Plant Epigenetics pp 73-79 | Cite as
Analysis of Global Genome Methylation Using the Cytosine-Extension Assay
Abstract
DNA methylation is a reversible covalent chemical modification of DNA intended to regulate chromatin structure and gene expression in a cell- and tissue-specific manner and in response to the environment. Cytosine methylation is predominantly occurring in plants, and cytosine nucleotides in plants can be methylated at symmetrical (CpG and CpHpG) and nonsymmetrical sites. Although there exists a number of various methods for the detection of cytosine methylation, most of them are either laborious or expensive or both. Here, we describe a quick inexpensive method for the analysis of global genome methylation using a cytosine-extension assay. The assay can be used for the analysis of the total level of CpG, CpHpG, and CpHpH methylation in a given sample of plant DNA.
Key words
Global genome methylation Cytosine extension Symmetrical methylation CpG CpHpG CpHpH Asymmetrical methylation HpaII MspIReferences
- 1.Law JA, Jacobsen SE (2010) Establishing, maintaining and modifying DNA methylation patterns in plants and animals. Nat Rev Genet 11(3):204–220CrossRefPubMedPubMedCentralGoogle Scholar
- 2.Saze H, Tsugane K, Kanno T, Nishimura T (2012) DNA methylation in plants: relationship to small RNAs and histone modifications, and functions in transposon inactivation. Plant Cell Physiol 53(5):766–784CrossRefPubMedGoogle Scholar
- 3.Shibuya K, Fukushima S, Takatsuji H (2009) RNA-directed DNA methylation induces transcriptional activation in plants. Proc Natl Acad Sci U S A 106(5):1660–1665CrossRefPubMedPubMedCentralGoogle Scholar
- 4.Bilichak A, Ilnystkyy Y, Hollunder J, Kovalchuk I (2012) The progeny of Arabidopsis thaliana plants exposed to salt exhibit changes in DNA methylation, histone modifications and gene expression. PLoS One 7(1):e30515CrossRefPubMedPubMedCentralGoogle Scholar
- 5.Boyko A, Blevins T, Yao Y, Golubov A, Bilichak A, Ilnytskyy Y, Hollunder J, Meins F Jr, Kovalchuk I (2010) Transgenerational adaptation of Arabidopsis to stress requires DNA methylation and the function of Dicer-like proteins. PLoS One 5(3):e9514CrossRefPubMedPubMedCentralGoogle Scholar
- 6.Cuozzo C, Porcellini A, Angrisano T, Morano A, Lee B, Di Pardo A, Messina S, Iuliano R, Fusco A, Santillo MR, Muller MT, Chiariotti L, Gottesman ME, Avvedimento EV (2007) DNA damage, homology-directed repair, and DNA methylation. PLoS Genet 3(7):e110CrossRefPubMedPubMedCentralGoogle Scholar
- 7.Schar P, Fritsch O (2011) DNA repair and the control of DNA methylation. Prog Drug Res 67:51–68PubMedGoogle Scholar
- 8.Kato M, Miura A, Bender J, Jacobsen SE, Kakutani T (2003) Role of CG and non-CG methylation in immobilization of transposons in Arabidopsis. Curr Biol 13(5):421–426CrossRefPubMedGoogle Scholar
- 9.Bassing CH, Swat W, Alt FW (2002) The mechanism and regulation of chromosomal V(D)J recombination. Cell 109(Suppl):S45–S55CrossRefPubMedGoogle Scholar
- 10.Bender J (1998) Cytosine methylation of repeated sequences in eukaryotes: the role of DNA pairing. Trends Biochem Sci 23(7):252–256CrossRefPubMedGoogle Scholar
- 11.Lan J, Hua S, He X, Zhang Y (2010) DNA methyltransferases and methyl-binding proteins of mammals. Acta Biochim Biophys Sin Shanghai 42(4):243–252CrossRefPubMedGoogle Scholar
- 12.Zhu JK (2009) Active DNA demethylation mediated by DNA glycosylases. Annu Rev Genet 43:143–166CrossRefPubMedPubMedCentralGoogle Scholar
- 13.Lippman Z, Gendrel AV, Black M, Vaughn MW, Dedhia N, McCombie WR, Lavine K, Mittal V, May B, Kasschau KD, Carrington JC, Doerge RW, Colot V, Martienssen R (2004) Role of transposable elements in heterochromatin and epigenetic control. Nature 430(6998):471–476CrossRefPubMedGoogle Scholar
- 14.Vaughn MW, Tanurdzic M, Lippman Z, Jiang H, Carrasquillo R, Rabinowicz PD, Dedhia N, McCombie WR, Agier N, Bulski A, Colot V, Doerge RW, Martienssen RA (2007) Epigenetic natural variation in Arabidopsis thaliana. PLoS Biol 5(7):e174CrossRefPubMedPubMedCentralGoogle Scholar
- 15.Wang X, Elling AA, Li X, Li N, Peng Z, He G, Sun H, Qi Y, Liu XS, Deng XW (2009) Genome-wide and organ-specific landscapes of epigenetic modifications and their relationships to mRNA and small RNA transcriptomes in maize. Plant Cell 21(4):1053–1069CrossRefPubMedPubMedCentralGoogle Scholar
- 16.Zilberman D, Gehring M, Tran RK, Ballinger T, Henikoff S (2007) Genome-wide analysis of Arabidopsis thaliana DNA methylation uncovers an interdependence between methylation and transcription. Nat Genet 39(1):61–69CrossRefPubMedGoogle Scholar
- 17.Cokus SJ, Feng S, Zhang X, Chen Z, Merriman B, Haudenschild CD, Pradhan S, Nelson SF, Pellegrini M, Jacobsen SE (2008) Shotgun bisulphite sequencing of the Arabidopsis genome reveals DNA methylation patterning. Nature 452(7184):215–219CrossRefPubMedPubMedCentralGoogle Scholar
- 18.Lister R, O’Malley RC, Tonti-Filippini J, Gregory BD, Berry CC, Millar AH, Ecker JR (2008) Highly integrated single-base resolution maps of the epigenome in Arabidopsis. Cell 133(3):523–536CrossRefPubMedPubMedCentralGoogle Scholar
- 19.Gehring M, Bubb KL, Henikoff S (2009) Extensive demethylation of repetitive elements during seed development underlies gene imprinting. Science 324(5933):1447–1451CrossRefPubMedPubMedCentralGoogle Scholar
- 20.Zemach A, Kim MY, Silva P, Rodrigues JA, Dotson B, Brooks MD, Zilberman D (2010) Local DNA hypomethylation activates genes in rice endosperm. Proc Natl Acad Sci U S A 107(43):18729–18734CrossRefPubMedPubMedCentralGoogle Scholar
- 21.Dowen RH, Pelizzola M, Schmitz RJ, Lister R, Dowen JM, Nery JR, Dixon JE, Ecker JR (2012) Widespread dynamic DNA methylation in response to biotic stress. Proc Natl Acad Sci U S A 109(32):E2183–E2191CrossRefPubMedPubMedCentralGoogle Scholar
- 22.Pogribny I, Yi P, James SJ (1999) A sensitive new method for rapid detection of abnormal methylation patterns in global DNA and within CpG islands. Biochem Biophys Res Commun 262(3):624–628CrossRefPubMedGoogle Scholar
- 23.McClelland M, Nelson M, Raschke E (1994) Effect of site-specific modification on restriction endonucleases and DNA modification methyltransferases. Nucleic Acids Res 22(17):3640–3659CrossRefPubMedPubMedCentralGoogle Scholar
- 24.Fujiwara H, Ito M (2002) Nonisotopic cytosine extension assay: a highly sensitive method to evaluate CpG island methylation in the whole genome. Anal Biochem 307(2):386–389CrossRefPubMedGoogle Scholar
- 25.Basnakian AG, James SJ (1996) Quantification of 3′OH DNA breaks by random oligonucleotide-primed synthesis (ROPS) assay. DNA Cell Biol 15(3):255–262CrossRefPubMedGoogle Scholar