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1998. 36 (5), 401-406
Adenine methylation at GATC sequences regulates activity of tobacco PR-1 and PR-2 promoters in electroporated protoplasts
Robert Brodzik, Jacek Hennig * Poland. Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Pawifiskiego 5a, 02-106 Warsaw, * Author to whom correspondence should be addressed (tel. 48 22 6597072; fax 48 39 121623; e-mail firstname.lastname@example.org) (Received September 8, 1997; accepted October 14, 1997) Abstract - Three different constructs, containing promoters of genes which belong to the Pathogenesis Related family (PR- 1a, PR-2d) and CaMV 35s promoter, fused with uidA gene (GUS) as a reporter, were tested individually for expression in tobacco protoplasts. DNA containing constructs were amplified in Escherichin coli K12 strains: DHSo: (wild type) or GM33 and GM48 defective in methylation systems (Dam and DamiDcm mutants, respectively). In contrast to 3% promoter, marked differences in transient activity of both PR promoters were observed depending on the methylation state of the templates. High levels of GUS expression driven by PR-la or PR-2d were observed only when the template was obtained from the DHSa strain. In vitro DNA methylation with Dam methylase, prior to protoplast transfection, restored high levels of activity to the investigated promoters. A gel mobility shift assay was used to check if there are any differences in binding of PR-la promoter fragments, containing GATC or GmATC sequences, with tobacco nuclear extract. Our data suggest that emethyl-deoxyadenine (6-mA), present in GATC sequences, may modulate the regulation of PR-s genes expression. 0 Elsevier, Paris. Metbylation / transient expression / Patbogenesis Related genes. / 6-mA, timethyl-deoxyadenine / 5-mC, N5methyl-deoxycyto-
CaMV, cauliflower mosaic virus / GUS, P-glucuronidase sine /PR, Pathogenesis Related genes
5-Methyl deoxycytosine (5mC) is the most common base modification found in eukaryotic DNAs. In plant genome up to 30 % of cytosine residues can be methylated [I]. The target motifs for cytosine methylation are CG, CNG and CNN. Two distinct methyltransferase activities have been identified in Arabidopsis thaliana and Pisum sutivum (pea) nuclear extracts [7, 221. The methyltransferase gene coding for a 1534 amino acid protein, was isolated from A. thaliana. The nucleic acid sequence of this gene has significant homology to prokaryotic and mammalian cytosine methyltransferases . In plants - as in animals - methylation of cytosine residues is clearly correlated with regulation of gene expression [3, 41. Transient expression studies in which DNA was specifically methylated in vitro prior to being introduced into cells, have shown that genes with specifically
Plant Physiol. Biochem., 098 I -9428/98/05/O Elsevier, Paris
methylated promoter regions are inactive when transformed into plant protoplasts [lo]. Silencing of even strong promoters in transgenic organisms is straightly correlated with an increase of 5-mC residues in the vicinity of silenced elements [6, 15, 18, 291. However, the mode of action of methylated cytosine residues on regulation of transcription is still poorly understood. DNA methylation could affect transcription initiation in different ways. Methylated cytosine residues might directly block interaction between transcription factors and specific cis-acting DNA sequences [2,27], In another scenario, access of transcription factors might be blocked by non-specific methylated DNA-binding proteins or by the inability to penetrate a particular chromatin conformation formed by the methylated sequences with associated proteins. The presence of DNA binding proteins and structured chromatin would affect transcription initiation as well as elongation, resulting in the negative
effect. It was found that, the presence of 5-mC residues in the coding region of uidA gene significantly decreased transient expression of this gene after transfection of plant protoplasts [ 121. In prokaryotic cells a common DNA modification is 6-methyl deoxyadenine (6-mA). Over 1 % of adenine residues in any random DNA sequence of bacteria is methylated. Dam methylation in bacterial cells plays an essential role in: DNA repair processes, regulation of plasmid replication and control of expression of some genes [for review see 201. The product of the Escherichia coli darn gene, coding for DNA adenine methylase, is responsible for methylation of deoxyadenine residues in GATC sequences. For a long time, modification of deoxyadenine residues was believed to be specific to prokaryotes. However, it was demonstrated that 6-mA is also present in plant nuclear DNA [ 17, 19, 211. Interestingly, Torres et al.  described that expression of GUS, as a reporter, in parsley protoplasts is positively regulated by presence of 6-mA in their constructs. In addition, Rogers and Rogers  clearly demonstrated that activation of the a-amylase promoter in bombarded intact barley aleurone layers is correlated with the presence of 6-mA in template DNA. In this paper, we show that the presence of 6-mA in tobacco PR- la and PR-2d promoters is critical for their activities in tobacco protoplasts.
We have investigated the effect of Dam and Dcm methylation on transcriptional activity of tobacco PRla, PR-2d and viral 35s promoters in a quantitative transient expression system after transfection of tobacco protoplasts. Results from two independent experiments are presented in table I. The presence of 6-mA and 5-mC residues in plasmid DNA drastically increased activity of PR- 1a and PR-2d promoters (over lOO- and 20-fold, respectively). Dcm methylation had little or no effect on the activity of PR promoters in transfected protoplasts. In contrast, the effect of Dam and Dcm methylation on the CaMV 3.5s promoter was not observed. GUS activity was undetectable when a promoterless construct was used as control (data not shown).
Table I. Effect of methylation on expression from different promoters. mAmC; represents DNA prepared from E. cofi DHSc(, -A-C; represents DNA prepared from DamfDcm mutant, -A”C; represents DNA prepared from strain carrying dam- mutation, “GUS activity generated in an aliquot of extract that contained IO6 relative light units of luciferase activity. In these experiments we used GUS as d test reporter and luciferase as an internal standard. Data represent the average of 6 results from 2 independent experiments where all variations of methylation were analysed. SD; standard deviation. Promoter 3% CaMV Methylation mAmC -A”C -A-C mAmC -AmC -A-C mAmC -AmC -A% GUS units” (mean + SD) 78356 t 25539 119028 k 39409 147075 f 55718 4988 r 20+ 36 f 1220 + 22* 53 * 1543 11 22 479 19 40
2.1. Transient expression and 35s promoters
of GUS driven by PR-s
We analysed the influence of Dam and Dcm methylation on expression of three plasmid constructs in transient assays in plant cells. These constructs consisted of plant or plant virus promoter regions fused to L&A (GUS) as a marker gene [ 131. Plasmids were amplified in the DHSa, GM33 or GM48 E. coli bacterial strains. DNA prepared from DHSa had A residues methylated at the GmATC sequence and C residues methylated at the CmC(A/T)GG sequence (Dam and Dcm methylation). Plasmids prepared from GM33 or GM48 strains lacked Dam methylation. In addition, GM48 strain is deficient in Dcm methylation. The presence of 6-mA and 5-mC residues in plasmid DNA was confirmed by restriction digest analysis with methylation-dependent endonucleases (data not shown).
Plant Physiol. Biochem.
2.2. In vitro methylation To test whether modification of deoxyadenine residues in GATC sequences may directly affect the expression of the gene, commercially available Dam methylase was used to modify GM48 derived plasmids containing PR-la/GUS or PR-2d/GUS constructs in vitro. After Dam methylation plasmid DNA was electroporated into tobacco protoplasts. As shown in figure 1, methylation of deoxyadenine resulted in increased expression of both PlUGUS genes. The presence of 6-mA residues in DNA restored about 60 % of the PR-la and 90 % of the PR-2d promoters activities as compared to template isolated from DHSa strain, where methylation of adenine residues occurred in vivo. To test whether the increase of PR/GUS
Effect of DNA methylation
on PR promoters
Figure 2. Gel mobility
shift assay to detect nuclear proteins binding with PR- I a promoter fragments. The in vitro Dam methylated and non-methylated fragments of PR-la promoter were end-labelled with [a-‘*P]dCTP using Klenow polymerase and incubated with nuclear extract as described in Methods. (A) lanes 2 and 3 protein binding with 108 bp long fragment of PR-la promoter, where DNA probe was non-methylated (lane 2) or Dam methylated in vitro (lane 3). Lane 1, represents control where DNA was incubated in buffer without protein extract. (B) protein binding assay with 125 bp long fragment of PR-la promoter. Analysis was performed as in (A).
Figure 1. Effect of in vitro Dam methylation on transient expression PR 1 a/GUS and PR2d/GUS gene fusions. A, expression of PR- 1a/GUS gene; B, expression of PR-Zd/GUS gene; C, expression of 3WGUS gene. mAmC; represents DNA prepared from E. co/i DHSc(, -A-C; represents DNA prepared from GM48 strain. The striped bars represent GUS expression from DNA isolated from GM48 strain and methylated in vitro with Dam methylase. GUS Units means GUS activity generated from an aliquot of protein extract that produced 10h relative light units of luciferase activity. Data represent the average of 5 results from I representative experiment. This experiment was repeated 3 times.
expression is a consequence of adenine methylation within a sequence of the promoter region, the 35S/ GUS construct was used as a control. In contrast to the PR constructs, GUS expression driven by 35s promoter was not markedly influenced by Dam methylation in vitro or in vivo cfigurel C and table r). The 35s promoter region used in these experiments contained two Dam methylation target sites (comparable to the PR-la promoter which contains three sites). Therefore, the number of Dam methylation sites does not seemto be critical for 3% promoter activity.
2.3. Interaction between a nuclear protein and methyl-dA or dA residues of the PR-la promoter
One of the possible mechanisms by which methyldA residues could affect transcription assumesthat
there are specific proteins that interact only with methylated DNA. To analyse the binding of tobacco nuclear proteins to fragments of PR- 1a promoter gel mobility-shift assayswere performed. Sequence analysis indicated that the PR-la promoter (903 bp from the 5’ flanking region) contains three GATC motifs, with the A residuesat positions: -254, -333 and -733 from the transcription start site. Nuclear extracts isolated from the healthy tobacco plants and a 108 bp (position -787 to -679) or a 125 bp fragment (-352 to -227) of the PR-la promoter with GATC motifs, were prepared and tested in these experiments. The results of the gel mobility-shift assay are presented in figure 2. Upon incubation with nuclear extract, some of the 108 bp probe was retarded in a more slowly migrating complex. There were no significant differences in binding of nuclear proteins to in vitro Dam methylated @gure 2 A, lane 3) and non-methylated labelled probe (figure 2 A, lane 2). A similar complex was formed when the binding assayswere done with nuclear extracts isolated from plants infected with the Tobacco mosaic virus (data not shown). Figure 2 B shows the results of a representative mobility-shift assay with the 125 bp fragment of the PR- la promoter. Under the conditions used, a portion of the labelled probe interacted with tobacco nuclear
vol. 36 (5) 1998
R. Brodzik, J. Hennig
proteins, resulting in two retarded bands with very similar mobility (figure 2 B, lane 2). The same complexes were formed when the binding assay was carried out with 125 bp fragment which had been Dam methylated in vitro; however, the amount of slower migrating complex was greatly reduced Qigure 2 B, lane 3). To determine whether the retarded bands represent DNA-protein interactions, the nuclear extract was treated to denature proteins. Heating of the nuclear extract for 10 min at 65 “C or treatment with 10 c(g of proteinase K for 15 min at 37 “C prior to incubation with the probe completely abolished the appearance of retarded bands, while treatment with 10 pg RNase A had no effect (data not shown).
3. DISCUSSION The results presented in this paper clearly indicate that modifications of template DNA, isolated from genetically different bacterial strains, may affect transient expression in plant cells. It was shown (t&e I and$gure 1) that Dam methylation is responsible for a substantial increase in PR/GUS genes expression. Similar findings have been described for light responsive and fungal elicitor-responsive parsley promoters  and for two u-amylase promoters . This effect appears to be specific to plants. The presence of 6-mA in the simian virus 40T antigen gene had no effect on its expression when it was injected into hamster cell nuclei, while expression of the herpes simplex virus thymidine kinase gene was actually decreased when methyl-dA residues were present in its promoter . Similarly, methylation of adenine in the 12 EIA promoter of adenovirus had no effect on its expression in HeLa cells [ 141. There are some possible explanations of how 6-mA might increase transient expression from plasmid in plant cells. First, the interaction of specific proteins with methyl-dA residues might somehow lead to more effective promoter activation. It was shown that wheat germ nuclear extracts contained a factor that preferentially interacted with methyl-dA residues . However, since we observed no dramatic differences in binding of nuclear proteins to methylated or nonmethylated DNA templates (figure 2 A), this seems an unlikely explanation for methyl-dA dependent transcription from the PR-1 a promoter. On the other hand, lack of qualitative but presence of quantitative differences detected in the binding assay with the 125 bp methylated and non-methylated probe (figure 2 B)
Plant Physiol. Biuchem.
could suggest that only the faster-migrating complex is transcriptionally active. Second, DNA lacking in 6-mA is unstable and more readily degraded in tobacco protoplasts. In order to verify that, GUS activities were measured at different times after electroporation of protoplasts with nonmethylated or Dam methylated PR/GUS or 35S/GUS constructs. In all cases, GUS activities increased to a maximum level 24 h after transfection. The ratio of GUS activities obtained from methylated and nonmethylated templates in three independent experiments remained constant over this period (data not shown). While the high stability of GUS protein in electroporated cells makes it difficult to judge if the template DNA is degraded, the constant ratio of GUS activities argues against differential degradation rates for methylated and non-methylated template DNA. Third, the presence of non-methylated dA residues may cause plasmid DNA to be selectively eliminated, thus decreasing the expression of the reporter gene product. As it was previously described  plasmid DNA lacking in 6-mA predisposed it to rearrangement and elimination from barley cells. However, other possibilities cannot be excluded. Further investigations are still required to clarify the mechanisms responsible for methylation dependent PR gene activation in transient assays 4. METHODS
4.1. Nucleic acid manipulations. The following
bacterial EC coli K12 strains were used to obtain DNA with various methylation patterns: dam ’ dcm + DNA was propagated in DH5cx (FsupE44, AlaciJl69, hsdR1, recA1, endAl, gyrA96, thi-I. relA1); dam - dcm ’ DNA was grown in GM33 (F-thr, leu, thi, lacY, galK, galT, ara, fivA, tsx, dam, supE44) and dam dcm - DNA’s were grown in GM48 (Fthr, leu, thi, lacY, galK, galT, ara,fhvA, tsx, dam, dcm, supE44). The mutant host strains were obtained from the Institute of Biochemistry and Biophysics Bacterial Stock Center. Plasmid DNA was prepared using standard alkali-lysis protocol  and purified using CsCl gradient method. 4.2. In vitro Dam methylation. Plasmid DNA, PR- 1a/GUS and PR-2d/GUS constructs  propagated in bacterial strain GM48, was subsequently methylated in vitro for 8 h at 37 “C, with Dam methylase (New England BioLabs) using manufacturer’s recommended conditions. Methylation status of DNA and efficiency of Dam methylation was checked by digestion of DNA with the Dam dependent restriction enzymes (DpnI and Mbol) and analysed by electrophoresis in agarose gels. Modified DNA was purified by phenol/chloroform extraction followed by ethanol precipitation,
Effect of DNA methylation 4.3. Plant material. Tobacco (Nicotiuna tabacum cv. Xanthi-nc) cell suspension culture was derived and maintained as previously described by Murashige and Skoog [ 161. Cells were cultured in MS medium supplemented with Gamborg’s vitamins, 3 % (w/v) sucrose, 0.5 g.L-’ hydrolisate of caseine and 1 mg.L-’ 2,4-D at 26°C in the dark, with continuous agitation. Five days after passage, cultures were used for isolation of protoplasts according to procedure described by Hilson et al. [ll]. Fifty pg CsCl purified plasmid DNA was transferred into freshly prepared protoplasts by electroporation using the standard Bio-Rad Gene Pulser system and conditions recommended by the company. Each sample contained 5 x lo5 protoplasts, in 1 ml electroporation buffer (10 mM HEPES pH 7.0, 150 mM NaCl, 4 mM CaCI, and 200 mM mannitol). Following addition of DNA, each sample was pulsed once with 750V cm-’ and 960 pF capacitive discharge. After the electric pulse the protoplasts were incubated 15 min at 4*C, diluted in 5 ml of protoplast culture medium and incubated at 28’C, 24 h in the dark. The protoplast culture medium consisted of: MS salts supplemented with Gamborg’s vitamins, 3 % (w/v) sucrose, 300 mM mannitol, 1 mg.L-’ NAA and 0.1 mg.L-’ BAP (pH 5.8). 4.4. Enzymatic assays. For quantitative GUS and luciferase enzymatic assays: PRluidA or 35SluidA (GUS) constructs were used as the reporter and 35Slluc (luciferase) construct as internal standard. Mixture of plasmids (in ratio 1:l) was electroporated into protoplasts. After 24 h incubation at 28 “C protoplasts were collected by centrifugation. Pellets were either processed immediately or stored frozen at -70°C. Protein extracts were prepared by sonication and resuspended in 100 mM Tris-acetate buffer pH 7.75, 2 mM EDTA. P-Glucuronidase and Iuciferase activities were assayed with 1 mM 4-methylumbelliferyl P-D-glucuronide and 0.5 mM D-lucyferin, as substrates for the reactions, as was previously described . Protein concentration was determined with a BioRad protein assay kit. GUS activity generated in an aliquot of protein extract that contained IO6 relative light units of luciferase was labeled as ‘GUS Units’. 4.5. Gel mobility-shift assay. Nuclear protein extracts were prepared from 6-week old N. tubacum cv. Xanthi-nc plants as described by Green et al. . Fragments of PR-la promoter (108 bp and 125 bp) with the (GATC) target sequences for Dam methylase were cloned into plasmid vector. After digestion, inserts were gel-purified and end-labelled with [a3’P]dCTP, using the Klenow fragment of DNA I polymerase. The probe (0.5 ng) was mixed with 2 pg of nuclear extract in a buffer containing 0.5 pg of poly(dA-dT)(dA-dT), 20 mM HEPES (pH 6,7), 40 mM KCl, 0.1 mM EDTA, 1 mM dithiothreitol, 10 % (v/v) glycerol, incubated at room temperature for 15 min, and electrophoresed on 5 % (w/v) polyacrylamide gel in 0.5 x TBE buffer [S]. Subsequently the gel was dried and exposed to X-ray film. Acknowledgements. We thank A. S. Buchel for providing facilities and assistance with gels shift assays. We would like to thank K. K. Wobbe and M. Krzymowska for comments on
on PR promoters
the manuscript and A. Talarczyk for help with preparing the figures. This research was supported by a grant from Komitet Badaii Naukowych (project no. 6P20302006).
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