Rapid Turnover of Acetyl Groups in the Four Core Histones of Simian Virus 40
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Proc. Natl. Acad. Sc. USA Vol. 76, No. 1, pp. 46-50, January 1979 Biochemistry Rapid turnover of acetyl groups in the four core histones of simian virus 40 minichromosomes (chromatin/histone acetylation) ANNICK CHESTIER AND MOSHE YANIV Departement de Biologie Molculaire, Institut Pasteur, 25 rue du Dr. Roux, 75015 Paris, France Communicated by Frangois Jacob, June 27, 1978 ABSTRACT The four core histones (H2a, H2b, H3, and H4) MATERIALS AND METHODS bound to simian virus 40 minichromosomnes isolated from in- fected cells contain rapidly labeled acetyl groups in internal Cells and Virus Infection. African green monkey kidney positions of the histone polypeptide chain. Upon chase, these cells, line CV1, were grown in Dulbecco's modified Eagle's acetyl residues decay with a half-life of less than 15 min. The medium supplemented with 5% fetal calf serum in 88-mm acetyl groups are incorporated in histones bound to mature plastic dishes in an air/CO2 incubator. Plaque-purified SV40 chromosomes and not in newly synthesized histones bound to was used to infect the cells at an input multiplicity of 30-50 replicating viral chromosomes. The rate of acetate incorporation plaque-forming units per cell. At the end of a 2-hr adsorption is not related to the degree of steady state acetylation of the individual viral or cellular histones. This rate is 4-fold higher period, 10 ml of medium containing 1% serum was added to for the viral chromatin than for its cellular counterpart isolated the infected cultures. from the same nuclei. The possible role for histone acetylation Labeling of Cells. [14C]Thymidine (52.8 mCi/mmol, in viral genome expression is discussed. Commissariat a l'Energie Atomique) (1 Ci = 3.7 X 1010 bec- querels), 5 gCi per plate, was used to label DNA for 16 hr; The circular double-stranded DNA of simian virus 40 (SV40) [3H]thymidine (35 Ci/mmol), 500 ,Ci per plate in 1 ml of is associated in virions and in the infected cells with histones medium, was used to label DNA for 10 min. For acetate la- H2a, H2b, H3, and H4 (1). The fifth histone, HI, is found only beling, sodium [,3H]acetate (20 Ci/mmol) was dissolved in fresh in viral nucleoprotein complexes.(minichromosomes) extracted medium and 9.3 mCi in 1 ml was added per plate for 10-min from infected cells (refs. 2 and 3 and the present study). Elec- labeling. tron microscopy and biochemical studies have shown that viral Purification of Nucleoprotein Complexes and of Cellular and cellular chromatins are very similar, if not identical (4-8). Histones. Cultures were pulse-labeled with [3H]acetate at 41 The replicating viral DNA is associated with histones as is the hr postinfection. After labeling, the medium containing the The radioisotopes was removed, the monolayer was washed twice replicating cellular DNA (9, 10). viral nucleoprotein with phosphate-buffered saline, the nuclei were isolated in the complexes are the template for viral RNA synthesis in the in- presence of 0.5% of Nonidet P-40, and the nucleoprotein fected cells (11, *). Hence, the viral DNA-histone complex is complex was extracted with 0.2% Triton X-100 and 0.2 M NaCl a useful model system for the study of the mechanisms of rep- (7) by two successive treatments for 30 min at 20'C. Approxi- lication and transcription of the cellular chromosome. mately 70%6 of the viral DNA was extracted by this procedure, Although the knowledge of the basic features of the nu- and the final DNA yield was 5-7 ug per plate. The Triton su- cleosome structure is rapidly progressing (12, 13), the possible pernatant was obtained after centrifugation (800 X g at 40C), modifications of this structure during transcription, replication, and the nucleoprotein complexes were partially purified by and mitosis are still obscure. Actively transcribed genes contain sedimentation on 5-20% (wt/vol) sucrose gradients in 0.01 M modified nucleosomes as revealed by their increased sensitivity Tris-HCl (pH 7.9)/0.001 M EDTA/0.2 M NaCl for 95 min at io the action of DNase 1 (14). Electron microscopic observations 40,000 rpm in an SW 41 rotor at 40C. Fractions were collected and biochemical studies indicate that, although histones are and assayed for radioactivity, and protein was precipitated with present on nonribosomal active genes, RNA polymerase can 20% trichloroacetic acid in the presence of 100 ug of protamine nevertheless elongate RNA chains in vivo and under certain as carrier. After centrifugation, the protein pellet was washed conditions in vitro (11, 13, *). successively with acidic acetone, acetone, and ether and dis- In an attempt to understand the role of histone modifications solved in the appropriate sample buffer for polyacrylamide gel in gene regulation, we have examined the acetylation of histones electrophoresis. The nuclear pellet remaining after the Triton bound to viral and cellular DNA in monkey cells infected with X-100 extraction was suspended in 0.5 ml of water by brief $V40. We report here that the core histones H2a, H2b, H3, and sonication, and the histones were then extracted with 0.2 M H4 contain rapidly turning over acetyl groups. Histones H3 and H2S04. After precipitation with 5 vol of ethanol, the histone H4 bound to the viral DNA are found to be more highly acet- pellet was dissolved in sample buffer. ylated than those bound to cellular DNA. The rate of acetylation Gel Electrophoresis. Three different methods were used for of the histones bound to the viral DNA is about 4-fold higher analysis of histones on 14-cm slab gels: 17.5% polyacrylamide than of those bound to the cellular DNA of the same infected gels m the presence of sodium dodecyl sulfate (NaDodSO4) and cells. acid urea, 15% gels containing 2.5 M urea as described (9), or The publication costs of this article were defrayed in part by page Abbreviations: SV40, simian virus 40; NaDodSO4, sodium dodecyl charge payment. This article must therefore be hereby marked "ad- sulfate. vertisement" in accordance with 18 U. S. C. §1734 solely to indicate * P. Gariglio, R. Llopis, P. Oudet, and P. Chambon, unpublished this fact. results. 46 Downloaded by guest on September 27, 2021 Biochemistry: Chestier and Yaniv Proc. Natl. Acad. Sci. USA 76 (1979) 47 acid urea gels containing Triton X-100 (15). Gels were stained daughter double helices (10). To test whether newly synthesized for 1 hr with Coomassie blue, destained, photographed, im- histones are found in higher acetylated forms, we labeled the pregnated with 2,5-diphenyloxazole (16), dried, and autora- infected cells for 10 min with [3H]acetate. As shown in Figs. 1 diographed with sensitized films by exposure at -70'C. and 2, the [3H]acetate counts cosedimented with the mature viral minichromosomes previously labeled with ['4C]thymidine, RESULTS not in the position of replicating complexes. In addition, acetate Viral Histones H3 and H4 Were More Highly Acetylated was incorporated in soluble nuclear nonhistone proteins found than Cellular Histones. Mild nonionic detergent treatment of on the top of the gradient. NaDodSO4/polyacrylamide gel nuclei isolated from SV40-infected cells permits the preferential analysis of proteins from a similar gradient of viral chromatin solubilization of native viral minichromosomes (4, 7). These labeled with [3H]acetate for 10 min in vivo is shown in Fig. 3. nucleoprotein complexes can be purified further by zonal Again, little acetate label was found in fraction 2, which con- sedimentation on sucrose gradients as shown in Fig. 1. Portions tained the replicating complexes. The majority of acetate label of the gradient were pooled, and their histone content was an- was found in fraction 3, which contained the mature complexes. alyzed on acid/urea/polyacrylamide gels. As shown in Fig. 2A, The specific activities of acetate label in replicating and mature the concentrations of the four core histones (H2a, H2b, H3, and complexes were calculated from the stain and the autoradi- H4) paralleled those of the viral DNA labeled with [14C]thy- ography of the gel and were found to be roughly equal. To midine for 16 hr. The H4 reveals that the viral chor- exclude artifactual acetylation arising during the isolation of pattern the viral minichromosomes, we mixed [3H]acetate pulse-labeled matin contained four different species of H4 histone. When noninfected cells with compared to cellular histones extracted from normal or buty- [14C]thymidine-labeled infected cells rate-treated cells the and extracted the viral DNA histone complex. No acetate label (Fig. 2A), viral DNA-bound H4 histone was found associated with the viral histones; only the cellular migrated identically to the highly acetylated H4 isolated from histones were labeled (results not butyrate-treated cells (17). Since H4 contains an N-acetylserine shown). in its NH2-terminal position (18, 19), the second, third, and Acetate Incorporation in H2a, H2b, H3, and H4, but Not fourth to in HI. The autoradiography of the [3H]acetate-labeled histones bands correspond one, two, and three internal acet- separated on the NaDodSO4/acrylamide shown in 3 ylations (17, 20). The nuclear pellet remaining after the ex- gel Fig. traction of viral and the scan of the same autoradiograph (Fig. 4 A and B) minichromosomes was sonicated, and the cel- demonstrate that only the four core histones H2a, H2b, H3, and lular histones were acid-extracted and analyzed on the same H4 and not Hi are rapidly labeled with acetate. Although the polyacrylamide gel. The cellular H4 contained only a mono- H2a and H2b are not separated in the fluorograph given, the acetylated species as shown in Fig. 2A. Although less well sep- width of the H2a and H2b band is twice that H3 or arated than H4 in this gel, the viral H3 histone contained of H4, forms suggesting that both H2a and H2b are acetylated.