTMs increase both H2A-H2B displacement and nucleosome disassembly.

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to disassembling them. The p300/CBP complex has been shown to acetylate lysine 122 of histone H3 (19). H3K122ac localizes to active promoters and enhances nucleosome eviction, and its levels are dynamically regulated at estrogen receptor targets (19). Highly correlated with these events is the mammalian SWI/SNF complex also being recruited by estrogen receptors to these target genes, and with our data this suggests that the molecular basis for H3K122ac increasing histone turnover is because H3K122ac enhances the nucleosome disassembly activity of SWI/SNF. Because of the known conservation between yeast and mammalian SWI/SNF (BAF) not only in the subunit architecture but also in the mechanism of remodeling, our studies with the more readily available yeast SWI/SNF helped reveal fundamental connections between these remodelers and the role of histone acetylation in the lateral surface of the nucleosome. ACKNOWLEDGMENTS This work was supported by the National Institutes of Health (GM048413 to B.B., GM083055 to M.G.P. and J.J.O., and GM088409 to K.L.). We declare we have no conflicts of interest.

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SNF.

Signaling associated with transcription activation occurs through posttranslational modification of histones and is best exemplified by lysine acetyla...
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