Real-time imaging of histone H4 hyperacetylationin living cellsKazuki Sasakia, Tamaki Itoa,b, Norikazu Nishinoc, Saadi Khochbind,e, and Minoru Yoshidaa,b,f,1

aChemical Genetics Laboratory/Chemical Genomics Research Group, RIKEN Advanced Science Institute, Wako, Saitama 351-0198, Japan; bGraduate School ofScience and Engineering, Saitama University, Saitama, Saitama 338-8570, Japan; cGraduate School of Life Science and Systems Engineering, Kyushu Instituteof Technology, Kitakyushu 808-0196, Japan; dInstitut National de la Sante et de la Recherche Medicale, U823, F-38706 Grenoble, France; eInstitut AlbertBonniot, Universit Joseph Fourier, F-38700 Grenoble, France; and fJapan Science and Technology Corporation, CREST Research Project, Kawaguchi, Saitama332-0012, Japan

Edited by Tom Misteli, National Cancer Institute, Bethesda, MD, and accepted by the Editorial Board July 30, 2009 (received for review March 2, 2009)

To visualize histone acetylation in living cells, we developed agenetically encoded fluorescent resonance energy transfer (FRET)-based indicator. Response of the indicator reflects changes in theacetylation state of both K5 and K8 in histone H4. Using thisacetylation indicator, we were able to monitor the dynamic fluc-tuation of histone H4 acetylation levels during mitosis, as well asacetylation changes in response to structurally distinct histonedeacetylase inhibitors.

BRDT � chromatin � FRET � HDAC inhibitor

Covalent modification of core histones plays an important rolein the modulation of chromatin structure and function.

Acetylation is a well-characterized modification regulated by twofamilies of evolutionarily conserved enzymes, histone acetyltras-ferases (HATs) and histone deacetylases (HDACs) (1, 2).Acetylation mainly occurs on lysine residues of core histoneN-terminal tails; in this context, the modification induces chro-matin conformational change by unfolding higher order chro-matin structure, and represents an epigenetic mark recognizedby regulatory factors including coactivators. Core histone acet-ylation influences gene expression by modifying chromatin con-formation and/or the recruitment of the regulatory factors. Theacetylation of histone H4 is thought to occur initially at K16, andthen propagates through K12, K8, and K5, progressing in anN-terminal direction (3). Thus, the simultaneous acetylation ofboth K5 and K8 in histone H4 is indicative of histone H4hyperacetylation (4, 5). Acetylated histones are recognized byregulatory proteins containing bromodomains, for example,PCAF, Brd2, Brd4, and BRDT (6). Recently, it has beensuggested that each distinct combination of covalent modifica-tions of histone tails functions as an epigenetic code by regu-lating the interaction of histone tails with chromatin-associatedproteins (7).

In vivo histone acetylation is reversibly and dynamicallyregulated. However, in most studies on protein acetylation,conventional biochemical methods such as immunostaining havebeen used. These methods do not always provide enough infor-mation about the temporal and spatial dynamics of proteinacetylation in living cells. In the case of other cellular dynamics,such as intracellular Ca2� and protein phosphorylation, visual-ization by fluorescence resonance energy transfer (FRET) in livecells has been used to successfully overcome the limitations ofconventional methods (8). Here, we report a FRET-basedindicator, named Histac, developed to allow visualization ofprotein acetylation in living cells.

ResultsA Fluorescent Indicator for Histone H4 Hyperacetylation. For use asan indicator, we developed a five-part tandem fusion proteinconsisting of an acetylation-binding domain, a f lexible linker,a substrate histone H4, and the two different-colored mutantsof GFP (CFP and Venus), which serve as the donor andacceptor f luorophores, respectively, for FRET (Fig. 1A).

Acetylation of the substrate histone H4 and subsequent bind-ing of the acetylated substrate histone H4 to the acetylation-binding domain induce a conformational change in the indi-cator protein. The acetylation-dependent conformationalchange in the indicator alters the distance and orientationbetween CFP and Venus, thus generating a change in intramo-lecular FRET. The bromodomain region of BRDT, whichcontains two bromodomains, was used as the acetylation-binding domain (9). The peptide pull-down analysis revealedthat this bromodomain region specifically bound to histone H4peptides in which both K5 and K8 were acetylated (Fig. 1B).We constructed 13 potential indicators containing variousVenus mutants with different domain organizations, andevaluated their ability to change the 480 nm/535 nm emissionratio in response to trichostatin A (TSA) (10), a potentinhibitor of HDACs, in live cells (Fig. S1). Among the candi-dates, six proteins showed the desired changes. We chose one,which we subsequently named Histac, for further study.

Author contributions: K.S. designed research; K.S. and T.I. performed research; N.N. andS.K. contributed new reagents/analytic tools; K.S. analyzed data; and K.S., S.K., and M.Y.wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission. T.M. is a guest editor invited by the Editorial Board.

1To whom correspondence should be addressed. E-mail: yoshidam@riken.jp.

This article contains supporting information online at www.pnas.org/cgi/content/full/0902150106/DCSupplemental.

AHistac

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Fig. 1. (A) Schematic representation of the domain structure of Histac. (B)Peptide pull-down assay using nonmodified or acetylated (Ac) histone H4N-terminal tail peptides (upper panel), or partly acetylated peptides (middleand lower panels). Pull-downs were analyzed by immunoblotting using anantibody against GFP.

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Histac colocalized with nuclear DNA stained by Hoechst33342 in COS7 cell (Fig. S2 A). The ratios of fluorescenceintensities between DNA and Venus determined at a number ofdistinct chromatin regions were essentially the same, suggestingthat Histac is uniformly incorporated into chromatin (Fig. S2B).On the other hand, a mutant lacking the C-terminal globulardomain of histone H4 (Histac-�C-H4) was located in both thenucleoplasm and the cytoplasm (Fig. S3A). To further investi-gate whether Histac was incorporated into nucleosomes, wefractionated extracts of the cells expressing Histac. Immunoblotanalysis of cytoplasmic (Cy), nucleoplasmic (Nu), and chromatin(Ch) fractions showed that Histac was localized in the chromatinfraction, whereas Histac-�C-H4 was present in both the cyto-plasmic and the nucleoplasmic fractions, indicating that thehistone H4 globular domain is responsible for its chromatinassociation (Fig. S2C). In addition, we examined the effect ofHistac expression on chromatin structure by determining thenucleosome repeat length. No difference between COS7 cellsexpressing Histac and the untransfected cells or those expressingHistac-�C-H4 was detected in the micrococcal nuclease diges-tion patterns (Fig. S2D).

We next verified acetylation of Histac by immunoblotanalysis. The acetylation could be detected within 3 h after theCOS7 cells expressing Histac were challenged with TSA (Fig.2A). The time course showed a similar kinetics of acetylationto that of endogenous histone H4. In contrast, Histac-�C-H4was hardly acetylated in response to TSA treatment (Fig. S3B),suggesting that, as with endogenous histones, incorporationinto the nucleosomes is required for the efficient acetylationof Histac.

Fig. 2B and Movie S1 show images of a COS7 cell expressingHistac after TSA challenge: the 480 nm/535 nm emission ratiois expressed as pseudocolored images. The response reached aplateau within 3 h after TSA treatment (Fig. 2C). The length oftime required for reaching the plateau was similar to that for

acetylation of Histac, as determined using immunoblotting (Fig.2 A and C). After the removal of TSA from the culture, bothemission ratio and acetylation of Histac rapidly returned to thebasal levels, indicating that Histac is a reversible indicator formonitoring acetylation of histone H4 in living cells (Fig. S4 andMovie S2). The cellular response in HeLa cells was essentiallythe same as in COS7 cells (Fig. S5). Photobleaching of Venusresulted in an increase in CFP fluorescence (Fig. S6), and thephotobleached indicator no longer responded to TSA (Fig. 2D).These results confirm that the change in emission ratio reflectsthe change in FRET from CFP to Venus.

To detect changes in acetylation levels by immunoblot, aminimum dose of 10 nM TSA is required (Fig. 3 A and B). Incontrast, the minimum concentration of TSA required to inducethe significant emission ratio change was approximately 1 nM(Fig. 3 C and D). Thus, Histac is more sensitive in detectinghistone acetylation than immunoblot analysis.

Mutational Analysis of the Acetylation-Binding Domain. To confirmthat the TSA-induced change in FRET is a result of the bindingof acetylated histone H4 domain to the acetylation-bindingdomain, we examined the effects of mutations in the Histacacetylation-binding domain (Fig. 4A). When two tyrosineresidues were replaced with alanine in the acetylation-bindingdomain (2YA), the double-bromodomain mutant became in-capable of binding to acetylated histone H4 (Fig. 4B) andfailed to respond to TSA (Fig. 4C). The single Y65A mutantin bromodomain 1 was also impaired in its ability to bind toacetylated histone H4 (Fig. 4B) and did not show any detect-able change in the emission ratio upon TSA treatment (Fig.4C). In contrast, the Y308A mutant in bromodomain 2 re-tained the ability to bind to acetylated histone H4 (Fig. 4B) andshowed a marked emission ratio change in response to TSA(Fig. 4C). No significant difference in the FRET response wasobserved between Histac and Histac-Y308A, suggesting thatthe major domain for recognizing acetylated histone H4 inBRDT is bromodomain 1.

Mutational Analysis of Acetylation Sites. To verify that the TSA-induced change in FRET reflects acetylation at the specific sites

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Fig. 2. (A) COS7 cells expressing Histac and nontransfected COS7 cells weretreated with 1 �M TSA. Immunoblot analyses were performed with antibodiesagainst histone H4 acetylated at Lys-5, 8, 12, and 16. (B and C) Pseudocoloredimages and a time course of the emission ratio in the nucleus of a COS7 cellexpressing Histac. TSA at a final concentration of 1 �M or vehicle alone wasadded to the culture at 0 min. (D) After photobleaching of Venus withinHistac, the cells were treated with 1 �M TSA for 3 h.

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Fig. 3. Immunoblot analysis was performed with antibodies against histoneH4 acetylated at Lys-5, 8, 12, and 16. COS7 cells were treated with variousconcentrations of TSA at 37 °C for 3 h (A and B). Emission ratio time courses (C)and changes in emission ratios (D) of cells expressing Histac treated with TSA.Asterisk indicates P � 0.05 compared with vehicle.

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of the histone H4 domain, we reciprocally tested the effects ofmutations of acetylation sites in the histone H4 domain. Sub-stitution of arginine for four lysines at the acetylation sites K5,K8, K12, and K16 of histone H4 in the indicator (Histac-4KR)caused a significant decrease in the response to TSA (Fig. 5A).We further examined the effects of each acetylation site muta-

tion on the emission ratio (Fig. 5A). Consistent with the peptidebinding analysis (Fig. 1B), replacement of both K5 and K8 witharginine resulted in a marked decrease in the response to TSAtreatment compared with Histac. Single substitutions at eitherK5 or K8 showed a decrease in the response to TSA treatment,but the decrease was smaller than that observed in the doublesubstitution mutant K5,8R. The emission ratio change was notaffected by K16R, indicating that acetylation of K16 does notcontribute to the binding of BRDT. Surprisingly, the responseintensity of K12R was almost the same as that of K5R. Sinceacetylated K12 is not the binding site for the bromodomains ofBRDT, this result suggests that K12 acetylation affects the K5acetylation. Indeed, Western blot analysis revealed that theK12R mutant was barely acetylated at K5, but fully acetylated atK8 (Fig. 5B). On the other hand, K5R, K8R and K16R did notaffect the acetylation of other sites. We further confirmed theseresults using histone H4 mutants bearing much smaller tags, torule out any effects of the fluorescent proteins fused to histoneH4 (Fig. 5C). Again, acetylation of K5 seemed to be largelydependent on acetylation of K12.

Live Imaging of Histac During Mitosis. There is no significantdifference in the acetylation level of K5 in histone H4 betweenmetaphase and interphase (11). On the other hand, HDACinhibitors cause G2/M cell cycle arrest and mitotic checkpointactivation (12, 13), suggesting that histone deacetylation isinvolved in the progression of mitosis. Recent analyses usingimmunof luorescence staining, immunoblotting and mass spec-trometry suggest that deacetylation at histone H4 K5 occurs inmitosis (14–16). However, because of technical limitations onthe biochemical methods used in these studies, the dynamicf luctuation of the K5 acetylation state in histone H4 duringmitosis remains controversial. We therefore used Histac todetermine whether histone H4 acetylation can be dynamicallyregulated during mitosis. Fig. 6 A and B show that the level ofhistone H4 acetylation started to decrease at the onset ofprophase, was minimized at anaphase, and recovered afterprogression into G1 (Movie S3). Chromatin condensationduring mitosis might cause a FRET response unrelated toacetylation. However, the decrease in the FRET emission ratioof Histac-4KR was not observed during mitosis (Fig. 6B andFig. S7). Furthermore, live cell chromatin compaction assaywas performed to see whether condensed chromatin structureaffects the FRET response (17). Time-lapse images wererecorded during 30 min after an osmolarity shift-up in themedium from 290 (physiological condition) to 570 mOsm (Fig.S8). The intensity of Venus was increased due to the chromatincompaction, but no significant change in the emission ratio wasobserved in the same regions in the nuclei. These data indicatethat the chromatin condensation itself has no apparent effecton the FRET response. The decrease in the level of histone H4acetylation during mitosis was validated by immunoblotting(Fig. 6C). Consistent with microscopic observations, theamount of acetylated histone H4 K5 was much lower in thenocodazole-arrested COS7 cells than in asynchronous cells.

Live Imaging of Histac During Interphase. To examine whetherdifferent FRET response occurs between nuclear subdomains,we analyzed the response to TSA in the interphase cells express-ing Histac by confocal f luorescence microscopy (Fig. S9). TheFRET responses in two distinct chromatin regions surroundedby blue and yellow lines were compared (Fig. S9A). No signif-icant difference between the two regions in a nucleus expressingHistac was observed upon 1 �M TSA treatment in this particularexperiment, suggesting that similar HAT activities are associatedin these regions. In contrast, after removal of TSA from culturea difference in the kinetics of histone deacetylation was detected

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Fig. 4. (A) The schematic representation shows the domain structure ofBRDT. YA is a bromodomain mutant, in which Tyr-65 and 308 of BRDT arereplaced with Ala. (B) Peptide pull-down assay using nonmodified or acety-lated (Ac) histone H4 N-terminal tail peptides. Pull-downs were analyzed byimmunoblotting using an antibody against GFP. (C) Changes in emission ratioof Histac mutants in response to 1 �M TSA for 3 h.

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Fig. 5. (A) Changes in emission ratio (mean � SD) of Histac mutants inresponse to 1 �M TSA for 3 h. Asterisk indicates P � 0.05 compared with Histac.(B) Acetylation of histone H4 mutants including Histac in COS7 cells wereanalyzed by immunoblotting using antibodies against histone H4 acetylatedat K5, K8, K12, or GFP. The cells were harvested 3 h after 1 �M TSA treatment.(C) Acetylation of histone H4 mutants bearing small affinity tags in COS7 cellswas analyzed by immunoblotting using antibodies against histone H4 acety-lated at K5, K12, or FLAG. We constructed histone H4 and H4K12R with twoFLAG epitopes and one hexahistidine tag (FFH). The cells were harvested 3 hafter 1 �M TSA treatment.

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(Fig. S9B). This observation suggests the existence of distinctregions with different HDAC activities in the nucleus.

Evaluation of HDAC Inhibitors in Living Cells. Histac is a highlysensitive imaging probe for monitoring the activity of HDACinhibitors in living cells. FK228, which is currently studiedclinically (18), is another potent HDAC inhibitor. FK228 has anintramolecular disulfide bond, and is activated in cells by reduc-tion to form two sulfhydryl groups, one of which can interact withthe HDAC active site (19). It was unclear, however, how muchtime is required for this process. Using Histac, we detectedFK228 activity immediately after the challenge (Fig. 7A), indi-cating that FK228 is rapidly incorporated and activated in thecells. CHAP31 (20) and SCOP402 (21), cyclic tetrapeptideshaving a hydroxamic acid and a disulfide as functional groups,respectively, also showed rapid FRET responses (Fig. 7 B and C).In contrast, SCOP304 (21), a dimer form of SCOP152 with adisulfide, gave significantly different kinetics (Fig. 7D). Itshowed a delayed response onset, and took more time than otherHDAC inhibitors to reach a plateau, probably due to poormembrane permeability. Based on these inhibitor data, it is clearthat Histac serves as a powerful assay tool for in vivo HDACinhibitor action.

DiscussionBefore the existence of Histac, Kanno et al. (22) developed amethod to screen for bromodomains that interact with acety-lated histone, using a flow cytometric adaptation of FRETtermed FC-FRET. This approach enabled one to measure thesteady-state interaction between bromodomain proteins andacetylated histones in living cells. However, because the state ofhistone acetylation is thought to be dynamically regulated in thecell, a real-time imaging probe for in vivo histone acetylation has

been long awaited. In this study, we showed that a FRET probefused tandemly with the BRDT bromodomain and histone H4enabled us to visualize the dynamic changes in histone H4acetylation in living cells. Indeed, we could demonstrate thedecrease in the level of histone H4 K5/K8 acetylation at meta-phase. Although early observation suggested no significantchange in acetylation of histone H4 K5, recent analyses usingimmunofluorescence staining, immunoblotting, and mass spec-trometry demonstrated the opposite (14–16). The present studyverified the dramatic decrease in K5 acetylation in mitosis. Inimmunofluorescence staining it cannot be ruled out that anantibody might be inaccessible to acetylated histones duringmitosis, due to chromatin condensation. Moreover, because cellsynchronization is required for immunoblotting and mass spec-trometry, stress induced by the agents for cell synchronizationmight affect the histone acetylation state. Histac can bypassthese technical challenges.

It is unclear why Histac-4KR still responded, albeit weakly, toTSA-induced hyperacetylation of histone H4 (Fig. 5A). It seemspossible that Histac may form a nucleosome together with anendogenous histone H4 molecule, and that the bromodomain inHistac-4KR can interact with acetylated lysine residues in theflexible tail of the endogenous histone H4 in the same nucleo-some. The X-ray structure analysis of a nucleosome showed thathistone H4 possesses an unstructured N-terminal tail, leaving the13 N-terminal residues in one of the two H4 molecules and sevenN-terminal residues in the other H4 disordered (23). The X-raystructure of the nucleosome (PDB ID: 1EQZ) indicates that thedistance between the residues of the two structured H4 N-terminal ends in a nucleosome is approximately 7.6 nm. Thebromodomain in Histac is fused to the flexible N-terminalextension from the structured end via a flexible 20-residue linkerin which the length per residue is approximately 0.38 nm (24).Therefore, it seems likely that the flexible intervening polypep-tide bridges the gap to allow access of the bromodomains to theacetylation sites of the endogenous H4.

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Fig. 6. (A) Pseudocolored images of the 480 nm/535 nm emission ratioobtained from a COS7 cell expressing the acetylation indicator during mitosis.(B) Time courses of the 480 nm/535 nm emission ratio of Histac (F) andHistac-4KR (E) during mitosis. (C) Acetylation of histone H4 at K5 and K8 ofasynchronous (A) and nocodazole-treated (N) COS7 cells was analyzed byimmunoblotting using antibodies against histone H4 acetylated at K5 and K8.Phosphorylated histone H3 (pS10), a mitotic marker, was analyzed by immu-noblotting using an antibody against phosphorylated histone H3. COS7 cellswere arrested in mitosis by treatment with 10 �g/mL nocodazole for 12 h.

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Fig. 7. Emission ratio time courses of cells expressing Histac treated with (A)FK228, (B) CHAP31, (C) SCOP402, and (D) SCOP304. Each HDAC inhibitor wasadded to the culture at 0 min.

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In vitro pull-down experiments show a strict requirement forthe acetylation at both K5 and K8 for the BRDT binding (Fig.1B). This requirement appears less strict in vivo, because of thepossible cross-talks between the bromodomain and acetylatedK5 and K8 in the endogenous H4 in the same nucleosomes (Fig.5A). The simultaneous presence of acetylation on both K5 andK8 is a signature of H4 hyperacetylation. A zip model supportsthe idea that acetylation propagates from K16 to K5 andsimultaneous acetylation at K5 and K8 should therefore indicatehyperacetylated H4 (3–5). Thus, Histac is a unique probe forimaging hyperacetylated histone H4. In this study, we demon-strate that the K12 acetylation is required for the efficient K5acetylation, which may be one of the mechanisms underlying thezip model. The acetylation of K12 may directly or indirectlyfacilitate the recruitment of a HAT to induce acetylation at K5in mammalian cells.

The ability of Histac to recognize the in vivo histone H4acetylation correlated well with the properties of the interactionof BRDT with histone H4 in vitro. BRDT contains two bromo-domains. The bromodomain structure consists of an atypicalleft-handed four-helix bundle (helices �Z, �A, �B, and �C), a longintervening loop between helices �Z and �A (termed the ZAloop), and a loop between helices �B and �C (termed the BCloop) (6). The ZA and BC loops form a hydrophobic pocket, towhich an acetyl-lysine residue binds. Y65 (bromodomain 1) andY308 (bromodomain 2) in the ZA loops in the BRDT bromo-domains are highly conserved throughout the large family ofbromodomains, including GCN5, TAFII250, CBP, p300, andBrd2. We found that the substitution of alanine for Y65 inbromodomain 1 is sufficient to impair its ability to bind toacetylated histone H4. On the other hand, Y308A did not abolishthe binding of BRDT to acetylated histone H4. These resultssuggest that bromodomain 1 in BRDT is the primary bindingdomain for acetylated histone H4, and bromodomain 2 is thesecondary domain. This idea is consistent with the observationthat BRDT containing a mutation in the bromodomain 1 (P50A,F51A, and V55A) could not induce chromatin remodeling, whileBRDT containing a mutation in bromodomain 2 (P293A,F294A, and V298A) retained TSA-dependent chromatin remod-eling activity (9).

In conclusion, we have developed an indicator for visualizinghistone H4 acetylation in living cells. Since our indicator rec-ognizes the acetylation of K5 and K8, the response reflects thehyperacetylation of histone H4 (3–5). It seems probable thatexchange of the acetylation-binding domain with other bromo-domains [e.g., Brd2 binds to acetylated K12 in histone H4 (22)]will allow monitoring of other acetylation sites. Our approachprovides a tool for spatial and temporal analysis of proteinacetylation, and will help to understand when, where, and how

histone H4 is acetylated in living cells, tissues, and transgenicanimals.

Materials and MethodsPlasmid Construction, Cell Culture, and Transfection. The cDNA of ECFP, Venus,histone H4, bromodomains of BRDT (BRDT1–443), and the linker were gener-ated using PCR and cloned into the restriction sites shown in Fig. 1A. EachcDNA was subcloned into the KpnI-XhoI site of a mammalian expressionvector, pcDNA3.1(�) (Invitrogen).

COS7 cells and HeLa cells were cultured in DMEM supplemented with 10%FCS, 1% penicillin/streptomycin, 1 mM sodium pyruvate, and 0.1 mM nones-sential amino acids at 37 °C in 5% CO2. These cells were transfected with aFuGENE 6 transfection reagent (Roche) and were then cultured for 24 h at37 °C in 5% CO2.

Imaging of Cells. After transfection, the culture medium was replaced withphenol red-free growth medium for imaging. Cells were observed at 37 °C in5% CO2 on an Olympus IX81 microscope with a UIC-QE cooled charged-coupled device camera (Molecular Devices). Images were collected by using aMetaFluor (Universal Imaging) with a 440AF21 excitation filter, a 455DRLPdichroic mirror, and two emission filters (480AF30 for CFP and 535AF26 forVenus). For Fig. S2, the images of Hoechst 33342 staining and Venus werecollected with FV1000 (Olympus) confocal microscope system.

Gel Electrophoresis and Immunoblot Analysis. For Figs. 1B and 4B, peptidepull-down assays were performed as described by Pivot-Pajot et al. (9). COS7cells were transiently transfected with monomeric Venus-BRDT or monomericVenus-BRDT-mutants. For Fig. S2C, mononucleosome core particles were pu-rified as described by Kanda et al. (25). The fractions were immunoprecipi-tated using an anti-GFP antibody (Takara Bio).

Immunoblot analysis was performed using standard procedures andvisualized using ECL Western Blotting Detection Reagents (GE HealthcareBio-Science Corp.). The antibodies that recognize acetyl-lysine 5, 8, 12, and16, respectively, of histone H4 were obtained from Upstate Biotechnology.The anti-histone H3, anti-histone H4, anti-GFP, and anti-FLAG antibodieswere purchased from Cell Signaling Technology, Abcam, takara Bio, andSigma, respectively. The anti-HDAC1 and anti-tubulin were obtained fromSigma.

Micrococcal Nuclease Digestion. Micrococcal nuclease digestion was carriedout essentially as described by Remboutsika et al. (26). The nucleus of COS7cells expressing the indicators were digested with increasing amounts ofmicrococcal nuclease (0.2, 0.8, or 3.2 units per 107 nucleus) (Sigma) at 37 °C for10 min. DNA was purified by phenol/chloroform extraction and ethanolprecipitation, and separated in a 2% agarose gel.

ACKNOWLEDGMENTS. We thank Atsushi Miyawaki (RIKEN, Japan) for pro-viding various Venus mutants and helpful discussion, A. Ganesan (University ofSouthampton and Karus Therapeutics Ltd., U.K.) for providing FK228, andAkihiro Ito for discussion; BSI’s Research Resources Center for providing DNAsequencing analysis and peptide synthesis; and RIKEN BSI-Olympus Collabo-ration Center for imaging equipment and software. This work was supportedin part by Grant-in-aid for Scientific Research on Priority Areas (to K.S.); CRESTResearch Project, JST, Grants-in-aid for Cancer Research from the Ministry ofEducation, Culture, Sports, Science, and Technology of Japan (to M.Y.), and‘‘ANR blanc’’ ‘‘Episperm’’ & ‘‘Empreinte, INCa,’’ and ‘‘ARC-ARECA’’ researchprograms (to S.K.).

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