Nuclei from hepatoma tissue culture (HTC) cells were isolated by standard methods and incubated in media commonly used for nuclease digestions (DNAase I and micrococcal nuclease) and for in vitro RNA synthesis. During the incubation, histones can be deacetylated from both control cells and cells treated with 6 mM sodium butyrate to enhance the levels of histone acetyla- tion. Deacetylation of histone is much more apparent in nuclei isolated from sodium butyrate-treated cells. Inclusion of 6 mM sodium butyrate in the incubation medium effectively inhibits the endogenous deacetylase activity acting on histones H3 and H4, whereas sodium acetate at the same concentra- tion has very little inhibitory effect.
Introduction
The role of histones in packaging eukaryotic DNA into discrete nucleo- protein complexes has been the subject of considerable investigation. It is now clear that the chromatin repeating unit, termed a nucleosome, consists of a compact core containing two each of the non-H1 histones (H2A, H2B, H3 and H4) associated with 140 base pairs of DNA [1,2], and a spacer region contain- ing H1 or non-histone proteins associated with 40--60 base pairs of DNA [3,4]. The binding of the histones to DNA includes electrostatic interactions between the positively charged residues in the amino-terminal end of the histones with the negatively charged phosphate backbone of the DNA [5--7], in addition to hydrophobic interactions between the histone and DNA [8,9].
Many of the lysine residues in the amino-terminal region of histom~s tt3 and H4 are subject to highly active post-synthetic acetylation and deacetylat ion [10--12] , with the half time of turnover about 3 min in vivo I121. Since histones H3 and H4 are more tightly bound to I)NA than are the other histones [8,9], and play a fundamental role in organizin~ both the DN.A and th(, other histones in the nucleosome [13--15] , it seems likely that the rapid acetylation and deacetylat ion of histones H3 and H4 may give rise to transient conforma- tional changes within the nucleosome. Histone acetylation has been postulated to be involved in both histone deposition [161 and in transcriptional activity [17,18].
Until recently it has been difficult to obtain chromatin or nuclei containing highly acetylated histones except by means of chemical modification [19]; however this approach is subject to the criticism that the chemical acylating agent may not be reacting with sites on histones that are acetylated in vivo. Thus results based on studies with in vitro modified histones must be inter- preted with caution. More recently, it has been shown that t rea tment of HeLa cells with sodium butyrate results in hyperacetylat ion of histones H3 and It4 in vivo [20,21]; fur thermore, it is now clear that the effect of butyrate is to inhibit histone deacetylat ion [22--27]. Thus it is possible to isolate chromatin containing histones that are hyperacetyla ted in vivo permitting a variety of studies on the effect of acetylation of histones on nucleosomal structure [24,28,291.
During the preparation and manipulation of nuclei from hepatoma tissue culture (HTC) cells, significant deacetylat ion of control and hyperacetylated histones was detected. We have studied the effects of nuclear isolation methods and nuclear incubation media on histone deacetylat ion and have determined conditions in which deacetylat ion is partially or completely inhibited.
Materials and Methods
Cell growth and nuclear isolation Hepatoma tissue culture (HTC) cells were grown in suspension culture as
described by Oliver et al., [30]. Log phase cells were either untreated or were treated with 6 mM sodium butyrate for 6 h prior to nuclear isolation. All cells were [3H]acetate labelled (5 mCi/1) for 1 h before isolation. It has been previ- ously shown that only acetylated histone species incorporate radiolabel during the 1 h labelling period [31]. Nuclei were isolated by one of two methods.
Method A. Nuclei were prepared by washing three times in 0.25 M sucrose, 10 mM Tris, 10 mM MgC12, 6 mM sodium butyrate, 1% Tri ton X-100, pH 7.0. The pellet was resuspended in the appropriate incubation buffer.
Method B. Nuclei were washed twice in 0.25 M sucrose, 60 mM KC1, 15 mM NaC1, 10 mM MgC12, 1 mM CaCl:, 15 mM MES (2-[N-morphol ino]ethane sulfonic acid), 6 m M sodium butyrate , 0.5% Tri ton X-100, pH 6.5, and resuspended in the appropriate incubation media.
Sodium butyrate was included during the isolation procedures to prevent histone deacetylation.
Gel electrophoresis o f histones 15% polyacrylamide acid-urea gels. Samples were made 0.2 M in H2SO4,
519
the DNA pelleted and the histones precipitated with 4 volumes of acetone. Histones were then electrophoresed on 23 cm acid-urea {2 M) gels for 48 h at 4°C as described by Panyim and Chalkley [32]. Gels were stained with amido black, destained electrophoretically and scanned at 600 nm. 2-mm gel slices were digested for 3 h with 0.3 ml H202 and counted in 5 ml of Bray's solution. Since only specific activity ratios are presented, specific activities were com- puted as the counts per minute for a histone including all modified forms, divided by the area under the curve for all five histones.
18% polyacrylamide-SDS histone gels. Nuclei were precipitated with 4 volumes of acetone, washed with acetone and dried under nitrogen. Samples were resuspended in 1% SDS {sodium dodecyl sulfate), 10% glycerol, 1% 2-mercaptoethanol, 10 mM Tris, 2 mM EDTA, 0.1 mM phenylmethylsulfonyl fluoride, 0.005% bromphenol blue, pH 7, and electrophoresed on polyacryl- amide gels containing SDS as described by Laemmli [33], and modified by Bonner and Pollard [34]. Samples were electrophoresed on 16-cm gels until the tracker dye reached the bottom. Gels were stained with amido black, diffusion destained and scanned at 600 nm. Bands were cut out and counted for 3H as described for the acid-urea gels. Specific activities were determined as the counts per minute divided by the amount of protein in the band.
Determination of the amount of protein in a band. A DuPont Curve Anal- yzer was used to resolve bands. Bands were then cut out, weighed, and the weights used as a measure of the amount of protein.
Results and Discussion
Dependence of deacetylation on incubation media While using nucleases as probes of chromatin structure, we observed a
decrease in the acetylated forms of the histones during the time course of nuclease digestion. To characterize and quantify this result, nuclei isolated by Method A, from cells pre-treated with sodium butyrate to enhance the levels of acetylation, were incubated at 37°C in 0.25 M sucrose, 10 mM cacodylic acid, I mM CaC12, pH 7.4, a buffer commonly used for micrococcal nuclease diges- tion of chromatin or nuclei [1--4]. Histones were rapidly deacetylated in this buffer and continued to be deacetylated during the 120 min time span of the incubation (Fig. 1). In Fig. 1A, histones were electrophoresed from left to right on long acid-urea gels, with the parent form of each of the four histones H3, H2B, H2A and H4 having the highest mobility for a given histone. On the gel scans and [3H]acetate profiles, all four of the H4 acetylated forms (mono- to tetra-acetylated forms abbreviated H4Acl to H4Ac4) can be visualized, three of the four H3 forms, and one each of H2B and H2A (H2BAcl is observed as a separate peak while H2AAcl is the fast migrating shoulder on the H2B parent band). As the incubation proceeds, a shift in counts is observed away from the more highly acetylated forms of H3 and H4, and a loss in counts from the region containing H2AAcl. The monoacetylated form of H2B is relatively unaffected. In conjunction with a shift and overall loss in counts (see Table I for specific activity changes), is a transfer in histone mass toward the parent forms of H3, H2A and H4. This shift for H3 and H4 was quantitated by resolv- ing and determining the areas under the curve for each form of these proteins
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F i g . 1. ( l e f t ) T i m e c o u r s e o f in s i t u d e a c c t y l a t i o n o f h e p a t o m a t i s s u e c u l t u r e ( H T C ) cell h i s t o n e s . L o g p h a s e ce l l s w e r e p r e - t r e a t e d f o r 6 h w i t h 6 m M s o d i u m b u t y r a t e to i n c r e a s e t h e l e v e l s o f a c e t y l a t i o n . N u c l e i w e r e i s o l a t e d b y M e t h o d A a n d i n c u b a t e d at 3 7 ° C in 0 . 2 5 M s u c r o s e , 10 m M c a c o d y l i c ac id , 1 m M C a C l 2 , p H 6 .5 , f o r ( A ) 0 r a in , (B) 3 0 ra in a n d ( C ) 1 2 0 ra in . H i s t o n e s w e r e a c i d e x t r a c t e d , e l e c t r o - p h o r e s e d o n 23 c m a c i d - u r e a ( 2 M) gels , s t a i n e d w i t h a m i d o b l a c k a n d s c a n n e d at 6 0 0 n m ( -) . Ge l s w e r e s l i c e d i n t o 2 - r a m s e c t i o n s to o b t a i n t h e [ 3 H ] a c e t a t e p r o f i l e s ( . . . . . . ). T h e ge l s c a n s a n d [ 3 H ] a c e t a t e p r o f i l e s are o n t h e s a m e s c a l e f o r all ge l s . ( r i g h t ) C o m p a r i s o n o f t h e a m o u n t o f p a r e n t or a c e t y l a t e d f o r m
o f H 3 o r H 4 w i t h i n c r e a s e d i n c u b a t i o n t i m e o f t h e n u c l e i , r e l a t i v e t o t h e s a m e s p e c i e s at z e r o t i m e . L] p a r e n t ; A m o n o a c e t y l a t e d ; A d i a c e t y l a t e d ; . , t r i a c e t y l a t e d a n d , t e t r a c e t y l a t e d f o r m s o f H3 a n d H 4 .
(Fig. 1B). With increased incubation time, the parent form of H3 gained mass at the expense of all three acetylated forms. For H4, the loss in H4Ac3 and H4Ac4 was most acute, resulting in an increase in mass of parent and mono- acetylated forms of H4.
it was apparent that meaningful nuclease digestion or RNA transcription reactions using isolated HTC nuclei containing hyperacetylated histones could not be carried out unless deacetylase activity was inhibited. A number of methods such as changes in ionic strength, pH, incubation temperature or addi- tion of chemical inhibitors are possible candidates for inhibition of deacetyla- tion. In particular, inclusion of acetate (product inhibition) or an acetate analog could be used to inhibit the enzyme(s) . Since it has been shown previ- ously that sodium butyrate blocks the deacetylase system in vivo [ 22--27 ], and acetate is one of the end products, both butyrate and acetate were tested as in vitro inhibitors. In agreement with observations in vivo [22] , acetate was a
521
poor inhibitor in vitro, and deacetylation occurred to the same extent as with- out acetate present. The results of an incubation carried out with nuclei and medium as in Fig. 1, except that 6 mM sodium butyrate was included, is shown in Fig. 2. In this case, it is noted in the [3H]acetate profiles that the counts shift for H3 and H4 is not observed, while the counts for monoacetylated H2A are lost in the same manner as in Fig. 1. Under these conditions, there was no measureable loss in total H3 and H4 specific activity (see Table I). Thus sodium butyrate appears to be a fairly effective inhibitor of in vitro H3 and H4 deacetylation, although the deacetylation of H2A was unaffected.
We also determined if deacetylase activity occurred to the same extent in other commonly used incubation media. These data are shown in Fig. 3 and Table I. In the first set of experiments, cells were treated with sodium butyrate, the nuclei isolated by Method A, and incubated in 0.25 M sucrose, 10 mM Tris, 10 mM NaC1, 3 mM MgC12, pH 7.4, a buffer commonly used for DNAase I digestions [35,36]. As demonstrated in Fig. 3A and 3B and in Table I, deacetylation is both rapid and continuous but occurs to a slightly lesser extent
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Fig. 2. ( l e f t ) T i m e course o f in situ d e a c e t y l a t i o n o f HTC cel l h i s tones in the presence o f 6 m M s o d i u m butyrate . Cells w e r e treated and histories ex trac ted , e l e c t r o p h o r e s e d and c o u n t e d as in Fig. 1. Nuc le i w e r e incubated at 3 7 ° C in 0 . 2 5 M s u c r o s e , 1 0 m M c ac od y l i c acid, 6 m M s o d i u m butyrate , 1 mM CaC12, pH 6 .5 ,
for ( A ) 0 r a i n , ( B ) 3 0 rain and ( C ) 1 2 0 m i n . - - , gel scan; . . . . . . , [ 3 H ] a c e t a t e prof i le . (r ight) Co mpa r- i son o f the a m o u n t o f parent or a c e t y l a t e d f o r m of H3 and H4 w i t h increased i n c u b a t i o n t i m e o f the nucle i in the presence o f 6 m M s o d i u m butyrate , relative to the same spec ies at zero t ime , D parent; 4 m o n o a c e t y l a t e d ; ~, d i a c e t y l a t e d ; e , t r iace ty la ted and o, t e t ra a ce ty la t ed f o r m s o f H3 and H 4 .
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T A B L E I
L O S S I N [ 3 H ] A C E T A T E S P E C I F I C A C T I V I T Y O F H I S T O N E S H 3 A N D H 4 A S A F U N C T I O N O F
C E L L T R E A T M E N T , I N C U B A T I O N M E D I A A N D I N C U B A T I O N T I M E
+, d e n o t e s n u c l e i i s o l a t e d f r o m cel ls t r e a t e d f o r 6 h w i t h 6 m M s o d i u m b u t y r a t e . - - , d e n o t e s n u c l e i f r o m u n t r e a t e d ce l l s . V a l u e s in p a r e n t h e s e s i n d i c a t e d a t a o b t a i n e d f r o m S D S - h i s t o n e gels . All o t h e r d a t a a re f r o m a c i d - u r e a gels . N u c l e i w e r e i s o l a t e d b y M e t h o d A .
I n c u b a t i o n Cell I n c u b a t i o n P e r c e n t i n i t i a l P e r c e n t i n i t i a l m e d i u m t r e a t m e n t t i m e ( r a i n ) s p e c i f i c a c t w i t h o u t s p e c i f i c a c t w i t h 6 m M
b u t y r a t e in i n c u b a - s o d i u m b u t y r a t e in
t i o n m e d i u m i n c u b a t i o n m e d i u m
H 3 H 4 H3 H 4
0 . 2 5 M s u c r o s e 1 0 m M + 3 0 8 2 ( 7 0 ) 6 8 ( 7 0 ) ( 8 8 ) ( 9 3 )
T r i s 10 m M NaC1, + 1 2 0 6 3 ( 5 1 ) 6 5 ( 5 9 ) ( 9 6 ) ( 1 0 4 ) 3 m M MgC12 , p H 7 . 4 - - 3 0 9 0 8 0 ( 1 0 5 ) ( 9 5 )
- - 1 2 0 82 77 ( 9 8 ) ( 1 0 5 )
0 . 2 5 M s u c r o s e , 10 m M + 3 0 6 4 6 5 9 4 96 c a c o d y l i c a c i d , 1 m M + 1 2 0 38 6 5 1 0 3 101
C a C I 2, p H 6 . 5
0.25 M sucrose, 60 mM + 30 103(90) 92(106) (98) (108) KCI, 15 mM NaCI, + 120 73(73) 98(94) (105) (107) 10 mM MgCI2, 1 mM -- 30 (95) (90) (105) (96) CaCI2, 15 mM MES, - 120 (90) (I01) (105) (98) o H 6 . 5
than in the previous cacodylate buffer. Shifts in the absorbance profiles of H3 and H4 are quite apparent, although the shift in H2A is not as noticable due to less resolution in this region of the gel. Again when sodium butyrate is included
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Fig . 3. I n s i tu d e a c e t y l a t i o n o f H T C cel l h i s t o n e s in v a r i o u s i n c u b a t i o n m e d i a . Ce l l s a n d h i s t o n e s w e r e
t r e a t e d as in F ig . 1; n u c l e i w e r e i s o l a t e d b y M e t h o d A. A l l s c a n s a re o n t h e s a m e sca le . N u c l e i ( a t 3 7 ° C ) w e r e i n c u b a t e d in 0 . 2 5 M s u c r o s e , 10 m M T r i s , 10 m M NaC1, 3 m M MgC12, p H 7 .4 , f o r ( A ) 0 r a in , (B) 1 8 0 m i n a n d f o r (C) 1 8 0 r a i n in t h e s a m e b u f f e r p l u s 6 m M s o d i u m b u t y r a t e . I n ( D ) , n u c l e i w e r e i n c u b a t e d f o r
1 8 0 m i n in 0 . 2 5 M s u c r o s e , 6 0 m M K C I , 15 m M NaC1, 10 m M MgC12, 1 m M C a C l 2, 15 m M M E S . p H 6 .5 .
523
in the incubation medium (Fig. 3C and Table I), deacetylation occurred to a very small extent, and could not be detected by monitoring the specific activity change of H3 and H4 on SDS histone gels.
Deacetylation was also monitored in two other buffer systems, differing mainly from those described previously by their increased ionic strength. In a buffer similar to that used by Hewish and Burgoyne [37] and in other nuclease digestion studies (both micrococcal nuclease and DNAase I [38] ), consisting of 0.25 M sucrose, 60 mM KC1, 15 mM NaC1, 10 mM MgC12, 1 mM CaC12, 15 mM MES, pH 6.5, deacetylation at 37°C occurred to a lesser extent (Fig. 3D and Table I), than the same nuclei incubated for the same length of time in 0.25 M sucrose, 10 mM Tris, 10 mM NaC1, 3 mM MgC12, pH 7.4 (Fig. 3B). Also deacetylation could not be detected in either acid-urea or SDS histone gels with the inclusion of 6 mM sodium butyrate (see Table I). It is concluded that such conditions are optimal for inhibiting deacetylase activity during nuclear incuba- tion. Similar results were obtained when histone deacetylation of nuclei from butyrate-treated and control cells was monitored at 25°C in the following RNA transcription medium [39] : 12.5% glycerol, 5 mM MgC12, 1 mM MnC12, 25 mM Tris-HC1 (pH 8.0), 0.05 mM EDTA, 0.4 mM ATP, CTP and GTP, 0.05 mM [3H]UTP, 0.15 M KCI, 12 mM 2-mercaptoethanol. Nuclei were suspended at 2.5 • 107/ml and incubated for up to 60 min without detectable loss of hyper- acetylated H3 and H4 as assayed by measurements of the area under the curve for the histone forms. In this case both ionic environment and the reduced temperature inhibit deacetylation in the absence of sodium butyrate.
Dependence of deacetylation on cell treatment In the previous section, log phase cells were treated with sodium butyrate to
enhance the levels of histone acetylation, allowing deacetylation to be easily visualized on acid-urea gels. Nuclei isolated by Method A from untreated cells and incubated in 10 mM Tris, 10 mM NaC1, 3 mM MgC12, pH 7.4, also demon- strated detectable amounts of deacetylation (see Table I), but to a lesser extent than nuclei from sodium butyrate-treated cells. This could be explained by dependence of the rate of deacetylation on the amount of substrate available. The loss in counts per minute occurs equally from each of the acetylated forms of H3 and H4 (data not shown}. Deacetylation in the same buffer in the presence of sodium butyrate could not be detected in nuclei from untreated cells.
That the deacetylation of histones in nuclei from untreated cells is signifi- cant is suggested by a statistical analysis of the data points in the right hand column of Table I. A standard deviation can be obtained from these values since neither H3 nor H4 showed any detectable change in specific activity or loss in hyperacetylated forms during incubation of nuclei in the presence of 6 mM sodium butyrate. The values obtained in the right hand column all therefore represent 100% of the control (zero time point) values. The standard deviation calculated from these values is +5% and therefore the values of 82% for H3 and 77% for H4 of the initial specific activity for nuclei from untreated cells represents a significant loss in activity.
Dependence of the rate of deacetylation on the method of nuclear isolation Nuclei, isolated by either Method A or B, from cells treated with sodium
524
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Fig . 4. T i m e c o u r s e o f in s i t u d e a c e t y l a t i o n o f H T C cel l h i s t o n c s H3 and H 4 as in Fig . 3 (A and B), e x c e p t
t h a t the loss i n spec i f i c a c t i v i t y o f t o t a l H 3 a n d H 4 was d e t e r m i n e d o n 18% p o l y a c r y l a m i d e - S D S h i s t o n e
ge ls (see M a t e r i a l s a n d M e t h o d s ) . N u c l e i were i s o l a t e d by M e t h o d B. ~, H3 ; • H4 .
butyrate, were incubated in 0.25 M sucrose, 10 mM Tris, 10 mM NaC1, 3 mM MgC12, pH 7.4. The rates of deacetylation in the above buffer were identical for nuclei isolated by either Method A or B. In Fig. 4, the rate of deacetylation is composed of two rates. Rapid deacetylation occurs at early incubation times, whereas the rate of deacetylation is considerably slower at longer incubation periods.
The above data, therefore, suggest the following conclusions: (1) Rapid deacetylation can occur when nuclei isolated from sodium
butyrate-treated cells are incubated in low ionic strength media. Deacetylation also occurs to a lesser extent during incubation of nuclei from untreated cells. Partial or complete inhibition of deacetylation occurs in media of slightly increased ionic strenth (I = 0.12--0.21).
(2) 6 mM sodium butyrate mostly inhibits in vitro deacetylation. Sodium acetate, at the same concentration, does not significantly inhibit this activity. To demonstrate that the deacetylase inhibitor, sodium butyrate, does not affect nucleolytic activity, we have tested micrococcal nuclease and DNAase I using nuclei from untreated cells in the above buffers with and without 6 mM sodium butyrate (data not shown). Enzyme activities were unaffected by the inclusion of sodium butyrate. The effects of acetylation on chromatin and nucleosomal structure can therefore be studied by a variety of techniques with- out deacetylation occurring during manipulation or incubation of samples.
(3) The deacetylase activity remains present whether nuclei are isolated at low (Method A) or moderate (I = 0.12; Method B) ionic strengths.
(4) The rate of deacetylation is greatest during the early part of in vitro incubations.
Acknowledgements
We wish to thank Mary Ellen Perry for help with the figures and Jack Liao and Tom Slattery for technical assistance. This work was supported by grants from the N.I.H. Cancer Institute Nos. Ca-10871 and Ca-20509.
525
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