In planta assays involving epigenetically silenced genes reveal inhibition of cytosine methylation by genistein

Plant Methods

Akira Kanazawa¹  Megumi Kasai¹  Sachiko Arase¹ 

[1] Research Faculty of Agriculture, Hokkaido University, Sapporo 060-8589, Japan

关键词: Transcriptional gene silencing;    RNA-directed DNA methylation;    Genistein;    Demethylating agents;    Cytosine methylation;   

Others  :  822590
DOI  :  10.1186/1746-4811-8-10

 received in 2012-01-16, accepted in 2012-03-19,  发布年份 2012

【 摘 要 】

Background

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.

Results

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.

Conclusions

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.

【 授权许可】

   
2012 Arase et al; licensee BioMed Central Ltd.

【 图 表 】

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【 参考文献 】

• [1]Zemach A, McDaniel IE, Silva P, Zilberman D: Genome-wide evolutionary analysis of eukaryotic DNA methylation. Science 2010, 328:916-919.
• [2]Bird A: DNA methylation patterns and epigenetic memory. Genes Dev 2002, 16:6-21.
• [3]Zhang X, Yazaki J, Sundaresan A, Cokus S, Chan SW, Chen H, Henderson IR, Shinn P, Pellegrini M, Jacobsen SE, Ecker JR: Genome-wide high-resolution mapping and functional analysis of DNA methylation in Arabidopsis. Cell 2006, 126:1189-1201.
• [4]Kato M, Miura A, Bender J, Jacobsen SE, Kakutani T: Role of CG and non-CG methylation in immobilization of transposons in Arabidopsis. Curr Biol 2003, 13:421-426.
• [5]Miura A, Yonebayashi S, Watanabe K, Toyama T, Shimada H, Kakutani T: Mobilization of transposons by a mutation abolishing full DNA methylation in Arabidopsis. Nature 2001, 411:212-214.
• [6]Singer T, Yordan C, Martienssen RA: Robertson's Mutator transposons in A. thaliana are regulated by the chromatin-remodeling gene Decrease in DNA Methylation (DDM1). Genes Dev 2001, 15:591-602.
• [7]Tsukahara S, Kobayashi A, Kawabe A, Mathieu O, Miura A, Kakutani T: Bursts of retrotransposition reproduced in Arabidopsis. Nature 2009, 461:423-426.
• [8]Goll MG, Bestor TH: Eukaryotic cytosine methyltransferases. Annu Rev Biochem 2005, 74:481-514.
• [9]Suzuki MM, Bird A: DNA methylation landscapes: provocative insights from epigenomics. Nat Rev Genet 2008, 9:465-476.
• [10]Zilberman D, Gehring M, Tran RK, Ballinger T, Henikoff S: Genome-wide analysis of Arabidopsis thaliana DNA methylation uncovers an interdependence between methylation and transcription. Nat Genet 2007, 39:61-69.
• [11]Matzke M, Kanno T, Daxinger L, Huettel B, Matzke AJ: RNA-mediated chromatin-based silencing in plants. Curr Opin Cell Biol 2009, 21:367-376.
• [12]Chan SW, Henderson IR, Jacobsen SE: Gardening the genome: DNA methylation in Arabidopsis thaliana. Nat Rev Genet 2005, 6:351-360.
• [13]Kanazawa A, Inaba J, Shimura H, Otagaki S, Tsukahara S, Matsuzawa A, Kim BM, Goto K, Masuta C: Virus-mediated efficient induction of epigenetic modifications of endogenous genes with phenotypic changes in plants. Plant J 2011, 65:156-168.
• [14]Kanazawa A, Inaba J, Kasai M, Shimura H, Masuta C: RNA-mediated epigenetic modifications of an endogenous gene targeted by a viral vector: a potent gene silencing system to produce a plant that does not carry a transgene but has altered traits. Plant Signal Behav 2011, 6:1090-1093.
• [15]Paszkowski J, Grossniklaus U: Selected aspects of transgenerational epigenetic inheritance and resetting in plants. Curr Opin Plant Biol 2011, 14:195-203.
• [16]Akimoto K, Katakami H, Kim HJ, Ogawa E, Sano CM, Wada Y, Sano H: Epigenetic inheritance in rice plants. Ann Bot 2007, 100:205-217.
• [17]Reinders J, Wulff BB, Mirouze M, Marí-Ordóñez A, Dapp M, Rozhon W, Bucher E, Theiler G, Paszkowski J: Compromised stability of DNA methylation and transposon immobilization in mosaic Arabidopsis epigenomes. Genes Dev 2009, 23:939-950.
• [18]Boyko A, Blevins T, Yao Y, Golubov A, Bilichak A, Ilnytskyy Y, Hollunder J, Hollander J, Meins F, Kovalchuk I: Transgenerational adaptation of Arabidopsis to stress requires DNA methylation and the function of Dicer-like proteins. PLoS One 2010, 5:e9514.
• [19]Hauben M, Haesendonckx B, Standaert E, Van Der Kelen K, Azmi A, Akpo H, Van Breusegem F, Guisez Y, Bots M, Lambert B, et al.: Energy use efficiency is characterized by an epigenetic component that can be directed through artificial selection to increase yield. Proc Natl Acad Sci USA 2009, 106:20109-20114.
• [20]Jones PA, Taylor SM: Cellular differentiation, cytidine analogs and DNA methylation. Cell 1980, 20:85-93.
• [21]Cheng JC, Matsen CB, Gonzales FA, Ye W, Greer S, Marquez VE, Jones PA, Selker EU: Inhibition of DNA methylation and reactivation of silenced genes by zebularine. J Natl Cancer Inst 2003, 95:399-409.
• [22]Baubec T, Pecinka A, Rozhon W, Mittelsten Scheid O: Effective, homogeneous and transient interference with cytosine methylation in plant genomic DNA by zebularine. Plant J 2009, 57:542-554.
• [23]Lee BH, Yegnasubramanian S, Lin X, Nelson WG: Procainamide is a specific inhibitor of DNA methyltransferase 1. J Biol Chem 2005, 280:40749-40756.
• [24]Selker EU: Trichostatin A causes selective loss of DNA methylation in Neurospora. Proc Natl Acad Sci USA 1998, 95:9430-9435.
• [25]Lawrence RJ, Earley K, Pontes O, Silva M, Chen ZJ, Neves N, Viegas W, Pikaard CS: A concerted DNA methylation/histone methylation switch regulates rRNA gene dosage control and nucleolar dominance. Mol Cell 2004, 13:599-609.
• [26]Kanazawa A, O'Dell M, Hellens RP: Epigenetic inactivation of chalcone synthase-A transgene transcription in petunia leads to a reversion of the post-transcriptional gene silencing phenotype. Plant Cell Physiol 2007, 48:638-647.
• [27]Januchowski R, Dabrowski M, Ofori H, Jagodzinski PP: Trichostatin A down-regulates DNA methyltransferase 1 in Jurkat T cells. Cancer Lett 2007, 246:313-317.
• [28]Chuang JC, Yoo CB, Kwan JM, Li TW, Liang G, Yang AS, Jones PA: Comparison of biological effects of non-nucleoside DNA methylation inhibitors versus 5-aza-2'-deoxycytidine. Mol Cancer Ther 2005, 4:1515-1520.
• [29]Fang MZ, Wang Y, Ai N, Hou Z, Sun Y, Lu H, Welsh W, Yang CS: Tea polyphenol (-)-epigallocatechin-3-gallate inhibits DNA methyltransferase and reactivates methylation-silenced genes in cancer cell lines. Cancer Res 2003, 63:7563-7570.
• [30]Fang M, Chen D, Yang CS: Dietary polyphenols may affect DNA methylation. J Nutr 2007, 137:223S-228S.
• [31]Fang MZ, Chen D, Sun Y, Jin Z, Christman JK, Yang CS: Reversal of hypermethylation and reactivation of p16INK4a, RARbeta, and MGMT genes by genistein and other isoflavones from soy. Clin Cancer Res 2005, 11:7033-7041.
• [32]King-Batoon A, Leszczynska JM, Klein CB: Modulation of gene methylation by genistein or lycopene in breast cancer cells. Environ Mol Mutagen 2008, 49:36-45.
• [33]Lee WJ, Zhu BT: Inhibition of DNA methylation by caffeic acid and chlorogenic acid, two common catechol-containing coffee polyphenols. Carcinogenesis 2006, 27:269-277.
• [34]Fini L, Selgrad M, Fogliano V, Graziani G, Romano M, Hotchkiss E, Daoud YA, De Vol EB, Boland CR, Ricciardiello L: Annurca apple polyphenols have potent demethylating activity and can reactivate silenced tumor suppressor genes in colorectal cancer cells. J Nutr 2007, 137:2622-2628.
• [35]Lee WJ, Shim JY, Zhu BT: Mechanisms for the inhibition of DNA methyltransferases by tea catechins and bioflavonoids. Mol Pharmacol 2005, 68:1018-1030.
• [36]Park OJ, Surh YJ: Chemopreventive potential of epigallocatechin gallate and genistein: evidence from epidemiological and laboratory studies. Toxicol Lett 2004, 150:43-56.
• [37]Otagaki S, Kawai M, Masuta C, Kanazawa A: Size and positional effects of promoter RNA segments on virus-induced RNA-directed DNA methylation and transcriptional gene silencing. Epigenetics 2011, 6:681-691.
• [38]Beisler JA: Isolation, characterization, and properties of a labile hydrolysis product of the antitumor nucleoside, 5-azacytidine. J Med Chem 1978, 21:204-208.
• [39]Mathieu O, Probst AV, Paszkowski J: Distinct regulation of histone H3 methylation at lysines 27 and 9 by CpG methylation in Arabidopsis. EMBO J 2005, 24:2783-2791.
• [40]Lindroth AM, Shultis D, Jasencakova Z, Fuchs J, Johnson L, Schubert D, Patnaik D, Pradhan S, Goodrich J, Schubert I, Jenuwein T, Khorasanizadeh S, Jacobsen SE: Dual histone H3 methylation marks at lysines 9 and 27 required for interaction with CHROMOMETHYLASE3. EMBO J 2004, 23:4286-4296.
• [41]Kikuno N, Shiina H, Urakami S, Kawamoto K, Hirata H, Tanaka Y, Majid S, Igawa M, Dahiya R: Genistein mediated histone acetylation and demethylation activates tumor suppressor genes in prostate cancer cells. Int J Cancer 2008, 123:552-560.
• [42]Christman JK: 5-Azacytidine and 5-aza-2'-deoxycytidine as inhibitors of DNA methylation: mechanistic studies and their implications for cancer therapy. Oncogene 2002, 21:5483-5495.
• [43]Gowher H, Jeltsch A: Mechanism of inhibition of DNA methyltransferases by cytidine analogs in cancer therapy. Cancer Biol Ther 2004, 3:1062-1068.
• [44]Jackson-Grusby L, Laird PW, Magge SN, Moeller BJ, Jaenisch R: Mutagenicity of 5-aza-2'-deoxycytidine is mediated by the mammalian DNA methyltransferase. Proc Natl Acad Sci USA 1997, 94:4681-4685.
• [45]Dhaubhadel S, Farhangkhoee M, Chapman R: Identification and characterization of isoflavonoid specific glycosyltransferase and malonyltransferase from soybean seeds. J Exp Bot 2008, 59:981-994.
• [46]Dixon RA, Steele CL: Flavonoids and isoflavonoids - a gold mine for metabolic engineering. Trends Plant Sci 1999, 4:394-400.
• [47]Winkel-Shirley B: Flavonoid biosynthesis. A colorful model for genetics, biochemistry, cell biology, and biotechnology. Plant Physiol 2001, 126:485-493.
• [48]Buer CS, Imin N, Djordjevic MA: Flavonoids: new roles for old molecules. J Integr Plant Biol 2010, 52:98-111.
• [49]Hutzler P, Fischbach R, Heller W, Jungblut T, Reuber S, Schmitz R, Veit M, Weissenbock G, Schnitzler J: Tissue localization of phenolic compounds in plants by confocal laser scanning microscopy. J Exp Bot 1998, 49:953-965.
• [50]Feucht W, Treutter D, Polster J: Flavanol binding of nuclei from tree species. Plant Cell Rep 2004, 22:430-436.
• [51]Buer CS, Muday GK: The transparent testa4 mutation prevents flavonoid synthesis and alters auxin transport and the response of Arabidopsis roots to gravity and light. Plant Cell 2004, 16:1191-1205.
• [52]Saslowsky DE, Warek U, Winkel BS: Nuclear localization of flavonoid enzymes in Arabidopsis. J Biol Chem 2005, 280:23735-23740.
• [53]Sarma A, Sharma R: Anthocyanin-DNA copigmentation complex: mutual protection against oxidative damage. Phytochemistry 1999, 52:1313-1318.
• [54]Metzlaff M, O'Dell M, Cluster PD, Flavell RB: RNA-mediated RNA degradation and chalcone synthase A silencing in petunia. Cell 1997, 88:845-854.
• [55]O'Dell M, Metzlaff M, Flavell R: Post-transcriptional gene silencing of chalcone synthase in transgenic petunias, cytosine methylation and epigenetic variation. Plant J 1999, 18:33-42.
• [56]Kasai M, Koseki M, Goto K, Masuta C, Ishii S, Hellens RP, Taneda A, Kanazawa A: Coincident sequence-specific RNA degradation of linked transgenes in the plant genome. Plant Mol Biol 2012, 78:259-273.
• [57]Napoli C, Lemieux C, Jorgensen R: Introduction of a chimeric chalcone synthase gene into petunia results in reversible co-suppression of homologous genes in trans. Plant Cell 1990, 2:279-289.
• [58]Ruiz MT, Voinnet O, Baulcombe DC: Initiation and maintenance of virus-induced gene silencing. Plant Cell 1998, 10:937-946.
• [59]Murashige T, Skoog F: A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 1962, 15:473-497.
• [60]Kanazawa A, O'Dell M, Hellens RP: The binding of nuclear factors to the as-1 element in the CaMV 35S promoter is affected by cytosine methylation in vitro. Plant Biol 2007, 9:435-441.