Oct 25, 2015 - Distribution of histone acetyltransferase activ- ity among salt- and size-resolved micrococcal nuclease produced fractions was almost 5-fold ...
Vol. 263,No , 30, Issue of October 25,PP. 15643-15651,1988 Printed in U.S. A.
OF BIOLOGICAL CHEMISTRY THEJOURNAL 0 1988 by The American Society for Biochemistry and Molecular Biology, Inc
Histone H3 Thiol Reactivity and Acetyltransferases Chicken in Erythrocyte Nuclei* (Received for publication, December 4, 1987)
Shirley Chan, Liliana Attisano, and Peter N. Lewis$ From the Department of Biochemistry, University of Toronto, Toronto, OntarioM5S lA8, Canada
Chicken erythrocytes have provided an excellent model for Chicken erythrocyte nuclei previouslyincubated separatelywithtwo novel mercury compounds ( N - the study of the relationship between chromatin structure chloromercuribenzoy1)-biocytin and bis(p(ch1oromer- and the transcriptional state of genes such as globin and c~ribenzoyl))-[~H]lysine diamide) were digested with ovalbumin whose sequence, organization, and transcriptional micrococcal nuclease and thedigest products fraction- condition are well known (1).Even so, a complete biochemical ated according to their solubility in 0.15 M NaCl and description of transcriptionally competent or active chromamolecular size. The identity and quantitation of the tin has not been achieved. Recent studies by Caplan et al. (2) chromatin fractions and proteins containing covalently indicate that large fragments (2-30 kilobase pairs) of globin bound mercury were determinedby Western blotting, chromatin generated by restriction enzyme digestion sediment autoradiography, and scintillation counting. The most more slowly in 100 mM NaCl than do ovalbumin fragments, highly acetylated species of histone H3 in the 0.15 M for example. This difference in sedimentation is not changed NaC1-soluble polynucleosome fraction also contained by the addition of linker histones and is attributed to a change the highest proportionof bound mercury. This fraction in the way that the linker histones interact with the histone contains hyperacetylated core histones, is depleted in octamers. Weintraub (3) has also reported that histones H1 linker histones, and enriched in nonhistone proteins. Histone H3 in the 0.15 M NaC1-soluble mononucleo- and H5 are bound differently in large (20-40 kilobase pairs) somes, which are unacetylated and lack linker his- globin domains. From these results and others (4) it can be tones, was 45% less labeledthan histone H3 in the 0.15 inferred that the transcriptionally competent globin domain in erythrocytes is compositionally different than bulk chroM NaCl-soluble polynucleosome fraction. In the 0.15 M matin and that this difference reflects itself in the ability of NaC1-insoluble polynucleosomes, which contain unacetylated histones and molar proportions of linker his- these active domains to form higher order structures. Recently, Allegra et al. ( 5 ) have reported that micrococcal tones, histone H3 was 63% less labeled. Allowing for the differential abundanceof these subfractions in the nuclease-generated chromatin fragmentsfrom rat liver, which nucleus, the relative H3 reactivities are 50, 7 , and 1 are soluble in 5 mM MgC12, contain histone H3 that can be for 0.15 M NaC1-soluble polynucleosomes, mononucleo- readily modified by iodoacetate. The greatest amount of labelsomes, and 0.15 M NaC1-insoluble polynucleosomes, ing occurs in tetracetylated histoneH3. These authorssuggest respectively. Thus a gradation of reactivities exists that unfolding of the nucleosome cores occurs in transcripwhich correlates with increasing hyperacetylation and tionally active chromatin thereby exposing Cys-110 of H3 linker histone depletion. High mobility group proteins which is normally buried in nucleosomes from bulk chromatin 1 and 2, found in subnucleosome particles in the 0.15 (6). An affinity chromatography method with mercury-SephM NaC1-soluble fraction, are extensively mercury-laarose was used to isolated these transcriptionally active fragbeled. Distribution of histone acetyltransferase activ- ments from micrococcal nuclease digests of nuclei from rat ity among salt- andsize-resolved micrococcal nuclease liver, induced cells from tissue culture, and Physarum polyproduced fractions was almost 5-fold greater in the cephalum, implying that the exposure of Cys-110 in H3 is a 0.15 M NaC1-soluble supernatant than in the 0.15 M general property of chromatin fragments obtained from nuNaC1-insoluble pellet, Furthermore, the acetyltransclease digestion of transcribing chromatin (7-9). ferase activity, which is tightly bound to undigested Moderate digestion of chicken erythrocyte nuclei by microchromatin, is rapidly releasedby both micrococcal nu- coccal nuclease yields EDTA extracts that can be fractionated clease and DNase I. For short digestion times the en- by physiological saline into a supernatant and a pellet. The zyme is associated with the salt-soluble polynucleosomes, but at longer times of digestion the enzyme salt-soluble supernatant, which contains about 10% of the appears to be free from intact nucleosomes. The en- total DNA, consists of mononucleosomes and a lesser amount zyme may be localized in the globin domain in eryth- of oligonucleosomes which are highly acetylated and somerocytes and maintains that region in a hyperacetylated what depleted in linker histones(10, 11).The insoluble pellet state which results inan altered linker histone binding contains predominantly oligonucleosomes and anormal comreflected in a change in the reactivity of the usually plement of linker histones (10, 11).The salt-soluble oligonucleosome fraction is up to 50-fold enriched in the transcripinaccessible H3 cysteine 110. tionally competent adult @ globin genesand depleted in genes * This research was supported by the Medical Research Council of such as ovalbumin and vitellogenin, which are transcriptionCanada Grant MT 5453 and studentship (to S. C.) The costs of ally quiescent even in transcriptionally active immature publication of this article were defrayed in part by the payment of erythrocytes (11).Nelson and co-workers (12, 13) have found page charges. This article must therefore be hereby marked “advertisement” in accordance with 18U.S.C. Section 1734 solelyto indicate that incubation of immature chicken erythrocytes with 10 mM n-butyrate results in the preferential solubilization in 3 this fact. a polynucleosome fraction enriched in tran$ T o whom correspondence and reprint requests should be ad- mMMgC12of dressed. scribed genes. Addition of yeast deacetylase to these fractions
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Histone H3 Thiol Reactivity and Acetyltransferases in Nuclei
causes precipitation, suggesting that histone hyperacetylation centrated to 10 mlby ultrafiltration with an Amicon PM-10 filter is a primary factor in the increased solubility of transcribed and filter apparatus. DNase I-Nuclei were suspended in a digest buffer containing 0.25 chromatin in buffersof physiological ionic strength (14). M sucrose, 10 mM Tris, pH 7.4, 6 mMMgC12, 1 mM PMSF at 50 We undertook our study to determine the accessibility of A ~ M ) ~DNase ~ / ~ I I. was added to 1 pg of DNase I/850 pg of DNA. The histone H3 in chicken erythrocyte nuclei to novel biotinylated reaction was stopped by the addition of EGTA to a final concentration and radiolabeled mercury compounds prior tonuclease diges- of 10 mM, and the samples were cooled on ice and then centrifuged tion. The bound reagents be can readily detected in the variousa t 17,000 X g for 1 min. The nuclear pellet was resuspended in 10 fractions obtained following the salt andsize fractionation of mM EDTA, pH 8, for 2 h and insoluble material pelleted at 17,000 X micrococcal nuclease digests. In view of the apparent role of g for 3 minutes. Both the reaction and EDTA supernatants were saved for further analysis. acetylation in the separation phenomenawe have also determined the location of a histone acetyltransferase activity in Gel Filtration Chromatography the various digest fractions. We find that the histone acetylBoth salt-soluble and insoluble samples from the micrococcal nutransferaseactivity is selectively associated with the saltclease Method l digests were fractionated into size classes bygel soluble oligonucleosome fraction and that the hyperacetylatedfiltration with Sephacryl S-300 (1.5 X lo6 upper exclusion limit, 3.2 H3 molecules contained therein are much more reactive to X 150 cm) equilibrated with TCE buffer (10 mM Tris-HC1, pH 8, 10 mM Na cacodylate, 0.7 mM EDTA). The column was pumped at 60 mercury compounds than they are in bulk chromatin. This result suggests that the hyperacetylated erythrocyte chroma- ml/h, 8-ml fractions were collected, and theDNA content was detertin exists in a conformationally distinct condition in nuclei mined from its absorbance a t 260 nm. The micrococcal nuclease Method 2 digest reaction EDTA supernatants, the DNase I reaction, from that of bulk chromatin and that this state is maintained and EDTA supernatants were resolved into nucleosome size classes by histone acetyltransferases bound nearby to linker or pro- by gel filtration on Sephacryl S-300 (1.5 X 80 cm) eluted with 10 mM tein free DNA. TCE. MATERIALSANDMETHODS
Isolation of Chicken Erythrocyte Nuclei Approximately 80 ml ofblood werecollected from live adult leghorn chickens into 20 ml of 0.085 M sodium citrate, 0.065 M citric acid, and 2% glucose, pH 7.4 (15). The bloodwas filtered through two layers of cheesecloth and spun down a t 1200 rpm in an H4000 swinging bucket rotor in an RC-3B centrifuge for 10 min at 4 "C. The cells were washed twice with Swim's S-77 medium containing 10% fetal bovine serum. Washed erythrocytes were lysedin 0.25 M sucrose, 10 mM Tris-HC1, pH 7.4, 6 mMMgC12, 10 mM butyrate, 0.4 mM PMSF,' and 0.5% (v/v) Nonidet P-40 (lysis buffer). Nuclear pellets were obtained by centrifugation at 8000 rpm for 10 min in an SS-34 rotor and subsequently washed three or four times with the lysis buffer until a beige pellet was obtained. Nuclei were resuspended in a wash buffer (lysis buffer without the detergent, Nonidet P-40) and spun down at 4000 rpm for 5 min in the SS-34 rotor. An estimate of the DNA content of a suspension of nuclei was made from the absorbance at 260 nm after solubilizing in 1%SDS. Unless stated otherwise all operations were performed at 4 "C and all buffers contained 10 mM Na butyrate to inhibit endogenous deacetylases (16). Nuclease Digestionof Erythrocyte Nuclei MicrococcalNuclease-In Method 1, 1000-1200 A 2 W n m units of nuclei were suspended in micrococcal nuclease digest buffer (0.25 M sucrose, 10 mM Tris-HC1, pH 7.4,lO mM Na butyrate, 0.4 mM PMSF) / ~ ~sample . was made to 1 mM at a concentration of40 A z w ~ ~The CaC12,preincubated at 37 "C for 10 min, and incubated at 37 "C for 20 min following the addition of 2 units of micrococcal nuclease per A2W,m of nuclei. Digestion was terminated by addition of EDTA to 10 mM. The nuclei were lysed by dialysis overnight against 4 litersof 1 mM EDTA, 1 mM PMSF 4 "C. Finally, the sample was clarified by centrifugation at 8000 rpm for 10 min in an SS-34 rotor at 4 "C. Method 2 was that described by Ridsdale and Davie (11) in which 2 mM MgClz was present during and after the micrococcal nuclease digestion. Salt Fractionation of Micrococcal Nuclease Digest Products-The resulting EDTA supernatant from a micrococcal nuclease digestion was adjusted to 0.15 M NaCl with 0.4 M NaCl and kept on ice for 30 min. The precipitate was spun down at 8000 rpm for 10 min in an SS-34 rotor at 4 "C. This precipitate was redissolved in 1 mM EDTA. The supernatant from the 0.15 M NaCl precipitation step was con-
' The abbreviations used are: PMSF, phenylmethylsulfonyl fluoride; MBB, N-(p-(chloromercuribenzoy1))-biocytin;MBL, bis@(chloromercuribenzoyl))-[3H]lysinediamide; TCE, Tris-cacodylateEDTA EGTA, [ethylenebis(oxyethylenenitrilo)]tetraacetic acid; SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis; HPLC, high performance liquid chromatography; HMG, high mobility group.
Electrophoresis 15%SDS-PAGE-Fractions obtained from the Sephacryl S-300 column were dialyzed against 1 mM PMSF and aliquots containing about 15 pgof histone were lyophilized prior to gel analysis. 15% SDS-polyacrylamide slab (0.1 X 14 X 19 cm) gels (17) were run for 4 h at 250 V, stained with Coomassie Brilliant Blue R-250 in 50% ethanol, 10% acetic acid overnight, and destained with 25% ethanol, 10% acetic acid. After destaining, the gels were transferred to 0.9 N acetic acid for storage at 4 'C. For biotin detection, the 15% SDSpolyacrylamide gelswere electroblotted onto nitrocellulose filters. The electroblotting was carried out for 1 h at 100 V and 50 mA in 0.05 M Tris-HC1, pH 8.3, 0.38 M glycine, 0.1% SDS, 20% methanol (18). 15% Acid-Urea-Triton-PAGE-Acetylated histones were resolved on 15% acid-urea-Triton gels (19). The sample buffer contained 8 M urea and 2% protamine sulfate to displace the histones from the DNA so that prior extraction of the histones from the nucleosome was unnecessary. Fluorograms of these gels were made by first soaking the fixed gel in water for 30 min and then for 45 min in 200 ml of 1 M Na salicylate, pH 6. The impregnated gels were vacuum-dried and then exposed to Fuji x-ray medical film at -90 "C for several days. Synthesis of N-(Chloromercuribenzoy1)-biocytin(MBB)-MBB was synthesized by reacting 25 pmol of biocytin (Sigma) with 25 pmol of the N-hydroxysulfosuccinimidyl ester of p-chloromercuribenzoate (see below). The reaction was carried out at 22 "C in 5 ml of 0.4 M NaHC03, pH 8.6, for 15 min. Following the reaction, the pH of the solution was dropped to 2 with 11N HCl, and theresulting precipitate was collected and washed with water to remove any unreacted biocytin. The precipitate was then dissolved by raising the pH to 8. Quantitative assays for mercury, biotin, and a single carboxyl group confirmed that each group was present at thesame molar ratio. MBB has a maximal solubility of about 3 mM in water a t pH 8. The N-hydroxysulfosuccinimidylester of p-chloromercuribenzoate was prepared by the condensation of 2.3 mmol of p-chloromercuribenzoate (Sigma) and 2.3 mmol of N-hydroxysulfosuccinimide (Pierce) with 2.5mmol of dicyclohexylcarbodiimide in 10 mlof dimethylformamide with stirring. After 16 h, 10 volumes of diethyl ether was added to the reaction solution causing the product to precipitate. The product was repeatedly washed with ether toremove unreacted dicyclohexylcarbodiimide and finally vacuum-dried. The crude N-hydroxysulfosuccinimidylester of p-chloromercuribenzoate was used as such in the reaction with biocytin described above. The major contaminants are unreacted p-chloromercuribenzoate, N-hydroxysulfosuccinimide,and dicyclohexylurea,none of which can react with biocytin under the conditions used. Synthesis of Bis(p-(chloromercuribenzoyl))-~Hllysine Diamide (MBW-An inexpensive but highly radiolabeled mercury compound was synthesized in situ by reacting [3H]lysine (77 Ci/mmol, ICN) with the N-hydroxysulfosuccinimidyl ester of p-chloromercuribenzoate as follows. Lysine (5 mCi, 65 mmol) was freeze-dried, and to this was added 5 p1 of 1 M sodium bicarbonate and a 10-fold molar excess of N-hydroxysulfosuccinimidyl ester of p-chloromercuriben-
Histone H3 Thiol Reactivity Acetyltransferases and in zoate dissolved in ice-cold distilled water (125 p1 of a 10.5 mM solution). This solution was left 5 h at 4 "C. After the reaction, the resulting solution contains the tritiated mono- and diamide adducts of lysine, unreacted lysine, and excess p-chloromercuribenzoate and N-hydroxysulfosuccinimide.The thiol specificity of this solution was testedon acid-extracted core histones and an immobilized thiolcontaining gel (Affi-Gel 401, Bio-Rad). The test reaction was done by adding 2.5 nmol of MBL to 50 pl of a 60 mg/ml histone solution (containing 50 nmol of H3) in 20 mM Na acetate, pH 5, for 62 h at room temperature. Following labeling, the histone mixture was precipitated with acidified acetone, washed with dry acetone, and then dissolved in distilled water. The histones were resolved by reverse phase HPLC using a Bio-Rad Hi-Pore RP318 (25 X 0.46 cm) column eluted with an increasing linear gradient of acetonitrile/water containing 0.06% trifluoroacetic acid at 1 ml/min. After subtraction of a blank, the H3 peak contained roughly 20 times as many counts as any of the other three core histones. The specific activity of the H3 recovered from the HPLCseparation was about 15,000 dpm/pg or 0.1 Ci/mmol. A time course of radiolabel incorporation was determined for MBL with the immobilized thiol. It was found that about 20% of the counts added were ultimately bound and subsequently released by treatment with 10 mM dithiothreitol. Thus, about 20% of the input lysine was incorporated into MBL during the reaction with Nhydroxysulfosuccinimidyl ester of p-chloromercuribenzoate.
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Histone Acetyltransferase Assay A solid-phase histone acetyltransferase assay described by Allis et al. (20) was carried out with the following modifications. Assay samples (
