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Specialized Technologies for Epigenetics in Plants

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Genomic Imprinting

Part of the book series: Methods in Molecular Biology ((MIMB,volume 925))

Abstract

Plants are excellent systems for discovering and studying epigenetic phenomena, such as transposon silencing, RNAi, imprinting, and DNA methylation. Imprinting, referring to preferential expression of maternal or paternal alleles, plays an important role in reproduction development of both mammals and plants. DNA methylation is critical for determining whether the maternal or paternal alleles of an imprinted gene is expressed or silenced. In flowering plants, there is a double fertilization event in reproduction: one sperm fertilizes the egg cell to form embryo and a second sperm fuses with the central cell to give rise to endosperm. Endosperm is the tissue where imprinting occurs in plants. MEDEA (MEA), a SET domain Polycomb group gene, was the first plant gene shown to be imprinted in endosperm, and its maternal expression is controlled by DNA methylation and demethylation. Recently there has been significant progress in identifying imprinted genes as well as understanding molecular mechanisms of imprinting in plants. Up to date, approximately 350 genes were found to have differential parent-of-origin expression in plant endosperm (Arabidopsis, corn, and rice). In Arabidopsis, many imprinted genes are regulated by the DNA METHYLTRANSFERASE1 (MET1) and the DNA-demethylating glycosylase DEMETER (DME), and/or their chromatin states regulated by Polycomb group proteins (PRC2). There are also maternally expressed genes regulated by unknown mechanisms in endosperm. In this protocol, we describe in detail how to perform a genetic cross, isolate the endosperm tissue from seed, determine the imprinting status of a gene, and analyze DNA methylation of imprinted genes by bisulfite sequencing in Arabidopsis.

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References

  1. McClintock B (1951) Chromosome organization and genic expression. Cold Spring Harbor Symp Quant Biol 16:13–47

    Article  PubMed  CAS  Google Scholar 

  2. McClintock B (1965) The control of gene action in maize. Brookhaven Symp Biol 18:162–184

    Google Scholar 

  3. Napoli C, Lemieux C, Jorgensen R (1990) Introduction of a chimeric chalcone synthase gene into petunia results in reversible co-suppression of homologous genes in trans. Plant Cell 2:279–289

    PubMed  CAS  Google Scholar 

  4. Wassenegger M, Heimes S, Sanger HL (1994) An infectious viroid RNA replicon evolved from an in vitro-generated non-infectious viroid deletion mutant via a complementary deletion in vivo. EMBO J 13:6172–6177

    PubMed  CAS  Google Scholar 

  5. Feil R, Berger F (2007) Convergent evolution of genomic imprinting in plants and mammals. Trends Genet 23:192–199

    Article  PubMed  CAS  Google Scholar 

  6. Haig D, Westoby M (1989) Parent-specific gene expression and the triploid endosperm. Am Nat 134:147–155

    Article  Google Scholar 

  7. Moore T, Haig D (1991) Genomic imprinting in mammalian development: a parental tug-of-war. Trends Genet 7:45–48

    PubMed  CAS  Google Scholar 

  8. Gregg C et al (2010) High-resolution analysis of parent-of-origin allelic expression in the mouse brain. Science 329:643–648

    Article  PubMed  CAS  Google Scholar 

  9. Constancia M, Kelsey G, Reik W (2004) Resourceful imprinting. Nature 432:53–57

    Article  PubMed  CAS  Google Scholar 

  10. Hsieh TF et al (2011) Regulation of imprinted gene expression in Arabidopsis endosperm. Proc Natl Acad Sci U S A 108:1755–1762

    Article  PubMed  CAS  Google Scholar 

  11. Jahnke S, Scholten S (2009) Epigenetic resetting of a gene imprinted in plant embryos. Curr Biol 19:1677–1681

    Article  PubMed  CAS  Google Scholar 

  12. Hermon P, Srilunchang KO, Zou J, Dresselhaus T, Danilevskaya ON (2007) Activation of the imprinted Polycomb Group Fie1 gene in maize endosperm requires demethylation of the maternal allele. Plant Mol Biol 64:387–395

    Article  PubMed  CAS  Google Scholar 

  13. Danilevskaya ON et al (2003) Duplicated fie genes in maize: expression pattern and imprinting suggest distinct functions. Plant Cell 15:425–438

    Article  PubMed  CAS  Google Scholar 

  14. He G et al (2010) Global epigenetic and transcriptional trends among two rice subspecies and their reciprocal hybrids. Plant Cell 22:17–33

    Article  PubMed  CAS  Google Scholar 

  15. Wolff P et al (2011) High-resolution analysis of parent-of-origin allelic expression in the arabidopsis endosperm. PLoS Genet 7:e1002126

    Article  PubMed  CAS  Google Scholar 

  16. Luo M, Taylor JM, Spriggs A, Zhang H, Wu X, Russell S, Singh M, Koltunow A (2011) A genome-wide survey of imprinted genes in rice seeds reveals imprinting primarily occurs in the endosperm. PLoS Genet 7:e1002125

    Article  PubMed  CAS  Google Scholar 

  17. Xiao W et al (2003) Imprinting of the MEA Polycomb gene is controlled by antagonism between MET1 methyltransferase and DME glycosylase. Dev Cell 5:891–901

    Article  PubMed  CAS  Google Scholar 

  18. Gehring M et al (2006) DEMETER DNA glycosylase establishes MEDEA polycomb gene self-imprinting by allele-specific demethylation. Cell 124:495–506

    Article  PubMed  CAS  Google Scholar 

  19. Makarevich G, Villar CB, Erilova A, Kohler C (2008) Mechanism of PHERES1 imprinting in Arabidopsis. J Cell Sci 121:906–912

    Article  PubMed  CAS  Google Scholar 

  20. Villar CB, Erilova A, Makarevich G, Trosch R, Kohler C (2009) Control of PHERES1 imprinting in Arabidopsis by direct tandem repeats. Mol Plant 2:654–660

    Article  PubMed  CAS  Google Scholar 

  21. Autran D et al (2011) Maternal epigenetic pathways control parental contributions to Arabidopsis early embryogenesis. Cell 145:707–719

    Article  PubMed  CAS  Google Scholar 

  22. Frommer M et al (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 89:1827–1831

    Article  PubMed  CAS  Google Scholar 

  23. Clark SJ, Harrison J, Paul CL, Frommer M (1994) High sensitivity mapping of methylated cytosines. Nucleic Acids Res 22:2990–2997

    Article  PubMed  CAS  Google Scholar 

  24. Rea M et al (2011) Determination of DNA methylation of imprinted genes in Arabidopsis endosperm. J Vis Exp 47, http://www.jove.com/index/Details.stp?ID=2327, doi: 10.3791/2327

  25. Paulin R, Grigg GW, Davey MW, Piper AA (1998) Urea improves efficiency of bisulphite-mediated sequencing of 5′-methylcytosine in genomic DNA. Nucleic Acids Res 26:5009–5010

    Article  PubMed  CAS  Google Scholar 

  26. Smyth DR, Bowman JL, Meyerowitz EM (1990) Early flower development in Arabidopsis. Plant Cell 2:755–767

    PubMed  CAS  Google Scholar 

  27. Kinoshita T, Yadegari R, Harada JJ, Goldberg RB, Fischer RL (1999) Imprinting of the MEDEA polycomb gene in the Arabidopsis endosperm. Plant Cell 11:1945–1952

    PubMed  CAS  Google Scholar 

  28. Rogers SO, Bendich AJ (1988) Extraction of DNA from plant tissues. Plant Mol Biol Manual A6:1–10

    Google Scholar 

  29. Jacobsen SE, Sakai H, Finnegan EJ, Cao X, Meyerowitz EM (2000) Ectopic hypermethylation of flower-specific genes in Arabidopsis. Curr Biol 10:179–186

    Article  PubMed  CAS  Google Scholar 

  30. Gehring M, Bubb KL, Henikoff S (2009) Extensive demethylation of repetitive elements during seed development underlies gene imprinting. Science 324:1447–1451

    Article  PubMed  CAS  Google Scholar 

  31. Cokus SJ et al (2008) Shotgun bisulphite sequencing of the Arabidopsis genome reveals DNA methylation patterning. Nature 452:215–219

    Article  PubMed  CAS  Google Scholar 

  32. Hsieh TF et al (2009) Genome-wide demethylation of Arabidopsis endosperm. Science 324:1451–1454

    Article  PubMed  CAS  Google Scholar 

  33. Lister R et al (2008) Highly integrated single-base resolution maps of the epigenome in Arabidopsis. Cell 133:523–536

    Article  PubMed  CAS  Google Scholar 

  34. Lister R et al (2009) Human DNA methylomes at base resolution show widespread epigenomic differences. Nature 462:315–322

    Article  PubMed  CAS  Google Scholar 

  35. Lister R et al (2011) Hotspots of aberrant epigenomic reprogramming in human induced pluripotent stem cells. Nature 471:68–73

    Article  PubMed  CAS  Google Scholar 

  36. Hayatsu H, Negishi K, Wataya Y (2009) Progress in the bisulfite modification of nucleic acids. Nucleic Acids Symp Ser 53:217

    Article  CAS  Google Scholar 

  37. Henderson IR, Chan SR, Cao X, Johnson L, Jacobsen SE (2010) Accurate sodium bisulfite sequencing in plants. Epigenetics 5:47–49

    Article  PubMed  CAS  Google Scholar 

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Acknowledgments

The author thanks colleagues in the lab for discussion and Dr. Tzung-Fu Hsieh for critical reading of the manuscript. This work is supported by startup fund from Saint Louis University and National Institutes of Health grants 1R15GM086846-01 and 3R15GM086846-01S1.

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Authors and Affiliations

  1. Department of Biology, Saint Louis University, St. Louis, MO, USA

    Wenyan Xiao

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  1. Wenyan Xiao
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Correspondence to Wenyan Xiao .

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Editors and Affiliations

  1. School of Medicine, Fels Institute, Temple University, N. Broad Street 3307, Philadelphia, 19140, Pennsylvania, USA

    Nora Engel

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Xiao, W. (2012). Specialized Technologies for Epigenetics in Plants. In: Engel, N. (eds) Genomic Imprinting. Methods in Molecular Biology, vol 925. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-011-3_16

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  • DOI: https://doi.org/10.1007/978-1-62703-011-3_16

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  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-62703-010-6

  • Online ISBN: 978-1-62703-011-3

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