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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.
REFERENCES
1. Allfrey, V.G., Faulkner, R., & Mirsky, A.E. (1964) Proc. Natl. Acad. Sci. U.S. 51, 786-794
2. Louie, A.J., Candido, E.P.M., & Dixon, G.H. (1973) Cold Spring Harbor Symp. Quant. Biol. 38, 803-819
3. Whitlock, J.P., Jr. & Simpson, R.T. (1977) J. Biol. Chem. 252, 6516-6520
4. Allfrey, V.G. (1977) in Chromatin and Chromosome Structure (Li, H.J. & Eckhardt, R.A., eds.) pp. 167-191, Academic Press, New York
5. Marushige, K. (1976) Proc. Natl. Acad. Sci. U.S. 73,3937-3941
6. Wallace, R.B., Sargent, T.D., Murphy, R.F., & Bonner, J. (1977) Proc. Natl. Acad. Sci. U.S. 74,
3244-32487. Davie, J.R. & Candido, E.P.M. (1977) J. Biol.
Chem. 252, 5962-59668. Hewish, D.R. & Burgoyne, L.A. (1973) Biochem.
Biophys. Res. Commun. 52, 504-5109. Noll, M., Thomas, J.O., & Kornberg, R.D. (1975) Science 187, 1203-1206
10. Noll, M. & Kornberg, R.D. (1977) J. Mol. Biol. 109, 393-404
11. Horiuchi, K. & Fujimoto, D. (1975) Anal. Biochem. 69,491-496
12. Thomas, J.O. & Kornberg, R.D. (1975) Proc. Natl. Acad. Sci. U.S. 72, 2626-2630
13. Bonner, W.M. & Loskey, R.A. (1974) Ear. J. Biochem. 46, 83-88
14. Vidali, G., Gershey, E.L., & Allfrey, V.G. (1968) J. Biol. Chem. 243, 6361-6366
15. Dixon, G.H., Candido, E.P.M., Honda, B.M., Louie, A.J., MacLeod, A.R., & Sung, M.T. (1975) in Structure and Function of Chromatin (Fitzsimons, D.W. & Wolstenholme, G.E.W., eds.) pp. 229-258, Associated Scientific Publishers, Amsterdam
16. Weintraub, H. & Van Lente, F. (1974) Proc. Natl. Acad. Sci. U.S. 71, 4249-4253
Vol. 84, No. 5, 1978