/ . Bwchem. 84, 1203-1207 (1978)
Enzymic Acetylation of Nucleosome Histone1 Kentaro HORIUCHI,* Daisaburo FUJIMOTO,* and Masanori FUKUSHTMA**-» •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°C for 5 min. Histones 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 al. reported that chemical acetylation of nucleosomes and chromatin changed their template activities (5) and physical properties (6). The acetylation of nucleosomes with ["C]-acetate has also been demonstrated in vivo (7). However, the nature of histone acetyltransferase in chromatin has not yet been elucidated. 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. 1 Present address: Aichj Cancer Center, Second Department of Internal Medicine, Chikusa-ku, Nagoya, Aichi 464 Abbreviations: EGTA, ethyleneglycol-bis-{£-aminoethylether>MN,N',yV'-tetraacetic acid; SDS, sodium dodecyl sulfate; acetyl-CoA, acetyl coenzyme A.
Vol. 84, No. 5, 1978
1203
MATERIALS AND METHODS Preparation of Nuclei—Rat liver nuclei were prepared by the method of Hewish and Burgoyne (8) with slight modifications. Wistar rats weighing 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 mM KC1, 15 mM NaCl, 0.15 mM spermine, 0.5 mM permidine, 15 mM 2-mercaptoethanol, 15 mM Tris-Cl, pH 7.4) containing 2 mM EDTA, 0.5 mM EGTA, and 0 . 3 4 M sucrose in a Teflon homogenizer. The homogenate was centrifuged at 500 x 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 L5-5O ultracentrifuge at 25,000 rpm for 1 h. The nuclei were stored at — 70°C with 2 volumes of glycerol. The amount of nuclei was estimated by measuring the absorption at 260 nm in 0.1 N NaOH.
1204
K. HORIUCHI, D. FUJIMOTO, and M. FUKUSHIMA
Preparation of Chromatin—The frozen nuclei with glycerol were resuspended in 0.34 M sucrosebuffer A, then the suspension (32 A^ units) was made up to 1 IDM in CaCl, and partially digested with micrococcal nuclease (40-50 units/ml, Sigma) for 1 min at 37°C Digestion was terminated and chromatin was extracted as described by Noll et at. (9). Preparation of Nucleosomes—The nuclei suspension (50 Auo units) was made up to 1 mM in CaQ, and extensively digested with micrococcal nuclease (200 units/ml) for 10 min at 37°C. Digestion was terminated and nucleosomes were extracted as described by Noll and Kornberg (JO). Nucleosomes 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 absorbance at 260 nm was determined for DNA estimation. The sedimentation coefficient of nucleosomes was estimated using catalase from beef liver (1 IS, Sigma) and /3-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 [l-14C]acetyl-CoA (25 nCi, 4.6 nmol, Radiochemical Centre, England), potassium phosphate (10 pmol, pH 6.8), and chromatin (50 //I) or nuclei (50 pi) in 0.16 ml. Incubations were carried out at 37°C for 60 min. The reaction was terminated by the addition of 50 fi\ of 1.0 N HC1 (final 0.25 N). The labeled histone was then extracted for 30 min at 4°C After addition of 20 ft\ of carrier histone solution.. j(10 mg/ml) the whole quantity of the solution was adsorbed onto P-cellulose paper (1.8 X 5.8 cm) and neutralized with 50 p\ 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 Acetylation and Nuclease Digestion of Chro-
matin—Chromatin (28 Auo units) was incubated with [l- 14 qacetyl-CoA (1.0 ^Ci) in 4.4 ml of potassium phosphate buffer (200 fimol, pH 7.4) at 37°C 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 (530%) and centnfuged. The other half was incubated with micrococcal nuclease (200 units), C a d i (1 mM), and EDTA (0 2 mM) in 1.2 ml of Tris-Cl (1 mM, pH 7.5) at 37°C 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 spectrophotometer (model LSC-651) with a toluene-based scintillator containing 33% Triton X-100, 0.4% 2,5-diphenyIoxazole and 0.01 % 1,4-bis[2-(4-methyl5-phenyloxazolyl)]benzene. Acetylation of Nucleosomes and Isolation of Acetylated Nucleosomes—Nucleosomes (17 Att0 units) were incubated with chromatin (25 Ail0 units) and [l-14C]acetyl-CoA (500 nCi) in 3.2 ml of potassium phosphate buffer (200 /imol, pH 6.8) at 37°C 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 Centriflo. The samples were then applied to a linear sucrose gradient (5-30%) and centrifuged. Fractionation, 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 proteins. Analysis of Proteins—Protein electrophoresis was carried out on a discontinuous SDS-18% polyacrylamide slab gel (10x10x0.2 cm) as described by Thomas and Kornberg (12). The sample volume applied was adjusted to give a protein concentration of 2-5 fig per protein band. After electrophoresis, the gel was cut into two. A half of the gel was stained with Coomassie /. 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 Att0 units/ml of 5 rriM Tris-Cl, pH 8, as described above. Trypsin digestion was carried out as described by Whitlock and Simpson (3). The incubation time was 120 min at 20°C. Gel filtration of trypsin-digested nucleosomes was carried out on a Sephacryl S-200 column (0.9x10 cm), eluting with 10 mM MgCl, in 5 mM Tris-Cl, pH 8.0. RESULTS AND DISCUSSION The present study was undertaken to obtain information about the properties of histone acetyltransferase of isolated chromatin. Figure 1 shows the time course of incorporation of radioactivity into histones of chromatin and nuclei. The isolated chromatin and the nuclei, when incubated with [l-14C]acetyl-CoA, catalyzed similar degrees of acetylation of histones relative to DNA. It was thus apparent that the nuclear histone acetyltransferase 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°C 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 (US) when the reaction mixture was analyzed by sucrose gradient centrifugation (Table II). 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 Vol. 84, No. 5, 1978
15
30
45
Time (Minutes) 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 Aw unit), chromatin (0.92 A^, unit), and chromatin heated at 65°C for 5 mm (0.92 AtK unit) were separately incubated with [l-uC]acetyl-CoA (25 nCi) in 0.16 ml of potassium phosphate buffer (10/imol, pH 6.8) at 37°C for the times indicated. The activity of histone acetyltransferase was assayed as described in "MATERIALS AND METHODS." .The radioactivity incorporated is normalized in terms of Aao. — • — , Nuclei; — o — . chromatin; , heated chromatin. TABLE I. Acetylation of nucleosomes by the chromatin-bound enzyme. Nucleosomes were prepared as described in " MATERIALS AND METHODS." Nucleosomes (0.85 Aw unit) and/or chromatin (1.2 /4,« unit) were incubated with [l-14C]acetyl-CoA (25 nCi) in 0.16 ml of potassium phosphate buffer (10 /imol, pH 6.8) at 37°C for 60 min. The incorporated radioactivity was assayed as described in " MATERIALS AND METHODS." Incubation conditions Nucleosomes
Chromatin
Radioactivity incorporated (cpm)
+ +
402 4,958 6,828
+ +
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.
1206
HORIUCHI, D. FUJIMOTO, and M. FUKUSHIMA
TABLE II. Radioactivity distribution in nucleosome acetylation. Chromatin (25 Aw units) was incubated with [l-14C]acetyl-CoA in the presence of exogenously added nucleosomes (17 Atn units). Chromatin (over 16S) and nucleosomes (about IIS) 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." (units)
Radioactivity (cpm)
Radioactivity//*,,,, (cpm/unit)
1 IS fractions (Nucleosomes)
12.1
70,000
5,800
Over 16S fractions (Chromatin)
23.8
126,000
5,300
a b e d H1
10
20
30
Fractions ( 03 ml each) Fig. 2. Sucrose gradient centrifugal analysis of acetylated chromatin. Chromatin (28 AtK units) was incubated with [l-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%) centnfugation, and the radioactivity and absorbance at 260 nm in each fraction were determined as described in " MATERIALS AND METHODS." 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 histories occurs mainly in the N-terminal regions in vivo (75) 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 analysis of acetylated chromatin and nucleosomes. Isolated labeled chromatin and nucleosomes (20 p\ each of 0.4 mg protein/ml solution) were subjected to SDS-polyacrylamide gel electrophoresis, and the radioactivity incorporated into various histones was analyzed using the fluorographic method as described in " MATERIALS AND METHODS." a, Proteins in chromatin; b, proteins in nucleosomes; c, radioactivity in chromatin; 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 2 5 % 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). Aoout 25% of the total radioactivity was incorporated into this fraction, suggesting that lysine residues in the trypsinresistant region may be acetylated to a lesser extent. The S value of the trypsin-digested nucleosomes was found to be approximately 9S by sucrose /. Biochem.
NUCLEOSOME ACETYLATION
1207 We would like to thank Prof. A. Ichiyama (Dept. of Biochemistry) for a critical reading of the manuscript, Dr. Y. Takahashi (Dcpt. 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
0
10
20
F r a c t i o n s (0.3 ml each) Fig. 4. a) Gel filtration profile of trypsin-digested nucleosomes. Labeled nucleosomes (10 A^, units) were digested with trypsin and subjected to gel filtration on Sephacryl S-200 as described in " MATERIALS AND METHODS." b) SDS-polyacrylamide gel electrophoretic analysis of nucleosomes and trypsin-treated nucleosomes. T, Proteins in trypsin-resistant nucleosomes; N, proteins in intact nucleosomes. gradient centrifugation analysis. These results are consistent with another report (5) 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 histone 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.
Vol. 84, No. 5, 1978
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) / . 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, 3244-3248 7. Davie, J.R. & Candido, E.P.M. (1977) / . Biol. Chem. 252, 5962-5966 8. Hewish, D.R. & Burgoyne, L.A. (1973) Biochem. Bwphys. Res. Commun. 52, 504-510 9. Noll, M., Thomas, J.O., & Kornberg, R.D. (1975) Science 187, 1203-1206 10. Noll, M. & Kornberg, R.D. (1977) / . 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. 11, 2626-2630 13 Bonner, W.M. & Loskey, R.A. (1974) Eur. J. Biochem. 46, 83-88 14 Vidali, G., Gershey, EX., & Allfrey, V.G. (1968) /. 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
Enzymic Acetylation of Nucleosome Histone1 Kentaro HORIUCHI,* Daisaburo FUJIMOTO,* and Masanori FUKUSHTMA**-» •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°C for 5 min. Histones 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 al. reported that chemical acetylation of nucleosomes and chromatin changed their template activities (5) and physical properties (6). The acetylation of nucleosomes with ["C]-acetate has also been demonstrated in vivo (7). However, the nature of histone acetyltransferase in chromatin has not yet been elucidated. 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. 1 Present address: Aichj Cancer Center, Second Department of Internal Medicine, Chikusa-ku, Nagoya, Aichi 464 Abbreviations: EGTA, ethyleneglycol-bis-{£-aminoethylether>MN,N',yV'-tetraacetic acid; SDS, sodium dodecyl sulfate; acetyl-CoA, acetyl coenzyme A.
Vol. 84, No. 5, 1978
1203
MATERIALS AND METHODS Preparation of Nuclei—Rat liver nuclei were prepared by the method of Hewish and Burgoyne (8) with slight modifications. Wistar rats weighing 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 mM KC1, 15 mM NaCl, 0.15 mM spermine, 0.5 mM permidine, 15 mM 2-mercaptoethanol, 15 mM Tris-Cl, pH 7.4) containing 2 mM EDTA, 0.5 mM EGTA, and 0 . 3 4 M sucrose in a Teflon homogenizer. The homogenate was centrifuged at 500 x 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 L5-5O ultracentrifuge at 25,000 rpm for 1 h. The nuclei were stored at — 70°C with 2 volumes of glycerol. The amount of nuclei was estimated by measuring the absorption at 260 nm in 0.1 N NaOH.
1204
K. HORIUCHI, D. FUJIMOTO, and M. FUKUSHIMA
Preparation of Chromatin—The frozen nuclei with glycerol were resuspended in 0.34 M sucrosebuffer A, then the suspension (32 A^ units) was made up to 1 IDM in CaCl, and partially digested with micrococcal nuclease (40-50 units/ml, Sigma) for 1 min at 37°C Digestion was terminated and chromatin was extracted as described by Noll et at. (9). Preparation of Nucleosomes—The nuclei suspension (50 Auo units) was made up to 1 mM in CaQ, and extensively digested with micrococcal nuclease (200 units/ml) for 10 min at 37°C. Digestion was terminated and nucleosomes were extracted as described by Noll and Kornberg (JO). Nucleosomes 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 absorbance at 260 nm was determined for DNA estimation. The sedimentation coefficient of nucleosomes was estimated using catalase from beef liver (1 IS, Sigma) and /3-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 [l-14C]acetyl-CoA (25 nCi, 4.6 nmol, Radiochemical Centre, England), potassium phosphate (10 pmol, pH 6.8), and chromatin (50 //I) or nuclei (50 pi) in 0.16 ml. Incubations were carried out at 37°C for 60 min. The reaction was terminated by the addition of 50 fi\ of 1.0 N HC1 (final 0.25 N). The labeled histone was then extracted for 30 min at 4°C After addition of 20 ft\ of carrier histone solution.. j(10 mg/ml) the whole quantity of the solution was adsorbed onto P-cellulose paper (1.8 X 5.8 cm) and neutralized with 50 p\ 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 Acetylation and Nuclease Digestion of Chro-
matin—Chromatin (28 Auo units) was incubated with [l- 14 qacetyl-CoA (1.0 ^Ci) in 4.4 ml of potassium phosphate buffer (200 fimol, pH 7.4) at 37°C 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 (530%) and centnfuged. The other half was incubated with micrococcal nuclease (200 units), C a d i (1 mM), and EDTA (0 2 mM) in 1.2 ml of Tris-Cl (1 mM, pH 7.5) at 37°C 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 spectrophotometer (model LSC-651) with a toluene-based scintillator containing 33% Triton X-100, 0.4% 2,5-diphenyIoxazole and 0.01 % 1,4-bis[2-(4-methyl5-phenyloxazolyl)]benzene. Acetylation of Nucleosomes and Isolation of Acetylated Nucleosomes—Nucleosomes (17 Att0 units) were incubated with chromatin (25 Ail0 units) and [l-14C]acetyl-CoA (500 nCi) in 3.2 ml of potassium phosphate buffer (200 /imol, pH 6.8) at 37°C 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 Centriflo. The samples were then applied to a linear sucrose gradient (5-30%) and centrifuged. Fractionation, 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 proteins. Analysis of Proteins—Protein electrophoresis was carried out on a discontinuous SDS-18% polyacrylamide slab gel (10x10x0.2 cm) as described by Thomas and Kornberg (12). The sample volume applied was adjusted to give a protein concentration of 2-5 fig per protein band. After electrophoresis, the gel was cut into two. A half of the gel was stained with Coomassie /. 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 Att0 units/ml of 5 rriM Tris-Cl, pH 8, as described above. Trypsin digestion was carried out as described by Whitlock and Simpson (3). The incubation time was 120 min at 20°C. Gel filtration of trypsin-digested nucleosomes was carried out on a Sephacryl S-200 column (0.9x10 cm), eluting with 10 mM MgCl, in 5 mM Tris-Cl, pH 8.0. RESULTS AND DISCUSSION The present study was undertaken to obtain information about the properties of histone acetyltransferase of isolated chromatin. Figure 1 shows the time course of incorporation of radioactivity into histones of chromatin and nuclei. The isolated chromatin and the nuclei, when incubated with [l-14C]acetyl-CoA, catalyzed similar degrees of acetylation of histones relative to DNA. It was thus apparent that the nuclear histone acetyltransferase 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°C 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 (US) when the reaction mixture was analyzed by sucrose gradient centrifugation (Table II). 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 Vol. 84, No. 5, 1978
15
30
45
Time (Minutes) 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 Aw unit), chromatin (0.92 A^, unit), and chromatin heated at 65°C for 5 mm (0.92 AtK unit) were separately incubated with [l-uC]acetyl-CoA (25 nCi) in 0.16 ml of potassium phosphate buffer (10/imol, pH 6.8) at 37°C for the times indicated. The activity of histone acetyltransferase was assayed as described in "MATERIALS AND METHODS." .The radioactivity incorporated is normalized in terms of Aao. — • — , Nuclei; — o — . chromatin; , heated chromatin. TABLE I. Acetylation of nucleosomes by the chromatin-bound enzyme. Nucleosomes were prepared as described in " MATERIALS AND METHODS." Nucleosomes (0.85 Aw unit) and/or chromatin (1.2 /4,« unit) were incubated with [l-14C]acetyl-CoA (25 nCi) in 0.16 ml of potassium phosphate buffer (10 /imol, pH 6.8) at 37°C for 60 min. The incorporated radioactivity was assayed as described in " MATERIALS AND METHODS." Incubation conditions Nucleosomes
Chromatin
Radioactivity incorporated (cpm)
+ +
402 4,958 6,828
+ +
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.
1206
HORIUCHI, D. FUJIMOTO, and M. FUKUSHIMA
TABLE II. Radioactivity distribution in nucleosome acetylation. Chromatin (25 Aw units) was incubated with [l-14C]acetyl-CoA in the presence of exogenously added nucleosomes (17 Atn units). Chromatin (over 16S) and nucleosomes (about IIS) 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." (units)
Radioactivity (cpm)
Radioactivity//*,,,, (cpm/unit)
1 IS fractions (Nucleosomes)
12.1
70,000
5,800
Over 16S fractions (Chromatin)
23.8
126,000
5,300
a b e d H1
10
20
30
Fractions ( 03 ml each) Fig. 2. Sucrose gradient centrifugal analysis of acetylated chromatin. Chromatin (28 AtK units) was incubated with [l-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%) centnfugation, and the radioactivity and absorbance at 260 nm in each fraction were determined as described in " MATERIALS AND METHODS." 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 histories occurs mainly in the N-terminal regions in vivo (75) 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 analysis of acetylated chromatin and nucleosomes. Isolated labeled chromatin and nucleosomes (20 p\ each of 0.4 mg protein/ml solution) were subjected to SDS-polyacrylamide gel electrophoresis, and the radioactivity incorporated into various histones was analyzed using the fluorographic method as described in " MATERIALS AND METHODS." a, Proteins in chromatin; b, proteins in nucleosomes; c, radioactivity in chromatin; 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 2 5 % 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). Aoout 25% of the total radioactivity was incorporated into this fraction, suggesting that lysine residues in the trypsinresistant region may be acetylated to a lesser extent. The S value of the trypsin-digested nucleosomes was found to be approximately 9S by sucrose /. Biochem.
NUCLEOSOME ACETYLATION
1207 We would like to thank Prof. A. Ichiyama (Dept. of Biochemistry) for a critical reading of the manuscript, Dr. Y. Takahashi (Dcpt. 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
0
10
20
F r a c t i o n s (0.3 ml each) Fig. 4. a) Gel filtration profile of trypsin-digested nucleosomes. Labeled nucleosomes (10 A^, units) were digested with trypsin and subjected to gel filtration on Sephacryl S-200 as described in " MATERIALS AND METHODS." b) SDS-polyacrylamide gel electrophoretic analysis of nucleosomes and trypsin-treated nucleosomes. T, Proteins in trypsin-resistant nucleosomes; N, proteins in intact nucleosomes. gradient centrifugation analysis. These results are consistent with another report (5) 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 histone 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.
Vol. 84, No. 5, 1978
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