A plant histone acetyltransferase specific for H3 in nucleosomes

Plant Science, 46 (1986) 189--194 Elsevier Scientific Publishers Ireland Ltd.

A PLANT HISTONE ACETYLTRANSFERASE

189

SPECIFIC FOR H3 IN NUCLEOSOMES

RAMON SENDRA, M. LUISA SALVADOR, GERARDO LOPEZ-RODAS, VICENTE TORDERA and LUIS FRANCO*

Department of Biochemistry, Faculties of Sciences, University of Valencia, Burjassot, Valencia (Spain) (Received April 21st, 1986) (Revision received June 10th, 1986) (Accepted July 4th, 1986) Two histone acetyltransferase activities have been found in embryonic axes of Pisum sativum. One of them corresponds to histone acetyltransferase B, whose properties have already been described by us (Salvador et al., FEBS Lett., 191 (1985) 55). The other enzyme readily catalyzes the transfer of acetate from acetylCoA to histone H3, and it is extremely specific for this histone when nucleosomes are used as substrate. In view of this fact and of other properties reported, the novel enzyme is identified as histone acetyltransferase A. Thus, the results show that plants do possess a set of acetyltransferases similar to those of other eukaryotes.

Key words: histone acetyltransferase A; histone acetyltransferase B; nucleosomes; histone H3; Pisum sativum

Introduction I t is c u r r e n t l y t h o u g h t t h a t h i s t o n e acetyla t i o n p l a y s a role in t h e c o n t r o l o f transc r i p t i o n in e u k a r y o t e s [ 1 , 2 ] . I n m o s t tissues a n d species t w o f o r m s o f t h e e n z y m e t h a t c a t a l y z e s t h e a c e t y l a t i o n o f h i s t o n e s are present, namely histone acetyltransferases A a n d B [ 3 ] . T h e latter, a c y t o p l a s m i c e n z y m e , is t h o u g h t t o a c e t y l a t e h i s t o n e s p r i o r to c h r o m a t i n a s s e m b l y [ 4 ] , b u t t h e role o f t h e f o r m e r , p r e s e n t in nuclei, s e e m s to be r e l a t e d t o t h e genetic activity o f chrom a t i n [ 5 ] . In s o m e cases, a t h i r d e n z y m e , closely a s s o c i a t e d w i t h c h r o m a t i n has b e e n described [6,7]. The knowledge of the regulation of plant gene e x p r e s s i o n is still far f r o m s a t i s f a c t o r y , a n d e x p e r i m e n t s are c u r r e n t l y m a d e to l o o k f o r genetic e l e m e n t s similar t o t h o s e d e s c r i b e d in animals.

*To whom correspondence should be sent. Abbreviations: MES, 2-(N-morpholino)ethanesulfonic acid.

We r e c e n t l y r e p o r t e d f o r the first t i m e the presence of histone acetyltransferase B in plants, b u t we f o u n d no evidence f o r t h e e x i s t e n c e o f o t h e r f o r m s o f t h e e n z y m e in the p e a seedlings used in o u r e x p e r i m e n t s

[81. This p a p e r describes a h i s t o n e acetylt r a n s f e r a s e A in d e v e l o p i n g p e a e m b r y o n i c axes, a d e v e l o p m e n t a l stage w h o s e genetic activity is higher t h a n t h a t o f seedlings. Materials a n d m e t h o d s

Materials P e a (Pisum sativum, cv. L i n c o l n ) seeds w e r e g e r m i n a t e d in m o i s t v e r m i c u l i t e at 28°C in t h e dark. E m b r y o n i c axes at 40 h of g e r m i n a t i o n w e r e used as a source of enzymes. Preparation o f histone acetyltransferases C r u d e a c e t y l t r a n s f e r a s e s were p r e p a r e d as described [8], except that the crude extract was sonified at 125 W f o r 4 p e r i o d s o f 55 s in a B r a u n L a b s o n i c 2 0 0 0 sonifier b e f o r e adding (NH4)2SO4. In s o m e e x p e r i m e n t s ,

0168-9452/86/$03.50 © 1986 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland

190 the a m m o n i u m sulphate step was substituted for a treatment with 20% polyethyleneglycol (Mw = 8000) in buffer B [8], and the proteins were allowed to precipitate for 45 min at 4°C. The precipitated proteins were dissolved, dialyzed and chromatographed on DEAE-Sepharose CL-6B as described previously [9]. Preparation su bstra tes

of

histone

and

nucleosome

Pea and chicken erythrocyte histones were obtained by the procedures given elsewhere [8]. To prepare pea oligonucleosomes, nuclei were obtained from pea seedlings [10] and suspended in 0.2 M sucrose, 8 mM CaC12, 60 mM NaC1, 4 mM 2-(N-morpholino)ethanesulfonic acid (MES) (pH 7.0) to a density of about 10 s nuclei/ml. Micrococcal nuclease (150 U/ml) was then added and the suspension was incubated at 37°C for 30 min. Reaction was stopped by adding EDTA to 10 mM and chilling on ice. The mixture was then dialyzed against 0.25 mM EDTA (pH 7.0) and centrifuged at 17000 × g for 20 min. The supernatant, further referred to as oligonucleosomes, was used in some experiments (see below), w i t h o u t any further purification, as substrate for the acetyltransferase reaction. Electrophoretic analysis [11] revealed that 66% of DNA was indeed in the form of oligonucleosomes (n ~_ 6). E n z y m a t i c assay

Histone acetyltransferase assay was carried out as described [9]. To study the acetylating activity towards nucleosomes, oligonucleosomes (about 0.8 mg DNA) were incubated with 800 pl of the enzymatic preparation and 0.3 ~Ci of labelled acetylCoA in a final volume of 1.1 ml. The remaining incubation conditions were as described for free histones [9]. The incubation was stopped by adding cold trichloroacetic acid to a final concentration of 25%. The precipitate was recovered by centrifugation, washed once with cold 25% trichloroacetic acid, twice with acetone:

37.5% HC1 (100:1, v/v) and twice with acetone and dried under vacuum. The electrophoresis and fluorography were carried out exactly as described [9]. The acetylating activity towards spermine and spermidine was also assayed. In these instances aliquots of the reaction mixture were dropped onto phosphocellulose filters (Whatman P-81). The filters were then washed twice in 50 mM NaHCO3-Na2CO3 (pH 9.2) at 40°C for 20 min each, once in methanol, dried at 70°C and counted. Results Two peaks with histone acetyltransferase activity were detected in the elution of the DEAE-Sepharose column (Fig. 1). These two peaks correspond to true enzymatic acetylation because, in contrast to chemical acetylation that occurs at low ionic strength [9], transfer of acetate from acetylCoA does not take place after heating the eluate (10 min at 70°C). The first peak elutes at 150 mM NH4C1, and the second one at 300 mM NH4C1. The histone acetyltransferase corresponding to the second peak was purified on Ultrogel AcA34 as described previously [8]. It elutes as a single peak with M r = 160 000 and, therefore, it coincides with the histone acetyltransferase B previously described in peas [8]. The activity found in the first peak of DEAE-Sepharose chromatography corresponds to a novel enzyme, which can be tentatively identified as histone acetyltransferase A. On purifying it on Ultrogel AcA34, a molecular weight of 120 000 was found (Fig. 2). Both enzymes are highly specific for histones as shown in Table I. These results also confirm that there actually exist two enzymes because of the different specificity. Histone acetyltransferase B does not acetylate protamine sulphate, but it can accept polyamines as substrates. Pea histone acetyltransferases A and B also differ in their specificity toward histone classes. Figure 3 shows that free histone H4 is the only histone substrate of acetyl-

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Fig. 1. Elution of histone acetyltransferases from DEAE-Sepharose CL-6B. A column of 3 x 18 cm was loaded with crude extract (see the text) from 45 g of embryonic axes. Elution was carried out with buffer B [9] and then with 1 1 of an NH,CI gradient (0.01--0.35 M) at a flow rate of 45 ml/h. Fractions of 7.5 ml were collected. (e) A2~0; (o) histone acetyltranferase activity (assayed with chicken erythrocyte histone; ( . . . . ) NH, CI gradient.

t r a n s f e r a s e B. This e n z y m e d o e s n o t c a t a l y z e the t r a n s f e r o f a c e t a t e to h i s t o n e s in t h e nucleosome. On the contrary, histone a c e t y l t r a n s f e r a s e A is able to a c e t y l a t e H 3 w h e n o l i g o n u c l e o s o m e s w e r e u s e d as substrates. M i c r o c o c c a l nuclease digestion s o m e t i m e s results in t h e release o f free histones. T h e r e f o r e , t h e possibility t h a t t h e labelled b a n d in lane 8 (Fig. 3) c o r r e s p o n d s t o free r a t h e r t h a n n u c l e o s o m a l H 3 s h o u l d be considered. T o rule o u t this p o s s i b i l i t y , an e x p e r i m e n t was carried o u t in w h i c h , a f t e r i n c u b a t i n g o l i g o n u c l e o s o m e s w i t h labelled a c e t y l C o A and a c e t y l t r a n s f e r a s e s as d e s c r i b e d above, the m i x t u r e was c e n t r i f u g e d at 2 4 0 0 0 0 X g f o r 6 h. U n d e r t h e s e c o n d i t i o n s ,

m o n o n u c l e o s o m e s s e d i m e n t , while free hist o n e s w o u l d r e m a i n in the s u p e r n a t a n t . O n c e e l i m i n a t e d t h e excess o f a c e t y l C o A , the activity o f this s u p e r n a t a n t was negligible. This d e m o n s t r a t e s t h a t the labelled h i s t o n e is a c t u a l l y n u c l e o s o m a l . Figure 3 also shows t h a t h i s t o n e H 3 is also the t a r g e t o f acetyla t i o n in e x p e r i m e n t s w i t h free p e a histories, b u t , in this instance, t h e specificity o f h i s t o n e a c e t y l t r a n s f e r a s e A seems to be less n a r r o w t h a n with n u c l e o s o m e s . In fact, s o m e o t h e r p o l y p e p t i d e s o f u n k n o w n n a t u r e were also a c e t y l a t e d {Fig. 3, lane 5} and, with c h i c k e n e r y t h r o c y t e h i s t o n e s as substrates, H4 and H5 w e r e t h e m a i n targets o f the a c e t y l a t i n g activity.

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Fig. 2. Purification of histone acetyltransferase A on Ultrogel AcA34. A c o l u m n of 120 × 1.5 cm, was equilibrated and eluted with buffer B [9] at a flow rate of 14 m l / h (3.1 ml/fraction). (e) A280; (o) histone acetyltransferase activity. Arrows, elution v o l u m e of M r markers: (a) dextran blue, M r 2000 000; (b) beef liver catalase, M r 240 000; (c) rabbit muscle aldolase, M r 158 000; (d) yeast invertase, M r 120 000; (e) egg yolk ovalbumin, M r 45 0 0 0 ; (f) egg ]ysozyme, M r 14 000.

Table I. A c e t y l a t i o n •f different pea histone acetyltransferases.

substrates

by

Substrate

Enzyme A(%)

Enzyme B(%)

Pea histones Chicken e r y t h r o c y t e histones Bovine serum albumin P r o t a m i n e sulphate Poly-L-lysine (Mr 25 000) Spermine Spermidine

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126 0 26 5 0 0

46 3 0 0 24 13

Discussion The results given in Table I and Fig. 2 strongly support the idea that two different histone acetyltransferases exist in embryonic axes. One of them, histone acetyltransferase B, had been previously described in plants [8]. It is n o t e w o r t h y that it does not acetylate nucleosomes at all, and this provides another indication in favour of its proposed role in the assembly of chromatin [7]. The second enzymatic activity reported

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Fig. 3. Specificity of histone acetylation by pea embryonic axes acetyltransferases. After incubation with [1-~4C]acetylCoA, histones were ;eparated by SDS-polyacrylamide gel electrophoresis. (A) Coomassie-stained gel. (B) Fluorogram. Substrates used: lanes 1--3, chicken histones; anes 4--6, pea histones; lanes 7--9, pea nucleosomes. In lanes 1, 4 and 7 the enzymatic extracts were substituted for buffer B [8 ] containing t l 0 mM NH4CI to check the extent of chemical unspecific acetylation. The enzyme was: lanes 2, 5 and 8, acetyltransferase A; lanes 3, 6, md 9, acetyltransferase B.

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194

in this p a p e r ' c a n be ascribed to h i s t o n e a c e t y l t r a n s f e r a s e A. A p a r t f r o m its characteristic early e l u t i o n in NH4C1 gradients, several features of the specificity o f this e n z y m e clearly p o i n t to t h a t identification. F o r instance, the b e h a v i o u r o f the p e a e n z y m e with p r o t a m i n e is similar to t h a t o f y e a s t h i s t o n e a c e t y l t r a n s f e r a s e A [ 9 ] , as it is the p r e f e r e n c e for H3 a m o n g free histones. Moreover, this pea e n z y m e does n o t a c e t y l a t e p o l y a m i n e s . But, a b o v e all, the ability o f the pea e n z y m e to a c c e p t n u c l e o s o m e s as substrates is t h e decisive p o i n t t h a t f a v o u r s its i d e n t i f i c a t i o n as h i s t o n e a c e t y l t r a n s f e r a s e A. Thus, a l t h o u g h the precise role o f nuclear h i s t o n e a c e t y l a t i o n is n o t k n o w n , the results p r e s e n t e d in this p a p e r add a n o t h e r similarity b e t w e e n p l a n t and animal c o n t r o l s o f genetic activity. Acknowledgements This w o r k has been s u p p o r t e d by g r a n t 1 1 0 1 / 8 1 f r o m C A I C Y T (Spain). References 1 H.R. Matthews and E.M. Bradbury, Cell cycle studies of histone acetylation and the structure

2 3

4 5 6 7 8 9

10 11

and function of chromatin, in: G.M. Padilla and K.S. McCarty (Eds.), Genetic Expression in the Cell Cycle, Academic Press, New York, 1982, p. 31. R. Reeves, Biochim. Biophys. Acta, 782 (1984) 343. K.S. McCarty, D.N. Kelner, K. Wilke and K.S. McCarty, Jr., Role of HMG-nucleosome complexes in eukaryotic gene activity, in: G.M. Padilla and K.S. McCarty (Eds.), Genetic Expression in the Cell Cycle, Academic Press, New York, 1982, p. 55. R.C. Wiegand and D.L. Brutlag, J. Biol. Chem., 256 (1981) 4578. R.L. Garcea and B.M. Alberts, J. Biol. Chem., 255 (1980) 11454. T. BShm, E. Schlaeger and R. Knippers, Eur. J. Biochem., 112 (1980) 353. D. Doenecke and D. Gallwitz, Mol. Cell. Biochem., 44 (1982) 113. M.L. Salvador, R. Sendra, G. L6pez-Rodas, V. Tordera and L. Franco, FEBS Lett., 191 (1985) 55. G. L6pez-Rodas, J.E. P6rez-Ortfn, V. Tordera, M.L. Salvador and L. Franco, Arch. Biochem. Biophys., 239 (1985) 184. M.A. Ull and L. Franco, Plant Mol. Biol., 7 (1986) 25. F. Estruch, J.E. P6rez-Ortfn and L. Franco, Cell. Mol. Biol., 32 (1986) 195.