Plant Meiosis pp 219-236 | Cite as

Chromatin Immunoprecipitation of Meiotically Expressed Proteins from Arabidopsis thaliana Flowers

  • Christophe Lambing
  • Kyuha Choi
  • Alexander R. Blackwell
  • Ian R. HendersonEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 2061)


During meiosis recombination occurs between homologous chromosomes which can result in reciprocal exchanges of genetic information, called crossovers. Crossover rate is heterogeneous within the genome, with local regions having a significantly higher recombination rate relative to the genome average. These regions are termed hotspots and typically occur with widths of kilobases. Therefore, there is a need to profile recombination factors at a similar resolution during meiosis via techniques such as chromatin immunoprecipitation (ChIP). Here we describe a ChIP protocol, combined with high throughput sequencing (ChIP-seq) optimised for analysis of meiotically expressed proteins in Arabidopsis thaliana flowers. We provide methods to (1) isolate nuclei and prepare the chromatin for shearing, (2) immunoprecipitate DNA molecules cross-linked to a protein of interest, (3) to size-select and purify immunoprecipitated DNA molecules, and (4) to prepare DNA sequencing libraries suitable for high-throughput sequencing. Together, these methods allow the detection of binding sites for meiotic proteins in the Arabidopsis genome at high resolution, which will provide insights into relationships between meiotic chromosome organization, chromatin and recombination.

Key words

Meiosis Chromatin immunoprecipitation Chromosome axis Recombination Arabidopsis 


  1. 1.
    Robert T, Nore A, Brun C, Maffre C, Crimi B, Bourbon HM, de Massy B (2016) The TopoVIB-like protein family is required for meiotic DNA double-strand break formation. Science 351:943–949CrossRefGoogle Scholar
  2. 2.
    Vrielynck N, Chambon A, Vezon D, Pereira L, Chelysheva L, De Muyt A, Mezard C, Mayer C, Grelon M (2016) A DNA topoisomerase VI-like complex initiates meiotic recombination. Science 351:939–943CrossRefGoogle Scholar
  3. 3.
    Panizza S, Mendoza MA, Berlinger M, Huang L, Nicolas A, Shirahige K, Klein F (2011) Spo11-accessory proteins link double-strand break sites to the chromosome axis in early meiotic recombination. Cell 146:372–383CrossRefGoogle Scholar
  4. 4.
    Stanzione M, Baumann M, Papanikos F, Dereli L, Lange J, Ramlal A, Trankner D, Shibuya H, de Massy B, Watanabe Y, Jasin M, Keeney S, Toth A (2016) Meiotic DNA break formation requires the unsynapsed chromosome axis-binding protein IHO1 (CCDC36) in mice. Nat Cell Biol 18:1208–1220CrossRefGoogle Scholar
  5. 5.
    Villeneuve AM, Hillers KJ (2001) Whence meiosis? Cell 106:647–650CrossRefGoogle Scholar
  6. 6.
    Yelina NE, Lambing C, Hardcastle TJ, Zhao X, Santos B, Henderson IR (2015) DNA methylation epigenetically silences crossover hot spots and controls chromosomal domains of meiotic recombination in Arabidopsis. Genes Dev 29:2188–2202CrossRefGoogle Scholar
  7. 7.
    Si W, Yuan Y, Huang J, Zhang X, Zhang Y, Zhang Y, Tian D, Wang C, Yang Y, Yang S (2015) Widely distributed hot and cold spots in meiotic recombination as shown by the sequencing of rice F2 plants. New Phytol 206:1491–1502CrossRefGoogle Scholar
  8. 8.
    Li X, Li L, Yan J (2015) Dissecting meiotic recombination based on tetrad analysis by single-microscope sequencing in maize. Nat Commun 24:6648CrossRefGoogle Scholar
  9. 9.
    Demirci S, van Dijk AD, Sanchez Perez G, Aflitos SA, de Ridder D, Peters SA (2017) Distribution, position and genomic characteristics of crossovers in tomato recombinant inbred lines derived from an interspecific cross between Solanum lycopersicum and Solanum pimpinellifolium. Plant J 89:554–564CrossRefGoogle Scholar
  10. 10.
    Kleckner N (2006) Chiasma formation: chromatin/axis interplay and the role(s) of the synaptonemal complex. Chromosoma 115:175–194CrossRefGoogle Scholar
  11. 11.
    Armstrong SJ, Caryl AP, Jones GH, Franklin FC (2002) Asy1, a protein required for meiotic chromosome synapsis, localizes to axis-associated chromatin in Arabidopsis and Brassica. J Cell Sci 115:3645–3655CrossRefGoogle Scholar
  12. 12.
    Nonomura K, Nakano M, Eiguchi M, Suzuki T, Kurata N (2006) PAIR2 is essential for homologous chromosome synapsis in rice meiosis I. J Cell Sci 119:217–225CrossRefGoogle Scholar
  13. 13.
    Ferdous M, Higgins JD, Osman K, Lambing C, Roitinger E, Mechtler K, Armstrong SJ, Perry R, Pradillo M, Cunado N, Franklin FC (2012) Inter-homolog crossing-over and synapsis in Arabidopsis meiosis are dependent on the chromosome axis protein AtASY3. PLoS Genet 8:e1002507CrossRefGoogle Scholar
  14. 14.
    Wang K, Wang M, Tang D, Shen Y, Qin B, Li M, Cheng Z (2011) PAIR3, an axis-associated protein, is essential for the recruitment of recombination elements onto meiotic chromosomes in rice. Mol Biol Cell 22:12–19CrossRefGoogle Scholar
  15. 15.
    Osman K, Yang J, Roitinger E, Lambing C, Heckmann S, Howell E, Cuacos M, Imre R, Durnberger G, Mechtler K, Armstrong S, Franklin FCH (2018) Affinity proteomics reveals extensive phosphorylation of the Brassica chromosome axis protein ASY1 and a network of associated proteins at prophase I of meiosis. Plant J 1:17–33CrossRefGoogle Scholar
  16. 16.
    Chambon A, West A, Vezon D, Horlow C, De Muyt A, Chelysheva L, Ronceret A, Darbyshire AR, Osman K, Heckmann S, Franklin FCH, Grelon M (2018) Identification of ASYNAPTIC4, a component of the meiotic chromosome axis. Plant Physiol 178:233–246CrossRefGoogle Scholar
  17. 17.
    Cai X, Dong F, Edelmann RE, Makaroff CA (2003) The Arabidopsis SYN1 cohesin protein is required for sister chromatid arm cohesion and homologous chromosome pairing. J Cell Sci 116:2999–3007CrossRefGoogle Scholar
  18. 18.
    Golubovskaya IN, Hamant O, Timofejeva L, Wang CJ, Braun D, Meeley R, Cande WZ (2006) Alleles of afd1 dissect REC8 functions during meiotic prophase I. J Cell Sci 119:3306–3015CrossRefGoogle Scholar
  19. 19.
    Lam WS, Yang X, Makaroff CA (2005) Characterization of Arabidopsis thaliana SMC1 and SMC3: evidence that AtSMC3 may function beyond chromosome cohesion. J Cell Sci 118:3037–3048CrossRefGoogle Scholar
  20. 20.
    Sanchez-Moran E, Santos JL, Jones GH, Franklin FC (2007) ASY1 mediates AtDMC1-dependent interhomolog recombination during meiosis in Arabidopsis. Genes Dev 21:2220–2233CrossRefGoogle Scholar
  21. 21.
    Zhang L, Wang S, Yin S, Hong S, Kim KP, Kleckner N (2014) Topoisomerase II mediates meiotic crossover interference. Nature 511:551–556CrossRefGoogle Scholar
  22. 22.
    Libuda DE, Uzawa S, Meyer BJ, Villeneuve AM (2013) Meiotic chromosome structures constrain and respond to designation of crossover sites. Nature 502:703–706CrossRefGoogle Scholar
  23. 23.
    Lambing C, Heckmann S (2018) Tackling plant meiosis: from model research to crop improvement. Front Plant Sci 9:829CrossRefGoogle Scholar
  24. 24.
    Lambing C, Osman K, Nuntasoontorn K, West A, Higgins JD, Copenhaver GP, Yang J, Armstrong SJ, Mechtler K, Roitinger E, Franklin FC (2015) Arabidopsis PCH2 mediates meiotic chromosome remodelling and maturation of crossovers. PLoS Genet 11:e1005372CrossRefGoogle Scholar
  25. 25.
    Kaufmann K, Muino JM, Osteras M, Farinelli L, Krajewski P, Angenent GC (2010) Chromatin immunoprecipitation (ChIP) of plant transcription factors followed by sequencing (ChIP-SEQ) or hybridization to whole genome arrays (ChIP-ChIP). Nat Protoc 5:457–472CrossRefGoogle Scholar
  26. 26.
    Kugou K, Ohta K (2009) Genome-wide high-resolution chromatin immunoprecipitation of meiotic chromosomal proteins in Saccharomyces cerevisiae. Methods Mol Biol 557:285–304CrossRefGoogle Scholar
  27. 27.
    Ma H (2006) A molecular portrait of Arabidopsis meiosis. Arabidopsis Book 4:e0095CrossRefGoogle Scholar
  28. 28.
    Smyth DR, Bowman JL, Meyerowitz EM (1990) Early flower development in Arabidopsis. Plant Cell 2:755–767PubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2020

Authors and Affiliations

  • Christophe Lambing
    • 1
  • Kyuha Choi
    • 1
    • 2
  • Alexander R. Blackwell
    • 1
  • Ian R. Henderson
    • 1
    Email author
  1. 1.Department of Plant SciencesUniversity of CambridgeCambridgeUK
  2. 2.Department of Life SciencesPohang University of Science and TechnologyPohangRepublic of Korea

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