Protocol: a beginner’s guide to the analysis of RNA-directed DNA methylation in plants

Plant Methods

Jian-Kang Zhu²  Zhaobo Lang¹  Cheng-Guo Duan¹  Bangshing Wang¹  Kai Tang¹  Huiming Zhang¹ 

[1] Department of Horticulture and Landscape Architecture, Purdue University, West Lafayette, IN 47907, USA;Shanghai Center for Plant Stress Biology, Shanghai Institute of Biological Sciences, Chinese Academy of Sciences, Beijing, Shijingshan, China

关键词: Nuclei fractionation;    Pol V;    Pol IV;    Scaffold RNA;    siRNA;    RNA-directed DNA methylation;    Epigenetics;   

Others  :  802424
DOI  :  10.1186/1746-4811-10-18

 received in 2014-05-15, accepted in 2014-06-05,  发布年份 2014

【 摘 要 】

Background

DNA methylation is a conserved epigenetic mark that controls genome stability, development and environmental responses in many eukaryotes. DNA methylation can be guided by non-coding RNAs that include small interfering RNAs and scaffold RNAs. Although measurement of DNA methylation and regulatory non-coding RNAs is desirable for many biologists who are interested in exploring epigenetic regulation in their areas, conventional methods have limitations and are technically challenging. For instance, traditional siRNA detection through RNA hybridization requires relatively large amount of small RNAs and involves radioactive isotopes. An alternative approach is RT-qPCR that employs stem loop primers during reverse transcription; however, it requires a prerequisite that the exact sequences of siRNAs should be known.

Results

By using the model organism Arabidopsis thaliana, we developed an easy-to-follow, integrative procedure for time-efficient, quantitative measurement of DNA methylation, small interfering RNAs, and scaffold RNAs. Starting with simplified nucleic acid manipulation, we examined DNA methylation levels by using Chop PCR (methylation-sensitive enzyme digestion followed by PCR), which allowed for fast screening for DNA methylation mutants without the need of transgenic reporters. We deployed a simple bioinformatics method for mining published small RNA databases, in order to obtain the nucleotide (nt) sequences of individual 24nt siRNAs within the regions of interest. The protocol of commercial TaqMan Small RNA Assay was subsequently optimized for reliable quantitative detection of individual siRNAs. We used nested qPCR to quantify scaffold RNAs that are of low abundance and without Poly-A tails. In addition, nuclei fraction enables separation of chromatin-associated scaffold RNAs from their cognate non-scaffold transcripts that have been released from chromatin.

Conclusions

We have developed a procedure for quantitative investigations on nucleic acids that are core components of RNA-directed DNA methylation. Our results not only demonstrated the efficacy of this procedure, but also provide lists of methylation-sensitive restriction enzymes, novel DNA methylation marker loci, and related siRNA sequences, all of which can be valuable for future epigenetic studies. Importantly, step-by-step protocols are provided in details such that the approaches can be easily followed by biologists with little experience in epigenetics.

【 授权许可】

   
2014 Zhang et al.; licensee BioMed Central Ltd.

【 图 表 】

Figure 1.

Figure 2.

【 参考文献 】

• [1]Matzke M, Kanno T, Daxinger L, Huettel B, Matzke AJM: RNA-mediated chromatin-based silencing in plants. Curr Opin Cell Biol 2009, 21:367-376.
• [2]Law JA, Jacobsen SE: Establishing, maintaining and modifying DNA methylation patterns in plants and animals. Nat Rev Genet 2010, 11:204-220.
• [3]Zhang H, Zhu JK: RNA-directed DNA methylation. Curr Opin Plant Biol 2011, 14:142-147.
• [4]Pikaard CS, Haag JR, Pontes OMF, Blevins T, Cocklin R: A transcription fork model for Pol IV and Pol V-dependent RNA-directed DNA methylation. Cold Spring Harb Symp Quant Biol 2012, 77:205-212.
• [5]Watanabe T, Tomizawa SI, Mitsuya K, Totoki Y, Yamamoto Y, Kuramochi-Miyagawa S, Iida N, Hoki Y, Murphy PJ, Toyoda A, Gotoh K, Hiura H, Arima T, Fujiyama A, Sado T, Shibata T, Nakano T, Lin H, Ichiyanagi K, Soloway PD, Sasaki H: Role for piRNAs and noncoding RNA in de novo DNA methylation of the imprinted mouse Rasgrf1 locus. Science 2011, 332:848-852.
• [6]Zhang H, Zhu JK: Active DNA demethylation in plants and animals. Cold Spring Harb Symp Quant Biol 2012, 77:161-173.
• [7]Saleh A, Alvarez-Venegas RUL, Avramova Z: An efficient chromatin immunoprecipitation (ChIP) protocol for studying histone modifications in Arabidopsis plants. Nat Protoc 2008, 3:1018-1025.
• [8]Kasinathan S, Orsi GA, Zentner GE, Ahmad K, Henikoff S: High-resolution mapping of transcription factor binding sites on native chromatin. Nat Methods 2014, 11:203-209.
• [9]Wierzbicki AT, Haag JR, Pikaard CS: Noncoding transcription by RNA polymerase Pol IVb/Pol V mediates transcriptional silencing of overlapping and adjacent genes. Cell 2008, 135:635-648.
• [10]He XJ, Hsu YF, Pontes O, Zhu J, Lu J, Bressan RA, Pikaard C, Wang CS, Zhu JK: NRPD4, a protein related to the RPB4 subunit of RNA polymerase II, is a component of RNA polymerases IV and V and is required for RNA-directed DNA methylation. Genes Dev 2009, 23:318-330.
• [11]Zhang H, Tang K, Qian W, Duan C, Wang B, Zhang H, Wang P, Zhu X, Lang Z, Yang Y, Zhu JK: An Rrp6-like protein positively regulates non-coding RNA levels and DNA methylation in Arabidopsis. Mol Cellin press
• [12]Xie Z, Johansen LK, Gustafson AM, Kasschau KD, Lellis AD, Zilberman D, Jacobsen SE, Carrington JC: Genetic and functional diversification of small RNA pathways in plants. PLoS Biol 2004, 2:E104.
• [13]Chen C, Ridzon DA, Broomer AJ, Zhou Z, Lee DH, Nguyen JT, Barbisin M, Xu NL, Mahuvakar VR, Andersen MR, Lao KQ, Livak KJ, Guegler KJ: Real-time quantification of microRNAs by stem-loop RT-PCR. Nucleic Acids Res 2005, 33:e179-e179.
• [14]Zhang X, Henderson IR, Lu C, Green PJ, Jacobsen SE: Role of RNA polymerase IV in plant small RNA metabolism. Proc Natl Acad Sci U S A 2007, 104:4536-4541.
• [15]Nakano M, Nobuta K, Vemaraju K, Tej SS, Skogen JW, Meyers BC: Plant MPSS databases: signature-based transcriptional resources for analyses of mRNA and small RNA. Nucleic Acids Res 2006, 34(Database issue):D731-5.
• [16]Mosher RA, Schwach F, Studholme D, Baulcombe DC: PolIVb influences RNA-directed DNA methylation independently of its role in siRNA biogenesis. Proc Natl Acad Sci U S A 2008, 105:3145-3150.
• [17]Zheng B, Wang Z, Li S, Yu B, Liu JY, Chen X: Intergenic transcription by RNA polymerase II coordinates Pol IV and Pol V in siRNA-directed transcriptional gene silencing in Arabidopsis. Genes Dev 2009, 23:2850-2860.
• [18]Zhang H, Ma ZY, Zeng L, Tanaka K, Zhang CJ, Ma J, Bai G, Wang P, Zhang SW, Liu ZW, Cai T, Tang K, Liu R, Shi X, He XJ, Zhu JK: DTF1 is a core component of RNA-directed DNA methylation and may assist in the recruitment of Pol IV. Proc Natl Acad Sci U S A 2013, 110:8290-8295.
• [19]Clarke JD: Cetyltrimethyl Ammonium Bromide (CTAB) DNA miniprep for plant DNA isolation. Cold Spring Harb Protoc 2009. doi: 10.1101/pdb.prot5177