In planta assays involving epigenetically silenced genes reveal inhibition of cytosine methylation by genistein
© Arase et al; licensee BioMed Central Ltd. 2012
Received: 16 January 2012
Accepted: 19 March 2012
Published: 19 March 2012
The Erratum to this article has been published in Plant Methods 2015 11:21
Cytosine methylation is involved in epigenetic control of gene expression in a wide range of organisms. An increasing number of examples indicate that changing the frequency of cytosine methylation in the genome is a feasible tool to engineer novel traits in plants. Although demethylating effects of compounds have been analyzed in human cultured cells in terms of suppressing cancer, their effect in plant cells has not been analyzed extensively. Here, we developed in planta assay systems to detect inhibition of cytosine methylation using plants that contain a transgene transcriptionally silenced by an epigenetic mechanism.
Seeds of two transgenic plants were used: a petunia line that has been identified as a revertant of the co-suppression of the chalcone synthase-A (CHS-A) gene and contains CHS-A transgenes whose transcription is repressed; Nicotiana benthamiana plants that contain the green fluorescent protein (GFP) reporter gene whose transcription is repressed through virus-induced transcriptional gene silencing. Seeds of these plants were sown on a medium that contained a demethylating agent, either 5-azacytidine or trichostatin A, and the restoration of the transcriptionally active state of the transgene was detected in seedlings. Using these systems, we found that genistein, a major isoflavonoid compound, inhibits cytosine methylation, thus restoring transgene transcription. Genistein also restored the transcription of an epigenetically silenced endogenous gene in Arabidopsis plants.
Our assay systems allowed us to assess the inhibition of cytosine methylation, in particular of maintenance of methylation, by compounds in plant cells. These results suggest a novel role of flavonoids in plant cells and that genistein is useful for modifying the epigenetic state of plant genomes.
Cytosine methylation is an epigenetic mark present in many eukaryotes, including plants, vertebrates and fungi , and plays an important role in various biological processes including regulation of gene expression, stability of the genome, cellular differentiation and development . Transposons and repeats are frequently methylated in a wide range of species . Loss of cytosine methylation induces reactivation and transposition of transposons [4–7], suggesting that cytosine methylation represents the primary mechanism of transposon suppression in host genomes . Cytosine methylation also functions to maintain a repressed chromatin state and stably silence promoter activity . A genome-wide analysis of Arabidopsis thaliana uncovered an interdependence between cytosine methylation and transcription .
Cytosine methylation in mammalian genomes occurs predominantly in the context of CG sequences. CG methylation is also the most common modification in plant genomes, but plant genomes also have cytosine methylation at CHG and CHH (where H is A, C or T) sequences. In plants, cytosine methylation can be established through RNA-directed DNA methylation (RdDM) , in which DOMAINS REARRANGED METHYLTRANSFERASE1 and 2 (DRM1 and DRM2), Arabidopsis orthologues of mammalian de novo methyltransferase DNMT3, are guided by small RNAs and targeted to DNA . Whether all de novo cytosine methylation in Arabidopsis is guided by small RNAs is not known . The maintenance of CG methylation requires METHYLTRANSFERASE1 (MET1), an orthologue of mammalian DNMT1, while that of non-CG methylation requires DRM1, DRM2 and the plant-specific methyltransferase CHROMOMETHYLASE3 (CMT3), which function redundantly . On the other hand, a family of DNA glycosylases can remove cytosine methylation in plants .
Modification of the epigenetic state, in particular the frequency of cytosine methylation, has become a feasible tool to engineer novel traits in plants. Transcriptional gene silencing (TGS) has been induced by targeting small RNA to a transgene promoter via RdDM . This approach has recently been applied to silence an endogenous gene using a viral vector, which led to the production of a plant that does not carry a transgene but has altered traits [13, 14]. In addition to inducing changes via RdDM, changes in epigenetic state can also be induced randomly in the genome by inhibiting cytosine methylation. Although a method for targeted demethylation has not been developed for any organisms, transgenerational inheritance of a state of decreased methylation with an increased transcriptional activity has been observed for limited loci in plants . These findings have prompted scientists to alter the cytosine methylation state of the genome that harbors novel epi-alleles. Examples of such attempts include a plant line that had a higher expression level of the Xa21G gene, which confers resistance against Xanthomonas oryzae, was produced in the progeny of rice plants treated with a demethylating agent ; various altered phenotypes were observed in a population of Arabidopsis recombinant inbred lines with epigenetically mosaic chromosomes consisting of wild-type and CG methylation-depleted segments that were derived from a cross between wild type and a met1 mutant ; exposure of Arabidopsis plants to various environmental stresses resulted in increased global genome methylation and a higher level of tolerance to stresses in the progeny . Similarly, plants from an isogenic canola population, selected on the basis of respiration intensity, were used to generate sublines that have distinct characteristics and different epigenetic states .
One of the methods to modify the frequency of methylcytosine in the genome is to treat the organism with demethylating agents. 5-Azacytidine (5-azaC; trade name Vidaza) and its deoxy derivative 5-aza-2'-deoxycytidine (Decitabine; trade name Dacogen) were first synthesized over 40 years ago and are commonly applied inhibitors of cytosine methylation in plants and animals . These reagents induce hypomethylation and reactivate transcriptionally repressed genes; hence, they have been used for cancer therapeutics to suppress hypermethylation of tumor suppressor genes in human. However, these toxic drugs are unstable in aqueous solution. More recently, 2-pyrimidine-1-β-D-riboside (Zebularine) induced the reactivation of hypermethylated genes in cultured human cells  and in plants . Although this compound is stable in aqueous solutions, it raises considerable concerns about toxicity as a nucleoside analog. Besides nucleoside analogs, procainamide has been identified as an inhibitor of the human DNA methyltransferase DNMT1 . Trichostatin A, known as a specific inhibitor of histone deacetylase, is also known to cause demethylation in Neurospora crassa  and plants [25, 26] and downregulates DNA methyltransferase in human cells . There is a growing list of cytosine methylation inhibitors in addition to these compounds (ref. ; and references therein).
In terms of finding compounds effective in suppressing cancer, natural products have been screened for inhibitory activity of cytosine methylation using human cancer cells. Through such analysis, plant secondary metabolites, polyphenols, were shown to be antagonists of cytosine methylation in cultured human cells. These compounds include (-)-epigallocatechin-3-O-gallate (EGCG) from green tea [29, 30], genistein from soybean [30–32], caffeic acid and chlorogenic acid from coffee , polyphenols from Annurca apples , and lycopene from tomato . In vitro studies revealed that some of these compounds inhibit DNA methyltransferase activity of human DNMT1 [31, 33, 35]. However, whether these compounds inhibit methylation in plant cells has not been examined.
In the present study, we focused on genistein, a major pharmacologically active isoflavone abundant in soybean  and examined whether genistein can function as a methylation inhibitor in plant cells. For that purpose, we developed two different in planta assay systems, both involving an epigenetically silenced transgene. In these systems, an increase in the mRNA level of the transgene, which is otherwise transcriptionally repressed, indicates inhibition of cytosine methylation. Through these assays, we demonstrated that genistein functions as a methylation inhibitor, thus identifying the first methylation inhibitor of plant origin that acts in plant cells.
Detection of inhibition of cytosine methylation by genistein in transgenic petunia plants that have a transcriptionally silenced transgene
Detecting inhibition of cytosine methylation by genistein in transgenic Nicotiana benthamiana plants that have a transcriptionally silenced transgene
We also analyzed the inhibitory effect of genistein on cytosine methylation using a different assay system (Figure 1, right). We previously developed a system that induces RNA-mediated heritable TGS using a virus vector [13, 14, 37]. In this system, a virus vector carrying a portion of a gene promoter efficiently induces cytosine methylation and TGS by targeting double-stranded RNA to the promoter, and the epigenetically silenced state of the gene is transmitted to subsequent generations. On the other hand, the virus that induced TGS is not transmitted to the next generation [13, 37]. We used seeds produced on transgenic N. benthamiana plants, in which TGS of the GFP gene was systemically induced by infecting the plants with a recombinant virus that contained a portion of the CaMV 35S promoter, which controlled transcription of the GFP gene (ref. ; for details, see also Methods). Seeds produced on such plants were sown on a medium for germination, and the resultant plants were grown in the medium for 1 month.
Genistein reactivates a transcriptionally silenced endogenous gene in Arabidopsis
In the in planta assay systems that we have established, we can detect the restoration of the transcriptionally active state of a gene whose transcription is epigenetically repressed. Petunia C002 line arose as a spontaneous revertant plant from a CHS-A co-suppressed plant line in which transcripts of both the CHS-A transgene and endogenous CHS-A gene are degraded. Because transcription of the CHS-A transgene is repressed in C002 plants and small RNAs corresponding to both the CHS-A coding region and CaMV 35S promoter are not detected in this plant line , it is not likely that cytosine methylation of the CaMV 35S promoter is established in each generation of this plant line through de novo methylation. Such a lack of de novo methylation is unequivocally true of the promoter of the silenced GFP transgene in N. benthamiana plants. In this plant, the virus that induced cytosine methylation of the CaMV 35S promoter is eliminated during the meiosis and is not transmitted to the subsequent generation. As a consequence, the progeny plants that were used for the assay in the present study do not have viral RNAs that trigger de novo methylation on the CaMV 35S promoter . Accordingly, reduction in the frequency of methylcytosine detected in the genistein-treated plants indicates inhibition of the process to maintain cytosine methylation rather than inhibition of de novo methylation. This notion is consistent with the fact that genistein inhibits the in vitro activity of human DNA methyltransferase DNMT1, an orthologue of Arabidopsis MET1 [31, 32]. Thus, this assay system allows the detection of a compound's inhibitory activity that is specific to the maintenance process of the cytosine methylation. In this regard, our in planta assay systems can be used to dissect the functions of demethylating compounds. In addition, our assay systems may be useful for screening natural or artificial compounds that can inhibit cytosine methylation.
Using these assay systems, we found that genistein induced the reduction of the methylation of cytosines in all sequence contexts, namely, CG, CHG and CHH. Genistein may affect the catalytic function of more than one DNA methyltransferase that controls the maintenance of cytosine methylation. In Arabidopsis, in addition to controlling CG methylation, MET1 is required to maintain non-CG methylation: pre-existing CG methylation, or a chromatin mark associated with it, might be able to attract non-CG methylation . Therefore, it is also possible that non-CG methylation is reduced as an indirect effect of inhibition of MET1 orthologue(s) by genistein. Alternatively, histone modification may be a target of genistein. Genetic studies have identified a link between histone and cytosine methylation: mutations in CMT3 and histone H3 lysine 9 (H3K9) methyltransferase gene KYP cause a reduction in CHG methylation (ref. ; and references therein). The CMT3 protein can interact with histone H3 when it is methylated at lysines 9 and 27, suggesting that these modifications recruit the CMT3 protein to target loci . In human cancer cells, genistein can induce demethylation and acetylation of H3K9 . Taking into account these observations, it is also conceivable that the observed reduction in cytosine methylation in plants treated with genistein is mediated by changes in histone modification, which result in failure to recruit DNA methyltransferase(s) to the targets. Such a link between cytosine methylation and histone modification is consistent with the restoration of transgene transcription in plants treated with TSA, an inhibitor of histone deacetylase. In fact, this compound can induce demethylation of cytosine in organisms including plants [24–26].
Genistein may serve as an agent to modify the epigenetic state of a plant genome
In terms of producing plants with novel traits, changes in methylation levels can be induced by crossing plants with methyltransferase mutants. However, cytosine methylation affected by a mutation in a single methyltransferase gene is confined to a particular sequence context because a particular methyltransferase is responsible for methylation of cytosine in a specific sequence context. Moreover, mutants in cytosine methylation are available only in limited plant species. Therefore, genistein, like cytidine analogs such as 5-azaC or zebularine, has the potential advantage over the use of mutants in efficiently producing a novel epi-allele in plants. In addition, polyphenols such as genistein have a potential advantage over cytidine analogs. The mechanism of inhibition of methyltransferase by cytidine analogs involves incorporation of these compounds into DNA [42, 43]. Incorporation of cytidine analogs results in a permanent alteration of the genome, often leading to mutation, whose process is also mediated by a DNA methyltransferase . On the other hand, polyphenols are not incorporated into DNA and, hence, likely inhibit cytosine methylation without inducing mutation. This notion is consistent with the result of computational modeling, which suggests that EGCG directly interacts with the catalytic site of human DNMT1 to inhibit its activity . Likewise, genistein might directly interact with MET1 orthologues and/or other methyltransferases without being incorporated into DNA in plants. Accordingly, in terms of inducing an epigenetic change rather than a mutation, the use of genistein may be preferred over nucleoside analogs, although the efficiency of the demethylation between genistein and nucleoside analogs may differ.
Genistein inhibition of cytosine methylation suggests a novel role for flavonoids in plant cells
The reduction in the frequency of methylation suggests that exogenously applied genistein was active in the nucleus of the plant cell. Isoflavonoids are mostly present as glycosides or malonyl glycosides in plant tissues such as soybean seeds . Whether these isoflavone glycoconjugates (e.g., genistin) or isoflavone malonyl glycoconjugates (e.g., malonylgenistin) affect cytosine demethylation the same as isoflavone aglycone (e.g., genistein) remains to be examined. Flavonoids, a major class of polyphenols, include flavonols, anthocyanins, proanthocyanidins (condensed tannins), and isoflavonoids. These compounds have diverse functions in plants, e.g., in flower pigmentation, UV protection, signaling, male fertility, and defense against pathogens [46–48].
Flavonoids are synthesized in the cytoplasm and are then deposited in a variety of cellular sites including the vacuole and cell wall or secreted to outside . In addition to these locations, flavonoids can also be present in plant cell nuclei [49–52]. However, the significance of the accumulation of flavonoids in nuclei has not been clear, although anthocyanin and DNA have been presumed to form a protective complex against oxidative damage . Here we found that genistein inhibited transcriptional repression not only of the transgene but also of an endogenous plant gene. The present results thus indicate the involvement of a flavonoid in the epigenetic control of transcription, a novel role for flavonoids in plant cells.
We developed an in planta assay systems using plants of transgenic petunia and N. benthamiana that contain epigenetically silenced transgenes, which allow the detection of inhibition of cytosine methylation by a particular compound. Transcriptional repression of the GFP gene in N. benthamiana plants was induced by a recombinant virus, which is not transmitted to the subsequent generation. Therefore, the assay for restoration of GFP expression in the progeny plants in our system is particularly useful for detecting the inhibition of the maintenance process of cytosine methylation. Using these assay systems, we found that genistein has such activity. Genistein also had restoring effects on an epigenetically silenced endogenous gene in Arabidopsis, suggesting that the compound is useful for modifying epigenetic state of plant genomes.
Seeds of a transgenic petunia (Petunia hybrida) line C002 [26, 54–56], designated CHS38P, were used. C002 is a line derived from a spontaneous revertant plant from the C001 line (designated as CHS38W), which was obtained by the transformation of wild-type plant V26 with the CHS-A transgene controlled by the CaMV 35S promoter and the NOS terminator . The C001 line produces white flowers as a consequence of stable co-suppression of the CHS-A genes , while revertant line C002 produces pigmented wild-type flowers as a consequence of transcriptional repression of the CHS-A transgene, which occurred via an epigenetic mechanism involving cytosine methylation .
Seeds of transgenic N. benthamiana line 16c plants infected with a recombinant Cucumber mosaic virus containing a segment of the transgene promoter  were also used. The N. benthamiana 16c line contains a single copy of the GFP transgene whose transcription is controlled by the CaMV 35S promoter . Plants of this line were infected with the recombinant virus containing the -208 to -89 region (positions are relative to the transcriptional initiation site) of the promoter. We have found that the resultant plants systemically lose GFP fluorescence through the induction of TGS mediated by RdDM of the promoter, and the silenced state is stably transmitted to the subsequent generations . In addition, seeds of A. thaliana ecotype Columbia (Col-0) were used for analyzing changes in the expression of endogenous genes.
Treatment of plants with demethylating agents
Surface-sterilized seeds of P. hybrida, N. bentahamiana or A. thaliana were sown on plates of medium containing 1/2 concentration of MS salts  and 10 g/l sucrose. The pH of the medium was adjusted to 5.7 before autoclaving. Media were solidified with 0.8% (w/v) agar, amended with final concentrations of 20 μM aqueous 5-azaC, 2-6 μM TSA in methanol or 10-50 μM genistein in 80% methanol just before plates were poured. After 10 or 20 seeds were sown per plate, plates were incubated at a photoperiod of 16-h light/8-h dark, 26°C. Development of plants treated with these compounds was slightly affected (e.g., delay in germination). Each treatment was done in triplicate.
Gene expression analysis
Isolation of total RNA and quantitative RT-PCR were done essentially as described previously . The following primer pairs were used for the PCR: for the CHS-A transgene, CHS-trans + 12 F (5'-CTCATTTCTCTATTACTTCAGCC-3') and 2350 (5'-GTGCTTTGATCAACACAGTTTG-3'); for the petunia α-tubulin gene, tub1110F (5'-GCCACCATCAAGACCAAGC-3') and tub201R (5'-ACCTCAGCAACACTGGTTGA-3'); for the GFP gene, mGFP + 148 F (5'-ACTGGAAAACTACCTGTTCC-3') and mGFP + 344R (5'-TCAAACTTGACTTCAGCACG-3'); for the N. benthamiana actin gene, Nb-actF (5'-GAAGATACTCACAGAAAGAGG-3'); and Nb-actR2 (5'-GGAGCTAATGCAGTAATTTGG-3'); for the Arabidopsis TSI gene, TSI-F1 5'-TAGAGCAGTTAACCCGAACC-3') and TSI-R1 (5'-TAGCTTACTTCACCTAGAGTC-3'); for the Arabidopsis eukaryotic initiation factor 4A-1 (eIF4A1) gene (At3g13920), eIF4A-F1 (5'-CATGTTGAAGAGGCAGTCTC-3') and eIF4A-R1 (5'-GAAGAACACACCAACTTGGA-3'). Differences in mRNA level between plants were statistically analyzed using Student's t-test or Bonferroni/Dunn test. GFP fluorescence was examined with an epifluorescence microscope (MVX10; Olympus, Tokyo, Japan) equipped with a GFP mirror unit (U-MGFP HQ/XL and U-MGFP/XL; Olympus).
Analysis of cytosine methylation
Cytosine methylation of the CaMV 35S promoter was analyzed by bisulfite sequencing. DNA was isolated from young seedlings grown on plates using Nucleon PhytoPure DNA extraction kit (Amersham Biosciences, Piscataway, NJ, USA). Bisulfite treatment of DNA, subsequent PCR amplification, and a control experiment to ensure the completion of the treatment were done as described previously .
We thank Shungo Otagaki for technical advice and seeds produced on virus-infected N. benthamiana plants. We also thank David Baulcombe for the original 16c N. benthamiana plants, and Neal Gutterson and Richard Jorgensen for transgenic petunia plants. We are grateful to Keisuke Kitamura and Chikara Masuta for helpful discussion on the roles of flanovoids in soybean and virus-induced gene silencing, respectively. Our work is supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan.
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