the op biological vol. 256, no. 18, issue of september 25 ... · vol. 256, no. 18, issue of...

Vol. 256, No. 18, Issue of September 25. pp. 9612-9621,1981 THE JOURNAL OP BIOLOGICAL CHEMISTRY

Prinfedin U.S.A.

Manifold Effects of Sodium Butyrate on Nuclear Function SELECTIVE AND REVERSIBLE INHIBITION OF PHOSPHORYLATION OF HISTONES H1 AND H2A AND IMPAIRED METHYLATION OF LYSINE AND ARGININE RESIDUES IN NUCLEAR PROTEIN FRACTIONS*

(Received for publication, February 3, 1981)

Lidia C. Boffa, Rosemarie J. Gruss, and Vincent G. Allfrey From The Rockefeller University, New York, New York 10021

ID addition to its known effect in suppressing the deacetylation of the nucleosomal core histones, sodium butyrate in the concentration range 0.6 to 15 m~ causes a selective inhibition of [S2P]phosphate incorporation into histones H 1 and H2A of cultured HeLa 53 cells. No commensurate general inhibition of phosphorylation is seen in the non-histone nuclear proteins of butyrate- treated cells, but phosphorylation patterns are altered and S2P-uptake may be stimulated, as well as inhibited, depending upon the protein fraction analyzed. The degree of inhibition of histone phosphorylation in intact cells increases progressively as the butyrate concentration is raised from 0.5 to 15 m ~ . The effect is time- dependent and fully reversible.

Butyrate has no effect on the kinetics of phosphate release from previously phosphorylated histones of cultured cells, nor does it significantly alter the rate of dephosphorylation of s2P-labeled histone H1 by endog- enous phosphatases in vitro. Despite the suppression of [32P]phosphate incorporation into histones H 1 and H2A of butyrate-treated cells, Na-butyrate does not inhibit the in uitm activities of either type I or type 11 cyclic AMP-dependent protein kinases, or the cAMP-independent HI kinase associated with cell cycle progression. This suggests that the butyrate effect on histone phosphorylation in vivo is indirect and may involve an alteration in substrate accessibility or a modulation of systems affecting kinase activities.

The poly(ADP)-ribosylation of HeLa histones is not inhibited by 6 m~ Na-butyrate. Cells exposed to butyrate show an impaired methylation of lysine and arginine residues in their histones and nuclear hnRNP particles, respectively.

Cells cultured in the presence of millimolar amounts of sodium butyrate show a progressive increase in the levels of acetylation of the nucleosomal core histones (1-7). There are concomitant changes in chromatin morphology (8) and in patterns of transcription (8-20). The butyrate effect on histone acetylation is attributable to its direct inhibition of histone deacetylase activities (2, 3-6, 21,221 without a corresponding inhibition of acetyl group transfer from acetylcoenzyme A to the €-amino groups of histone lysine residues. As a consequence, the dynamic equilibrium between acetylation and deacetylation is shifted to favor accumulation of the multiacetylated forms of the core histones.

* This work was supported in part by Grant GM 17383 and Grant CA 14908 from the United States Public Health Service, National Institutes of HeaIth. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The positive charge neutralization associated with acetylation of the €-amino groups of the lysine residues clustered in the NHz-terminal regions of the histones weakens the electro- static interactions with the negatively charged phosphate groups of the enveloping DNA strand. This release of constraints would be expected to increase DNA accessibility and to potentiate its template functions (23-26). The changes in nucleosome structure in butyrate-treated cells are evident in a greater accessibility of core DNA sequences to DNase I attack (2,3,27-29), and in an increased availability of histone H3 to phosphorylation by a nuclear Ca2+-dependent protein kinase (30). It has also been observed that the 5”terminal phosphates at the ends of the core DNA are removed 2- to 3- fold faster during micrococcal nuclease digestions of hyperacetylated core particles than from core particles of control cells (3). The release of constraints on the DNA increases its template function in RNA synthesis, as judged by higher rates of RNA chain initiation and elongation in nucleosomes from butyrate-treated cells as compared to nucleosomes from un- treated cells (31).

Butyrate has also been shown to increase the levels of acetylation of the high mobility group proteins, HMG-14 and HMG-17 (32). To what extent are the chromatin structural changes and altered patterns of transcription of butyrate- treated cells a direct or exclusive consequence of histone or high mobility group protein hyperacetylation?, or does butyrate have other effects on the structure or metabolism of nuclear proteins which would also modify the phenotype and growth potential of the cell? To investigate these questions, we have analyzed the effects of butyrate treatment on four postsynthetic modifications of chromosomal proteins in HeLa cells: acetylation, phosphorylation, poly(ADP)-ribosylation, and methylation. We have also assessed the effects of Na butyrate on the synthesis of histones and other sets of non- histone nuclear proteins in relation to the inhibition of DNA replication in the same culture.

The results show that butyrate has a broad spectrum of kinetically overlapping effects; it not only blocks histone deacetylation, but appears to inhibit selectively the phospho rylation of histones H1 and H2A. Its effects on histone phosphorylation are concentration- and time-dependent, and they are reversible. Butyrate also influences the phosphorylation of non-histone nuclear proteins, but in a complex way; z.e. 32P- incorporation may be stimulated, unaffected, or inhibited, depending upon the protein analyzed. As butyrate concentrations are increased, the biosynthesis and methylation of histones and proteins associated with hnRNP’ particles are also inhibited. Some implications of these findings for studies of

* The abbreviations used are: hnRNP, 40 S ribonucleoprotein par-

PMSF, phenylmethyhulfonyl fluoride; SDS, sodium dodecyl sulfate. ticles containing newly synthesized heterogeneous nuclear RNA;

9612

Butyrate Inhibition of Histone Phosphorylation 9613

gene activation, enzyme induction, and cell cycle arrest in butyrate-treated cells are discussed.

EXPERIMENTAL PROCEDURES’

HeIa S3 cells were maintained in suspensim culture a t 2-5 ml by daily dilution with Jdrlik-nodified Eagle’s minimal

esspltial medim CQIfaining Wribiotics (CIBCO, Grand Island, N.Y.) a d supplerented with 5% fetaicalf s a .

&Mate Effects on p the stock HeIa culture (100 ml) were adjusted to butyrate coxentrations

- Aupuots of

‘=%Y hxmtcuem, ard dcm saplea were with&- for i s o t o p i c - ~ ~ P& tyrate solutim. After culture for 15 h. the cells vere m t e d in a

hglwd NrL. Inc., Bostcn. &I); 4 u c i of luethyl-3H thymtdtne of BIperiments using 10 u a / d of cwier-free 32P-orrhophosphate ( ~ e w

apedfic activity 20 per mole; 4 u a / m l of [me*l-&]ethtnine of specific activity 5-15 ci per mmle; or 4 sCi/d of a 3 H - t d ~ acid mLrmre of specific actidty 5.6 mci per ug. After lncuhtim for 1 h at 37’ C., the cell suspensicm were rapidly drilled in ice a d cmtrifuged

UM Na bisulfite, hameatpi by cmtrifugatim, and frozen at -80’ C. .prfor at 2.m x g for 10 ndn. Ihe cells were wash& with cold 140 mM NaC1- x)

to d v s i s .

fmnOtoMnHbytheeddit ionof t h e ~ c & a t e w l u r e s o f 1 M

of Wlei - HeLB cell nuclei were isolated by a nndificatim of the nrthod of Hwcock (34) wing Ne bisulfite to inhibit protein pbsphatase activity (35) and Na buryrate to W b i t deacetylase activity (21). Ihe wsshed, frozen cells were rhwed in 140 mM NaC1-50 mM Na bisulfi te0.5 mM Na Lutyrate-0.1 mM PPSF. e x r i f q e d , wd in 5 ml of Bo Id.I NaCl-

mM EDTA-50 mM Na bisulfite-0.5 mM Na b.qrate0.1 mM “1% M t m

with 20 stmkes of a Darvletype glass hnngenizer with a tight pestle X-100. pH 7.0 ( d m A). After 10 nbn, the cells uw brrken by shearing

(type B: htes, kc., V i r d a d , N . J . ) . Ihe inrcgenate was mntrifuped a t

A mima the detergent. 2 .000xgfor5mintope l l e t the~ le i .whichwere tknwas~fnmedim

prot wereextrac ect y Reparatim of klear prorein Ractim - Histones and otkr acid-soluble

N HC1. lb reaidd nrletrr pellet was ZQacted with 6 y Z - O . & S x 1 - 0 . 1 M Na phosphare buff?, pH 7.4 (buffer B)(36) wd cenaihged at 100,WO x for 15 h. Both the a u d - soluble and residual protem fractions m & pdfied by h-ertchwge dPn0atCgr-h~ m *Rex 70 (Bic-M,Inc. , M M , C 4 ) . Protein smqles were suspeded in buffer B CCmWniq 0.1% 2-mereaptoethwol and applied to 0.5 x 10 an mlum of Bl0-k 70, Mo-600 mesh, VhLch had been equilibrated with buffer B. tL7W.s- proteins w e eluted first in buffer B and the histones were subsqcmtly displaced in 4 M g u n ” H C l (36). Ihe eluates were dialyzed against water and lyophilized.

G e o r g i e v w d ~ ( 3 7 ) , b u t i n t h e ~ o f o . l m M ~ . A f t e r c e n t r i - WleaT IrmP particles were extracted essentially 86 described by

of hJINp particles SedImPn a t apprax. 4OS uas mllected for analysis. figaticn a t 23,oOO x g for 15 h in 15-3CfL 1- maose gradients, th? peak

p b s p b a t a e e a s s a y s . Itwasprqaredfrcm800dofaHeLecells

phosphate for 3 h. P, cells m e collected by cgltrifrgatm wd rnrlei pqmredasdeaaibed. Afteruas~in140mMNaClamtainirg50mMNa bisulfite end 0.5 mM Na butyrate. the -lei -e -acted mice with 1 ml portions of 5% perchloric acid. Ihe 5% HClQ exuacts were ccmbitd, CM- fied. a& adjusted to 0 .2 H in WW, before precipitation of the proteins in 10 ml of acetone (38). Ihe precipitate was dissolved fn 0.1 M Na mfer, pH 7.0-% Mer c) and appm to a 1 x 1€2%% of Rlo-Rex 70 been equilibrated in buffet C. After was- the alum with 3 wl of =fer C, histme Hl was eluted with 0.1 M Na ptcqhate

utcm Hl labem wi th%-pmte was wed as a substrate for protein

CQlfaintng 3 x ld cells/ml xhi& had been inarbated with 25 mcimF-

Portions of this paper (including “Experimental Procedures” and Tables I to V) are presented in miniprint as prepared by the authors. Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are available from the Journal of Biological Chemistry, 9650 RockviUe Pike, Bethesda, MD 20014. Request Doc- ument No. 81M-254, cite author(s), and include a check or money order for $3.60 per set of photocopies. Full size photocopies are also included in the microfilm edition of the Journal that is available from Waverly Press.

Electrwbreric Anslysis of 32P-Labeled NuElw Proteins - iiista-es and nmbistone nuclear proteins, sepsra w a l y d (11 SlE-polyaaylaucide g e l s ? a ? a z a l r L ’ E a i t d by laemnli (411, but uskg 9-15% aayhdd? adients and stairhg with Ccrmassie Brilliant Blue R2M. Histme sub!?&ctims vhich differed in degree of acetylation were separated m long acetic acid-urea-plyaaylmide gels

0- o n a u w @ v . =e

(42.43) wd grained with 0.1% Addo BLsck 1OB in 25% me-l-% acetic

Electrwbreric Anslysis of 32P-Labeled NuElw Proteins - iiista-es and nmbistone nuclear proteins, sepsra w a l y d (11 SlE-polyaaylaucide g e l s ? a ? a z a l r L ’ E a i t d by laemnli (411, but uskg 9-15% aayhdd? adients and stairhg with Ccrmassie Brilliant Blue R2M. Histme sub!?&ctims vhich differed in degree of acetylation were separated m long acetic acid-urea-plyaaylmide gels

0- o n a u w @ v . =e

(42.43) wd grained with 0.1% Addo BLsck 1OB in 25% me-l-% acetic

9614 Butyrate Inhibition of Histone Phosphorylation phosphate OJor-t~n) as substrate Ik effects of inmeas butyrate c a r c e n t r a m m a- ph0sphatar;e were also tested -2fy using ~ e ~ e tmmgmates or calf intestinal alkaline phosphatases. NO kriribid~n w18 &served at butyrate mncenmaticns between 0.5 and 40 uM. DNA w t - lhe RU mtmt of representatiw a U p m of the cell

m~dification of the made ree~& (57). C U l ~ o r ~ i ~ o f ~ O l a t e d ~ ~ ~ ~ b y ~ ~

RESULTS

Selective Inhibition of Histone Phosphorylation by Nu Butyrate-The effects of butyrate on the incorporation of [3zP]phosphate into the histones and non-histone nuclear proteins of HeLa cells was first tested in cultures which had been exposed to 5 lll~ Na-butyrate for 15 h, a concentration known to substantially increase the levels of acetylation of the nucleosomal core histones (1-7). After a 1-h ''pulse'' with ["PI phosphate, nuclei were isolated and the histones were extracted in acid and purified by ion exchange chromatography. Equal amounts of histone from butyrate-treated and control cells were analyzed by electrophoresis in SDS-polyacrylamide gels. It was found that the proportions of the various histone classes, as determined by quantitative densitometry of the stained gels, were not altered as a consequence of butyrate treatment (Table I). The 3ZP-labeled histone bands were lo- cated by autoradiography and their relative 32P-activities were determined by quantitative densitometry of the autoradi- ograms. Comparisons of the 32P-spe~ific activities of the histones from butyrate-treated and control cells are shoyn in Fig. U. It is clear that the incorporation of [32P]phosphate into histones H1 and H2A is virtually eliminated in HeLa cells cultured in 5 lll~ Na-butyrate. A similar inhibition of C3'P] phosphate incorporation into histone H1 (but not histone H2A) has been reported for butyrate-treated Chinese hamster ovary cells (7).

Comparisons of [3zP]phosphate uptake into the nuclear non-histone proteins of control and butyrate-treated HeLa cells show a complex response, varying from stimulation to inhibition, depending upon the proteins analyzed. Some proteins, e.g. an acid-soluble protein of molecular weight 11O,OOO, are more highly phosphorylated after butyrate treatment, doubling the 32P-activity seen in control cultures, without any change in the amount of protein present (Fig. 1A). Other non- histone nuclear proteins, e.g. a band of molecular weight 92,000, seem unaffected by 5 m~ Na-butyrate, while the phosphorylation of proteins at 55,000 and 39,000 daltons is definitely inhibited (Fig. 1B). (Equal weights of proteins from control and butyrate-treated cells were compared in these experiments, and it was established that the relative amounts

TABLE I

contrasa Effecta of Na Butyrate on the Ftodwrylatim of Histaw

and a 110 Kllodelton kclear Rotein of S3 Cells

Cells e culhrred in 5 mM butyrate for the indicated rimes d 'pulsed" with 3fpnosphate for 1 h Ik add-aoluble k proteins were separated elecPophcaeHcally wd & pmportiars and%-acHviHes de- BB described in Eqeiuental RccehYs.

Protein fraction analvzed

horns 0 16.3$.8 100. 2 5 . e . 9 loo. 3 . e . 5 100.

6 18.65.7 51.013 2 5 . w . 7 5 1 . t 2 3.3-10.4 139.% 3 18.s+o.5 87.e2 25 .15 .6 60.012 3 . 2 5 . 5 1 1 8 . t 5

11 18 .15 .7 7 . t 2 25 .75 .9 l3.33 3 . 1 s . 6 201.016 9 18.3-10.7 41.013 25.3-10.8 3 8 . e 2 3 . e . 5 1 7 1 . w

15 1 8 . w . 7 2 . e 2 25.7-Lo.7 9 . e 2 3.3-10.8 214.013

a . "sed as 7. of total absorberre in the demitaredc tradng of t k

BI CONTROL

MW X 103

FIG. 1. Effects of Na-butyrate (6 m ~ ; 15 h) on phosphorylation of histones and non-histone nuclear proteins 0 of HeLa 53 cells. A, comparison of the =P-activities of histones H1, H2A, and other acid-soluble nuclear proteins of control and butyrate- treated cells, as determined by quantitative autoradiography of acid- urea-polyacrylamide gels. Note that =P-uptake into histones H1 and H2A is suppressed while the phosphorylation of a protein of M, 110,OOO (arrows) is stimulated by butyrate. B, comparison of the =P- activities of the non-histone proteins of control and butyrate-treated cells after separations on SDS-polyacrylamide gels. The vertical, dashed lines indicate the positions of protein bands at 92,000, 55,000, and 39,OOO daltons referred to in the text.

TABLE I1

V ~ I & a F w ? e S o f D i f f e . r € l l t t & l f ! a r ~ ~ t o

Incseasim Ccwxnfzathm of Na attyrate

&.la cells ad- in the preaare of the indicated a n c e n t r a m o f N a b r t y r a t e f o r 1 5 h ~ p l l s e - ~ w i e h 3 2 P - p h o s p h a t e f c a l h . "e a t m e nuclear protsins m separated e l e c ~ r i c a l l y d W%-acHdties de- as described in -tal -.

92 KD Protein fract ion

55 m 39 m btyrate

raM 0

0.5 27.75.5 100. 16 .79 .5 100. 2 . 7 5 . 4 100.

2.0 28 .15 .4 9w 17 .15 .5 95 .25 2 .810 .3 78 .w

5.0 2 7 . 9 . 5 95t6 1 6 . w . 4 7 7 . 7 5 2 . w . 4 5 8 . e 2 7 . w . 5 low 1 7 . w . 5 7 6 . e 2 . e . 4 5 6 . ~

10.0 15.0

28.010.3 lo? 1 7 . w . 3 5O.w 2 . 1 5 . 4 46.e 2 7 . w . 4 1% l 6 . w . 5 19.014 2 . v . 4 1 9 . e

m u a * -P lhant AcHvlty praant kavity W i C spedfic

a. &pressed as X of total abaorbwce in the densitaredc ts- of the

b. Rpre.ss~~7. of tk specific 32P-acUvity obecrved in ttw stained 1 + S.D.

pmlm of omml cells S.D.

of the proteins at 92,000, 55,000, and 39,000 daltons were unchanged (Table II).) In no case did the inhibition of ["PI phosphate incorporation into the non-histone proteins equal that observed for histones H1 and H2A in cells exposed to 5 m~ butyrate for 15 h.

Concentration Dependence of Butyrate Effects on Phos- phoryzation-Parallel cultures of HeLa S3 cells were incubated for 15 h in media containing increasing amounts of Na- butyrate, and each culture was then pulse-labeled with ["PI phosphate for 1 h. Comparisons of the specific 32P-activities of the electrophoretically purified histones from control and butyrate-treated cells showed that the phosphorylation of histones H1 and H2A was suppressed by 80 to 90% at butyrate concentrations as low as 2 nm. A slight further decline in %P- uptake occurred at Concentrations between 2.5 and 15 m ~ . Under these conditions, the phosphorylation of histone H1 was more inhibited than that of histone H2A, which persisted


at about 10% of the control level even in 15 m~ butyrate (data not shown).

The effects of butyrate on the phosphorylation of the non- histone nuclear proteins varied with concentration as shown in Fig. 2. JAW concentrations, e.g. 2 m ~ , had little effect on the phosphorylation of these proteins as compared to the extensive inhibition of histone phosphorylation under the

I

I I [ I I I I 110 80 68 55 45 25 18.6

MW x IO3 FIG. 2. Effects of increasing butyrate concentrations on the

phosphorylation of HeLa nuclear non-histone proteins. The uppermost (dashed) curve shows the densitometric tracing of the stained protein bands after electrophoresis in SDS-polyacrylamide gelq this pattern is not detectably different in the butyrate-treated cells. The lower curves show the corresponding "P-distributions in the nuclear proteins from control cells and from cells grown at the indicated Concentrations of Na-butyrate. Quantitation of the results for proteins a t 92,000,55,000, and 39,000 daltons is presented in Table 11.

same conditions. Butyrate concentrations in excess of 10 m~ did lead to a general suppression of [32P]phosphate uptake, but the phosphorylation of particular protein bands, e.g. at 92,000 daltons, remained unaffected (Fig. 2). The densitometric analysis of the protein banding patterns showed little if any change in the relative proportions of the non-histone proteins as a consequence of butyrate treatment (Table 11).

Time Dependence of Butyrate Effects-The kinetics of inhibition of histone H1 and H2A phosphorylation were stud- ied in HeLa cells cultured in 5 m~ Na butyrate for periods ranging from 0 to 15 h. Aliquots of the cell suspension were withdrawn at the indicated times and "pulsed" with ["PI phosphate for 1 h before isolation and analysis of the histones. Fig. 3 compares the specific activities of histones H1 and H2A at each time point, and reveals two interesting differences in the effects of butyrate upon their phosphorylation. First, the inhibition of [32P]phosphate incorporation at early times is much greater for histone H2A than for H1, but at later times H1 phosphorylation is more inhibited. A second distinction is evident in the shapes of the curves; the suppression of H1 phosphorylation is almost linear with time of exposure to 5 m~ Na-butyrate, but the decline in H2A phosphorylation is nonlinear, with a more resistant component of phosphorylation persisting at later times. This difference is in accord with the results showing that H2A phosphorylation cannot be suppressed completely, even at butyrate concentrations as high as 15 m.

Fig. 3 also depicts the time course of accumulation of the acetylated forms of histone H4, expressed here as the declining percentage of H4 molecules remaining in the nonacetylated form. Note that the inhibitory effects of sodium butyrate on H2A phosphorylation are at least as rapid as the consequences of its inhibition of histone deacetylase activities.

Coordinate studies of C3H]thymidine incorporation into

- 60

- 5 0 0 r w z

- 4 0 fl 52 r

-30 f a J

-* -20 a z =I

-10 z IO ''>T.

'..>& r , , , , , , , , , , , , , t 2 4 6 8 IO 12 14

TI ME (hours)

FIG. 3. Time dependency of butyrate inhibition of histone H1 and H2A phosphorylation, deacetylation of histone H4, and ['HJthymidine incorporation into DNA. Parallel cultures of HeLa 53 cells were incubated in the presence of 5 m~ Na butyrate for the indicated periods and then pulsed with [32P]phosphate or [3H]thymidine for 1 h. The histones were electrophoretically separated as in Fig. 1. The 32P-activities of histone H1 (A) and H2A (A) are expressed relative to the corresponding activities of those histones in cells incubated in butyrate-free medium. The progressive accumulation of the multiacetylated forms of histone H4 is indicated by the declining proportion of H4 molecules in the nonacetylated form (0). [3H] Thymidine uptake into the DNA of butyrate-treated cells is expressed relative to that observed in butyrate-free medium at the Same time points (0). Note that the phosphorylation of H2A is more rapidly inhibited than that of H1, but eventually persists at a higher level. Vertical bars at each time point indicate the standard deviation from the mean.

9616 Butyrate Inhibition of Histone Phosphorylation

DNA at successive times during exposure of HeLa cells to 5 m~ Na-butyrate show very little effect at 3 h and a rapid decline thereafter (Fig. 3). From 6 h on, the decline in DNA synthetic capacity is roughly parallel to the decreasing rate of histone HI phosphorylation.

Changes in the patterns of phosphorylation of the nuclear non-histone proteins have also been observed in HeLa cells at different times during subculture in 5 m~ butyrate. These will not be reported in any detail, except to note a remarkable contrast in the response of an acid-soluble protein at 110,OOO daltons which continues to increase its incorporation of [”PI phosphate while the phosphorylation of the histones declines progressively (Table I).

Reversibility of Butyrate Inhibition of Histone Phospho- rylation-In tests for reversibility of the butyrate effect, cells cultured in 5 m~ Na-butyrate for 15 h were transferred to an equal volume of butyrate-free medium. Aliquots were then withdrawn at intervals between 0 and 24 h and pulsed with [32P]phosphate before isolation and analysis of the histones. The specific 32P-activities of histones H1 and HZA, after a slight lag at the outset, increased linearly with time and reached control levels within 10 h. No further change in rates of histone phosphorylation was observed up to 24 h (data not shown).

Absence of a Butyrate Effect on Histone Dephosphoryla- tion-The previous assessments of butyrate effects on phosphorylation have all been based on measurements of [32P] phosphate incorporation during a 1-h pulse. In order to deter- mine whether the diminished [32P]phosphate content of the histones in butyrate-treated cells is entirely due to a suppression of phosphate uptake, or whether is might be due in part to a stimulation of [32P]phosphate release from the histones,

looh

comparisons were made of the rates of dephosphorylation of histones H1 and H2A in control and butyrate-treated cells. The histones were labeled with [32P]phosphate under normal growth conditions and the cells were then transferred to a nonradioactive medium in the presence or absence of 5 m~ Na-butyrate. A plot of histone specific activities during the “cold chase” shows that there is no appreciable difference in the rates of dephosphorylation of either histone whether butyrate had been present or not (Fig. 4). The more rapid turnover of phosphate groups on histone H2A, as compared with histone H1, is in accord with previous observations on HeLa cells (58).

The absence of a significant difference in rates of histone dephosphorylation in butyrate-treated and control cells was also confirmed in comparisons of histone H1 phosphatase activities of extracts of control cells and of cells which had been cultured in 5 m~ Na-butyrate for 15 h. The assay was carried out in two ways; by measuring retention of [32P]phosphate activity during enzymatic treatment of a 32P-labeled H1 substrate, and by monitoring the release of inorganic [32P] phosphate into the medium. The results summarized in Fig. 5 show that the histone phosphatase activity of the butyrate- treated cell extracts is only slightly higher than that observed in the controls. The difference is not likely to account for the large differences in 32P-a~tivity of the histones after short term pulse-labeling experiments (Fig. 3).

Extracts of control and butyrate-treated HeLa cells were also examined for alkaline phosphatase activity following in- cubation for 15 h in media containing 0 to 50 m~ Na-butyrate. No s i m k a n t differences in alkaline phosphatase activity were observed (data not shown). This result is in accord with earlier studies showing that the induction of alkaline phos-

1 2 3 4 5 6

TIME (hours)

N R

a

8 z

10 20 30 40 50 60

FIG. 4 (left). Comparisons of [32P]phosphate ‘‘turnover” in histones H1 and H2A of HeLa cells subcultured in the presence or absence of 6 m~ Na-butyrate. The histones were labeled with [32P]phosphate under normal growth conditions, and equal numbers of cells were then transferred to nonradioactive media containing 0 or 5 m~ Na-butyrate. Aliquots of each cell suspension were withdrawn at the indicated times for analysis of the residual 32P-activities of histones H1 and H2A. The speciftc activity is plotted versus time for the H1 of control (0) and butyrate-treated (0) cells, and for histone H2A of control (A) and butyrate-treated (A) cells. Note that butyrate does not affect the rate of release of [32P]phosphate release from either histone. Each point represents the average of two experiments. Standard deviations ranged from 1 to 3% of the mean.

FIG. 5 (right). Comparisons of the histone phosphatase activities of control and butyrate-treated HeLa cells. Extracts of cells cultured for 15 h in the presence and absence of 5 mM Na butyrate were tested for their capacity to dephosphorylate 32P-labeled histone HZ. The assay was carried out in two ways: by measuring the retention of [3ZP]phosphate by the H1 substrate at successive times (upper curues; control (O), butyrate-treated (O)), and by monitoring the release of inorganic [32P]phosphate into the medium (lower curues; control (A), butyrate-treated (A)). The H1 phosphatase activities of the preparations agree within about 10%. Each point represents the average of three experiments, with standard deviation ranging from 1 to 3% of the mean.


phatase by butyrate requires 24 to 36 h and is not seen earlier (9). Finally, the direct addition of 0.5 to 50 m sodium butyrate to the phosphatase assay system had no effect on the activity of an alkaline phosphatase prepared from calf intestinal mucosa (data not shown). Thus, there is no evidence that the low ["Plphosphate of the histones in butyrate-treated cells is due to butyrate enhancement of phosphatase activities or accelerated rates of release of [3zP]phosphate from H1 or H2A.

Tests for Butyrate Effects on Protein Kinase Actiuities- Given the inhibitory effects of Na-butyrate on 32P-incorporation into the histones of intact HeLa cells, we tested to see whether butyrate can inhibit directly the activities of kinases known to be involved in the phosphorylation of histone HI. A chromatin-bound kinase with high specificity for histone H1 has been closely linked to cell cycle progression (59-64). This growth-associated kinase was prepared from control and butyrate-treated HeLa cells essentially as described by Langan (50), and each preparation was tested for its ability to phos- phorylate histone H1 in uitro. No difference in activity was noted between the enzymes from butyrate-treated and un- treated cells, nor was either preparation inhibited by the addition of 5 m Na-butyrate to the assay system (Table 111).

Although most of the phosphorylations of histone H1 in growing cells are mediated by cyclic AMP-independent kinases (64-66), the serine residue at position 38 of histone HI is known to be phosphorylated by a cyclic AMP-dependent protein kinase (67, 68). For this reason, we tested the effects of Na butyrate on type I and type 11 CAMP-dependent protein kinases (from calf heart muscle), using purified calf histone H1 as the substrate. Neither of the kinases was inhibited by butyrate; on the contrary, both enzymes were slightly stimulated by the addition of Na butyrate to the assay system. As shown in Table 111, type I activity was enhanced by about 20% in 5 m and 30 m butyrate, while type I1 activity was 14% higher in 5 ~ l l ~ and 24% higher in 15 and 30 m butyrate than in butyrate-free buffer.

Thus, the observed suppression of [32PJphosphate incorporation into histones H1 and H2A of butyrate-treated cells does not appear to be due to a direct inhibition by butyrate of either type I or type I1 cyclic AMP-dependent kinases or the CAMP-independent growth-associated kinase. While it is pos- sible that other kinases involved in histone phosphorylations are inhibited by butyrate, the negative evidence strongly

TABLE 111

15 1.8 30 1.8 50 1.7

0 5

1.9 2.0

30 15 2.3

50 2.3 2.1 "

TABLE IV

Testa for -ate Effect6 m AtS"Rib0nylation of HeLa Eistones

cells m e -14 with %-ademsine or ZP- hosphate in t k presence

w t m e fractFon d the %:activities of electrophoretidly-+ied ~d absence of 5 m~ butyr e spec~fic #-activities of the total

@waptdieaterase as described in -a1 Roc&wres. HldH2AwereQteminedbeforewdaftertreatnmtwithsnakevenrm

Conditions of Precursor Histone H1 Histone H2A Total Hist. Experiment Used -h. +Ez. -Ru. +Fhz. -h. +Bz.

Specific Activitya

conhol 5 nM butyrate control 5 nM butyrate

3%-ptosphete 100. 92.011.6 1W. 88.&1.2 - - 3H"ine - " - 27.9c.2 0

" - 25.15.2 0

2.5-11 2 . E l . 1 9 . 2 1 8.E1.2 - -

a. 3- P-activities expressed an % of the ~peciflc activity of the ademsine incorporadon expressed as qxn!yg total hlstore.

correspading histone of mntrol cells +- S.D.

suggests that the butyrate effect on histone phosphorylation is indirect and may involve changes in substrate accessibility or altered equilibria between active and inactive forms of histone-specific kinases.

Lack of Butyrate Effect on Histone ADP-ribosylation- Parallel cultures of HeLa cells were labeled with C3H]adenosine (33) or C3'P]phosphate in the presence and absence of 5 m Na-butyrate, as described under "Experimental Proce- dures." The nature of the histone modification was investi- gated using phosphatase-free snake venom phosphodiesterase (44) as a probe for ADP-ribosylation. It was found that all of the C3H]adenosine incorporated into the acid-soluble protein fraction was released by phosphodiesterase treatment and presumably represents ADP-ribosylation. The uptake of C3H] adenosine into the nuclear proteins of butyrate-treated cells was only slightly lower than that observed in the control cultures (Table IV).

Studies of [32P]phosphate uptake and enzymatic release under the same conditions showed that butyrate effectively lowered 32P-incorporation, as expected, and that little of the 32P-a~tivity of the histones could be attributed to ADP-ribosylation, since phosphodiesterase treatment reduced H1 specific activity by only 8 to 9% and H2A activity by only 12 to 14% (Table IV).

Butyrate Efiects on DNA Synthesis and Cell Prolifera- tion-DNA synthesis is suppressed in HeLa cells exposed to 5 mM Na-butyrate (17,69) (Fig. 3). An analysis of the effects of increasing concentrations of Na-butyrate on r3H]thymidine incorporation into DNA shows that DNA synthesis is almost completely inhibited at butyrate concentrations higher than 2.5 m (Fig. 6, left panel). As a consequence of butyrate treatment, most cells are arrested in cell cycle progression and accumulate at the G1/S boundary (17, 69). This accounts for the lower total DNA content of the HeLa cultures maintained in 2.5 to 25 lll~ Na-butyrate (Fig. 6, leftpanel). Also, the total number of cells in butyrate-treated cultures at 15 h is consid- erably less than that observed in control cultures in which cell cycle progression is not inhibited (Fig. 6, right panel). The reduction in cell number in butyrate-treated cultures relative to that in control cultures is a function of time and butyrate concentration (Fig. 6, right panel). This must be taken into account in interpreting changes in other biosynthetic activities during butyrate treatment. Our approach to this problem has generally been to compare equal numbers of cells rather than equal volumes of control and butyrate-treated cultures, and to analyze equal amounts of histones or other goteins for [32P]phosphate incorporation or %-labeled amino acid activities.

Butyrate Effects on Protein Synthesis-HeLa cells subcultured in 5 m Na-butyrate show a progressive decline in their rates of amino acid incorporation into total cellular proteins.

9618 Butyrate Inhibition of Histone Phosphorylation

I - 5 p I

- 4 2 I t '"". L3.5 d -1

-15.5

-4.5

I V

- " . A 3 5 IO 15 20

m M BUTYRATE

FIG. 6. Effects of increasing butyrate concentrations on [$HI thymidine incorporation into DNA, DNA content, and cell number in HeLa 53 cultures. Parallel cultures were incubated for 15 h in the presence of the indicated concentrations of Na butyrate and pulsed with [3H]thymidine for 1 h. The specific activity of the DNA is plotted as a function of the butyrate concentration in the left panel (0- - -0). The corresponding DNA content of the butyrate- treated cultures, relative to that observed in butyrate-free controls is also plotted in the left panel (A-A). The right panel compares the cell concentrations after 15 h of culture at the indicated butyrate concentrations (W).

After 15 h, the uptake of [methyl-3H]methionine or a mixture of 3H-labeled amino acids is reduced by about 60% (data not shown). Studies of the concentration dependence of this effect indicate a reduction of about 40% at low butyrate concentrations (1 to 2 m ~ ) . The inhibition plateaus at about 60% at butyrate concentrations higher than 30 m ~ . This represents the protein synthesis capacity of the HeLa cell population under conditions in which DNA synthesis is almost completely blocked (Fig. 3).

Are there differences in the effects of butyrate on the synthesis of histones as compared to other proteins of the nucleus? This question was examined in cells which were pulse-labeled with [methyb3H]methionine at successive times during subculture in 5 m~ Na-butyrate. The results summarized in Fig. 7A show clear differences between the response of the histones and the non-histone proteins associated with the 40 S hnRNP particles; e.g. histone synthesis is progressively inhibited after the 1st h, whereas the uptake of C3H] methionine into the proteins associated with nascent hnRNA chains is accelerated at first and then declines to about 70% of that seen in control cultures (Fig. 7A). This suggests that the synthesis of proteins involved in the processing and transport of nascent RNA molecules continues at a high level in the GI- arrested cell population. It is also of interest that histone synthesis in the butyrate-treated cells persists at a much higher rate than does DNA synthesis under the same conditions (compare Fig. 7A with Fig. 3). As butyrate concentrations are increased, the capacity for nuclear protein synthesis continues to decline, but even in 15 m~ butyrate the specific activity of the histones is about 40% of that observed in control cultures (Fig. 7B).

Butyrate Effects on Nuclear Protein Methylation-When HeLa cells are subcultured in 50 m~ Na butyrate to favor accumulation of the hyperacetylated forms of the histones, the methylation of the histones and of non-histone nuclear proteins is inhibited. This effect was examined in cells pulsed with [methyl-3H]methionine after 15-h exposure to butyrate. The histones and non-histone protein fractions were extracted and purified by chromatography on Bio-Rex 70. The 40 S hnRNP particles were also prepared, and each fraction was hydrolyzed in acid to release the methylated derivatives of arginine and lysine. The specific activities of E-N-methyllysine,

NG-monomethylarginine, and NG,NG-dimethylarginine of each fraction from control and butyrate-treated cells are compared in Table V. It is clear that methylation of the histone lysine residues is strongly inhibited, as is the formation of mono- and dimethylarginines in the non-histone proteins. The highest specific activity of NG,NG-dimethylarginine is observed in proteins associated with W N P particles, in agree- ment with earlier observations on their extensive modification in this way (48, 70).

The differences in [3H]methyl incorporation into the proteins of control and butyrate-treated cells is not attributable to butyrate inhibition of the formation of S-adenosyl[methyG 3H]methionine. Analysis of the S-adenosylmethionine "pools" of normal cells incubated with [n~ethyl-~H]methionine showed a specific activity of 9,730 cpm/pnol, while the corresponding activity in the butyrate-treated cells was 11,640 cpm/pmol.

n I

IL 0

z t 5 IO I5 IO 20 30 40

TIME (hours) mM BUTYRATE

FIG. 7. Differential effects of Na-butyrate on amino acid incorporation into the histones, hnRNP particle proteins, and residual non-histone proteins of HeLa nuclei. A, cells were cultured in 5 m~ Na-butyrate for the indicated times and pulsed with [methyL3H]methionine for 1 h before preparation of the histones (A), residual non-histone nuclear p r o t e k (O), and proteins associated with 40 S hnRNP particles (0). Note that [3H]methionine incorporation into the histones is not inhibited as effectively as is [3H] thymidine uptake into DNA (Fig. 6), and that labeling of hnRNP particles is accelerated at early times and is only slightly inhibited thereafter. B, cells were cultured for 15 h in the presence of the indicated concentrations of Na butyrate and then pulsed with ['HI methionine for 1 h before preparation of the nuclear protein fractions. Note that labeling of the histones (A) is not completely suppressed even in 40 m~ butyrate, and that hnRNP protein synthesis remains relatively resistant until butyrate concentrations exceed 20 m ~ . The activities of each fraction are expressed relative to the corresponding 3H-activities of proteins from cells incubated in butyrate-free media. Each point represents the average of three esperiments with standard deviations of 4% or less from the mean.

procek, Fraction -t l y 8 h arginine Histams cartrol 8.120.

5 nN butyrate 3,020.

5mMburyrare 60. 220. 940.

5 butyrate 2.680. 80. €03.

FyPprofeinS crntrol 120. 440. 2,080.

Residusl protein Gmtrol 3,960. m. 1,420.

a. Specific activity expeessed an cpm per mule.


DISCUSSION

The experiments described show a broad spectrum of re- sponses of cultured HeLa cells to butyrate treatment. These include a rapid and reversible inhibition of phosphorylation of histones HI and H2A, accumulation of the hyperacetylated forms of the nucleosomal core histone H4, inhibitions of DNA synthesis and cell proliferation, and an overall reduction in the rates of nuclear protein synthesis and methylation. There is no appreciable effect of butyrate on histone ADP-ribosylation.

The effects of butyrate on nuclear protein phosphorylation, unlike those on acetylation, are not associated with a change in the rate of removal of the mocllfying group. The kinetics of [32P]phosphate release during a “cold chase” are not altered by butyrate treatment, nor is there any indication that cells grown in 5 m~ butyrate for 15 h have abnormally high levels of histone phosphatase or alkaline phosphatase activities. As judged by pulse-labeling experiments with C3’P]phosphate, the phosphorylation of histones H1 and H2A is suppressed as long as butyrate is present and restored when butyrate is removed. Presumably, this is due to an inhibition of histone kinase activities, or to an altered accessibility of the histone substrates, or to a combination of both factors. Since the specific =€”activities of some nuclear non-histone proteins are much higher in butyrate-treated cells than in the controls, the low =P-activities of the histones cannot be attributed to an inhibition of raP]ATP synthesis during butyrate treatment. The reason for the butyrate inhibition of [32P]phosphate incorporation into histones H1 and H2A remains to be clari- fied. In view of the varied possibilities for modulation of protein kinase activities (71-74), the effects of butyrate may be due to altered states of activation of the enzymes. In any case, not all kinase reactions in the HeLa nucleus are inhibited in the presence of butyrate. Phosphorylation of a 110,000- dalton phosphoprotein increases as butyrate concentration is raised. (The identity of this HeLa nuclear protein with a DNA-binding protein of the same molecular weight found in a variety of tumor tissues (75) is under investigation.) Other nuclear phosphoproteins, e.g. a protein band at 55,000 daltons which has an exceptionally high rate of phosphate turnover during the cell cycle of synchronized HeLa cells (76), show reduced 32P-uptakes after butyrate treatment. Phosphopro- teins of this size class include a polypeptide which stimulates transcription until it is phosphorylated (77), and a protein (M, 55,000, PI 6.5) which is selectively phosphorylated at very early stages in chemical carcinogenesis of the colonic epithe- lium?

The consequences of impaired phosphorylation of histones H1 and H2A deserve comment. Certain phosphorylations of histone H1 have been linked to enzyme induction mecha- nisms. Phosphorylation of serine 37 in rat liver H1 is stimulated by cyclic AMP and by peptide hormones, such as glu- cagon, that activate adenylate cyclase (67,78-81). The CAMP- mediated modification of histone H1 is observed as an early event in hormonal induction of enzymes in the liver (81-84) and in gangllal cells responding to the nerve growth factor (85). It is not yet known whether the suppression of histone H1 phosphorylation in butyrate-treated cells affects the phosphorylation of serine 37 (or serine 38), but in view of the almost total inhibition of =P-uptake, this seems likely. If this site-specific modification of H1 must precede some types of gene activation, its inhibition might account for phenomena such as the butyrate suppression of the induction of glycerol

L. C. Boffa, R. J. Gruss, and V. G. AUfrey, manuscript in preparation.

phosphate dehydrogenase in glial cells treated with glucocorticoids (86), or the inhibition by butyrate of hormonal induction of the ovalbumin and transferrin genes in the chick oviduct (87). The effects of butyrate on the phosphorylation of histone H2A may also be relevant to gene activation by glucocorticoids, because dexamethasone has been shown to stimulate H2A phosphorylation (88). We have observed that butyrate suppression of H2A phosphorylation is a rapid and reversible phenomenon, as is the butyrate block of the hormonal induction of ovalbumin mRNA in the oviduct (87).

The impairment of histone phosphorylation may also be associated with the massive changes in chromatin structure and increased nuclease sensitivity of the DNA of butyrate- treated cells (2,3,8,27-29,89). Correlations have been noted between high levels of H2A phosphorylation and heterochro- matin contents of Peromyscus cell lines (W), and a clumping of chromatin is associated with an increase in H2A phosphorylation in dimethyl sulfoxide-treated erythroleukemia cells (91). If this is generally the case, the inhibition of H2A phosphorylation by butyrate would favor dispersal of hetero- chromatic clumps, which has been observed (8). A similar argument applies for H1 phosphorylation, which has been linked repeatedly to the formation of higher orders of chromatin structure, as well as to chromatin compaction during mitosis (35, 58-62, 92-94). In the case of histone H1, the problem is complicated by the existence of primary sequence variants with multiple sites of phosphorylation which differ in their degree of modification throughout the cell cycle (50,58, 59,62,65,79,91,95). Superphosphorylation events, mediated by growth-associated kinases (50,59,95,96) seem to correlate with chromatin compaction during mitosis, while the subse- quent dispersal of chromatin in early GI is associated with a dephosphorylation of histone H1 (58, 59, 62, 91). Conse- quently, the inhibition of growth-associated phosphorylations by butyrate would also be expected to favor dispersal of the chromatin, this remains to be established as a causational effect. An alternative explanation is that butyrate, by blocking cell cycle progression at the Gl/S boundary (7, 17), prevents the cells from reaching mitosis and thus diminishes the number of multiple phosphorylation events associated with chromatin compaction. The rate of decline of H1 phosphorylation in HeLa cells placed in 5 m~ Na-butyrate (Fig. 3) is not inconsistent with this view.

There is an extensive literature relating H1 phosphorylation to cell cycle progression (25, 58-65, 91, 95, 96). A kinase- mediated advance of mitosis in Physarum suggests a causational role of H1 phosphorylation in chromatin compaction during the Gz to M transition (61, 97), and there is evidence that H1 phosphorylation at the GI/S boundary is required (but not sufficient) for initiation of the S-phase (98). Butyrate inhibition of H1 phosphorylation at this stage could be a factor in the observed GI-arrest of HeLa cells (17) and Chinese hamster ovary cells (7).

Finally, the consequences of butyrate treatment are far more complex than an inhibition of histone deacetylase activities would suggest (2,4-6,21,22). They include alterations in the phosphorylation and methylation of histones and other nuclear proteins, arrest of the cell cycle, and changes in chromatin morphology and function. Therefore, it is prema- ture to conclude that histone hyperacetylation, per se, is the major triggering mechanism responsible for the alterations in phenotype and G1-anest of butyrate-treated cells.

Acknowledgments-We are greatly indebted to Dr. Francois Go- deau for the gi f t of the ADP-ribosylated histone H1 and the phosphatase-free phosphodiesterase, and to Eben Weitzman for perform- ance of the phosphatase assays reported herein.

9620 Butyrate Inhibition of

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