Proc. Natl. Acad. Sci. USA Vol. 75, No. 12, pp. 5773-5777, December 1978 Biochemistry Displacement and aberrant methylation in vitro of H-1 histone in rat liver nuclei after half-saturation of chromatin with polycations (polylysine/protamine/methyllysine/nucleosome) PAUL BYVOET*, C. STUART BAXTERf, AND DAVID F. SAYRE* *Veterans Administration Hospital and Department of Pathology, University of South Florida, Tampa, Florida 33612; and tDepartment of Environmental Health, Kettering Laboratory, University of Cincinnati Medical Center, Cincinnati, Ohio 45267 Communicated by George K. Davis, July 26,1978 ABSTRACT Radiomethyl incorporation in vitro into Ne- MATERIALS AND METHODS methyllysine of histones from rat liver nuclei incubated in the presence of S-adenosyl[methyl-3H]methionine is stimulated if Di-a-L-lysine and tri-a-L-lysine (neutral hydrochloride salts), the lycations polylysines, protamines, or histones are added poly-a-L-lysines (degrees of polymerization = 9, 12, and 350 to the incubation mixture. Maximal stimulation occurs at a residues; neutral hydrobromide salts), and protamine sulfate cation/nucleotide ratio of 0.5. Past this point stimulation dro s, or chloride (clupeine) were purchased from Sigma. Penta-a- except in the case of very lysine-rich histone H-1, for which the maximal level of incorporation remains constant upon further L-lysine pentaacetate was from Cyclo. H-1 histone was pre- addition of this histone. Bio-Gel P-10 chromatography, differ- pared from rat spleen according to the methods described ential precipitation, and gel electrophoresis of radiomethylated below. Rat liver nuclei used in these studies were obtained from histones indicate that although the usual incorporation of ra- Holtzman rats housed in facilities accredited by the American diomethyl into histone H-3 is not affected, active methylation Association for the Accreditation of Laboratory Animal Care of H-1 occurs in the presence of polycations. Column chroma- and provided by the Tampa Veterans Administration tographic amino acid analysis reveals that the methylation of Hospital H-1 will specifically generate Ne-monomethyllysine. Except for Research Service. this condition, H-1 is never methylated in vivo or in incubated Histone Methylation in Nuclei. Rat liver nuclei were in- cell nuclei. Because H-1 is the weakest bound histone in chro- cubated in the presence of S-adenosyl[methyl-3H]methionine matin, the above phenomena may be explained by assuming and histones were isolated and assayed for incorporation of that, within the chromatin, polycations displace the lysine-rich radiomethyl as described elsewhere (11). Briefly, nuclear pellets histone towards the nucleosome, which results in its aberrant containing approximately 0.5 of DNA were in methylation, assuming that the native nucleosome is the seat mg suspended of the histone incubation medium (0.25 M sucrose/0.02 M KCI/0.01 M lysine methyltransferase. MgCl2/0.01 M mercaptoethanol/0.05 M Tris-HCI, pH 8.5/2.5 Methylation of r-NH2 groups on lysine side chains in histones ,uCi of [3H]AdoMet) (1 Ci = 3.7 X 1010 becquerels) and incu- occurs in situ in chromatin at the end of S phase (1-3) on newly bated for 12 min unless stated otherwise. The reaction was synthesized histones (4). The lysine methyltransferase respon- stopped by chilling and addition of saline/EDTA (0.08 M sible for this process utilizes S-adenosylmethionine as methyl NaCl/0.02 M EDTA), pH 7.2. The pellet was washed twice donor (1) and may introduce up to three methyl groups into the with the same solution and extracted with 0.25 M HCI to obtain c-N position of specific lysine residues in H-S and two in H-4. whole histone, or it was treated first with 0.5 M HC104 to extract All methylated lysine residues are located in the polar regions the H-i histones and then with 0.25 M HCI to extract the of these nucleosomal (5) histones. Methylated lysine residues nucleosomal histones. Extracts were centrifuged at 20,000 X appear to be situated 3 or 4 amino acid residues from an acet- g for 15 min, supernatants were made 0.2 M in H2SO4, and ylated lysine (6) and in H-3 next to a phosphorylated serine (7). histones were precipitated by addition of 3 vol of acetone. Ra- In contrast to acetylation and phosphorylation, however, dioactivity was determined by liquid scintillation counting and methylation has been found to be irreversible (4). A function protein concentration, by the method of Lowry et al. (12). for this process has eluded investigators so far (8). In those experiments in which polycations were added to the Previous studies (8, 9) in this laboratory using rat liver nuclei incubation medium, nuclear pellets were suspended in 0.25 M indicate that uptake of radiomethyl into histones will occur sucrose and distributed over a series of samples containing during incubation in vitro of rat liver nuclei or chromatin in 0.5-1.0 mg of DNA. The exact amount of DNA was then de- the presence of S-adenosyl[methyl-3H]methionine ([3H]Ado- termined by measurement of A20 of hot 0.5 M HC104 extracts Met) and that this uptake is practically confined to that into H-3, of the pellets obtained after two washings with saline/EDTA, the uptake into H-4 being much less. pH 7.2, and two with cold 0.5 M HCI04. The amount of poly- We have reported (9) that a number of carcinogenic agents cations needed was then calculated from the values obtained were inhibitory, to this process, whereas planar compounds able and added before the label. to intercalate between DNA base pairs appeared to be stimu- Bio-Gel P-10 Chromatography. Alkylation was carried out latory (10). according to Candido and Dixon (13) by dissolving the histones We now report that saturation of chromatin with polycations in 400 ,ul of 0.2 M sodium borate buffer, pH 8.9, containing 0.02 (cation/base-pair ratio = 1) stimulates radiomethylation of M dithiothreitol and 5 M urea. After incubation at room tem- histones in vitro. We also present evidence indicating that this perature for 90 min, iodoacetamide was added to a concen- stimulation is due to displacement of H-1 histone within the tration of 0.08 M and incubation was continued for another 90 chromatin, resulting in its subsequent aberrant methylation. min. The solution was then carefully layered on the surface of a 3 X 140 cm Bio-Gel P-10 column and eluted at a flow rate of The publication costs of this article were defrayed in part by page 30 ml/hour in fractions of 1.5 ml, with 0.02 M HCI/0.05 M charge payment. This article must therefore be hereby marked "ad- NaCI/0.02% NaN3 (14). vertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact. Abbreviation: AdoMet, S-adenosylmethionine. 5773 Downloaded by guest on October 1, 2021 5774 Biochemistry: Byvoet et al. Proc. Natl. Acad. Sci. USA 75 (1978) 150 0.75 C x 80 100 0.5 E 0 c 0 4- a 50I[ 0.25 u 20 40 60 80 -40- Fraction Lysine/nucleotide FIG. 2. Bio-Gel P-10 chromatography of in vitro radiomethylated histones from rat liver nuclei incubated in the presence of [3H]AdoMet 12*0 B and a half-saturating amount of oligolysine (n = 9; lysine/nucleotide ratio = 0.5). The breakthrough fraction consists of nonhistone pro- The first peak with high specific ra- C teins and aggregated material. *ro 80- dioactivity represents lysine-rich histone H-1; the second A230 peak represents H-2a, H-2b, and H-3; and the third A230 peak (shoulder), E / \ H-4. The second radioactive peak is situated at the right of the H- 2a-H-2b-H-3 complex, where H-3 is eluted. Due to its low molecular 40- weight, oligolysine eluted following H-4. No radioactivity was present in oligolysine. of histones (18-20). Results obtained with protamines, recorded 0.4 0.8 1.2 1.6 in Fig. 1B, were similar to those with higher-order polylysines, Arginine/nucleotide the cationic group in this case being arginine rather than ly- FIG. 1. Stimulation by polycations of radiomethyl uptake into sine. histones in vitro. The polycations were added as neutral salts. All Absence of Direct Stimulation of Lysine Methyltransfer- points were duplicate experiments. (A) Stimulation of radiomethyl ase. Because stimulation of radiomethyl incorporation into uptake as a function ofthe lysine/nucleotide (polylysine/DNA) ratio. histones in vitro may result from direct stimulation of the A, (Lys)5; 0, (Lys)g; X, (Lys)12; 0, (Lys)320. (B) Stimulation of ra- diomethyl uptake as a function of the arginine/nucleotide (prota- methyltransferase, a semi-purified preparation of this enzyme mine/DNA) ratio. (21) was incubated with [3H]AdoMet and whole histone in the presence of various polycations free in solution. The results, Gel Electrophoresis. Gel electrophoresis of histones was summarized in Table 1, indicate that, in contrast to the results performed in 15% acrylamide gel containing 2.5 M urea, ac- with whole nuclei, higher-order polylysines appeared to inhibit cording to the method of Panyim and Chalkley (15). Gel di- the in vitro radiomethylation of histones under these conditions. mensions were 0.6 X 20 cm. Samples were dissolved in up to 50 Negligible incorporation into trichloroacetic acid-precipitable ,ul of 8 M urea/0.5 M mercaptoethanol/30% sucrose before material occurred in the absence of either enzyme or his- application. The gels were run at room temperature at 1 mA tone. per gel for 18 hr, during which time the bands in the cluster Bio-Gel P-10 Chromatography of Methyl-Labeled His- typical of whole histone migrate over approximately 65% of the tones.