these conditions, chemical acetylation with acetic anhydride is a useful ... is 5 mM Tris/HCl, pH 7.9; 5 mM MgCI2; 0.1 mM EDTA; 0.5 mM DTT; and 25% glycerol.
Bioscience Reports 4, 155-163 (1984) Printed in Great Britain
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T r a n s c r i p t i o n of c h e m i c a l l y a c e t y l a t e d c h r o m a t i n w i t h h o m o l o g o u s RNA p o l y m e r a s e B A. CSORDAS*, I. MULTHAUP and H. GRUNICKE Institute for Medical Chemistry and Biochemistry, University of Innsbruck, Innsbruck, Austria (Received 6 January 1984)
Homologous RNA polymerase B was used to examine the template properties of rat liver chromatin modified by acetic anhydride. Transcription of chromatin was s t r o n g l y s t i m u l a t e d on the c h e m i c a l l y a c e t y i a t e d template. Under conditions of reinitiation inhibition there was an approximately two-fold increase in the number of initiation sites on the acetylated chromatin. A new method of chemical acetylation of histones, with a high degree of specificity, is presented. The role of histone acetylation and deacetyiation in gene 9eguiation has been studied extensively in recent years (for reviews see 1,2,3). For several years n-butyric acid has been used as an inhibitor of the h i s t o n e d e a c e t y l a s e ( s ) , in order to produce and conserve a h y p e r a c e t y l a t e d s t a t e of histones. However, a large number of physiological effects were reported in addition to inhibition of the histone deacetylases. These manifold effects are documented in a r e v i e w by 3. Kruh ( # ) . The most severe effects of n-butyrate treatment of cells on chromatin structure, which possibly interfere with template properties in transcription, are the alterations in the phosphorylation and ADP-ribosylation pattern of chromosomal proteins. Altered phosphorylation of histones and non-histones, after butyrate treatment, was reported by several laboratories (5,6,7). Cells exposed to butyrate were found to have impaired methyiation of histones and nuclear hnRNP particles (7). In other systems butyrate treatment has been shown to stimulate the ADP-ribosylation of specific proteins (8). It is d i f f i c u l t to establish a causal connection between degree of a c e t y l a t i o n of histories and template function of chromatin, when several m o d i f i c a t i o n reactions changing the microheterogeneity of histones are perturbed simultaneously. Therefore caution has to be e x e r c i s e d in the i n t e r p r e t a t i o n of changes observed in chromatin function after treatment with butyrate. Sufficiently pure acetyltransferases are not yet available. Under these conditions, chemical acetylation with acetic anhydride is a useful t o o l to e s t a b l i s h a h i g h e r degree of a c e t y l a t i o n of histones in *To whom correspondence should be addressed, at Universita't I~nsbruck, Institut fur Medizinische Chemie und Biochemie, A-6020 Innsbruck, Fritz-Pregl-Str. 3, Austria. 01984
The Biochemical Society
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chromatin. A l t h o u g h c h e m i c a l acetylation with acetic anhydride p r o d u c e s , in a d d i t i o n to the e n z y m a t i c acetylation sites, a few non-physiological acetylations, it has the advantage of introducing only one type of protein modification into chromatin. The acetylation of chromatin takes place as a rapid reaction at 0~ For the s t u d i e s p r e s e n t e d in this paper, a new m e t h o d of a c e t y l a t i o n was used, which does not seem to a f f e c t the histone integrity of chromatin. A n a l y s i s of h i s t o n e s by Triton-urea polyacrylamide-gel electrophoresis (9) revealed a highly specific pattern of discrete classes of acetylated histones. Materials
and Methods
Chromatin preparation
Nuclei were prepared according to Blobel and Potter (10) from the unfrozen livers of female Sprague-Dawley rats, 200-300 g body weight. B e f o r e being s a c r i f i c e d t h e r a t s were s t a r v e d overnight. The chromatin preparation was described previously (l 1). Chromatin was kept unfrozen on ice and used within 4g h. Purification of RNA polymerase B
RNA p o l y m e r a s e B was p r e p a r e d from 70- to 100-g female S p r a g u e - D a w l e y r a t s which were s t a r v e d o v e r n i g h t . For RNA p o l y m e r a s e B, n u c l e i were prepared according to the Krebs and Chambon modification of the Chaveau procedure (12). The extraction of RNA-polymerase B from the nuclei was done according to Seifart (13). The DEAE-Sephadex fractions were made 5096 with glycerol, frozen, and stored at -90~ In vitro acetylation of chromatin
The acetylation procedure was carried out while keeping chromatin at 0~ on ice. Chromatin (300/pg DNA/ml) was made 5 mM with 1 M Tris/HCl buffer pH g.5, by adding dropwise the appropriate amount, with vigorous stirring. After l0 min, the chromatin solution was made 0.7 mM with acetic anhydride by adding the proper amount of a 5% (v/v) solution in twice-distilled water which was prepared immediately before use. After 20 min more stirring, the acetylated chromatin and an equal amount of controJ chromatin which was made 5 mM with T r i s / H C l pH g.5 were d i a l y s e d overnight against 2 1 of 10 mM Tris/HCl pH g.0. RNA-synthesis without reinitiation
a) According to Cedar and Felsenfeld (14): Chromatin containing 10 pg of DNA was p r e i n c u b a t e d with varying amounts of RNA polymerase B at 37~ for 15 min. The preincubation buffer contained 2 mM of each of CTP, GTP, and ATP; 1.2 mM MnCI2; l0 mM Tris/HCl, pH 7.9; 0.01 mM EDTA; and 0.2 mM DTT. The enzyme was in TGMED buffer which was made 5096 with glycerol. TGMED buffer is 5 mM Tris/HCl, pH 7.9; 5 mM MgCI2; 0.1 mM EDTA; 0.5 mM DTT; and 25% glycerol. Volume at preincuSation was 290 lal. At the end of the preincubation period RNA synthesis was started for another 15
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min with the addition of 50 lal of 2 M (NH~)2SO ~ in TGMED and 20 lal (20 laCi) of [ 3 H ] U T P ( A m e r s h a m ) , s p e c i f i c a c t i v i t y 1.59 T B q / m m o l . The final c o n c e n t r a t i o n of UTP was 0.1 mM. The t o t a l volume of the assay was 350 lal. b) Using essentially the same assay as above, the volume for the p r e i n c u b a t i o n was 290 lal~ with 100 IJl of e n z y m e and 50 lal (15 lag DNA) of c h r o m a t i n . R i f a m y c i n AF/013 was a generous gift of Dr. G. Lancini, Gruppo L e p e t i t , Milan~ Italy. A f t e r p r e i n c u b a t i o n for 15 min at 37~ t r a n s c r i p t i o n was s t a r t e d by addition of 20 tal (20 laCi) of [ 3 H ] U T P , 1.59 T B q / m m o l , and t~g lag of r i f a m y c i n AF/013 in 20 lal of d i m e t h y l sulfoxide to give a final c o n c e n t r a t i o n of [50 tag/ml AF/013. T h e f i n a l v o l u m e was 320 lal. The r e a c t i o n was stopped by the addition of 10 ml of ice-cold 5% t r i c h l o r o a c e t i c acid. This m e t h o d of inhibition of r e i n i t i a t i o n was introduced by Meilhac et al. (15). Analysis of histones
Histones were e x t r a c t e d from c h r o m a t i n , which was s e d i m e n t e d at 12 000 g, with 0.4 N H2SO~, p r e c i p i t a t e d overnight at -20~ with t~ volumes of absolute ethanol, and c o l l e c t e d by c e n t r i f u g a t i o n at I0 000 g for 30 min. The histones were a n a l y z e d by p o t y a c r y l a m i d e - g e l e l e c t r o p h o r e s i s (12% p o l y a c r y l a m i d e - 5% a c e t i c acid - g M urea 0.37% T r i t o n X-100) a c c o r d i n g to A l f a g e m e e t al. (9). Samples were e l e c t r o p h o r e s e d in a slab-gel e l e c t r o p h o r e s i s c h a m b e r (2g c m ) at 350 V for 4g h at 4~ The gels were then stained in 1% Coomassie blue - 50% TCA for at least 1 h and destained in 7% a c e t i c - 20% ethanol. Results Marushige was the first who reported that chemical acetylation of calf thymus chromatin leads to an increased template a c t i v i t y for Escherichia coli R N A
polymerase (16).
Previous results from our l a b o r a t o r y have shown t h a t in rat liver c h r o m a t i n , c h e m i c a l a c e t y l a t i o n is followed by a t w o - f o l d increase in t h e n u m b e r of initiation sites for E. c o Z i RNA p o l y m e r a s e when m e a s u r e d with the r i f a m p i c i n challenge assay (l 1). Using c h r o m a t i n dissociation and r e c o n s t i t u t i o n technique, we have shown with E. c o ! i RNA p o l y m e r a s e t h a t the increase in initiation sites is exclusively a c o n s e q u e n c e of the a c e t y l a t i o n of the histone f r a c t i o n , and c h e m i c a l acetylation of n o n - h i s t o n e and DNA f r a c t i o n s had no e f f e c t on c h r o m a t i n - d i r e c t e d t r a n s c r i p t i o n (11,17). It is well known t h a t e u k a r y o t i c RNA p o l y m e r a s e B has d i f f e r e n t specificity of i n i t i a t i o n from the prokaryotic E . c o l i RNA polymerase (lg). While the E. coZi enzyme prefers double-stranded DNA, the e u k a r y o t i c RNA polymerase B is specific for single-stranded DNA. Furthermore, chromatin-directed RNA synthesis with the E. coZi RNA polymerase may have serious limitations (19,20). For instance, only the e u k a r y o t i c RNA polymerase B was found to have f i d e l i t y of genetic control when mouse fetal liver chromatin was transcribed in vitro (21). In Fig. 1 the t e m p l a t e a c t i v i t y of c h r o m a t i n a c e t y l a t e d with 0.7 mM a c e t i c a n h y d r i d e is c o m p a r e d with control c h r o m a t i n . This
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Fig. I. Effect of acetylation on chromatin-direeted RNA synthesis. Increasing amounts of rat liver RNA polymerase B were added to control chromatin ( 9 and acetylated chromatin (O). Assay mixture: 1.2 mM MnCI2; 50 pl chromatin (15 pg DNA); ATP, CTP and GTP e a c h 2mM; 20 D1 (20 ~Ci) [3H]UTP (1.59 TBq/mmol); 0.i mM UTP, i0 mM Tris/HCl pH 7.9, 0.01 mM E~TA, and 0.2 mM DTT; I00 ~i RNA polymerase B. Total vol. 310 ~i. After 30 min the reaction was stopped with i0 ml ice-cold 5% trichloroacetic acid.
concentration of acetic anhydride was applied by Marushige (16) and us (l l) using E. c o ] i RNA polymerase. The level of acetylation introduced under these conditions corresponds roughly to the biological level of acetyl groups. In Fig. 1 the t o t a l synthesis was determined during a 30-min incubation period with the homologous rat liver RNA polymerase B. Under t h e s e c o n d i t i o n s , r e i n i t i a t i o n is possible and the observed increase in template activity could be either a result of an increased number of initiation sites, or a consequence of an increased elongation rate and higher frequency of initiations with unchanged number of initiation sites. In order to clarify whether the number of initiation sites is altered by acetylation of chromatin, the following two experiments were done under conditions when reinitiation is not possible. Fig. 2 shows the experiment in which according to Cedar and Felsenfeld (14) high salt concentration is used to prevent reinitiation. The c h r o m a t i n t e m p l a t e is p r e i n c u b a t e d with RNA-polymerase t3 without nucleoside triphosphates at low ionic strength, to permit the
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RNA POLYMERASE B Fig. 2. Effect of aces on chromatin-directed RNA synthesis without reinitiation. Increasing amounts of RNA polymerase B were preincubated for 15 min at 37~ with chromatin to permit initiation. At the end of the preincubation period RNA synthesis was initiated by the addition of [3H]UTP and (NH4)2SO 4. After a consecutive incubation of 15 min, RNA synthesis was terminated. ~ See also Materials and Methods. Control chromatin ( | and acetylated chromatin ( O ) .
f o r m a t i o n of an initiation complex. After this preincubation, the reaction is started by addition of the nucleoside triphosphate and a concentration of ammonium sulfate which inhibits initiation but permits elongation. Fig. 2 shows the titration of acetylated and control chromatin with 0.3 M (NH~)zSO ~ as reinitiation inhibitor. In the acetylated chromatin about twice the amount of enzyme is required to reach saturation. We do not yet know why under conditions of high ionic strength the total RNA synthesis measured is not higher with the acetylated chromatin. A possible explanation would be the preferential inhibition of transcription of hyperacetyJated chromatin by ammonium sulfate. Such an effect has been described by Dobson and Ingram (22), who studied the transcription of hyperacetylated chromatin obtained by treatment of HeLa cell cultures with n-butyrate. Note that in Fig. t with reinitiation and in Fig. 3 without reinitiation the total synthesis is about twice that of the untreated control. As shown in Fig. 3 t h e p r o p e r t i e s of chemically acetylated c h r o m a t i n were f u r t h e r investigated by inhibiting reinitiation with rifamycin AF/013. Rifamycin AF/013 prevents initiation of eukaryotic class B polymerase as a function of polymerase concentration (15,23) without inhibiting elongation. Therefore in this experiment a constant ratio of enzyme to chromatin was maintained. After preincubation for 15 min, the reaction was started by adding the nucleoside triphosp h a t e s and r i f a m y c i n AF/013 (150 pg/ml) to prevent reinitiation. T h e r e is a p p r o x i m a t e l y t w i c e as much RNA synthesis with the
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a c e t y l a t e d chromatin under conditions of reinitiation inhibition. After a few minutes a saturation level is reached because only one copy of transcript is possible. It should be emphasized that the acet yl at i on procedure leading to the functional alterations of chromatin, as described in the present experiments, is di f f er e nt from the previous methods used by Simpson (24), Marushige (16), and us (11) in earlier work. In the original m e t h o d , a c h r o m a t i n p r e c i p i t a t e in 0.15 N NaC1 was a c e t y l a t e d , causing a partial loss of histone H1 by 0.15 N NaCl (16,25). According to the new method, introduced in this paper, chromatin is a c e t y l a t e d in the homogenous phase, in Tris/HCl buffer, pH g.5, w i t h o u t any a d d i t i o n a l s a l t (see also the Materials and Methods s ectio n ) . Under these conditions no loss of histones was observed and the acety la t i on appears to be highly specific. The histone analysis by T r i t on- urea polyacrylamide gel is shown in Fig. 4. Histones e x t r a c t e d from control chromatin are compared with histones e x t r a c t e d from the a c e t y l a t e d chromatin. The a c e t y l a t e d histones appear as discrete bands as they are known f r o m e n z y m a t i c studies, though there are some differences in the acetylation pattern. Histone HI is a c e t y l a t e d in a very specific m a n n e r m ovi ng to a discrete, slightly-slower-migrating band. The h i s t o n e s H2b, H3, and H2a are converted with high yield to the a c e t y l a t e d f o r m s , a v e r y l i m i t e d n u m b e r of a c e t y l a t i o n s being introduced per nucleosome. In contrast to the other core histones, the acetylation of histone H# took place to an almost negligible extent.
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Fig. 4. Analysis of chemically acetylated histones by Triton-urea polyacrylamide-gel electrophoresis. According to Alfageme et al. (9) 12% polyacrylamide, 5% acetic acid, 8 M urea, and 0.37% Triton X-100. Slab gel (28 cm) (see also Materials and Methods). Lanes I, 3, 5, and 7: histones extracted from chemically acetylated chromatin. Lanes 2, 4, 6, and 8: histones extracted from control chromatin.
Discussion
Wallace et al. (26) e x a m i n e d the physical p r o p e r t i e s of rat liver c h r o m a t i n a c e t y l a t e d with a c e t i c anhydride and observed, in addition to increased thermal denaturation, s t r o n g e f f e c t s on magnesium solubility and nuclease sensitivity. With the t e c h n i q u e of c h r o m a t i n r e c o n s t i t u t i o n we have shown t h a t the m o d i f i c a t i o n of the histone f r a c t i o n is responsible for the t r a n s c r i p t i o n a l stimulation ( l l , 1 7 ) . In this paper we show which histone groups are a c e t y l a t e d and to what e x t e n t . Our studies were p e r f o r m e d with sheared c h r o m a t i n which was s e d i m e n t e d at 12 000 g. It remains the subject of f u r t h e r studies to d e t e r m i n e w h e t h e r c h e m i c a l a c e t y l a t i o n of nucleosomes, nuclei, and m i c r o c o c c u s nuclease digests of nuclei leads to similar a l t e r a t i o n s in the s t r u c t u r e and function of c h r o m a t i n as r e p o r t e d here with sheared r a t liver c h r o m a t i n . The question w h e t h e r a c e t y l a t i o n of histones plays a role in gene r e g u l a t i o n is still c o n t r o v e r s i a l (27-30). Our working hypothesis is t h a t a c e t y l a t i o n could be a mechanism of gene expression in tissues like liver where s h o r t - t e r m expression and subsequent repression of genes is required as a response, for instance, to hormonal stimuli. A r e c e n t r e p o r t of Pasqualini (31) is in a g r e e m e n t with this hypothesis.
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He found that acetylation of histones in fetal guinea-pig uterus is s t i m u l a t e d by e s t r o g e n a d m i n i s t r a t i o n and the estrogeD e f f e c t is blocked by progesterone and tamoxifen. Thus it is suggested that histone ace t yl a t i on is an early step induced by estrogen action during intrauterine life. Acetylation could be a very flexible mechanism of gene expression, in contrast with DNA methylation, which is a more p e r s i s t e n t modification. So far as rat liver is concerned it was reported that undermethylation at the 5' end of the albumin gene is necessary but not sufficient for albumin production by rat hepatoma ceils (32). While being aware of the limitations of the non-enzymatic system we used, we would like to consider it as a model which should s t i m u l a t e f u r t h e r experiments about the biological role of histone acetylation. Acknowledgements These studies were supported by Fonds der Gewerblichen Wirtschaft T i r o l s 1992, P r o j e c t No. 9952, and Fonds zur F S r d e r u n g s der W i s s e n s c h a f t l i c h e n Forschung (Austrian Science Foundation) Project No. P 5097. References i. Allfrey VG (1980) Cell Biology: A Comprehensive Treatise (David M. Prescott & Lester Goldstein, eds), pp 347-437, Academic Press. 2. Mathis D, Oudet P & Chambon P (1980) Progress in Nucleic Acid Research and Molecular Biology 24, 1-55. 3. Doenecke D & Gallwitz D (1982) Molec. and Cellul. Biochem. 44, 113-128. 4. Kruh J (1981) Molec. and Cellul. Biochem. 42, 65-82. 5. D'Anna JA, Tobey RA & Gurley LR (1980) BiochemiNtry 19, 2656-2671. 6. Whitlock JP Jr, Augustine R & Schulman H (1980) Nature 287, 74-76. 7. Boffa LC, Gruss RJ & Allfrey VG (1981) J. Biol. Chem. 256, 9612-9621. 8. Ingram VM, Hagopian HK, Riggs MG, Neumann JR, Dobson ME, Owens BB & Maniatis GM (1979) Cellular and Molecular Regulation of Hemoglobin Switching, pp 471-489, Grune and Stratton, New York. 9. Alfageme CR, Zweidler A, Mahowald A & Cohen LH (1974) J. Biol. Chem. 249, 3729-3736. i0. Blobel G & Potter V (1966) Science 154, 1662. II. Oberhauser H, Csordas A, Puschendorf B & Grunicke H (1978) Biochem. Biophys. Res. Commun. 84, 110-116. 12. Krebs G & Chambon P (1976) Eur. J. Biochem. 61, 15-25. 13. Seifart KH, Benecke BJ & Juhasz PP (1972) Arch. Biochem. Biophys. 151, 519-532. 14. Cedar H & Felsenfeld G (1973) J. Mol. Biol. 77, 237-257. 15. Meilhac M, Tysper Z & Chambon P (1972) Eur. J. Biochem. 28, 291-300.
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16. Marushige K (1976) Proc. Natl. Acad. Sci. U.S.A. 73, 3937-3941. 17. Csordas A, Oberhauser H, Puschendorf B & Grunicke H (1978) Hoppe-Seyler's Z. Physiol. Chem. 359, 1070. 18. Lewis MK & Burgess RR (1982) Eukaryotic RNA polymerases, in: the Enzymes, 3rd ed, vol 15B (Boyer PD, ed), pp 109-153, New York, Academic Press. 19. Zasloff M & Felsenfeld G (1977) Biochemistry 16, 5135-5145. 20. Giesecke K, Sippel A, Nguyen-Huu MC, Groner B, Hynes ErE, Wurtz T & Schutz G (1977) Nucleic Acids Res. 3943-3958. 21. Draper KG & Riggsby WS (1981) Biochim. Biophys. Acta 656, 213-219. 22. Dobson ME & Ingram VM (1980) Nucleic Acids Res. 8, 4201-4219. 23. Rose KM, Rauch PA & Jacob S (1975) Biochemistry 14, 35983604. 24. Simpson RE (1971) Biochemistry i0, 4466-4470. 25. Wong TK & Marushige K (1976) Biochemistry 15, 2041-2046. 26. Wallace RB, Sargent TD, Murphy RF & Bonner J (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 3244-3248. 27. Mathis DM, Oudet P, Wasylik B & Chambon P (1978) Nucleic Acids Res. 5, 3523-3547. 28. Lilley DM & Berendt AR (1979) Biochem. Biophys. Res. Commun. 90, 917-924. 29. Vidali G, Boffa LC, Bradbury EM & Allfrey VG (1978) Proc. Natl. Acad. Sci. U.S.A. 75, 2239-2243. 30. Nelson D, Covault J & Chalkley R (1980) Nucleic Acids Res. 8, 1745-1763. 31. Pasqualini JR, Cosquer-Clavreaul C & Gelli C (1983) Biochim. Biophys. Acta 739, 137-140. 32. Ott MO, Sperling L, Cassio D, Levilliers J, Sala-Trepat J & Weiss MC (1982) Cell 30, 825-833.
