Arabidopsis Protocols pp 285-298 | Cite as
Global DNA Methylation Analysis Using Methyl-Sensitive Amplification Polymorphism (MSAP)
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Abstract
DNA methylation is a crucial epigenetic process which helps control gene transcription activity in eukaryotes. Information regarding the methylation status of a regulatory sequence of a particular gene provides important knowledge of this transcriptional control. DNA methylation can be detected using several methods, including sodium bisulfite sequencing and restriction digestion using methylation-sensitive endonucleases. Methyl-Sensitive Amplification Polymorphism (MSAP) is a technique used to study the global DNA methylation status of an organism and hence to distinguish between two individuals based on the DNA methylation status determined by the differential digestion pattern. Therefore, this technique is a useful method for DNA methylation mapping and positional cloning of differentially methylated genes. In this technique, genomic DNA is first digested with a methylation-sensitive restriction enzyme such as HpaII, and then the DNA fragments are ligated to adaptors in order to facilitate their amplification. Digestion using a methylation-insensitive isoschizomer of HpaII, MspI is used in a parallel digestion reaction as a loading control in the experiment. Subsequently, these fragments are selectively amplified by fluorescently labeled primers. PCR products from different individuals are compared, and once an interesting polymorphic locus is recognized, the desired DNA fragment can be isolated from a denaturing polyacrylamide gel, sequenced and identified based on DNA sequence similarity to other sequences available in the database. We will use analysis of met1, ddm1, and atmbd9 mutants and wild-type plants treated with a cytidine analogue, 5-azaC, or zebularine to demonstrate how to assess the genetic modulation of DNA methylation in Arabidopsis. It should be noted that despite the fact that MSAP is a reliable technique used to fish for polymorphic methylated loci, its power is limited to the restriction recognition sites of the enzymes used in the genomic DNA digestion.
Key words
DNA methylation MSAP Mutant lines 5-azaC and zebularineReferences
- 1.Ehrlich M, Wang RY (1981) 5-Methylcytosine in eukaryotic DNA. Science 212:1350–1357PubMedCrossRefGoogle Scholar
- 2.Doerfler W (1983) DNA methylation and gene activity. Annu Rev Biochem 52:93–124PubMedCrossRefGoogle Scholar
- 3.Riggs AD, Jones PA (1983) 5-Methylcytosine, gene regulation, and cancer. Adv Cancer Res 40:1–30PubMedCrossRefGoogle Scholar
- 4.Yaish MW, Colasanti J, Rothstein SJ (2011) The role of epigenetic processes in controlling flowering time in plants exposed to stress. J Exp Bot 62:3727–3735PubMedCrossRefGoogle Scholar
- 5.Boyko A et al (2010) Transgenerational adaptation of Arabidopsis to stress requires DNA methylation and the function of Dicer-like proteins. PLoS One 5:e9514PubMedCrossRefGoogle Scholar
- 6.Chan SW, Henderson IR, Jacobsen SE (2005) Gardening the genome: DNA methylation in Arabidopsis thaliana. Nat Rev Genet 6:351–360PubMedCrossRefGoogle Scholar
- 7.Molinier J et al (2006) Transgeneration memory of stress in plants. Nature 442:1046–1049PubMedCrossRefGoogle Scholar
- 8.Bartee L, Bender J (2001) Two Arabidopsis methylation-deficiency mutations confer only partial effects on a methylated endogenous gene family. Nucleic Acids Res 29:2127–2134PubMedCrossRefGoogle Scholar
- 9.Singer T, Yordan C, Martienssen RA (2001) Robertson’s mutator transposons in A. thaliana are regulated by the chromatin-remodeling gene decrease in DNA methylation (DDM1). Genes Dev 15:591–602PubMedCrossRefGoogle Scholar
- 10.Santi DV, Garrett CE, Barr PJ (1983) On the mechanism of inhibition of DNA-cytosine methyltransferases by cytosine analogs. Cell 33:9–10PubMedCrossRefGoogle Scholar
- 11.Yaish MW, Peng M, Rothstein SJ (2009) AtMBD9 modulates Arabidopsis development through the dual epigenetic pathways of DNA methylation and histone acetylation. Plant J 59:123–135PubMedCrossRefGoogle Scholar
- 12.Borowska N, Idziak D, Hasterok R (2011) DNA methylation patterns of Brachypodium distachyon chromosomes and their alteration by 5-azacytidine treatment. Chromosome Res 19:955–967PubMedCrossRefGoogle Scholar
- 13.Castilho A et al (1999) 5-Methylcytosine distribution and genome organization in triticale before and after treatment with 5-azacytidine. J Cell Sci 112(Pt 23):4397–4404PubMedGoogle Scholar
- 14.Cheng JC et al (2003) Inhibition of DNA methylation and reactivation of silenced genes by zebularine. J Natl Cancer Inst 95:399–409PubMedCrossRefGoogle Scholar
- 15.Zilberman D, Henikoff S (2007) Genome-wide analysis of DNA methylation patterns. Development 134:3959–3965PubMedCrossRefGoogle Scholar
- 16.Vos P et al (1995) AFLP: a new technique for DNA fingerprinting. Nucleic Acids Res 23: 4407–4414PubMedCrossRefGoogle Scholar
- 17.Beaulieu J, Jean M, Belzile F (2009) The allotetraploid Arabidopsis thaliana–Arabidopsis lyrata subsp. petraea as an alternative model system for the study of polyploidy in plants. Mol Genet Genomics 281:421–435PubMedCrossRefGoogle Scholar
- 18.Madlung A et al (2002) Remodeling of DNA methylation and phenotypic and transcriptional changes in synthetic Arabidopsis allotetraploids. Plant Physiol 129:733–746PubMedCrossRefGoogle Scholar
- 19.Murashige T, Skoog F (1962) A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol Plant 15:473–497CrossRefGoogle Scholar