Abstract
5-Methylcytosine is the major epigenetic modification occurring on DNA. It is known to be involved not only in gene expression regulation but also in the control of chromatin structure. However, this modification is also found on different types of RNA, including mRNA. Generally, biomolecular techniques are applied for studying the epigenetic profile of nucleic acids. Here, we describe the ultrastructural detection of 5-methylcytosine as an unusual approach to localize this modification on chromatin regions and/or RNA single molecules. This tool requires a careful sample preparation to preserve antigen epitopes that will be revealed immunocytochemically by a specific anti-5-methylcytosine antibody. The multiple staining procedures that can be adopted allow the identification of both DNA or RNA. A semiquantitative analysis can also be carried out.
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References
Cmarko D, Verschure PJ, Martin TE, Dahmus ME, Krause S, Fu XD, van Driel R, Fakan S (1999) Ultrastructural analysis of transcription and splicing in the cell nucleus after bromo-UTP microinjection. Mol Biol Cell 10(1):211–223
Cmarko D, Verschure PJ, Otte AP, van Driel R, Fakan S (2003) Polycomb group gene silencing proteins are concentrated in the perichromatin compartment of the mammalian nucleus. J Cell Sci 116(Pt 2):335–343
Puvion E, Puvion-Dutilleul F (1996) Ultrastructure of the nucleus in relation to transcription and splicing: roles of perichromatin fibrils and interchromatin granules. Exp Cell Res 229(2):217–225. https://doi.org/10.1006/excr.1996.0363
Spector DL (1996) Nuclear organization and gene expression. Exp Cell Res 229(2):189–197. https://doi.org/10.1006/excr.1996.0358
Choy MK, Movassagh M, Goh HG, Bennett MR, Down TA, Foo RS (2010) Genome-wide conserved consensus transcription factor binding motifs are hyper-methylated. BMC Genomics 11:519. https://doi.org/10.1186/1471-2164-11-519
Ferguson LR, Tatham AL, Lin Z, Denny WA (2011) Epigenetic regulation of gene expression as an anticancer drug target. Curr Cancer Drug Targets 11(2):199–212
Ngo TT, Yoo J, Dai Q, Zhang Q, He C, Aksimentiev A, Ha T (2016) Effects of cytosine modifications on DNA flexibility and nucleosome mechanical stability. Nat Commun 7:10813. https://doi.org/10.1038/ncomms10813
Jimenez-Useche I, Yuan C (2012) The effect of DNA CpG methylation on the dynamic conformation of a nucleosome. Biophys J 103(12):2502–2512. https://doi.org/10.1016/j.bpj.2012.11.012
Motorin Y, Lyko F, Helm M (2010) 5-methylcytosine in RNA: detection, enzymatic formation and biological functions. Nucleic Acids Res 38(5):1415–1430. https://doi.org/10.1093/nar/gkp1117
Squires JE, Preiss T (2010) Function and detection of 5-methylcytosine in eukaryotic RNA. Epigenomics 2(5):709–715. https://doi.org/10.2217/epi.10.47
Liu J, Jia G (2014) Methylation modifications in eukaryotic messenger RNA. J Genet Genomics 41(1):21–33. https://doi.org/10.1016/j.jgg.2013.10.002
Squires JE, Patel HR, Nousch M, Sibbritt T, Humphreys DT, Parker BJ, Suter CM, Preiss T (2012) Widespread occurrence of 5-methylcytosine in human coding and non-coding RNA. Nucleic Acids Res 40(11):5023–5033. https://doi.org/10.1093/nar/gks144
Dominissini D, Rechavi G (2017) 5-methylcytosine mediates nuclear export of mRNA. Cell Res 27(6):717–719. https://doi.org/10.1038/cr.2017.73
Harrison A, Parle-McDermott A (2011) DNA methylation: a timeline of methods and applications. Front Genet 2:74. https://doi.org/10.3389/fgene.2011.00074
Gehrke CW, McCune RA, Gama-Sosa MA, Ehrlich M, Kuo KC (1984) Quantitative reversed-phase high-performance liquid chromatography of major and modified nucleosides in DNA. J Chromatogr 301(1):199–219
Bestor TH, Hellewell SB, Ingram VM (1984) Differentiation of two mouse cell lines is associated with hypomethylation of their genomes. Mol Cell Biol 4(9):1800–1806
Frommer M, McDonald LE, Millar DS, Collis CM, Watt F, Grigg GW, Molloy PL, Paul CL (1992) A genomic sequencing protocol that yields a positive display of 5-methylcytosine residues in individual DNA strands. Proc Natl Acad Sci U S A 89(5):1827–1831
Weber M, Davies JJ, Wittig D, Oakeley EJ, Haase M, Lam WL, Schubeler D (2005) Chromosome-wide and promoter-specific analyses identify sites of differential DNA methylation in normal and transformed human cells. Nat Genet 37(8):853–862. https://doi.org/10.1038/ng1598
Huang TH, Perry MR, Laux DE (1999) Methylation profiling of CpG islands in human breast cancer cells. Hum Mol Genet 8(3):459–470
Gitan RS, Shi H, Chen CM, Yan PS, Huang TH (2002) Methylation-specific oligonucleotide microarray: a new potential for high-throughput methylation analysis. Genome Res 12(1):158–164. https://doi.org/10.1101/gr.202801
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):215–219. https://doi.org/10.1038/nature06745
Maunakea AK, Nagarajan RP, Bilenky M, Ballinger TJ, D'Souza C, Fouse SD, Johnson BE, Hong C, Nielsen C, Zhao Y, Turecki G, Delaney A, Varhol R, Thiessen N, Shchors K, Heine VM, Rowitch DH, Xing X, Fiore C, Schillebeeckx M, Jones SJ, Haussler D, Marra MA, Hirst M, Wang T, Costello JF (2010) Conserved role of intragenic DNA methylation in regulating alternative promoters. Nature 466(7303):253–257. https://doi.org/10.1038/nature09165
Santos F, Hendrich B, Reik W, Dean W (2002) Dynamic reprogramming of DNA methylation in the early mouse embryo. Dev Biol 241(1):172–182. https://doi.org/10.1006/dbio.2001.0501
Kobayakawa S, Miike K, Nakao M, Abe K (2007) Dynamic changes in the epigenomic state and nuclear organization of differentiating mouse embryonic stem cells. Genes Cells 12(4):447–460. https://doi.org/10.1111/j.1365-2443.2007.01063.x
Hussain S, Aleksic J, Blanco S, Dietmann S, Frye M (2013) Characterizing 5-methylcytosine in the mammalian epitranscriptome. Genome Biol 14(11):215. https://doi.org/10.1186/gb4143
Moyne G (1980) Methods in ultrastructural cytochemistry of the cell nucleus. Prog Histochem Cytochem 13(1):1–72
Masiello I, Biggiogera M (2017) Osmium ammine for staining DNA in electron microscopy. Methods Mol Biol 1560:261–267. https://doi.org/10.1007/978-1-4939-6788-9_19
Biggiogera M, Masiello I (2017) Visualizing RNA at electron microscopy by terbium citrate. Methods Mol Biol 1560:277–283. https://doi.org/10.1007/978-1-4939-6788-9_21
Dundr M, Raska I (1993) Nonisotopic ultrastructural mapping of transcription sites within the nucleolus. Exp Cell Res 208(1):275–281. https://doi.org/10.1006/excr.1993.1247
Trentani A, Testillano PS, Risueno MC, Biggiogera M (2003) Visualization of transcription sites at the electron microscope. Eur J Histochem 47(3):195–200
Bernhard W (1969) A new staining procedure for electron microscopical cytology. J Ultrastruct Res 27(3):250–265
Vazquez-Nin GH, Biggiogera M, Echeverria OM (1995) Activation of osmium ammine by SO2-generating chemicals for EM Feulgen-type staining of DNA. Eur J Histochem 39(2):101–106
Biggiogera M, Fakan S (1998) Fine structural specific visualization of RNA on ultrathin sections. J Histochem Cytochem 46(3):389–395. https://doi.org/10.1177/002215549804600313
Masiello I, Biggiogera M (2017) Ultrastructural localization of 5-methylcytosine on DNA and RNA. Cell Mol Life Sci 74(16):3057–3064. https://doi.org/10.1007/s00018-017-2521-1
Acknowledgments
The authors would like to thank Ms. Francine Flach for excellent technical skill in preparing the ultrathin sections.
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Masiello, I., Biggiogera, M. (2019). Electron Microscope Detection of 5-Methylcytosine on DNA and RNA. In: Wajapeyee, N., Gupta, R. (eds) Epitranscriptomics. Methods in Molecular Biology, vol 1870. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-8808-2_12
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DOI: https://doi.org/10.1007/978-1-4939-8808-2_12
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