J. Biochem. 84, 1203-1207 (1978)

Enzymic Acetylation of Nucleosome Histonel

Kentaro HORIUCHI,* Daisaburo FUJIMOTO,*

and Masanori FUKUSHIMA**,

*Department of Chemistry, and **Department of Biochemistry,

Hamamatsu University School of Medicine,

Hamamatsu, Shizuoka 431-31

Received for publication, June 17, 1978

Rat liver chromatin prepared from purified nuclei catalyzed the acetylation of histones

in nucleosomes at the same level as that of nuclei. The activity of histone acetyltransferase

in chromatin was destroyed by heat treatment at 65•Ž for 5 min. Histories in exogenously

added nucleosomes also served as substrate for the enzyme. The sites of acetylation in the

nucleosomes appeared to be in the trypsin-digestable N-terminal regions of histones H4, H3,

and H2A, as has been reported in an in vivo system.

The acetylation of histones is thought to be a

possible mechanism for the regulation of the

template activity of chromatin in eukaryotes and

may exert its effect by weakening the interaction

of DNA with the histone core (1-4). Recently,

Marushige and Wallace et ƒ¿l. reported that chemi

cal acetylation of nucleosomes and chromatin

changed their template activities (5) and physical

properties (6). The acetylation of nucleosomes

with [14C]-acetate has also been demonstrated in

vivo (7). However, the nature of histone acetyl

transferase in chromatin has not yet been eluci

dated. In the study described here we observed

the enzymic acetylation of nucleosomes in vitro.

1 This work was supported in part by a grant (201014)

from the Ministry of Education, Science and Culture

of Japan.2 Present address: Aichi Cancer Center , Second De

partment of Internal Medicine, Chikusa-ku, Nagoya, Aichi 464

Abbreviations: EGTA, ethyleneglycol-bis-(ƒÀ -amino

ethylether)-N, N, N•Œ, N•Œ-tetraacetic acid; SDS, sodium

dodecyl sulfate; acetyl-CoA, acetyl coenzyme A.

MATERIALS AND METHODS

Preparation of Nuclei-Rat liver nuclei were

prepared by the method of Hewish and Burgoyne

(8) with slight modifications. Wistar rats weigh-

ing 250 to 350 g were killed by decapitation and

their livers were removed after perfusion with 20 ml

of ice-cold 0.9 % NaCl per rat. The livers (50 g)

were homogenized in 300 ml of buffer A (60 nmt

KCl, 15 mm NaCl, 0.15 mm spermine, 0.5 mm

permidine, 15 mm 2-mercaptoethanol, 15 mm

Tris-G, pH 7.4) containing 2 mm EDTA, 0.5 mm

EGTA, and 0.34 M sucrose in a Teflon homog

enizer. The homogenate was centrifuged at 500•~

g for 5 min. The nuclear pellet was dispersed

in 7 volumes of buffer A containing 2.4 M sucrose,

0.1 mm EDTA, and 0.1 mm EGTA, layered over

an equal volume of the same buffer and centrifuged

in an SW 27 rotor of a Beckman model L5-50

ultracentrifuge at 25,000 rpm for 1 h. The nuclei

were stored at -70•Ž with 2 volumes of glycerol.

The amount of nuclei was estimated by measuring

the absorption at 260 nm in 0.1 N NaOH.

Vol. 84, No. 5, 1978 1203

1204 K. HORIUCHI, D. FUJIMOTO, and M. FUKUSHIMA

Preparation of Chromatin-The frozen nuclei

with glycerol were resuspended in 0.34m sucrose-

buffer A, then the suspension (32 A260 units) was

made up to 1 mm in CaCl2 and partially digested

with micrococcal nuclease (40-50 units/ml, Sigma)

for 1 min at 37•Ž. Digestion was terminated and

chromatin was extracted as described by Noll

et al. (9).

Preparation of Nucleosomes-The nuclei sus

pension (50 A260 units) was made up to I mm in

CaCl2 and extensively digested with micrococcal

nuclease (200 units/ml) for 10 min at 37•Ž. Diges

tion was terminated and nucleosomes were extracted

as described by Noll and Kornberg (10). Nucleo

somes were identified by linear sucrose gradient

centrifugation (5-30% sucrose containing 0.2 mm

EDTA, pH 7) and by electron microscopy. The

centrifugation was carried out in an SW 40 Ti

rotor at 36,000 rpm for 17 h. Fractions of 0.3 ml

were collected from the bottom via a capillary

tube carefully inserted from the top and the ab

sorbance at 260 nm was determined for DNA

estimation. The sedimentation coefficient of

nucleosomes was estimated using catalase from

beef liver (11S, Sigma) and ƒÀ-galactosidase from

E. coli (16S, Boehringer) as markers.

Assay of Chromatin Acetylation-The activity

of histone acetyltransferase was analyzed in terms

of the amount of radioactivity incorporated into

histones as determined by the P-cellulose paper

disk method described previously (11), with slight

modifications. The reaction mixture contained

[1-14C]acetyl-CoA (25 nCi, 4.6 nmol, Radiochemi

cal Centre, England), potassium phosphate (10

ƒÊ mol, pH 6.8), and chromatin (50 ƒÊl) or nuclei

(50 ƒÊl) in 0.16 ml. Incubations were carried out

at 37•Ž for 60 min. The reaction was terminated

by the addition of 50 hl of 1.0 N HCl (final 0.25 N).

The labeled histone was then extracted for 30 min

at 4•Ž. After addition of 20 ƒÊl of carrier histone

solution (10 mg/ml) the whole quantity of the

solution was adsorbed onto P-cellulose paper (1.8 •~

5.8 cm) and neutralized with 50 pl of 0.2 M sodium

carbonate buffer (pH 9.2). The paper was soaked

in 0.05 M sodium carbonate buffer (pH 9.2) and

then washed three times with acetone. After

drying in warm air the radioactivity, trapped on

the paper was measured as described previously

(11).

Acetylation and Nuclease Digestion of Chro

- matin-Chromatin (28 A260 units) was incubated

with [1-14C]acetyl-CoA (1.0 ƒÊCi) in 4.4 ml of

potassium phosphate buffer (200 ƒÊmol, pH 7.4) at

37•Ž for 60 min. The reaction was terminated

by dilution with ice-cold water. The acetylated

chromatin was concentrated and washed with

0.2 mm EDTA (pH 7) using a Centriflo (CF-25,

Amicon). A half of the acetylated chromatin

was then applied to a linear sucrose gradient (5-

30%) and centrifuged. The other half was incu

bated with micrococcal nuclease (200 units), CaCl2

(1 mm), and EDTA (0.2 mm) in 1.2 ml of Tris-Cl

(1 mm, pH 7.5) at 37•Ž for 1 min. The reaction

was terminated by the addition of 0.2 ml of 10 mm

EDTA (pH 7). The nuclease-treated chromatin

was concentrated and washed with 0.2 mm EDTA

(pH 7) using a Centriflo, applied to a linear sucrose

gradient (5-30%) and centrifuged. Fractionation

and DNA estimation were carried out as described

above. The radioactivity of each fraction was

measured with an Aloka liquid scintillation spectro

photometer (model LSC-651) with a toluene-based

scintillator containing 33% Triton X-100, 0.4

2,5-diphenyloxazole and 0.01 % 1,4-bis[2-(4-methyl-

5-phenyloxazolyl)]benzene.

Acetylation of Nucleosomes and Isolation of

Acetylated Nucleosomes-Nucleosomes (17 A260

units) were incubated with chromatin (25 A260

units) and [1-14C]acetyl-CoA (500 nCi) in 3.2 ml of

potassium phosphate buffer (200 ƒÊmol, pH 6.8) at

37•Ž for 60 min. The reaction was terminated by

dilution with ice-cold water. The acetylated

nucleosomes and chromatin were concentrated and

washed with 0.2 mm EDTA (pH 7) using a Cen

triflo. The samples were then applied to a linear

sucrose gradient (5-30%) and centrifuged. Frac

tionation, DNA estimation, and measurement of

the radioactivity were carried out as described

above. The acetylated nucleosome or chromatin

fractions were pooled and washed with 5 mM

Tris-Cl, pH 8, using a Centriflo to analyze pro

teins.

Analysis of Proteins-Protein electrophoresis

was carried out on a discontinuous SDS-18

polyacrylamide slab gel (10•~10•~0.2 cm) as de

scribed by Thomas and Kornberg (12). The

sample volume applied was adjusted to give a

protein concentration of 2-5 leg per protein band.

After electrophoresis, the gel was cut into two.

A half of the gel was stained with Coomassie

J. Biochem.

NUCLEOSOME ACETYLATION 1205

brilliant blue to analyze protein as described by Thomas and Kornberg (12). The other half of the gel was prepared for fluorographic analysis of the radioactivity by the procedure of Bonner and

Loskey (13). The film used was Fuji Rx Medical and the exposure time was 2 weeks.

Trypsin Digestion of Labeled Nucleosomes-

Acetylated nucleosomes or chromatin were adjusted

to a concentration of 10 A210 units/ml of 5 mm

Tris-0, pH 8, as described above. Trypsin diges

tion was carried out as described by Whitlock and

Simpson (3). The incubation time was 120 min

at 20•Ž. Gel filtration of trypsin-digested nucleo

somes was carried out on a Sephacryl S-200 column

(0.9 x 10 cm), eluting with 10 MM MgC12 in 5 mm

Tris-Cl, pH 8.0.

RESULTS AND DISCUSSION

The present study was undertaken to obtain

information about the properties of histone acetyl

transferase of isolated chromatin. Figure 1 shows

the time course of incorporation of radioactivity

into histones of chromatin and nuclei. The iso

lated chromatin and the nuclei, when incubated

with [1-14C]acetyl-CoA, catalyzed similar degrees

of acetylation of histones relative to DNA. It

was thus apparent that the nuclear histone acetyl

transferase was almost exclusively recovered in

chromatin. The histone acetyltransferase activity

of chromatin had a pH optimum at about 7.5 and

the activity of chromatin was destroyed by heat

treatment at 65•Ž for 5 min.

The labeled chromatin was digested with

micrococcal nuclease and analyzed by sucrose

gradient centrifugation. The radioactivity was

mainly recovered in the nucleosome fraction (Fig.

2). When nucleosomes were added exogenously,

the total incorporation of radioactivity increased

(Table I), and the increase was found to be entirely

accounted for by the radioactivity recovered in the

nucleosome fraction (11S) when the reaction

mixture was analyzed by sucrose gradient centrifu

gation (Table ‡U). These results indicate that the

exogenously added nucleosomes were used as

acetylation acceptors. SDS-polyacrylamide gel

electrophoretic analysis showed that the acetylation

occurred in H4, H3, and H2A of chromatin and

exogenously added nucleosomes (Fig. 3). The

pattern of acetylation of histones was consistent

Fig. 1. Time course of acetylation of histones in nuclei

or chromatin. Nuclei and chromatin were prepared as

described in " MATERIALS AND METHODS."

Nuclei (2.7 A280 unit), chromatin (0.92 Also unit), and

chromatin heated at 65•Ž for 5 min (0.92 Also unit) were

separately incubated with [1-14C]acetyl-CoA (25 nCi) in

0.16 ml of potassium phosphate buffer (10 ƒÊmol, pH

6.8) at 37•Ž for the times indicated. The activity of

histone acetyltransferase was assayed as described in

" MATERIALS AND METHODS." The radioac

tivity incorporated is normalized in terms of A260.

-•œ-, Nuclei; -•ü-. chromatin; ......, heated

chromatin.

TABLE I. Acetylation of nucleosomes by the chro

matin-bound enzyme. Nucleosomes were prepared

as described in " MATERIALS AND METHODS."

Nucleosomes (0.85 A260 unit) and/or chromatin (1.2 A260

unit) were incubated with [1-14C]acetyl-CoA (25 nCi) in

0.16 ml of potassium phosphate buffer (10 ƒÊmol, pH

6.8) at 37•Ž for 60 min. The incorporated radioactivity

was assayed as described in " MATERIALS AND

METHODS."

with the results obtained in vivo by other workers

(14). Thus, histone acetyltransferase bound to chromatin catalyzed histone acetylation of not only endogenous nucleosomes but also exogenously added nucleosomes in vitro.

Vol. 84, No. 5, 1978

1206 K. HORIUCHI, D. FUJIMOTO, and M. FUKUSHIMA

TABLE II. Radioactivity distribution in nucleosome acetylation. Chromatin (25 A200 units) was incubated with [114C]acetyl-CoA in the presence of exogenously added nucleosomes (17 A260 units). Chromatin (over 16S) and nucleosomes (about 11S) were then separated by sucrose gradient centrifugation and the radioactivity and absorbance at 260 nm of each fraction were determined as described in " MATERIALS AND METHODS."

Fig. 2. Sucrose gradient centrifugal analysis of acetylated chromatin. Chromatin (28 A260 units) was incubated with [1-14C]acetyl-CoA, and a half of the acetylated chromatin was further digested with micrococcal nuclease as described in " MATERIALS AND METHODS." The acetylated chromatin and the nuclease-treated chromatin were fractionated by sucrose

gradient (5-30%) centrifugation, and the radioactivity and absorbance at 260 nm in each fraction were deter-mined as described in " MATERIALS AND METH-ODS." a), Sucrose gradient centrifugal analysis of acetylated chromatin. b), Sucrose gradient centrifugal analysis of nuclease-treated acetylated chromatin.

It is generally accepted that the acetylation of histones occurs mainly in the N-terminal regions in vivo (15) and the regions which are susceptible to trypsin digestion are also N-terminal (16). Therefore, the acetylated nucleosomes were digested with trypsin and analyzed. As shown in Fig.

Fig. 3. SDS-polyacrylamide gel electrophoretic analy

sis of acetylated chromatin and nucleosomes. Isolated

labeled chromatin and nucleosomes (20 ƒÊl each of 0.4

mg protein/ml solution) were subjected to SDS-poly

acrylamide gel electrophoresis, and the radioactivity

incorporated into various histones was analyzed using

the fluorographic method as described in " MATE-

RIALS AND METHODS." a, Proteins in chromatin;

b, proteins in nucleosomes; radioactivity in chro

matin; d, radioactivity in nucleosomes.

4-a, over 70 % of the radioactivity in the nucleosome digest was recovered in the small molecular weight fraction while about 25 % of the total radioactivity was co-eluted with bulk DNA in the void volume. When the void volume fraction was analyzed by SDS-polyacrylamide gel electrophoresis it was apparent that trypsin-resistant nucleosomal protein comprised 4 main bands near the H4 fraction (Fig. 4-b). About 25 % of the total radioactivity was incorporated into this fraction, suggesting that lysine residues in the trypsin-resistant region may be acetylated to a lesser extent. The S value of the trypsin-digested nucleosomes was found to be approximately 9S by sucrose

J. Biochem.

NUCLEOSOME ACETYLATION 1207

Fig. 4. a) Gel filtration profile of trypsin-digested

nucleosomes. Labeled nucleosomes (10 A260 units)

were digested with trypsin and subjected to gel filtration

on Sephacryl S-200 as described in " MATERIALS

AND METHODS." b) SDS-polyacrylamide gel elec

trophoretic analysis of nucleosomes and trypsin-treated

nucleosomes. T, Proteins in trypsin-resistant nucleo

somes; N, proteins in intact nucleosomes.

gradient centrifugation analysis. These results are consistent with another report (3) and indicate the integrity of the trypsin-digested nucleosomes. Thus the enzyme bound to chromatin was shown to acetylate mainly the N-terminal regions of histones H4, H3, and H2A integrated in the his-tone core of nucleosomes in vitro, as has been reported in vivo (14-16).

The results obtained in this study provide enzymic evidence for the hypothesis that the acetylation of N-terminal regions of histone fractions decreases the interaction with DNA, changes the shape of nucleosomes and may regulate the template activity of chromatin (5, 6). Preliminary results indicate that the sedimentation of the acetylated nucleosomes was retarded compared with that of control nucleosomes, and a detailed study of the effects of enzymic acetylation on the

physical and biochemical properties of nucleosomes and chromatin is now in progress.

We would like to thank Prof. A. Ichiyama (Dept. of Biochemistry) for a critical reading of the manuscript, Dr. Y. Takahashi (Dept. of Pathology) for carrying out electron microscopic observations of nucleosomes, and Prof. E. Nakano, Nagoya University, and Dr. N. Uto (Dept. of Biology) for their fluorographic observations. We are particularly grateful to Dr. A. Inoue, Osaka City University, for helpful discussions.

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