Gene, 74 (1988) 117-121 Elsevier
DNA methylation in plants * (Nucleotide sequence; Smethylcytosine; methyladenine; cycle; phytohormone action; wheat seedlings)
Boris F. Vanyushin and M&hail D. Kirnos A.N. Belozersky Laboratory of Molecular Biology and Bioorganic Chemktty, Moscow State University, Moscow 119899 (U.S.S.R.) Received 6 June 1988 Accepted 15 August 1988 Received by publisher 23 August 1988
Both replicative and postreplicative nuclear DNA (nDNA) methylation, with the formation of Smethylcytosine (mC) residues, occurs in plants. These two types of enzymatic DNA modification are different in amount and nucleotide sequence of methylatable sites, as well as in sensitivity to phytohormones, temperature and various inhibitors of DNA methylation, transcription and replication. The role of DNA methylation in regulation of replication, gene expression and cell differentiation is discussed.
5-Methylcytosine (mC) has been detected in nDNA of animals and higher plants (Vanyushin, et al., 1970; 1971). In addition mA has been shown to exist in higher plant DNA (Vanyushin et al., 197 1; 1988). DNA mC and mA originate from selective methylation cytosine and adenine residues, respectively, at the polynucleotide level. This enzymatic reaction is carried out by DNA methyltransCorrespondence to: Dr. B.F. Vanyushm, A.N. Belozersky Laboratory of Molecular Biology and Bioorganic Chemistry, Moscow State University, Moscow 119899 (U.S.S.R.) Tel. 9395412. * Presented at the New England Biolabs Workshop on Biological DNA Modification, Gloucester, MA (U.S.A.) 20-23 May 1988.
Abbreviations: AdoMet, S-adenosylmethionine; 6-BAP, 6-benzylaminopurine; C, cytosine; 2,4-D, 2,4-dichlorophenoxyacetic
0378-l 119/88/$03.SO 0 1988 Elsevier
ferases which use AdoMet as the donor of CH, groups. It was suggested that in plant cells there may be a few different DNA methyltransferases (Kirnos et al., 198 l), which methylate the cytosine residues in sequences with two different types of symmetry; in CG and CNG sequence (Kimos et al., 1981; Gruenbaum et al., 1981). The methylation of DNA is one possible mechanism of regulation of gene expression and cell differentiation in eukaryotes (Razin and Riggs, 1980; acid; GA,, gibberellic acid A,; IAA, indole acetic acid; mA, N6-methyladenine; mC, 5-methylcytosine; ML, DNA methylation level: lOOxmC/(C + mC); mt, mitochondrial; N, any nucleotide; nDNA, nuclear DNA; nsDNA, newly synthesized DNA; RIF, DNA replication intermediate fragments; Se, S,, S,, early, late, mid stages of S phase; SIBA; 5’-S-isobutyladenosine; SSM, site specificity of DNA methylation (the y0 oftotal mC found in the Pu-mC-Pu sequence).
Vanyushin, 1984). U~o~~ately, at the present time, almost nothing is known about how DNA is methylated in the cell cycle in plants. In the present work we studied the level and specificity of methylation in replicative DNA intermediates formed at the various stages of S phase (S,, S,, and S,) in cells of the initial leaf of etiolated wheat seedlings. The effects of different ph~oho~ones (IAA, cytokinms, gibberellin) on replicative and postreplicative methylation of nDNA was also studied.
MATERIALS AND METHODS
Etiolated seedlings were grown from seeds of wheat of the Mironovskaya 808 variety and their growth was synchronized as described earlier (Kirnos et al., 1983b). A radioactive label was introduced into seedlings (whole or cut plants) during the interphase and S-phase of meristem cells of the initial leaf incubated in a medium with a radioactive precursor of DNA synthesis. For the study of DNA synthesis and methylation, the cut seedlings were incubated with [2-‘4C]orotic acid (0.5 mCi/ml; Izotop, U.S.S.R., sp. radioactivity 43.4 mCi/mmol) or 0.5 mCi/ml L-[methylJ4C]methionine (Institute of Isotopes, Hungary; sp. radioactivity 21.8 mCi/~ol) or in the presence of 10 @i/ml of [ 2-*4C]thymidine, sp. radioactivity 0.1 Cilmmol). PARERTAL DNA STRARD II
MLC25 SSM =40-42
ML = zo-73 SSM = 80- 82
DAUWl’ER ML < 25 DRA STRARD SSU =40-42
* METRYLATED SITE 0 URMETRYLATED SIK
Fig. 1. Different methylation types of DNA duplexes (I-III) in the cells of wheat seedling initial ieaf. ML, methylation level [ 100x mC/(C + mC)] ; S SM, site specificity of DNA methylation (the % of total mC found in Pu-mC-Pu).
For the study of nonreplicative DNA methylation, seedlings were incubated for 30 min in 25 mM hydroxyurea and then in a solution containing 25 mM hydroxyurea and labeled DNA precursors. After incubation with radioactive substances in the presence or absence of phytohormones, the initial leaves from seedlings were isolated. DNA from these leaves was isolated and fractionated by the alkaline sucrose and CsCl density gradient centrifugations (Kirnos et al., 1983a,b; 1984b). Hydrolysis of DNA to bases and two-d~ension~ thin-layer chromatographic analyses were performed using methods developed earlier (Kirnos et al., 1983a,b). Hydrolysis of DNA to pyrimidine tracts and analysis of the dist~bution of labeled mC in these tracts were carried out as described previously (Kios et al., 1981; 1983b).
RESULTS AND DISCUSSION
(a) Methylation of wheat DNA We have found that nDNA synthesis is periodic and synchronous in the first leaf of developing etiolated wheat seedlings (Kirnos et al., 1983b). With the completion of coleoptile formation, nDNA synthesis practi~~y ceases in it, while mtDNA continues in synchronous cycles (Kimos et al., 1983b). Mitochondrial DNA does not contain mC and differs in base composition (GC = 55-56x) from nDNA (GC = 45%). (1) DNA synthesis phase
Seedlings (in mid S-phase of initial leaf cells) were incubated for 30 min with [2-14C]orotic acid, and after mild lysis of the plant material (first leaf) and purification of nDNA, by equilibrium CsCl density gradient centrifitgation, it was fractionated into individual RIF by zonal centrifugation in alkaline sucrose gradients. Radioactive mC and C from 5S, 8S, 12s and > 12s DNA fragments were separated and quantitated (Kimos et al., 1984b). Thus, in wheat seedlings, as in a suspension culture of tobacco cells (Bashkite et al., 1980), the Okazaki fragments are me~ylat~. The ML (100 mC/ (C + mC) = 7.4 f. 0.5) in Okazaki fragments (< 5s) in etiolated seedlings was three to four
times lower than that in total wheat nDNA. After ligation of Okazaki fragments, leading to formation of long RIF (8S, Z 12S), the ML remained at almost the same level as the Okazaki fragments; therefore, recently replicated DNA is significantly undermethylated. Thus, replication generates undermethylated DNA molecules. DNA duplexes formed during replication exhibit sharply pronounced asymmetry of the mC distribution along the complementary - parent and daughter - DNA chains (Kirnos et al., 1984b). In the whole plant, in contrast to cell cultures (Bashkite et al., 1980) the second step of replicative DNA methylation (methylation of ligation stretches) is less clearly expressed. After ~cubation of wheat seedlings with L-[methyl-i4C]methionine, we have found radioactive mC in Okazaki fragments (Kirnos et al., 1984b). In ligated (> 12s) and mature nDNA, up to 40% of all the mC residues are located in the Pu-mC-Pu sequence; whereas, in the Okazaki fragments, this sequence contains only 20% of all the mC (Kirnos et al., 1984b); (Fig. 1). This suggests that there may be a DNA-methyltransferase associated with the replication fork that is different from the one methylating the long RIF; and these activities recognize and methylate different nucleotide sequences. (2) Interphase Si~i~c~t incor~ration of label from [5methyl-3H]thymidine, [6-3H]uridine, [8-14C]adenine, L-[methyl-i4C] or L-[methyl-3H]methionine into nDNA takes place in the interphase between replication cycles (S-phases; Kirnos et al., 1984a); this incorporation is not associated with de novo replication. Thus, non-replicative (reparative) synthesis and post-rep~cative methylation of nDNA occur in plants. The level of nDNA methylation during reparative synthesis in the presence of hydroxyurea (ML = 11.6) is 50% that in total wheat nDNA. Thus, reparative synthesis in cells of intact plants, like replication, is another pathway of mC content reduction in DNA. The specificity of DNA methylation (the mC dis~bution among padre tracts in DNA) during repair synthesis is similar to that during methylation of total wheat nDNA (Kimos et al., 1984a). It has been established that during the cell cycle, in addition to methylation of repaired sites, nDNA also undergoes intensive postreplicative methylation. This leads to a sharp increase in ML
(22.9 + 0.6) and, thereby, to a decrease in the number of hemimethylated sites by the end of the cell cycle. Thus, replicative and post-replicative DNA methylation in the plant cell nucleus appear to be different. Finally, the methylation level (ML = 7.9-8.2) of newly synthesized DNA (nsDNA) labeled with [2-‘4C]orotic acid for one hour in first leafof control wheat shoots was practically unchanged at various times during S-phase (Table I). (b) Effect of phytohormones
Addition of a phytohormone inhibited replicative me~ylation to various degrees, depending on the concentration and according to time of addition. The strongest (up to 50 %) inhibition of replicative DNA methylation was observed in S, and S, phases of the cell cycle (Table I). A weak, stimulatory effect was exerted by 6-BAP, 2,4-D, GA, and kinetin during prolonged (20 h) incubation of cut-off pulselabeled shoots (Kirnos et al., 1986). The genomic methylation measured by the incorporation of 14CH3 groups from L-[methylJ4C]methionine into mC per pmol of the total nDNA adenine sharply declined in the S, phase in the presence of all phytohormones TABLE I Effect of phytohormones on replicative DNA methylation in first leaf cells of wheat seedlings at various stages of S-phase Incubation conditions a
nsDNA methylation level, ML f. ub
(M) Sn Control 2,4-D Kinetin 6-BAP GA,
1O-4 10-s 1O-4 10-s 1o-4 1o-5 10-s 10-e
7.9 + 7.0 + 8.0 7.9 8.5 2 4.5 f 1.9
8.0 f 0.8 6.6 f 0.6 6.2 f 0.7 4.2 f 0.2 6.7 + 0.1 7.4 5.5 k 0.7 7.6 & 0.7
8.2 f 6.6 + 7.8 f 6.5 c 6.5 f 5.5 f 6.3 + 5.3 + 6.0 f
0.7 0.4 0.2 0.5 0.7 0.3 0.2 0.5 0.2
a Shoots aged 74 h (Sn), 78 h (S,), and 83 h (S,) were cut and incubated for 30 mm with phytohormones and then for 1 h with [2-r4C]orotic acid. b DNA from the fust leafwas isolated, hydrolyzed to bases, and the radioactivities in mC and C were determined. u, standard deviation. For ML, see Abbreviations and section al.
Fig. 2. Possible formation of unmethylated adenine sites following replication of DNA containing cytokinin (CK; incorporated during previous replicated cycle).
studied, whereas in S, and S, phases this inhibition of DNA methylation was much weaker (Kirnos et al., 1986). In the presence of exogenous phytohormones the specificity of DNA methylation changed also. Short-term incubation (30 min) of wheat
seedlings with r.-[methyl-14C]methionine and GA, (10V6 mol) resulted in an increase (approx. 40%), while long-term incubation (3 h) resulted in a decrease, in the amount of mC in Pu-mC-Pu sequences (Kirnos et al., 1986). After blocking replication by hydroxyurea (50 mM), pronounced post-replicative DNA methylation proceeds (Bashkite et al., 1980; Kimos et al., 1987). The various phytohormones studied do not influence postreplicative DNA methylation (Kirnos et al., 1987). Postreplicative nDNA methylation in first leaf is insensitive to seedling incubation in the presence of cycloheximide (20 pg/ml), ethidium bromide, actinomycin D (100 pg/ml) and 5-azacytidine (100 pg/ml); it is strongly inhibited by SIBA (1.5 mM) and completely blocked during seedling incubation at 40’ C (Kimos et al., 1987). In contrast, the replicative DNA methylation in the first leaf is very sensitive to 5-azacytidine but almost insensitive to SIBA (Kirnos et al., 1988). This indicates that replicative and postreplicative DNA methylation differ in their response to phytohormones, temperature, inhibitors of repli-
CELL CYCLE I
_L CELL CYCLE li T
REPLICATION PERMITTED +
B t Q 3
REPLICATION II PROHIBITED 1
. METUYLATED SITE 0 UNMETWYLATED SITE
Fig. 3. Possible regulation of the replication and transcription orders of nucleotide sequences by their methylation (demethylation) in subsequent cell cycles. I-III, different methylation types of DNA duplexes (as indicated in Fig. 1); &-DNA, early replicating DNA compartments; S,-DNA, lately replicating DNA compartments. We suggest that only the symmetrically methylated DNA duplexes are permitted to be replicated. So, in the early S phase the completely methylated genome compartments (Sn DNA) may be replicated. In contrast, nucleotide sequences which should enter into replication in the S, phase (&-DNA) are methylated asymmetrically and their replication in S, phase is prohibited. With the termination of the SE-DNA replication, the newly formed S, duplexes are distinctly asymmetric as to the mC content in complementary DNA strands; their transcription seems to be permitted but repeated replication in the same cell cycle is prohibited. As a result of the persistent process of postreplicative methylation, the S, sequences from the preceding cell cycle gradually become symmetrically methylated: as a result, the transcription of corresponding (late) genes is terminated and they enter into replication. By the onset of a new S phase, SE- and &-DNA sequences will be methylated to the same extent as before the preceding cycle of DNA synthesis. S, and S, duplexes attain this level depending on the rate of postreplicative DNA methylation, coordinated with the duration ofthe cell cycle. Thus, the periodic modulation ofthe asymmetry ofmethylated sites in nDNA in sequential cell cycles, via replication and replicative or postreplicative methylation, may be regarded as a mechanism regulating the periodicity and fidelity of gene replication in the cell cycle.
cation and transcription, suggesting they are carried out by different enzymes. Thus, modulation of DNA methylation may be an important mechanism of action by which phytohormones affect gene expression and cell differentiation. We suspect that natural plant growth regulators such as cytokinins (N6-substituted adenine derivatives) may also influence replicative methylation of adenine residues in DNA of eukaryotes which are known to contain m6A (protozoa, fungi, algae, higher plants). It has been found recently that labelled 6-BAP as well as zeatin, appear to be incorporated in Tetrahymenapyrifrmis DNA (Mazin and Vanyushin, 1986) and in wheat seedling DNA (Kudryashova and Vanyushin, 1986). If cytokinin is incorporated in a nucleotide sequence recognized by the plant DNA adenine methyltransferase, then methylation of adenine in this particular sequence will not occur. Thus, incorporation into DNA and induction of the formation of unmethylated adenine sites may be another mechanism for regulation of replication, transcription and cell differentiation by cytokinins (Vanyushin, 1984) (Fig. 2). (c) Conclusions
DNA post-replicative methylation may regulate duration of the expression of various gene groups replicated at the different times in S-phase. Moreover, it may also serve as a control mechanism for the fidelity and temporal order of DNA replication in S phase (Fig. 3).
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